Compositions and methods for amplifying or detecting varicella zoster virus

Abstract

To provide compositions and methods for amplifying or detecting Varicella zoster virus.SOLUTION: Disclosed herein is a reagent and a method for amplifying part of DNA polymerase gene or glycoprotein B gene of Varicella zoster virus (VZV). Disclosed are an oligonucleotide composition comprising an oligonucleotide probe and a primer pair and a method for providing highly sensitive and specific detection of a target nucleic acid sequence of VZV or an amplicon formed from the target nucleic acid sequence when Varicella zoster virus (VZV) is present in a sample. The presence of VZV in a sample can be detected by using the oligonucleotide, the composition and the method.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority of U.S. Provisional Application No. 62 / 739,571, filed on October 1, 2018, which is incorporated herein by reference.

[0002] Sequence Listing The sequence listing filed as 536442_SeqListing_ST25.txt, which is 17 kilobytes in size, was created on September 30, 2019, and is incorporated herein by reference.

[0003] Embodiments herein are directed to the field of detecting infectious pathogens. Specifically, the claimed compositions, kits, methods, formulations, and reaction mixtures are designed to detect viruses such as varicella - zoster virus.

Background Art

[0004] Varicella - zoster virus (VZV) is a highly infectious human virus belonging to the family Alphaherpesviridae. The VZV genome is a linear double - stranded DNA molecule 124,884 nucleotides in length. Primary infection via skin lesions or direct exposure through airborne transmission causes chickenpox. After infection, the virus persists latently in the nervous system of the infected person. Subsequently, VZV can reactivate later in life and induce secondary infections such as shingles. In some cases, VZV infection can also cause additional complications such as hepatitis, pancreatitis, pneumonia, encephalitis, bronchitis, and bacterial superinfection. Currently, there is a need to detect VZV with high sensitivity, specificity, and speed.

Summary of the Invention

Means for Solving the Problems

[0005] Provided herein are amplification oligonucleotides, oligonucleotide compositions, kits, reaction mixtures, formulations, and methods for the sensitive and specific amplification and / or detection of VZV or VZV target nucleic acid sequences. The amplification oligonucleotides include amplification primers for amplifying the target nucleic acid sequences and detection probes for detecting the target sequences or amplified sequences. The amplification oligonucleotides, oligonucleotide compositions, kits, reaction mixtures, and formulations described are suitable for use in nucleic acid-based detection techniques, which include, but are not limited to, amplification techniques such as polymerase chain reaction (PCR) and real-time PCR techniques. The amplification oligonucleotides, oligonucleotide compositions, kits, reaction mixtures, formulations, and methods described provide for the rapid detection and / or quantification of VZV. The present disclosure is aimed at meeting these needs, providing other benefits, or at least providing useful alternatives to the public.

[0006] Definitions To aid in the understanding of aspects of the present disclosure, some terms used herein are defined in more detail. All other scientific and / or technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the relevant art, or the same meaning as provided in the Dictionary of Microbiology and Molecular Biology, 2nd ed. (Singleton et al., 1994, John Wiley & Sons, New York, NY) and The Harper Collins Dictionary of Biology (Hale & Marham, 1991, Harper Perennial, New York, NY). Unless otherwise noted, the techniques used or contemplated herein are standard methods well known to one of ordinary skill in molecular biology.

[0007] Before describing the present teachings in detail, it is to be understood that the present disclosure is not limited to particular compositions or process steps, as these may vary. It should be noted that, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. For example, as used herein, "nucleic acid" is understood to represent one or more nucleic acids. Thus, the terms "a" (or "an"), "one or more", and "at least one" may be used interchangeably herein. Accordingly, references to "oligomer" may include plural oligomers. The conjunction "or" should be interpreted in an inclusive sense (e.g., as equivalent to "and / or") unless the context clearly dictates otherwise.

[0008] It will be understood that, as used herein, there is an implied "about" associated with the temperatures, concentrations, times, etc. discussed in the present disclosure, such that slight and non-substantive deviations are within the scope of the present teachings. In general, the term "about" indicates a non-substantive variation in the amount of a component of a composition that does not significantly affect the activity or stability of the composition. All ranges should be interpreted to include the endpoints, unless a clear exclusion such as "excluding the endpoints" is specified. For example, "within 10 to 15" includes the values 10 and 15. Further, to the extent practical, ranges include all whole integers and fractional values between the endpoints. Any material incorporated by reference is superseded by the explicit content of the present disclosure to the extent that the incorporated material is inconsistent therewith.

[0009] Unless otherwise specified, embodiments of this specification that recite "comprising" various components are also contemplated as "consisting of" or "consisting essentially of" the recited components, and embodiments of this specification that recite "consisting of" various components are also contemplated as "comprising" or "consisting essentially of" the recited components, and embodiments of this specification that recite "consisting essentially of" various components are also contemplated as "consisting of" or "comprising" the recited components (this interchangeability does not apply to the use of these terms in the claims). "Consisting essentially of" means that additional component(s), composition(s), or method step(s) that do not substantially change the basic and novel features of the compositions and methods described herein may be included in these compositions or methods. Such features include the ability to detect a target nucleic acid sequence located within a target nucleic acid region from a VZV nucleic acid sequence in a sample, thereby indicating the presence of VZV in the sample as contrasted with other known viruses.

[0010] "Sample" includes any specimen that contains, or is suspected of containing, VZV, or a component thereof, such as a nucleic acid, nucleic acid fragment, or nucleic acid derived from VZV. The sample can be derived from any source, for example, but not limited to, biological specimens, clinical specimens, and environmental sources. A biological sample includes any tissue or material derived from a mammal or organism (alive or dead) that may contain VZV or a target nucleic acid sequence derived from VZV, for example, respiratory tissue or effusion such as bronchoscopy, bronchoalveolar lavage (BAL) or lung biopsy, sputum, saliva, peripheral blood, plasma, serum, lymph nodes, gastrointestinal tissue, feces, urine, semen or other body fluids or materials or lesion swabs. In some embodiments, laboratories may test plasma / serum or lesion swabs to test for VZV. Tests such as plasma / serum tests can be performed before and / or after a medical or surgical procedure, which includes, for example, but is not limited to, transplantation. In some embodiments, lesion swabs can be used to assess the presence of VZV, such as in chickenpox. A biological sample can be physically, chemically, or mechanically treated to disrupt the tissue or cell structure to release intracellular components into solution. The solution further includes enzymes, buffers, salts, surfactants, etc., which are used to prepare biological samples for analysis using standard methods. In some embodiments, the sample can include a processed sample such as a sample obtained by passing the sample through a filtration device, or a sample obtained after centrifugation, or a sample obtained by attachment to a medium, matrix, or support.

[0011] The term "analog" is used to define two or more structures that have common features. The term "structural analog" refers to an object such as a chemical compound that shares a similar structural architecture with another compound. Despite showing common structural similarity, each analog may have different biochemical properties. Alternatively, a "functional analog" refers to two or more objects such as compounds that may have structurally different analogs but share the same mechanism of action (or biochemical properties).

[0012] The term "portion" is used to indicate a group or functional group within a molecule that is involved in one or more distinguishable biochemical properties of the molecule.

[0013] As used interchangeably herein, "nucleic acid" or "polynucleotide" refers to a polymeric compound composed of nucleotides (or nucleotide analogs). Conventional examples of polynucleotides include ribonucleic acid (RNA), deoxyribonucleic acid (DNA), mixed RNA-DNA, and polymers (substances having a molecular structure consisting of repeating nucleotide subunits). A polynucleotide "backbone" can be composed of various linkages including a sugar-phosphate diester linkage, a peptide-nucleic acid linkage, peptide nucleic acid (PCT WO95 / 32305), a phosphorothioate linkage, a methylphosphonate linkage, or one or more combinations thereof. When referring to the length range of a polynucleotide, or other oligonucleotide, it will be understood that the range includes all integers (e.g., a length of 19 to 25 consecutive nucleotides includes 19, 20, 21, 22, 23, 24, and 25).

[0014] A "nucleotide" is a compound that includes a single five-carbon (pentose) sugar moiety, a nitrogen heterocyclic base, and one to three phosphate groups. As the basic unit, nucleotides are covalently bonded to form nucleic acids. The sugar moiety of each nucleotide is ribose (RNA), 2'-deoxyribose (DNA), or analogs thereof, including analogous compounds having substitutions (e.g., 2'-methoxy substitution or 2'-halide substitution). In addition to the pentose sugar moiety, each nucleotide contains a nitrogen heterocyclic base bonded to the pentose ring via a glycosidic bond. Conventional examples of nitrogen heterocyclic bases include purines (e.g., adenine (A) and guanine (G)) and pyrimidines (e.g., cytosine (C), thymine (T), and uracil (U)). Purine bases are composed of a six-atom ring and a five-atom ring joined by two shared atoms. Pyrimidine bases are composed of a six-atom ring. Generally, deoxyribonucleotide triphosphates (dNTPs) are used generically when considering the four deoxyribonucleotides, namely, dATP, dCTP, dGTP, and dTTP. The nitrogen heterocyclic base is also its non-conventional analog (e.g., inosine (I) or others, see The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11th ed., 1992), or can be an analogous derivative of purines or pyrimidines. Furthermore, a polynucleotide may contain one or more "abasic" residues, in which case the backbone does not contain a nitrogenous base at the position(s) of the polymer (U.S. Patent No. 5,585,481). In addition to conventional polynucleotide formation, a polynucleotide may form an analog that includes one or more locked nucleic acid (LNA) nucleotide monomers having a bicyclic furanose unit locked in a sugar conformation that mimics RNA, and the conformation enhances the hybridization affinity for complementary RNA and DNA sequences (Vester and Wengel, 2004, Biochemistry 43(42):13233-41). Embodiments of oligomers that can affect the stability of the hybridization complex include peptide nucleic acid oligomers, oligomers containing 2'-methoxy or 2'-fluoro substituted RNA, oligomers that affect the overall charge, charge density, and steric association of the hybridization complex (including oligomers containing charged linkages such as phosphorothioates), or neutral groups (e.g., methylphosphonate). Unless otherwise indicated, 5-methylcytosine can be used with any of the aforementioned backbones / sugars / linkages including an RNA or DNA backbone (or mixtures thereof).

[0015] "Oligomer", "oligonucleotide", or "oligo" refers to a polymer composed of two or more linked nucleoside subunits or nucleic acid base subunits. Oligonucleotides can be DNA and / or RNA and their analogs. In some embodiments, the oligomer is in a size range having a lower limit of 5 to 21 nucleic acid bases and an upper limit of 18 to 500 nucleic acid bases. In some embodiments, the oligomer is in a size range of 10 to 100 nucleic acid bases, 10 to 90 nucleic acid bases, 10 to 80 nucleic acid bases, 10 to 70 nucleic acid bases, or 10 to 60 nucleic acid bases. In some embodiments, the oligomer is in a size range having a lower limit of about 15, 16, 17, 18, 19, 20, or 21 nucleic acid bases and an upper limit of about 18 - 50 or 18 - 100 nucleic acid bases. In some embodiments, the oligomer is in a size range having a lower limit of about 10 - 21 nucleic acid bases and an upper limit of about 18 - 100 nucleic acid bases. The oligomer is not composed of wild-type chromosomal DNA or its in vivo transcription products. The oligomer can be synthetically made by using any well-known in vitro chemical or enzymatic method and can be purified after synthesis by using standard methods, such as high-performance liquid chromatography (HPLC). The oligomer can be referred to by a functional name (e.g., detection probe or amplification primer). The term oligonucleotide is generally used to encompass all such reagents described herein and does not indicate any particular function for the reagent.

[0016] The term "annealing" or "anneal" describes the process by which two complementary strands of nucleic acid bind by base pair (bp) hybridization. Generally, one of ordinary skill in molecular biology will understand that annealing (for PCR-related) is possible at a temperature 5°C lower than the calculated melting temperature (Tm) during the exponential phase of the amplification reaction.

[0017] The term "hybridization" or "hybridizing" describes the formation of hydrogen bonds between nucleotide subunits of two complementary strands of nucleic acid.

[0018] The term "nucleic acid hybrid" or "hybrid" or "duplex" refers to a nucleic acid structure consisting of a double-stranded region joined via hydrogen bonds (base pairing), where each strand is sufficiently complementary to the other. Examples of hybrids include RNA:RNA, RNA:DNA, or DNA:DNA double-stranded molecules.

[0019] The terms "complementary" or "sufficiently complementary" refer to a particular nucleotide base pairing relationship between two single-stranded polynucleotides (e.g., an amplification oligonucleotide and a target nucleic acid sequence), or between two different regions of the same single-stranded polynucleotide (e.g., a molecular beacon), which allows hybridization (e.g., the formation of a stable double-stranded hybrid). Complementary sequences need not be completely complementary (100% complementary) to form a stable double-stranded. In some embodiments, partially complementary (less than 100% complementarity, due to mismatches with standard nucleic acid base pairing) sequences remain sufficiently complementary if the polynucleotide sequences can anneal. The percentage of complementarity indicates the percentage of bases in a continuous strand in a first nucleic acid sequence that can form hydrogen bonds (e.g., Watson-Crick base pairs) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, or 10 out of 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary). The percentage of complementarity is calculated in a similar manner to the percentage of identity. Purine bases bind to pyrimidine bases according to base pairing rules indicating pairs of adenine with thymine or uracil (A and T or U) and only cytosine with guanine (C and G). It should be noted that base pairing can also occur between bases that are not members of these conventional (e.g., "canonical") pairs. Non-canonical base pairing is well known to those skilled in the art of molecular biology (see, e.g., R.L.P. Adams et al., The Biochemistry of the Nucleic Acids (11th ed. 1992)). Appropriate hybridization conditions are well known to those skilled in the art of molecular biology and can be predicted based on sequence composition or determined empirically using routine testing (e.g., §§ 1.90-1.91, 7.37-7.57, 9.47-9.51, and 11.47-11.57 of Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed., particularly §§ 9.50-9.51, 11.12-11.13, 11.45-11.47, and 11.55-11.57).

[0020] Sequence identity can be determined by using algorithms such as BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0 (Genetics Computer Group, 575 Science Dr., Madison, Wis.), aligning the sequences using the default gap parameters, or by inspection and the best alignment (i.e., resulting in the highest percentage of sequence similarity over the entire comparison window). The percentage of sequence identity is calculated by comparing two optimally aligned sequences over the entire comparison window, determining the number of positions where identical residues occur in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions of matched and mismatched positions without counting the gaps (i.e., the window size) within the comparison window, and multiplying the result by 100 to yield the percentage of sequence identity. Unless otherwise indicated, the comparison window between two sequences is defined by the full length of the shorter of the two sequences.

[0021] “Self-complementarity” refers to an oligonucleotide containing internal complementary sequences that can hybridize to each other, creating a double-stranded structure or region within the oligonucleotide. Depending on the position of the complementary sequences within the oligonucleotide, hybridization of the sequences may lead to the formation of a hairpin loop, a junction, a bulge, or an internal loop. In some embodiments, the self-complementary sequences can each be 4 to 6 nucleobases in length. In some embodiments, the self-complementary sequences are located at the 5' and 3' termini of the oligonucleotide. In some embodiments, the self-complementary sequences can be added to the 5' or 3' terminus of an oligonucleotide such as a detection probe.

[0022] The term "configured to specifically hybridize" means that the specific intention and intended use of certain particular oligonucleotides are explicitly selected based on the desire to amplify or detect a target nucleic acid sequence of VZV. For example, amplification primers configured to generate a specific amplicon from a specific target nucleic acid sequence utilize specific forward and reverse amplification oligos that provide accurate hybridization to the target sequence if a target oligohybridization sequence located within the target nucleic acid region of VZV is present in the sample, and generate the targeted PCR product (e.g., amplicon). One of ordinary skill in the art understands that being configured to specifically hybridize does not mean hybridizing exclusively, as some low level of hybridization to non-target nucleic acids can occur.

[0023] "Preferentially hybridize" or "preferential hybridization" means that under stringent hybridization conditions, an amplification oligonucleotide can hybridize to its target nucleic acid to form a stable oligonucleotide:target hybrid, but is unable to form a sufficient number of stable oligonucleotide:non-target hybrids. Amplification oligonucleotides that preferentially hybridize to a target nucleic acid are useful for the amplification and detection of the target nucleic acid, but not particularly useful for non-target nucleic acids, especially in organisms that are phylogenetically closely related. Thus, the amplification oligonucleotide hybridizes to the target nucleic acid to a sufficiently greater extent than to non-target nucleic acids, enabling one of ordinary skill in the art to accurately amplify and / or detect the presence (or absence) of nucleic acids derived from a particular VZV, as needed. In general, decreasing the degree of complementarity between an oligonucleotide sequence and its target sequence will reduce the specificity or ratio of oligonucleotide hybridization to that target region. However, including one or more non-complementary nucleosides or nucleobases can enhance the ability of the oligonucleotide to discriminate against non-target organisms.

[0024] Preferred hybridization can be evaluated using techniques known in the art and described herein, such as in the examples provided below. In some embodiments, there is at least a 10-fold difference, at least a 20-fold difference, at least a 50-fold difference, at least a 100-fold difference, at least a 200-fold difference, at least a 500-fold difference, or at least a 1,000-fold difference between the target hybridization signal and the non-target hybridization signal in the test sample. In some embodiments, the non-target hybridization formation signal in the test sample is below the background signal level.

[0025] The term "stringent hybridization conditions" means conditions that allow an oligomer to preferentially hybridize to a target nucleic acid sequence rather than to nucleic acids derived from closely related non-target nucleic acids. The reaction environment that can be used for stringent hybridization can vary depending on factors including the GC content and length of the oligomer, the degree of similarity between the oligomer sequence and non-target nucleic acid sequences that may be present in the test sample, and the target sequence. Hybridization conditions include temperature and the composition of the hybridization reagents or solutions. Specific hybridization assay conditions are described later in the Examples section. Other acceptable stringent hybridization conditions can be readily identified by those skilled in the art.

[0026] As used herein, the term "substantially corresponding" means a situation where an oligomer can anneal to a complementary oligohybridization sequence in a target nucleic acid and enables accurate hybridization or detection to the target nucleic acid sequence in the sample (in the presence of other nucleic acids found in the test sample). In certain embodiments, the oligonucleotide "substantially corresponds" to an oligohybridization sequence with a complementarity base pairing in the range of 100% to about 80%, 100% to about 85%, or 100% to about 90%, or 100% to about 95%. The degree of complementarity can also be described in terms of the number of nucleotide substitutions or nucleotide mismatches within the sequence.

[0027] A "homologous sequence" is a continuous nucleotide sequence that is similar to the continuous nucleotide sequence of a target nucleic acid sequence but ultimately is not the intended target of an amplification primer or detection probe. Therefore, when designing amplification oligonucleotides for real-time PCR, by selecting an oligohybridization sequence unique to the target nucleic acid sequence, the possibility that the amplification oligonucleotide anneals to and amplifies a homologous sequence is reduced.

[0028] The terms "non-target-specific sequence" or "non-target hybridization sequence" refer to regions of an oligomer that do not anneal to a complementary oligohybridization sequence of a target nucleic acid under standard hybridization conditions. Such non-target-specific sequences can be complementary to a part of the target-specific sequence of an oligonucleotide. Examples of oligomers having non-target-specific sequences include, but are not limited to, molecular beacons.

[0029] "Sense" and "antisense" are used to describe two complementary polynucleotide strands that run in opposite directions (sequenced 5' to 3'). As an example, double-stranded DNA is composed of a sense strand and an antisense strand of antiparallel strands. The antisense strand functions as a template for transcription and contains a nucleotide sequence complementary to the transcribed mRNA.

[0030] Generally, one of ordinary skill in the art of molecular biology will understand that the phrase "or its complement", or "RNA equivalent", or "its DNA / RNA chimera" associated with a DNA sequence includes (in addition to the reference DNA sequence) the complement of the DNA sequence, the RNA equivalent of the reference DNA sequence, the RNA equivalent of the complement of the reference DNA sequence, the DNA / RNA chimera of the reference DNA sequence, and the DNA / RNA chimera of the complement of the reference DNA sequence. Similarly, the phrase "or its complement", or "RNA equivalent", or "its DNA / RNA chimera" associated with an RNA sequence includes (in addition to the reference RNA sequence) the complement of the RNA sequence, the DNA equivalent of the reference RNA sequence, the DNA equivalent of the complement of the reference RNA sequence, the DNA / RNA chimera of the reference RNA sequence, and the DNA / RNA chimera of the complement of the reference RNA sequence.

[0031] The acronym VZV refers to the varicella-zoster virus, a human virus belonging to the family Alphaherpesviridae. According to the National Center for Biotechnology Information (NCBI), the laboratory strain of VZV is 124,884 nucleotides in length. VZV can cause a primary infection (such as chickenpox) and a secondary infection (such as shingles).

[0032] As used herein, the term "VZV nucleic acid sequence" refers to the entire varicella-zoster virus. Specifically, the VZV nucleic acid sequence is used herein to describe the entire laboratory strain of VZV (124,884 nucleotides in length) as defined by the NCBI.

[0033] As used herein, the term "target nucleic acid region" refers to a specific gene or region within the VZV nucleic acid sequence.

[0034] "Target nucleic acid" or "target" is a nucleic acid containing a target nucleic acid sequence. "Target nucleic acid sequence", "target sequence" or "target region" is a continuous nucleotide sequence to which an amplification oligonucleotide anneals (within a larger continuous target nucleic acid region) and contains the nucleotide sequence of a target organism such as VZV to be amplified. The target sequence or its complement contains a sequence that hybridizes to an amplification primer and / or a detection probe used for amplification and / or detection of the target nucleic acid. The target nucleic acid may contain other sequences other than the target sequence that may not be amplified. The target nucleic acid may be DNA or RNA and may be either single-stranded or double-stranded. The target nucleic acid can be, but is not limited to, genomic nucleic acid, transcribed nucleic acid such as rRNA, or nucleic acid derived from genomic nucleic acid or transcribed nucleic acid. The continuous nucleotide sequence between the forward amplification primer and the reverse amplification primer defines the polynucleotide to be amplified.

[0035] The term "oligo hybridizing sequence" or "oligo hybridization sequence" refers to a position (e.g., a continuous nucleotide sequence) within a larger target nucleic acid sequence to which an amplification primer or a detection probe binds (i.e., anneals or hybridizes). In some cases, reference to an oligo hybridizing sequence includes both the sense and antisense sequences.

[0036] The term "region" refers to a subset of continuous nucleotides contained within a larger VZV nucleic acid sequence, and the continuous subset contains fewer nucleotide base pairs than the larger polynucleotide. As a non-limiting example where the polynucleotide is a target nucleic acid sequence, the term "region" can be used to mean a smaller oligo hybridizing sequence.

[0037] "Amplification" refers to any known procedure for obtaining multiple copies of a target nucleic acid sequence, or its complement, or a fragment thereof. Known amplification methods include thermal amplification methods and isothermal amplification methods. Polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), transcription-mediated amplification (TMA, e.g., as described in Kacian and Fultz, U.S. Patent No. 5,888,779 and International Patent Application Publications WO2007 / 146154A1 and WO2006 / 026388A2), and nucleic acid sequence-based amplification (NASBA) are non-limiting examples of polynucleotide amplification methods. Replicase-mediated amplification uses self-replicating RNA molecules and a replicase such as Qβ replicase (e.g., U.S. Patent No. 4,786,600). In PCR amplification, a DNA polymerase, a primer pair, and thermal cycling are used to synthesize multiple copies of double-stranded DNA from a template or target double-stranded DNA (dsDNA) or complementary DNA (cDNA) (e.g., U.S. Patents Nos. 4,683,195, 4,683,202, and 4,800,159). LCR amplification uses four or more different oligonucleotides and multiple cycles of hybridization, ligation, and denaturation to amplify a target and its complementary strand (e.g., U.S. Patent Nos. 5,427,930 and 5,516,663). SDA uses a restriction endonuclease and a primer containing a recognition site for an endonuclease that nicks one strand of an incompletely modified (hemimodified) DNA duplex containing the target sequence, thereby causing amplification in a series of primer extension and strand displacement steps (e.g., U.S. Patent Nos. 5,422,252, 5,547,861, and 5,648,211). An "amplicon" or "amplification product" is the nucleic acid molecule(s) generated in a nucleic acid amplification reaction and is a nucleic acid molecule derived from the target nucleic acid. An amplicon or amplification product contains a target nucleic acid sequence that can be in the same and / or opposite sense as the target nucleic acid.

[0038] The "Polymerase Chain Reaction" (PCR) refers to a cyclic amplification method that copies and replicates a specific sequence of target DNA or cDNA. Using amplification oligonucleotides, a thermostable DNA polymerase, and thermal cycling, the PCR reaction generates many copies of a specific target nucleic acid sequence of a polynucleotide (e.g., an amplicon). Since PCR amplifies exponentially (the number of target nucleic acid sequences doubles with each amplification cycle), a 40-cycle PCR can generate millions of copies of the target nucleic acid. PCR consists of the following three steps: (1) Denaturation where high temperature is used to "melt" dsDNA into single strands (the temperature may increase if the template has a high GC content, but it is usually carried out at about 95°C), (2) Annealing where the amplification primers can anneal to the target nucleic acid sequence (generally carried out at a temperature about 5°C lower than the calculated melting temperature (Tm) of the amplification primers), and (3) Extension where the thermostable polymerase is used to generate the amplicon (e.g., 70 - 72°C). The length of the amplicon is usually less than 1000 bases. In some embodiments, the amplicon is 60 - 200 bases in length. In some embodiments, the detection and quantification of the amplicon are carried out after the PCR reaction is complete and involve the use of an agarose gel and image analysis.

[0039] "Real-time amplification", "real-time detection", or "real-time PCR" refers to the real-time detection of amplicons during amplification. Real-time PCR uses specific amplification oligonucleotides configured to target nucleic acid sequences. Real-time PCR enables the quantification of amplicon products in real time (at the end of each amplification cycle). Thus, real-time PCR further incorporates a detection probe for the real-time quantification of amplicons present in a sample. In some embodiments, the detection probe includes a fluorophore. The fluorescence level is a direct measure of the amount of amplification product present during the reaction. Fluorescence can be measured continuously during the amplification reaction or at the end of each cycle. By plotting relative fluorescence versus cycle number, an amplification plot can be created to show the amount of amplification product generated over time. Any of the known real-time detection methods, systems, and / or apparatuses known in the art can be used in conjunction with the described amplification oligonucleotides.

[0040] "Amplification primer" or "primer" (e.g., first amplification primer, second amplification primer, forward amplification primer, second amplification primer, forward primer, and reverse primer) refers to an amplification oligonucleotide that hybridizes to a target nucleic acid, or its complement, and is involved in a nucleic acid amplification reaction. The amplification primer has a 3'-OH (3'-hydroxyl) group that can hybridize to a template nucleic acid and be extended by polymerization. In some embodiments, the amplification primer is single-stranded. In some embodiments, the amplification primer is predominantly single-stranded and has 5 or fewer base pairs. In some embodiments, the amplification primer is 19 to 50, 19 to 40, or 19 to 30 nucleic acid bases in length. In some embodiments, the amplification primer is 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleic acid bases in length. The amplification primer includes a target hybridization sequence that anneals to the target nucleic acid sequence. The target hybridization sequences of the forward and reverse amplification primers hybridize to complementary nucleotide sequences on the target nucleic acid sequence. The forward and reverse amplification primers hybridize to specific oligohybridization sequences within the target nucleic acid sequence and are adjacent to the target nucleic acid sequence to be amplified. The target hybridization sequence of the amplification primer can be at least about 80%, at least about 90%, at least about 95%, or completely (100%) complementary to its oligohybridization sequence within the target nucleic acid sequence. The amplification primer can further include a non-target hybridization sequence. Such non-target hybridization sequences include tags, adapters, barcodes, promoters, self-complementary regions, and other nucleic acid components, as understood in the art.

[0041] In a periodic amplification method (e.g., real-time PCR) for detecting amplicons in real time, the term "baseline" refers to the measurable signal level detected during the first amplification cycle. This low-level signal is often referred to as "background" or "noise" and will vary depending on the experimental conditions. Through the initial cycles (generally amplification cycles: 1 - 15), there is little variation in the fluorescence signal. However, as the reaction progresses (usually cycle 15+), the measured fluorescence values begin to increase exponentially with each cycle. In calculating the baseline, typically, the cycles in which the measured amplification signal begins to rise above the background are excluded.

[0042] The term "threshold" is related to the point at which the measured fluorescence signal is considered statistically greater than the baseline (e.g., background) signal, thereby distinguishing the measurable amplification signal from noise. In some embodiments, the threshold is set to 10 times the standard deviation of the fluorescence value of the baseline.

[0043] The term "threshold cycle" (Ct) is the specific number of cycles at which the fluorescence signal of the reaction exceeds the threshold. Notably, since the Ct value is inversely proportional to the starting amount of the target, Ct can be used to calculate the initial DNA copy number. Given the same amount of input, the Ct of one amplification / detection system may be lower than that of another amplification / detection system. The cause of this Ct difference is the sensitivity of the primers. Similarly, reaction components (non-nucleic acids) can potentially modify the Ct.

[0044] In real-time PCR reactions, the "standard curve" refers to a mathematical formula that compares the actual effectiveness of amplification (measured efficiency) to the theoretical effectiveness. Various methods are used to calculate the standard curve, but typically, the standard curve is generated by creating a dilution series of the target nucleic acid sequence and performing real-time PCR (the amplification primers function under the theory that they need to generate a proportional dose-response curve). In some embodiments, the dilution range of the standard curve spans the concentration range predicted for the experimental samples. When the results are plotted on a graph (using the Ct value on the y-axis), a slope is generated and used to compare the reaction efficiency. Since the theoretical efficiency of PCR needs to be 100% (indicating that the template doubles after each cycle during exponential amplification), the efficiency data provides useful information about the reaction. Importantly, experimental factors such as primer length, primer composition (and the presence of secondary structure), and the GC content of the amplicon can cause a decrease in efficiency.

[0045] The term "normalization" is used herein to describe a process by which relative Ct values (indicating biological differences between samples) are not erroneously affected by non-biological factors (such as differences in sample preparation or salt concentration in the solution). Thus, normalization reduces the impact of experimental variability and can be used as an internal control. In general, those skilled in the art of molecular biology will understand various methods for normalization, including normalization to sample volume, normalization to RNA or DNA amount, or normalization to a reference gene. Typically, normalization to a reference gene, such as a housekeeping gene (an endogenous control) that provides consistent expression between samples, is used to address the variability of real-time PCR. Commonly used endogenous normalization groups include, but are not limited to, genes encoding cytoskeletal components such as β-actin, ribosomal subunits such as 18S rRNA, serine-threonine phosphatase inhibitors such as cyclophilin A, and glycolytic pathway proteins such as glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Common housekeeping genes are described in BioTechniques 29:332 (2000) and J Mol Endocrinol 25:169 can be found in (2000).

[0046] An "internal control" (IC) is a nucleic acid sequence that is amplified in parallel with the sample, which can indicate whether the assay steps and / or assay conditions were performed appropriately and / or whether the reagents and equipment functioned. The IC can be either exogenous or endogenous. An exogenous cell source can include cells that, when added to the sample, are exposed to the same sample processing procedures as the sample, amplified, and detected using the same amplification primers and detection probes, if necessary. Detection of a signal from the amplified IC (without detecting a signal from the target nucleic acid sequence of interest) indicates that the assay was performed appropriately and that the sample was negative for VZV. An endogenous IC is typically a cell source associated with / seen in the sample specimen (e.g., a housekeeping gene such as β-actin). The endogenous cell source is similarly processed and amplified and detected using the same amplification primers and / or detection probes, if necessary. Similarly, detection of a signal from the amplified IC in the absence of a signal from the target nucleic acid sequence of interest indicates an appropriate experimental design and that the sample was negative for VZV (see, for example, Poljak et al., J.Clin.Virol, 25:S89-97, 2002, U.S. Patent No. 6,410,321, and U.S. Patent Application Publication No. 2004-0023288, each of which is incorporated herein by reference). Further, if quantitative results are desired, the IC can also be used as an internal standard for the assay. The IC for the primers and probes can be constructed using any of a variety of well-known methods, provided that detection of the amplified IC sequence is possible under the same assay conditions used to amplify and detect amplicons from the target nucleic acid sequence derived from VZV and that the primers and probes function to amplify the IC target sequence.

[0047] "Relative Fluorescence Unit" ("RFU") is a unit for measuring fluorescence intensity. RFU varies depending on the characteristics of the detection means used. RFU can be used to comparatively quantify PCR products of samples and / or controls. Samples containing more amplified products will have higher corresponding RFU values.

[0048] "Specificity" refers to the degree of hybridization between a specific arrangement of consecutive nucleotides containing oligonucleotides such as primers and / or detection probes, and a specific arrangement of consecutive nucleotides containing an oligo-hybridization formation sequence on the target nucleic acid sequence (for example, specificity is the ability to distinguish between a target sequence and a non-target sequence). Regarding nucleic acid amplification, specificity generally refers to the ratio of the number of specific amplicons generated to the number of by-products or non-target amplicons (for example, signal-to-noise ratio). Regarding detection, specificity generally refers to the signal related to the binding affinity of the detection probe for the target nucleic acid sequence of interest, compared to the signal generated from non-target nucleic acids.

[0049] "Melting curve analysis" measures the change in fluorescence when dsDNA dissociates into single-stranded DNA (ssDNA) and can be used to measure the specificity of primers. Fluorescence is detectable when dsDNA separates into single-stranded DNA at the melting temperature (Tm) and the subsequent process involved in amplification normally cleaves the detection probe. The resulting fluorescence can be measured and plotted against temperature (-ΔF / ΔT). Similar PCR products are often compared using melting characteristics.

[0050] The term "sensitivity" is used herein to define the accuracy with which amplified products can be detected and / or quantified. The sensitivity of an amplification reaction is generally a measure of the minimum copy number of the target nucleic acid sequence that can be reliably detected. Generally, 2 - 10 copies are considered the minimum number of target nucleic acid sequences that can be consistently quantified.

[0051] A "detection probe" (also referred to as a "detection oligomer" or "probe") refers to an oligonucleotide containing a target hybridization-forming sequence that anneals to a specific oligohybridization-forming sequence under conditions that promote hybridization. Specifically, a detection probe is used to identify the presence of a target nucleic acid sequence or amplicon. Detection can be direct (e.g., a contiguous nucleotide sequence containing the detection probe directly hybridizes to a complementary contiguous nucleotide sequence containing the oligohybridization-forming sequence on the target nucleic acid) or indirect (e.g., the probe hybridizes to an intermediate structure that links the target nucleic acid sequence, such as a hairpin structure, to the probe (e.g., U.S. Pat. Nos. 5,118,801, 5,312,728, 6,835,542, and 6,849,412)). The detection probe is designed to anneal to the target nucleic acid sequence between a forward amplification primer and a reverse amplification primer. The detection probe may further include a non-target hybridization-forming sequence. Such non-target hybridization-forming sequences include self-complementary regions, tags, and other nucleic acid components, as understood in the art. Generally, those skilled in the art of molecular biology will understand that the probe can be generated by various techniques such as chemical synthesis or by in vitro or in vivo expression from recombinant nucleic acid molecules. The detection probe can be a DNA or RNA oligomer, or an oligomer containing a combination of DNA and RNA nucleotides, or a synthetic oligomer having a modified backbone (e.g., an oligomer containing one or more 2'-methoxy-substituted ribonucleotides). Generally, a detectable label binds to the detection probe. In some embodiments, the detection probe is 20 to 50, 20 to 45, 20 to 40, 20 to 35, or 20 to 30 nucleic acid bases in length. In some embodiments, the amplification primer is 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleic acid bases in length.

[0052] "Label" or "detectable label" refers to a moiety or compound that is directly (or indirectly) attached to a detectable probe or a probe that generates a detectable signal. Labels can be attached to the probe by a variety of means including covalent bonding, chelation, and ionic interactions. For example, TaqMan™ probes utilize covalent bonding to attach a reporter dye and a common quencher dye to the 5' and 3' termini. Indirect attachment of a label may use a bridging moiety or linker (e.g., an antibody or additional oligonucleotide(s)) to amplify the detectable signal. Detectable labels include, but are not limited to, radionuclides, ligands (e.g., biotin or avidin), enzymes, enzyme substrates, reactive groups, chromophores (e.g., dyes, or particles such as latex or metal beads), luminescent compounds (e.g., bioluminescent, phosphorescent, or chemiluminescent compounds), and fluorescent compounds (e.g., fluorophores). Detectable labels include compounds that emit a detectable light signal (e.g., fluorophores), or luminescence that can be detected in a homogeneous mixture (e.g., chemiluminescent compounds). A particular probe may have multiple labels, or multiple types of labels. Detection may rely on using a probe mixture in which each probe is labeled with a compound that generates a detectable signal (see, e.g., U.S. Patent Nos. 6,180,340 and 6,350,579, each of which is incorporated herein by reference). There are many real-time fluorescence PCR chemistries, but generally, fluorescence detection probes that utilize 5' nuclease activity in combination with a quencher molecule that absorbs light when in proximity to a fluorophore are most widely used. In addition to TaqMan™ probes, examples of other commonly utilized labels include molecular torches, and molecular beacons. In some embodiments, TaqMan™ probes, molecular torches, or molecular beacons include a non-fluorescent acceptor (quencher) that does not emit fluorescence directly from quencher excitation.

[0053] "Fluorescence Resonance Energy Transfer" (FRET) describes the interaction between a first fluorescent dye (e.g., a "reporter dye") on the 5' domain of a detection probe and a second fluorescent dye (e.g., a "quencher") on the 3' domain. In the native state of the detection probe, the quencher (which includes a longer wavelength) absorbs the higher energy emitted from the shorter wavelength of the reporter dye. However, during PCR, due to the 5' nuclease activity of DNA polymerase (and subsequent enzymatic degradation of the detection probe), the 5' reporter is consequently separated from the 3' quenching dye, thus losing the ability of the quencher to absorb the fluorescent signal emitted from the reporter dye. Therefore, in the state where the quencher is no longer in proximity, the signal emitted from the higher energy reporter can be measured. Detection probes containing both a fluorescent label and a quencher such as TaqMan™ detection probes are particularly useful because they can quantify the relative amount of amplicon product in a quantitative real-time PCR reaction by using the release of the fluorescent label (such as a reporter dye) at the 5' domain and the subsequent increase in fluorescence. Specific variations of such detection probes include, for example, TaqMan™ detection probes (Catalog No.: 401846, Thermo Fisher Scientific, developed by Roche Molecular Diagnostics, Pleasanton, CA, U.S. Patent Nos. 5,723,591, 5,801,155, and 6,084,102). It is well known to those skilled in the art of molecular biology that a combination of mismatched fluorophores and quenchers can lead to an increase in background fluorescence. Synthetic techniques and methods for binding labels to nucleic acids and detecting the labels are well known in the art (e.g., Sambrook et al., Molecular Cloning. A Laboratory Manual. 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), Chapter 10, U.S. Patent No. 5,658,737 to Nelson et al., U.S. Patent No. 5,656,207 to Woodhead et al., U.S. Patent No. 5,547,842 to Hogan et al., U.S. Patent No. 5,283,174 to Arnold et al., U.S. Patent No. 4,581,333 to Kourilsky et al.), and European Patent Application No. 0747706 to Becker et al.).

[0054] A "molecular beacon" is a single-stranded, two-labeled, fluorescent probe that exhibits self-complementarity and forms a hairpin loop conformation. The labeled portion of the molecular beacon includes a first portion containing a fluorophore and a second portion containing a quencher. The stem of the hairpin loop is bound by self-complementary base pairing of the 5' and 3' ends of the probe containing the reporter molecule and the quencher molecule. In some embodiments, the molecular beacon includes at the 5' end a sequence of 4 to 6 nucleotides that is complementary to and can hybridize with a sequence of 4 to 6 nucleotides at the 3' end. In some embodiments, either the 5' or 3' complementary sequence is a non-target hybridizing sequence (also called a target closing domain). In some embodiments, the 3' end 4 to 6 nucleotide sequence that is complementary to and can hybridize with the 4 to 6 nucleotides at the 5' end is linked to the molecular beacon via a linker. In some embodiments, the linker is a C1 to C16 linker. In some embodiments, the linker is a C9 linker. The molecular beacon is designed such that the target binding domain preferentially hybridizes to the target sequence over the target closing domain. In some embodiments, the molecular beacon includes a fluorescent molecule bound to the 5' end and a quencher bound to the 3' end. Alternatively, the fluorescent molecule may be bound to the 3' end of the torch and the quencher may be bound to the 5' end of the detection oligomer. Upon hybridization, the hairpin loop structure opens, separating the reporter from the quencher (rendering the quencher ineffective). Since the quencher is no longer in proximity to the reporter, fluorescence can be measured. The fluorescence emitted is directly proportional to the amount of target DNA. The molecular beacon is described in detail in U.S. Patent No. 5,925,517.

[0055] A "molecular torch" can be used to indicate whether an amplicon is present in a sample. The molecular torch contains regions of different self-complementarity. When exposed to a target, two self-complementary regions (fully or partially complementary) of the molecular torch melt, allowing individual nucleotides (including the target-binding domain) to hybridize to complementary consecutive nucleotides on the target nucleic acid sequence. It is important that the molecular torch is designed such that the target-binding domain has a preference for hybridization to the target sequence over the target-blocking domain. In contrast to when the molecular torch hybridizes to the target nucleic acid sequence, the target-binding domain and the target-blocking domain of the molecular torch contain interaction labels (e.g., a fluorescent dye and a quencher) such that different signals are generated when the molecular torch self-hybridizes (thereby enabling the detection of probe:target duplexes in a test sample in the presence of non-hybridized probes). Methods of synthesizing labels, methods of attaching labels to nucleic acids, and methods of detecting signals from labels are well known in the art (e.g., Chapter 10 of Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), as well as U.S. Patent Nos. 5,658,737, 5,656,207, 5,547,842, 5,283,174, and 4,581,333, and European Patent Application No. 0747706).

[0056] "Delta G" or "ΔG" represents the amount of energy required to melt or dissociate a hybrid. The larger the ΔG (the larger the negative value), the greater the amount of energy required to dissociate two hybridizing sequences. A low ΔG number (a negative value close to zero) indicates that less energy is required to melt or dissociate the hybrid. It is important that energy is proportional to temperature (higher temperatures are required as ΔG increases).

[0057] References to "SEQ ID NO:_", unless otherwise indicated, refer to the contiguous nucleotide sequence of the corresponding Sequence Listing entry, and do not require identity of backbone (e.g., RNA, 2'-O-Me RNA, or DNA) or base modification (e.g., methylation of cytosine residues).

[0058] The term "isolated" is used herein with respect to nucleic acids obtained from their natural environment, but the term does not imply any degree of purification.

[0059] "Sample preparation" refers to any step or method necessary to prepare a sample for amplification and / or detection. Sample preparation can include any known method for concentrating components such as polynucleotides from a larger sample volume, e.g., by filtration of airborne or waterborne particles from a larger volume sample, or by isolation of microorganisms from a sample using standard microbiological methods. Sample preparation can include physical and / or mechanical and / or chemical lysis of cell components to release intracellular components substantially into an aqueous or organic phase, and removal of debris. Sample preparation can also include the use of polynucleotides to selectively or non-specifically capture a target nucleic acid and separate it from other sample components (e.g., as described in U.S. Patent No. 6,110,678 and International Patent Application Publication No. WO2008 / 016988, each incorporated herein by reference).

[0060] The term "separating" or "purifying" refers to removing one or more components of a mixture from one or more other components in the mixture, such as a sample. Sample components can include nucleic acids, cell fragments, proteins, carbohydrates, lipids, and other compounds. Separation or purification does not imply a particular degree of purification. In some embodiments, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the target nucleic acid or amplification product is separated or removed from other components in the mixture.

[0061] A "degenerate" base refers to a nucleotide that can form a base pair or hybridize with multiple nucleic acid bases. A "wobble base pair" is a pairing between two nucleotides in an RNA molecule that does not follow the Watson-Crick base pair rules (e.g., a bond between pyrimidines (C and T) or purines (A and G)). Since an imperfect hybrid can potentially form a moderately stable double-strand, the presence of degenerate bases does not necessarily prevent the formation of a stable hybrid. 5-Nitroindole is an example of a degenerate base and can pair with all four natural bases.

[0062] Any of the described amplification oligonucleotides can contain at least one modified nucleotide. The modified nucleotide can be, but is not limited to, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, or 5-methylcytosine. In some embodiments, the 2'-O-methyl modified nucleotide is a 2'-OMe ribonucleotide. In some embodiments, the amplification oligonucleotide contains two or more modified nucleotides. In some embodiments, all nucleotides in the amplification oligonucleotide are modified. The two or more modified nucleotides can be the same or different. In some embodiments, any of the described amplification oligonucleotides can contain one or more 5-methylcytosines. The amplification oligonucleotide can have 1, 2, 3, 4, 5, 6, 7 or more 5-methylcytosines. In some embodiments, all cytosine nucleotides in the amplification oligonucleotide are 5-methylcytosine modified nucleotides. The amplification oligonucleotide can have 1, 2, 3, 4, 5, 6, 7 or more 2'-OMe ribonucleotides. In some embodiments, all nucleotides in the amplification oligonucleotide are 2'-OMe ribonucleotides. In some embodiments, thymidine nucleotides can be replaced with uridine nucleotides. In some embodiments, all thymidine nucleotides can be replaced with uridine nucleotides. In some amplification oligonucleotides, the use of 5-methyl-2'-deoxycytosine bases can increase the stability of the double strand by raising the Tm of each 5-methyl-2'-deoxycytosine (5-Me-dC) incorporated into the oligonucleotide (compared to the corresponding non-methylated amplification oligonucleotide) by about 0.5 °C to 1.3 °C.

[0063] The term "assay conditions" is used to indicate conditions that allow for stable hybridization of an oligonucleotide to a particular oligohybridization-forming sequence. Assay conditions do not require preferential hybridization of the oligonucleotide to the target nucleic acid.

[0064] The term "stable" or "stable to detection" refers to the temperature of the reaction mixture that is lower than the temperature at which the nucleic acid double strand denatures. In certain embodiments, for example, the following items are provided. (Item 1) A composition for amplifying a varicella-zoster virus (VZV) target nucleic acid sequence, comprising: (a) a forward amplification primer having a length of 19 to 50 nucleic acid bases and comprising 19 to 23 consecutive nucleic acid bases having at least 90% identity to a sequence of 19 to 23 nucleotides present in SEQ ID NO: 38 or its complement or SEQ ID NO: 39 or its complement; and (b) a reverse amplification primer having a length of 19 to 50 nucleic acid bases and comprising 19 to 23 consecutive nucleic acid bases having at least 90% identity to a sequence of 19 to 23 nucleotides present in SEQ ID NO: 38 or its complement or SEQ ID NO: 39 or its complement. (Item 2) The composition according to item 1, wherein the forward amplification primer comprises the nucleic acid base sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 23, 24, 25, 26, or 27. (Item 3) The composition according to any one of items 1 to 2, wherein the reverse amplification primer comprises the nucleic acid base sequence of SEQ ID NO: 16, 17, 18, 19, 20, 21, 22, 34, 35, 36, or 37. (Item 4) (a) The forward amplification primer comprises the nucleic acid base sequence of SEQ ID NO: 1, and the reverse amplification primer comprises the nucleic acid base sequence of SEQ ID NO: 16. (b) The forward amplification primer comprises the nucleic acid base sequence of SEQ ID NO: 1, and the reverse amplification primer comprises the nucleic acid base sequence of SEQ ID NO: 17. (c) The forward amplification primer comprises the nucleic acid base sequence of SEQ ID NO: 2, and the reverse amplification primer comprises the nucleic acid base sequence of SEQ ID NO: 17. (d) The forward amplification primer comprises the nucleic acid base sequence of SEQ ID NO: 3, and the reverse amplification primer comprises the nucleic acid base sequence of SEQ ID NO: 18. (e) The forward amplification primer contains the nucleobase sequence of SEQ ID NO: 4, and the reverse amplification primer contains the nucleobase sequence of SEQ ID NO: 19, (f) The forward amplification primer contains the nucleobase sequence of SEQ ID NO: 5, and the reverse amplification primer contains the nucleobase sequence of SEQ ID NO: 20, (g) The forward amplification primer contains the nucleobase sequence of SEQ ID NO: 6, and the reverse amplification primer contains the nucleobase sequence of SEQ ID NO: 21, (h) The forward amplification primer contains the nucleobase sequence of SEQ ID NO: 7, and the reverse amplification primer contains the nucleobase sequence of SEQ ID NO: 22, (i) The forward amplification primer contains the nucleobase sequence of SEQ ID NO: 23, and the reverse amplification primer contains the nucleobase sequence of SEQ ID NO: 34, (j) The forward amplification primer contains the nucleobase sequence of SEQ ID NO: 24, and the reverse amplification primer contains the nucleobase sequence of SEQ ID NO: 34, (k) The forward amplification primer contains the nucleobase sequence of SEQ ID NO: 25, and the reverse amplification primer contains the nucleobase sequence of SEQ ID NO: 35, (l) The forward amplification primer contains the nucleobase sequence of SEQ ID NO: 26, and the reverse amplification primer contains the nucleobase sequence of SEQ ID NO: 36, or (m) The forward amplification primer contains the nucleobase sequence of SEQ ID NO: 27, and the reverse amplification primer contains the nucleobase sequence of SEQ ID NO: 37, The composition according to any one of items 1 to 3. (Item 5) Further comprising a detection probe for detecting an amplified varicella-zoster virus (VZV) target nucleic acid sequence, the detection probe comprising at least one detectable label, The composition according to any one of items 1 to 4. (Item 6) The composition according to item 5, wherein the detection probe contains the nucleobase sequence of SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 28, 29, 30, 31, 32, or 33. (Item 7) (a) The detection probe contains the nucleobase sequence of SEQ ID NO: 8 or 9, the forward amplification primer contains the nucleobase sequence of SEQ ID NO: 1, and the reverse amplification primer contains the nucleobase sequence of SEQ ID NO: 16 or 17. (b) The detection probe contains the nucleobase sequence of SEQ ID NO: 9, the forward amplification primer contains the nucleobase sequence of SEQ ID NO: 2, and the reverse amplification primer contains the nucleobase sequence of SEQ ID NO: 17. (c) The detection probe contains the nucleobase sequence of SEQ ID NO: 10, the forward amplification primer contains the nucleobase sequence of SEQ ID NO: 3, and the reverse amplification primer contains the nucleobase sequence of SEQ ID NO: 18. (d) The detection probe contains the nucleobase sequence of SEQ ID NO: 11 or 12, the forward amplification primer contains the nucleobase sequence of SEQ ID NO: 4, and the reverse amplification primer contains the nucleobase sequence of SEQ ID NO: 19. (f) The detection probe contains the nucleobase sequence of SEQ ID NO: 13, the forward amplification primer contains the nucleobase sequence of SEQ ID NO: 5, and the reverse amplification primer contains the nucleobase sequence of SEQ ID NO: 20. (g) The detection probe contains the nucleobase sequence of SEQ ID NO: 14, the forward amplification primer contains the nucleobase sequence of SEQ ID NO: 6, and the reverse amplification primer contains the nucleobase sequence of SEQ ID NO: 21. (h) The detection probe contains the nucleobase sequence of SEQ ID NO: 15, the forward amplification primer contains the nucleobase sequence of SEQ ID NO: 7, and the reverse amplification primer contains the nucleobase sequence of SEQ ID NO: 22. (i) The detection probe contains the nucleobase sequence of SEQ ID NO: 28, the forward amplification primer contains the nucleobase sequence of SEQ ID NO: 23 or 24, and the reverse amplification primer contains the nucleobase sequence of SEQ ID NO: 34. (j) The detection probe contains the nucleobase sequence of SEQ ID NO: 29 or 30, the forward amplification primer contains the nucleobase sequence of SEQ ID NO: 25, and the reverse amplification primer contains the nucleobase sequence of SEQ ID NO: 35. (k) The detection probe contains the nucleotide sequence of SEQ ID NO: 31, the forward amplification primer contains the nucleotide sequence of SEQ ID NO: 26, the reverse amplification primer contains the nucleotide sequence of SEQ ID NO: 36, or (l) The detection probe contains the nucleotide sequence of SEQ ID NO: 32 or 33, the forward amplification primer contains the nucleotide sequence of SEQ ID NO: 27, the reverse amplification primer contains the nucleotide sequence of SEQ ID NO: 37, The composition according to item 6. (Item 8) The composition according to any one of items 1 to 7, wherein the forward amplification primer, the reverse amplification primer, and / or the detection probe contains at least one modified nucleotide. (Item 9) The composition according to item 8, wherein the modified nucleotide contains a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, or 5-methylcytosine. (Item 10) The at least one detectable label is (a) a chemiluminescent label, (b) a fluorescent label, (c) a quencher, or (d) a combination of two or more of (a), (b), and (c), The composition according to any one of items 5 to 9, selected from the group consisting of. (Item 11) The composition according to item 10, wherein the at least one detectable label contains the fluorescent label, the quencher, or both the fluorescent label and the quencher. (Item 12) The composition according to any one of items 1 to 11, wherein the detection probe contains a 5' non-target hybridization formation sequence that base pairs with the 3' end of the detection probe, or a 3' non-target hybridization formation sequence that base pairs with the 5' end of the detection probe. (Item 13) The composition according to item 12, wherein the detection probe contains a molecular beacon or a molecular torch. (Item 14) The composition according to any one of items 1 to 13, further comprising one or more of a buffer, a salt, dNTP, a surfactant, and an enzyme. (Item 15) The composition according to item 14, wherein the enzyme comprises a thermostable DNA polymerase, a reverse transcriptase, an RNA polymerase, or any combination of two or more of a thermostable DNA polymerase, a reverse transcriptase, and an RNA polymerase. (Item 16) The composition according to any one of items 1 to 15, wherein the amplification primer is present in an aqueous solution, frozen, or lyophilized. (Item 17) The composition according to any one of items 1 to 16, wherein the composition comprises two or more pairs of amplification primers and / or two or more detection probes, and each pair of amplification primers consists of a forward amplification primer and a reverse amplification primer. (Item 18) The composition according to item 17, wherein the two or more pairs of amplification primers and / or two or more detection probes amplify target nucleic acid sequences of the same or different organisms. (Item 19) The composition according to any one of items 1 to 18, further comprising an internal standard target nucleic acid sequence, an oligomer for amplifying and / or detecting the internal standard target nucleic acid sequence, or a combination thereof. (Item 20) A detection probe for detecting a VZV target nucleic acid sequence, comprising an oligonucleotide comprising the nucleic acid base sequence of SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 28, 29, 30, 31, 32, or 33, wherein the oligonucleotide comprises one or more detectable labels. (Item 21) The detection probe according to item 20, wherein the detection probe comprises at least one modified nucleotide. (Item 22) The detection probe according to item 21, wherein the modified nucleotide comprises a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, or 5-methylcytosine. (Item 23) One or more of the detectable labels are (a) a chemiluminescent label, (b) a fluorescent label, (c) a quencher, or (d) a combination of two or more of (a), (b), and (c), and is selected from the group consisting of, the detection probe according to any one of Items 20 to 22. (Item 24) One or more of the detectable labels include the fluorescent label, the quencher, or both the fluorescent label and the quencher, the detection probe according to Item 23. (Item 25) The detection probe includes a 5' non-target hybridization-forming sequence that base pairs with the 3' end of the detection probe, or a 3' non-target hybridization-forming sequence that base pairs with the 5' end of the detection probe, the detection probe according to any one of Items 20 to 24. (Item 26) The detection probe includes a molecular beacon or a molecular torch, the detection probe according to Item 25. (Item 27) A method for amplifying a VZV target nucleic acid sequence, comprising: (a) obtaining a sample containing or suspected of containing a VZV target nucleic acid sequence; and (b) contacting the sample with the composition according to any one of Items 1 to 19; and (c) providing conditions sufficient to amplify the target nucleic acid sequence, and generating an amplification product of the VZV target nucleic acid sequence if the VZV target nucleic acid sequence is present in the sample. (Item 28) The method further includes contacting the sample with the detection probe according to any one of Items 20 to 26 to determine the presence or absence of the amplification product, the method according to Item 27. (Item 29) A method for determining the presence or absence of VZV in a sample, comprising: (a) obtaining a sample containing or suspected of containing a VZV target nucleic acid sequence; and (b) contacting the sample with the composition according to any one of Items 1 to 19; (c) generating an amplification product by providing conditions sufficient to amplify the target nucleic acid sequence; (d) detecting the presence or absence of the amplification product, a method comprising the steps of. DETAILED DESCRIPTION OF THE INVENTION

[0065] The present disclosure provides amplification oligonucleotides, oligonucleotide compositions, kits, methods, formulations, and reaction mixtures for detecting VZV in a sample. Further, the oligonucleotide compositions, kits, methods, formulations, and reaction mixtures are further useful for generating amplicons from the target nucleic acid sequence if present in the sample. Amplification and detection of VZV can be used for diagnosis. Diagnosis can be used to facilitate effective treatment to limit the spread of the virus. Therefore, the amplification oligonucleotides, oligonucleotide compositions, kits, methods, formulations, and reaction mixtures are useful for screening individuals at risk of VZV infection (regardless of whether they exhibit symptoms), or individuals at high risk of serious complications from VZV infection (e.g., young, old, or immunocompromised). Therefore, the disclosed oligonucleotide compositions, kits, methods, formulations, and reaction mixtures meet the need for rapid, sensitive, and specific testing of clinical samples from patients who may have been infected or exposed to VZV.

[0066] In certain embodiments, the oligonucleotide compositions, kits, and methods disclosed herein include amplification primers for amplifying a target nucleic acid sequence within a VZV nucleic acid sequence. In some embodiments, the oligonucleotide compositions, kits, and methods disclose detection probes for detecting VZV. In some embodiments, the amplification primers and detection probes are two separate products. In some embodiments, the amplification primers and detection probes are provided in a kit. In certain embodiments, the present disclosure relates to oligonucleotide compositions, kits, and methods for contacting a sample with at least one amplification primer pair and performing an in vitro nucleic acid amplification reaction, wherein any target nucleic acid sequence present in the sample can be used as a template for generating an amplification product. In some embodiments, the present disclosure relates to oligonucleotide compositions, kits, and methods for contacting a sample with at least one detection probe, wherein any target nucleic acid sequence present in the sample or its amplification product can hybridize to the detection probe to facilitate detection.

[0067] In certain embodiments, the oligonucleotide compositions, kits, and methods disclosed herein provide guidance for utilizing at least one amplification primer pair to generate an amplicon from a target nucleic acid sequence within a specific target nucleic acid region of a VZV nucleic acid sequence. In certain embodiments, the oligonucleotide compositions, kits, and methods disclosed herein provide guidance for utilizing at least one detection probe to detect VZV in a sample. Any application of a particular combination of amplification primers or detection probes should similarly be understood as a disclosed method for amplification or detection of a target nucleic acid sequence of VZV.

[0068] In certain embodiments, the VZV amplification oligonucleotides disclosed herein are configured to specifically hybridize to complementary nucleotide subunits within the target nucleic acid sequence, thus minimizing cross-reactivity with other non-VZV nucleic acids (if present) in the sample.

[0069] In certain embodiments, the oligonucleotide compositions, kits, and methods disclosed herein include at least one amplification primer. In certain embodiments, the oligonucleotide compositions, kits, and methods include one or more sets or pairs of amplification primers. In some embodiments, a set of amplification primers includes a first amplification primer and a second amplification primer. In some embodiments, a set of amplification primers includes a forward amplification primer and a reverse amplification primer. In certain embodiments, the oligonucleotide compositions, kits, and methods include a single set of forward and reverse amplification primers that generate a single amplicon of a target nucleic acid sequence from a target nucleic acid region. In certain embodiments, the oligonucleotide compositions, kits, and methods include two or more sets of amplification primers that generate two or more amplicons. The two or more amplicons can be from two or more regions within a single target nucleic acid, two or more target nucleic acids, or combinations thereof. The two or more target nucleic acids can be from the same organism or different organisms.

[0070] In certain embodiments of the oligonucleotide compositions, kits, and methods, the amplification oligonucleotides are configured to specifically anneal to oligohybridization-forming sequences within the target nucleic acid regions of SEQ ID NO: 38 and SEQ ID NO: 39 of the VZV nucleic acid sequence (if present) in the sample.

[0071] In certain embodiments of the oligonucleotide compositions, kits, and methods, the target nucleic acid region is SEQ ID NO: 38, and the forward and reverse amplification primers are, independently of each other, about 19 to about 23 nucleotides in length and are configured to generate an amplicon about 89 to about 127 nucleotides in length from the target nucleic acid region of SEQ ID NO: 38.

[0072] In certain embodiments of the oligonucleotide compositions, kits, and methods, the target nucleic acid region is SEQ ID NO: 38, the forward amplification primer is selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7, the reverse amplification primer is about 19 to about 23 nucleotides in length, and the forward and reverse amplification primers are configured to generate an amplicon from the target nucleic acid region within SEQ ID NO: 38 that is about 89 to about 127 nucleotides in length. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the forward amplification primer comprises the sequence of SEQ ID NO: 1. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the forward oligomer comprises the sequence of SEQ ID NO: 2. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the forward oligomer comprises the sequence of SEQ ID NO: 3. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the forward oligomer comprises the sequence of SEQ ID NO: 4. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the forward oligomer comprises the sequence of SEQ ID NO: 5. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the forward oligomer comprises the sequence of SEQ ID NO: 6. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the forward oligomer comprises the sequence of SEQ ID NO: 7.

[0073] In certain embodiments of the oligonucleotide composition, kit, and method, the target nucleic acid region is SEQ ID NO: 38, the forward amplification primer is selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, and 7, and the reverse amplification primer is about 19 to about 23 nucleotides in length and is selected from the group consisting of SEQ ID NO: 16, 17, 18, 19, 20, 21, and 22. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the reverse oligomer comprises the sequence of SEQ ID NO: 16. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the reverse oligomer comprises the sequence of SEQ ID NO: 17. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the reverse oligomer comprises the sequence of SEQ ID NO: 18. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the reverse oligomer comprises the sequence of SEQ ID NO: 19. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the reverse oligomer comprises the sequence of SEQ ID NO: 20. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the reverse oligomer comprises the sequence of SEQ ID NO: 21. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the reverse oligomer comprises the sequence of SEQ ID NO: 22.

[0074] In certain embodiments of the oligonucleotide composition, kit, and method, the target nucleic acid region is SEQ ID NO: 38, the reverse amplification primer is selected from the group consisting of SEQ ID NO: 16, 17, 18, 19, 20, 21, and 22, and the forward amplification primer is about 20 to about 23 nucleotides in length and is configured to generate an amplicon from the target nucleic acid sequence within SEQ ID NO: 38 that is about 89 to about 127 nucleotides in length.

[0075] In certain embodiments of the oligonucleotide compositions, kits, and methods, the target nucleic acid region is SEQ ID NO: 38, the forward amplification primer is selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7, the reverse amplification primer is selected from the group consisting of SEQ ID NOs: 16, 17, 18, 19, 20, 21, and 22, and the forward and reverse amplification primers are configured to generate an amplicon from the target nucleic acid sequence within SEQ ID NO: 38 selected from the group consisting of lengths of 89, 93, 100, 102, 119, 123, and 127 nucleotides. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the amplicon is 89 nucleotides in length. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the amplicon is 93 nucleotides in length. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the amplicon is 100 nucleotides in length. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the amplicon is 102 nucleotides in length. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the amplicon is 119 nucleotides in length. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the amplicon is 123 nucleotides in length. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the amplicon is 127 nucleotides in length.

[0076] In certain embodiments of the oligonucleotide compositions, kits, and methods, the target nucleic acid region is SEQ ID NO: 38, the forward amplification primer is selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7, the reverse amplification primer is selected from the group consisting of SEQ ID NOs: 16, 17, 18, 19, 20, 21, and 22, and the forward and reverse amplification primers substantially contain the target nucleic acid sequence corresponding to the oligohybridization-forming sequences of (a) SEQ ID NO: 1 and SEQ ID NO: 16, (b) SEQ ID NO: 1 and SEQ ID NO: 17, (c) SEQ ID NO: 2 and SEQ ID NO: 17, (d) SEQ ID NO: 3 and SEQ ID NO: 18, (e) SEQ ID NO: 4 and SEQ ID NO: 19, (f) SEQ ID NO: 5 and SEQ ID NO: 20, (g) SEQ ID NO: 6 and SEQ ID NO: 21, (h) SEQ ID NO: 7 and SEQ ID NO: 22.

[0077] In certain embodiments of the oligonucleotide compositions, kits, and methods, the target nucleic acid region is SEQ ID NO: 38, and (a) the forward amplification primer and the reverse amplification primer are configured to generate an amplicon from a target nucleic acid sequence of at least about 89 nucleotides in length adjacent between SEQ ID NO: 3 and SEQ ID NO: 18 within the target nucleic acid region, (b) the forward amplification primer and the reverse amplification primer are configured to generate an amplicon from a target nucleic acid sequence of at least about 93 nucleotides in length adjacent between SEQ ID NO: 4 and SEQ ID NO: 19 within the target nucleic acid region, (c) the forward amplification primer and the reverse amplification primer are configured to generate an amplicon from a target nucleic acid sequence of at least about 100 nucleotides in length adjacent between SEQ ID NO: 2 and SEQ ID NO: 17 within the target nucleic acid region, (d) the forward amplification primer and the reverse amplification primer are configured to generate an amplicon from a target nucleic acid sequence of at least about 102 nucleotides in length adjacent between SEQ ID NO: 7 and SEQ ID NO: 22 within the target nucleic acid region, (e) the forward amplification primer and the reverse amplification primer are configured to generate an amplicon from a target nucleic acid sequence of at least about 119 nucleotides in length adjacent between SEQ ID NO: 6 and SEQ ID NO: 21 within the target nucleic acid region, (f) the forward amplification primer and the reverse amplification primer are configured to generate an amplicon from a target nucleic acid sequence of at least about 123 nucleotides in length adjacent between SEQ ID NO: 1 and SEQ ID NO: 17 within the target nucleic acid region, (g) the forward amplification primer and the reverse amplification primer are configured to generate an amplicon from a target nucleic acid sequence of at least about 127 nucleotides in length adjacent between SEQ ID NO: 1 and SEQ ID NO: 16 or between SEQ ID NO: 5 and SEQ ID NO: 20 within the target nucleic acid region.

[0078] In certain embodiments of the oligonucleotide composition, kit, and method, the target nucleic acid region is SEQ ID NO: 39, the forward amplification primer and the reverse amplification primer are about 20 to about 23 nucleotides in length, and the forward and reverse amplification primers are configured to generate an amplicon from the target nucleic acid sequence within SEQ ID NO: 39 that is about 89 to about 143 nucleotides in length.

[0079] In certain embodiments of the oligonucleotide composition, kit, and method, the target nucleic acid region is SEQ ID NO: 39, the forward amplification primer is selected from the group consisting of SEQ ID NOs: 23, 24, 25, 26, and 27, the reverse amplification primer is about 20 to about 22 nucleotides in length, and the forward and reverse amplification primers are configured to generate an amplicon from the target nucleic acid region within SEQ ID NO: 39 that is about 89 to about 143 nucleotides in length. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39 and the forward oligomer comprises the sequence of SEQ ID NO: 23. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39 and the forward oligomer comprises the sequence of SEQ ID NO: 24. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39 and the forward oligomer comprises the sequence of SEQ ID NO: 25. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39 and the forward oligomer comprises the sequence of SEQ ID NO: 26. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39 and the forward oligomer comprises the sequence of SEQ ID NO: 27.

[0080] In certain embodiments of the oligonucleotide composition, kit, and method, the target nucleic acid region is SEQ ID NO: 39, the forward amplification primer is selected from the group consisting of SEQ ID NO: 23, 24, 25, 26, and 27, the reverse amplification primer is about 20 to about 22 nucleotides in length, and is selected from the group consisting of SEQ ID NO: 34, 35, 36, and 37. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39, and the reverse oligomer comprises the sequence of SEQ ID NO: 34. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39, and the reverse oligomer comprises the sequence of SEQ ID NO: 35. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39, and the reverse oligomer comprises the sequence of SEQ ID NO: 36. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39, and the reverse oligomer comprises the sequence of SEQ ID NO: 37.

[0081] In certain embodiments of the oligonucleotide composition, kit, and method, the target nucleic acid region is SEQ ID NO: 39, the reverse amplification primer is selected from the group consisting of SEQ ID NO: 34, 35, 36, and 37, the forward amplification primer is about 20 to about 23 nucleotides in length, and the forward and reverse amplification primers are configured to generate an amplicon from the target nucleic acid region within SEQ ID NO: 39 that is about 89 to about 143 nucleotides in length.

[0082] In certain embodiments of the oligonucleotide composition, kit, and method, the target nucleic acid region is SEQ ID NO: 39, the forward amplification primer is selected from the group consisting of SEQ ID NO: 23, 24, 25, 26, and 27, the reverse amplification primer is selected from the group consisting of SEQ ID NO: 34, 35, 36, and 37, and the forward and reverse amplification primers are configured to generate an amplicon from the target nucleic acid sequence within SEQ ID NO: 39, which is selected from the group consisting of lengths of 89, 99, 109, 126, and 143 nucleotides. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39 and the amplicon is 89 nucleotides in length. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39 and the amplicon is 99 nucleotides in length. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39 and the amplicon is 109 nucleotides in length. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39 and the amplicon is 126 nucleotides in length. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39 and the amplicon is 143 nucleotides in length.

[0083] In certain embodiments of the oligonucleotide composition, kit, and method, the target nucleic acid region is SEQ ID NO: 39, the forward amplification primer is selected from the group consisting of SEQ ID NO: 23, 24, 25, 26, and 27, the reverse amplification primer is selected from the group consisting of SEQ ID NO: 34, 35, 36, and 37, and the forward and reverse amplification primers substantially contain the target nucleic acid sequence corresponding to the oligohybridization formation sequences of (a) SEQ ID NO: 23 and SEQ ID NO: 34, (b) SEQ ID NO: 24 and SEQ ID NO: 34, (c) SEQ ID NO: 25 and SEQ ID NO: 35, (d) SEQ ID NO: 26 and SEQ ID NO: 36, (e) SEQ ID NO: 27 and SEQ ID NO: 37.

[0084] In certain embodiments of the oligonucleotide compositions, kits, and methods, the target nucleic acid region is SEQ ID NO: 39, and (a) the forward amplification primer and the reverse amplification primer are configured to generate an amplicon from a target nucleic acid sequence at least about 89 nucleotides in length adjacent between SEQ ID NO: 25 and SEQ ID NO: 35 within the target nucleic acid region, (b) the forward amplification primer and the reverse amplification primer are configured to generate an amplicon from a target nucleic acid sequence at least about 99 nucleotides in length adjacent between SEQ ID NO: 24 and SEQ ID NO: 34 within the target nucleic acid region, (c) the forward amplification primer and the reverse amplification primer are configured to generate an amplicon from a target nucleic acid sequence at least about 109 nucleotides in length adjacent between SEQ ID NO: 23 and SEQ ID NO: 34 within the target nucleic acid region, (d) the forward amplification primer and the reverse amplification primer are configured to generate an amplicon from a target nucleic acid sequence at least about 126 nucleotides in length adjacent between SEQ ID NO: 27 and SEQ ID NO: 37 within the target nucleic acid region, (e) the forward amplification primer and the reverse amplification primer are configured to generate an amplicon from a target nucleic acid sequence at least about 143 nucleotides in length adjacent between SEQ ID NO: 26 and SEQ ID NO: 36 within the target nucleic acid region.

[0085] In certain embodiments of the oligonucleotide compositions, kits, and methods, at least one amplification primer is configured to anneal to the target nucleic acid sequence in a forward orientation, at least one amplification primer is configured to anneal to the target nucleic acid sequence in a reverse orientation, and the forward and reverse amplification primers specifically hybridize to a contiguous nucleotide sequence comprising an oligo-hybridization forming sequence on the target nucleic acid sequence that is amplified within the target nucleic acid region of SEQ ID NO: 38 or SEQ ID NO: 39 of the VZV nucleic acid sequence in the sample, if present.

[0086] In some embodiments of the oligonucleotide compositions, kits, and methods, the composition for determining the presence (or absence) of a target nucleic acid sequence of VZV in a sample comprises (1) at least one forward amplification primer configured to specifically hybridize to an oligohybridization-forming sequence within the target nucleic acid region of SEQ ID NO: 38 or SEQ ID NO: 39, and (2) at least one reverse amplification primer configured to specifically hybridize to an oligohybridization-forming sequence within the target nucleic acid region of SEQ ID NO: 38 or SEQ ID NO: 39.

[0087] In certain embodiments of the oligonucleotide compositions, kits, and methods, the forward amplification primer comprises at least one modified nucleobase. In certain embodiments, the modified nucleobase is selected from the group consisting of (a) 2'-O-methyl, (b) 5-methylcytosine, (c) 2'-fluoro, and (d) combinations of two or more of (a), (b), and (c).

[0088] In certain embodiments of the oligonucleotide compositions, kits, and methods, the forward amplification primer comprises from 2 to 6 modified nucleobases. The 2 to 6 modified nucleobases may be the same or different. In certain embodiments, the forward amplification primer comprises from 2 to 6 5-methylcytosine residues. In certain embodiments, the forward amplification primer comprises 2 5-methylcytosine residues. In some embodiments, the forward amplification primer comprises 3 5-methylcytosine residues. In certain embodiments, the forward amplification primer comprises 4 5-methylcytosine residues. In certain embodiments, the forward amplification primer comprises 5 5-methylcytosine residues. In certain embodiments, the forward amplification primer comprises 6 5-methylcytosine residues. In certain embodiments, the forward amplification primer comprises from 2 to 6 2'-O-methyl residues. In certain embodiments, the forward amplification primer comprises 2 2'-O-methyl residues. In some embodiments, the forward amplification primer comprises 3 2'-O-methyl residues. In certain embodiments, the forward amplification primer comprises 4 2'-O-methyl residues. In certain embodiments, the forward amplification primer comprises 5 2'-O-methyl residues. In certain embodiments, the forward amplification primer comprises 6 2'-O-methyl residues.

[0089] In certain embodiments of the oligonucleotide compositions, kits, and methods, the reverse amplification primer further comprises at least one modified nucleobase. In certain embodiments, the modified nucleobase is selected from the group consisting of (a) 2'-O-methyl, (b) 5-methylcytosine, (c) 2'-fluoro, and (d) combinations of two or more of (a), (b), and (c).

[0090] In certain embodiments of the oligonucleotide compositions, kits, and methods, the reverse amplification primer comprises from 2 to 6 modified nucleobases. The 2 to 6 modified nucleobases may be the same or different. In certain embodiments, the reverse amplification primer comprises from 2 to 6 5-methylcytosine residues. In certain embodiments, the reverse amplification primer comprises 2 5-methylcytosine residues. In some embodiments, the reverse amplification primer comprises 3 5-methylcytosine residues. In some embodiments, the reverse amplification primer comprises 4 5-methylcytosine residues. In some embodiments, the reverse amplification primer comprises 5 5-methylcytosine residues. In some embodiments, the reverse amplification primer comprises 6 5-methylcytosine residues. In certain embodiments, the reverse amplification primer comprises from 2 to 6 2'-O-methyl residues. In certain embodiments, the reverse amplification primer comprises 2 2'-O-methyl residues. In some embodiments, the reverse amplification primer comprises 3 2'-O-methyl residues. In some embodiments, the reverse amplification primer comprises 4 2'-O-methyl residues. In some embodiments, the reverse amplification primer comprises 5 2'-O-methyl residues. In some embodiments, the reverse amplification primer comprises 6 2'-O-methyl residues.

[0091] In certain embodiments of the oligonucleotide compositions, kits, and methods, the third oligomer is configured to specifically anneal to an amplified target nucleic acid sequence within the target nucleic acid regions of SEQ ID NO: 38 and SEQ ID NO: 39 of the VZV nucleic acid sequence (if present) in the sample. In certain embodiments, the third oligomer hybridizes to the oligohybridization-forming sequence within SEQ ID NO: 38. In some embodiments, the third oligomer hybridizes to the oligohybridization-forming sequence within SEQ ID NO: 39. In certain embodiments of the oligonucleotide compositions, kits, and methods, the third oligomer is a detection probe.

[0092] In certain embodiments of the oligonucleotide composition, kit, and method, the target nucleic acid region is SEQ ID NO: 38, and the detection probe is from about 23 to about 27 nucleotides in length.

[0093] In certain embodiments of the oligonucleotide composition, kit, and method, the target nucleic acid region is SEQ ID NO: 38, and the detection probe is selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, and 15. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38, and the detection probe comprises the sequence of SEQ ID NO: 8. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38, and the detection probe comprises the sequence of SEQ ID NO: 9. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38, and the detection probe comprises the sequence of SEQ ID NO: 10. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38, and the detection probe comprises the sequence of SEQ ID NO: 11. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38, and the detection probe comprises the sequence of SEQ ID NO: 12. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38, and the detection probe comprises the sequence of SEQ ID NO: 13. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38, and the detection probe comprises the sequence of SEQ ID NO: 14. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38, and the detection probe comprises the sequence of SEQ ID NO: 15.

[0094] In certain embodiments of the oligonucleotide compositions, kits, and methods, the target nucleic acid region is SEQ ID NO: 38, and the detection probe comprises a target nucleic acid sequence that substantially corresponds to the following oligohybridization forming sequence. When the forward and reverse amplification primers are (I) SEQ ID NO: 1 and SEQ ID NO: 16 or (II) SEQ ID NO: 1 and SEQ ID NO: 17, the sequence is SEQ ID NO: 8; when the forward and reverse amplification primers are (I) SEQ ID NO: 1 and SEQ ID NO: 16 or (II) SEQ ID NO: 1 and SEQ ID NO: 17 or (III) SEQ ID NO: 2 and SEQ ID NO: 17, the sequence is SEQ ID NO: 9; when the forward and reverse amplification primers are SEQ ID NO: 3 and SEQ ID NO: 18, the sequence is SEQ ID NO: 10; when the forward and reverse amplification primers are SEQ ID NO: 4 and SEQ ID NO: 19, the sequence is SEQ ID NO: 11; when the forward and reverse amplification primers are SEQ ID NO: 4 and SEQ ID NO: 19, the sequence is SEQ ID NO: 12; when the forward and reverse amplification primers are SEQ ID NO: 5 and SEQ ID NO: 20, the sequence is SEQ ID NO: 13; when the forward and reverse amplification primers are SEQ ID NO: 6 and SEQ ID NO: 21, the sequence is SEQ ID NO: 14; when the forward and reverse amplification primers are SEQ ID NO: 7 and SEQ ID NO: 22, the sequence is SEQ ID NO: 15.

[0095] In certain embodiments of the oligonucleotide compositions, kits, and methods, the target nucleic acid region is SEQ ID NO: 38, and (a) when the forward amplification primer and the reverse amplification primer are configured to generate an amplicon of a target nucleic acid sequence having a length of at least about 89 nucleotides from SEQ ID NO: 3 and SEQ ID NO: 18 on the target nucleic acid region, the detection probe comprises the sequence of SEQ ID NO: 10; (b) when the forward amplification primer and the reverse amplification primer are configured to generate an amplicon of a target nucleic acid sequence having a length of at least about 93 nucleotides from SEQ ID NO: 4 and SEQ ID NO: 19 on the target nucleic acid region, the detection probe comprises the sequence of SEQ ID NO: 11 or SEQ ID NO: 12; (c) when the forward amplification primer and the reverse amplification primer are configured to generate an amplicon of a target nucleic acid sequence having a length of at least about 100 nucleotides from SEQ ID NO: 2 and SEQ ID NO: 17 on the target nucleic acid region, the detection probe comprises the sequence of SEQ ID NO: 9; (d) when the forward amplification primer and the reverse amplification primer are configured to generate an amplicon of a target nucleic acid sequence having a length of at least about 102 nucleotides from SEQ ID NO: 7 and SEQ ID NO: 22 on the target nucleic acid region, the detection probe comprises the sequence of SEQ ID NO: 15; (e) when the forward amplification primer and the reverse amplification primer are configured to generate an amplicon of a target nucleic acid sequence having a length of at least about 119 nucleotides from SEQ ID NO: 6 and SEQ ID NO: 21 on the target nucleic acid region, the detection probe comprises the sequence of SEQ ID NO: 14; (f) when the forward amplification primer and the reverse amplification primer are configured to generate an amplicon of a target nucleic acid sequence having a length of at least about 123 nucleotides from SEQ ID NO: 1 and SEQ ID NO: 17 on the target nucleic acid region, the detection probe comprises the sequence of SEQ ID NO: 8 or SEQ ID NO: 9; (g) when the forward amplification primer and the reverse amplification primer are configured to generate an amplicon of a target nucleic acid sequence having a length of at least about 127 nucleotides from SEQ ID NO: 1 and SEQ ID NO: 16 on the target nucleic acid region, the detection probe comprises the sequence of SEQ ID NO: 8 or SEQ ID NO: 9, or when the forward amplification primer and the reverse amplification primer are from SEQ ID NO: 5 and SEQ ID NO: 20 on the target nucleic acid region,When configured to generate an amplicon of a target nucleic acid sequence that is at least about 127 nucleotides in length, the detection probe comprises the sequence of SEQ ID NO: 13.

[0096] In certain embodiments of the oligonucleotide compositions, kits, and methods, the target nucleic acid region is SEQ ID NO: 39 and the detection probe is from about 22 to about 27 nucleotides in length.

[0097] In certain embodiments of the oligonucleotide compositions, kits, and methods, the target nucleic acid region is SEQ ID NO: 39 and the detection probe is selected from the group consisting of SEQ ID NOs: 28, 29, 30, 31, 32, and 33. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39 and the detection probe comprises the sequence of SEQ ID NO: 28. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39 and the detection probe comprises the sequence of SEQ ID NO: 29. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39 and the detection probe comprises the sequence of SEQ ID NO: 30. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39 and the detection probe comprises the sequence of SEQ ID NO: 31. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39 and the detection probe comprises the sequence of SEQ ID NO: 32. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39 and the detection probe comprises the sequence of SEQ ID NO: 33.

[0098] In certain embodiments of the oligonucleotide compositions, kits, and methods, the target nucleic acid region is SEQ ID NO: 39, and the detection probe comprises a target nucleic acid sequence that substantially corresponds to the following oligohybridization-forming sequence. When the forward and reverse amplification primers are (I) SEQ ID NO: 23 and SEQ ID NO: 34 or (II) SEQ ID NO: 24 and SEQ ID NO: 34, SEQ ID NO: 28; when the forward and reverse amplification primers are SEQ ID NO: 25 and SEQ ID NO: 35, SEQ ID NO: 29; when the forward and reverse amplification primers are SEQ ID NO: 25 and SEQ ID NO: 35, SEQ ID NO: 30; when the forward and reverse amplification primers are SEQ ID NO: 26 and SEQ ID NO: 36, SEQ ID NO: 31; when the forward and reverse amplification primers are SEQ ID NO: 27 and SEQ ID NO: 37, SEQ ID NO: 32; when the forward and reverse amplification primers are SEQ ID NO: 27 and SEQ ID NO: 37, SEQ ID NO: 33.

[0099] In certain embodiments of the oligonucleotide compositions, kits, and methods, the target nucleic acid region is SEQ ID NO: 39, and (a) when the forward amplification primer and the reverse amplification primer are configured to generate an amplicon of a target nucleic acid sequence of at least about 89 nucleotides from SEQ ID NO: 25 and SEQ ID NO: 35 on the target nucleic acid region, the third oligomer comprises the sequence of SEQ ID NO: 29 or SEQ ID NO: 30; (b) when the forward amplification primer and the reverse amplification primer are configured to generate an amplicon of a target nucleic acid sequence of at least about 99 nucleotides from SEQ ID NO: 24 and SEQ ID NO: 34 on the target nucleic acid region, the third oligomer comprises the sequence of SEQ ID NO: 28; (c) when the forward amplification primer and the reverse amplification primer are configured to generate an amplicon of a target nucleic acid sequence of at least about 109 nucleotides from SEQ ID NO: 23 and SEQ ID NO: 34 on the target nucleic acid region, the third oligomer comprises the sequence of SEQ ID NO: 28; (d) when the forward amplification primer and the reverse amplification primer are configured to generate an amplicon of a target nucleic acid sequence of at least about 126 nucleotides from SEQ ID NO: 27 and SEQ ID NO: 37 on the target nucleic acid region, the third oligomer comprises the sequence of SEQ ID NO: 32 or SEQ ID NO: 33; (e) when the forward amplification primer and the reverse amplification primer are configured to generate an amplicon of a target nucleic acid sequence of at least about 143 nucleotides from SEQ ID NO: 26 and SEQ ID NO: 36 on the target nucleic acid region, the third oligomer comprises the sequence of SEQ ID NO: 31.

[0100] In certain embodiments, the oligonucleotide compositions, kits, and methods for determining the presence (or absence) of VZV in a sample as described herein include at least one detection probe configured to specifically anneal to a target nucleic acid region of SEQ ID NO: 38 or SEQ ID NO: 39, and the detection probe is adjacent between the forward amplification primer and the reverse amplification primer.

[0101] In certain embodiments of the oligonucleotide compositions, kits, and methods, the detection probe comprises at least one detectable label. In certain embodiments, the detection probe further comprises a second label that interacts with a first label, such as a quencher.

[0102] In certain embodiments of the oligonucleotide compositions, kits, and methods, the label is selected from the group consisting of (a) a chemiluminescent label, (b) a fluorescent label, (c) a quencher, and (d) combinations of two or more of (a), (b), and (c). In certain embodiments, the oligonucleotide compositions, kits, and methods comprise a fluorescent label. In certain embodiments, the oligonucleotide compositions, kits, and methods comprise a quencher. In certain embodiments, the oligonucleotide compositions, kits, and methods comprise both a fluorescent label and a quencher.

[0103] In certain embodiments of the oligonucleotide compositions, kits, and methods, the detection probe is linear and does not exhibit any degree of self-complementarity retained by intramolecular bonding. In such embodiments, the linear detection probe comprises a fluorophore as a label. In some embodiments, the linear detection probe comprises both a fluorophore and a quenching moiety (e.g., TaqMan™ probe).

[0104] In certain embodiments of the oligonucleotide compositions, kits, and methods, the detection probe exhibits at least some degree of self-complementarity and is used to facilitate detection of probe:target duplexes in a sample without the need to first hybridize and remove unhybridized probes prior to detection.

[0105] In certain embodiments of the oligonucleotide compositions, kits, and methods, a hairpin detection probe that exhibits at least some degree of self-complementarity is a molecular beacon or a molecular torch.

[0106] In certain embodiments of the oligonucleotide compositions, kits, and methods, the labeled detection probe is non-extendable. For example, the labeled detection probe can have a 3'-terminal 3'-deoxynucleotide (e.g., a terminal 2',3'-dideoxynucleotide), or a 3'-terminal inverted nucleotide (e.g., the last nucleotide is inverted such that it is attached to the second-to-last nucleotide by a 3' to 3' phosphodiester bond or an analog thereof (such as phosphorothioate)), or have a conjugated fluorophore, quencher, or other label that interferes with extension (which may be attached via the 3'-position of the terminal nucleotide, but not necessarily), and can be made non-extendable by 3'-phosphorylation. In certain embodiments, the 3'-terminal nucleotide is not methylated.

[0107] In certain embodiments of the oligonucleotide compositions, kits, and methods, the detection probe further comprises at least one modified nucleobase. In certain embodiments, the modified nucleobase is selected from the group consisting of (a) 2'-O-methyl, (b) 5-methylcytosine, (c) 2'-fluorine, and (d) combinations of two or more of (a), (b), and (c).

[0108] In certain embodiments, the oligonucleotide compositions, kits, and methods can further comprise additional reagents suitable for performing in vitro amplification, such as, for example, buffers, salts, various dNTPs, and / or enzymes.

[0109] In certain embodiments, the oligonucleotide compositions, kits, and methods can be packaged in a variety of different embodiments, and one of ordinary skill in the art will understand that the present disclosure encompasses many different kit configurations.

[0110] In certain embodiments, the oligonucleotide composition is aqueous or can be frozen or lyophilized.

[0111] The present disclosure provides formulations for the detection or amplification of VZV in a sample. In certain embodiments, the formulations disclosed herein include amplification primers for amplifying a target nucleic acid sequence within a VZV nucleic acid sequence. In some embodiments, the formulations disclose a detection probe for detecting VZV. In some embodiments, the amplification primer formulation and the detection probe are provided as two separate products or in separate vials.

[0112] In certain embodiments, the oligonucleotide formulation is configured to specifically hybridize to complementary nucleotide subunits within the target nucleic acid sequence, thus minimizing cross-reactivity with other non-VZV nucleic acids (if present) in the sample.

[0113] In certain embodiments, the formulations disclosed herein include at least one amplification primer. In certain embodiments, the formulation includes a set of amplification primers. In some embodiments, the formulation includes a set of amplification primers, wherein the first amplification primer includes a forward amplification primer and the second amplification primer includes a reverse amplification primer. In certain embodiments, the formulation includes a single set of forward and reverse amplification primers that generate a single amplicon of the target nucleic acid sequence from the target nucleic acid region. In certain embodiments, the formulation includes multiple sets of amplification primers that generate multiple amplicons from various target nucleic acid sequences within various target nucleic acid regions. In certain embodiments, the formulation includes multiple sets of amplification primers that generate multiple amplicons from various target nucleic acid sequences within a single target nucleic acid region.

[0114] In certain embodiments of the formulation, the amplification primer is configured to specifically anneal to an oligohybridization sequence within the target nucleic acid region of SEQ ID NO: 38 or SEQ ID NO: 39 of the VZV nucleic acid sequence (if present) in the sample.

[0115] In certain embodiments of the formulation, the target nucleic acid region is SEQ ID NO: 38, and the forward and reverse amplification primers are, independently of each other, about 19 to about 23 nucleotides in length, and the forward and reverse amplification primers are configured to generate an amplicon of about 89 to about 127 nucleotides in length from the target nucleic acid region of SEQ ID NO: 38.

[0116] In certain embodiments of the formulation, the target nucleic acid region is SEQ ID NO: 38, the forward amplification primer is selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7, the reverse amplification primer is about 19 to about 23 nucleotides in length, and the forward and reverse amplification primers are configured to generate an amplicon from the target nucleic acid region within SEQ ID NO: 38 that is about 89 to about 127 nucleotides in length. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the forward amplification primer comprises the sequence of SEQ ID NO: 1. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the forward oligomer comprises the sequence of SEQ ID NO: 2. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the forward oligomer comprises the sequence of SEQ ID NO: 3. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the forward oligomer comprises the sequence of SEQ ID NO: 4. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the forward oligomer comprises the sequence of SEQ ID NO: 5. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the forward oligomer comprises the sequence of SEQ ID NO: 6. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the forward oligomer comprises the sequence of SEQ ID NO: 7.

[0117] In certain embodiments of the formulation, the target nucleic acid region is SEQ ID NO: 38, the forward amplification primer is selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6 and 7, and the reverse amplification primer is about 19 to about 23 nucleotides in length and comprises the nucleic acid base sequences of SEQ ID NO: 16, 17, 18, 19, 20, 21 and 22. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the reverse oligomer comprises the sequence of SEQ ID NO: 16. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the reverse oligomer comprises the sequence of SEQ ID NO: 17. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the reverse oligomer comprises the sequence of SEQ ID NO: 18. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the reverse oligomer comprises the sequence of SEQ ID NO: 19. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the reverse oligomer comprises the sequence of SEQ ID NO: 20. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the reverse oligomer comprises the sequence of SEQ ID NO: 21. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the reverse oligomer comprises the sequence of SEQ ID NO: 22.

[0118] In certain embodiments of the formulation, the target nucleic acid region is SEQ ID NO: 38, the reverse amplification primer is selected from the group consisting of SEQ ID NO: 16, 17, 18, 19, 20, 21 and 22, and the forward amplification primer is about 20 to about 23 nucleotides in length and is configured to generate an amplicon from the target nucleic acid sequence within SEQ ID NO: 38 that is about 89 to about 127 nucleotides in length.

[0119] In certain embodiments of the formulation, the target nucleic acid region is SEQ ID NO: 38, the forward amplification primer is selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7, the reverse amplification primer is selected from the group consisting of SEQ ID NOs: 16, 17, 18, 19, 20, 21, and 22, and the forward and reverse amplification primers are configured to generate an amplicon from the target nucleic acid sequence within SEQ ID NO: 38, which is selected from the group consisting of lengths of 89, 93, 100, 102, 119, 123, and 127 nucleotides. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the amplicon is 89 nucleotides in length. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the amplicon is 93 nucleotides in length. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the amplicon is 100 nucleotides in length. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the amplicon is 102 nucleotides in length. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the amplicon is 119 nucleotides in length. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the amplicon is 123 nucleotides in length. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the amplicon is 127 nucleotides in length.

[0120] In certain embodiments of the formulation, the target nucleic acid region is SEQ ID NO: 38, the forward amplification primer is selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7, the reverse amplification primer is selected from the group consisting of SEQ ID NOs: 16, 17, 18, 19, 20, 21, and 22, and the forward and reverse amplification primers substantially contain the target nucleic acid sequence corresponding to the oligohybridization forming sequences of (a) SEQ ID NO: 1 and SEQ ID NO: 16, (b) SEQ ID NO: 1 and SEQ ID NO: 17, (c) SEQ ID NO: 2 and SEQ ID NO: 17, (d) SEQ ID NO: 3 and SEQ ID NO: 18, (e) SEQ ID NO: 4 and SEQ ID NO: 19, (f) SEQ ID NO: 5 and SEQ ID NO: 20, (g) SEQ ID NO: 6 and SEQ ID NO: 21, (h) SEQ ID NO: 7 and SEQ ID NO: 22.

[0121] In certain embodiments of the formulation, the target nucleic acid region is SEQ ID NO: 38, and (a) the forward amplification primer and the reverse amplification primer are configured to generate an amplicon from a target nucleic acid sequence at least about 89 nucleotides in length adjacent between SEQ ID NO: 3 and SEQ ID NO: 18 within the target nucleic acid region, (b) the forward amplification primer and the reverse amplification primer are configured to generate an amplicon from a target nucleic acid sequence at least about 93 nucleotides in length adjacent between SEQ ID NO: 4 and SEQ ID NO: 19 within the target nucleic acid region, (c) the forward amplification primer and the reverse amplification primer are configured to generate an amplicon from a target nucleic acid sequence at least about 100 nucleotides in length adjacent between SEQ ID NO: 2 and SEQ ID NO: 17 within the target nucleic acid region, (d) the forward amplification primer and the reverse amplification primer are configured to generate an amplicon from a target nucleic acid sequence at least about 102 nucleotides in length adjacent between SEQ ID NO: 7 and SEQ ID NO: 22 within the target nucleic acid region, (e) the forward amplification primer and the reverse amplification primer are configured to generate an amplicon from a target nucleic acid sequence at least about 119 nucleotides in length adjacent between SEQ ID NO: 6 and SEQ ID NO: 21 within the target nucleic acid region, (f) the forward amplification primer and the reverse amplification primer are configured to generate an amplicon from a target nucleic acid sequence at least about 123 nucleotides in length adjacent between SEQ ID NO: 1 and SEQ ID NO: 17 within the target nucleic acid region, (g) the forward amplification primer and the reverse amplification primer are configured to generate an amplicon from a target nucleic acid sequence at least about 127 nucleotides in length adjacent between SEQ ID NO: 1 and SEQ ID NO: 16 or between SEQ ID NO: 5 and SEQ ID NO: 20 within the target nucleic acid region.

[0122] In certain embodiments of the formulation, the target nucleic acid region is SEQ ID NO: 39, the forward amplification primer and the reverse amplification primer are about 20 to about 23 nucleotides in length, and the forward and reverse amplification primers are configured to generate an amplicon from a target nucleic acid sequence within SEQ ID NO: 39 that is about 89 to about 143 nucleotides in length.

[0123] In certain embodiments of the formulation, the target nucleic acid region is SEQ ID NO: 39, the forward amplification primer is selected from the group consisting of SEQ ID NO: 23, 24, 25, 26 and 27, the reverse amplification primer is about 20 to about 22 nucleotides in length, and the forward and reverse amplification primers are configured to generate an amplicon from the target nucleic acid region within SEQ ID NO: 39 that is about 89 to about 143 nucleotides in length. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39 and the forward oligomer comprises the sequence of SEQ ID NO: 23. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39 and the forward oligomer comprises the sequence of SEQ ID NO: 24. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39 and the forward oligomer comprises the sequence of SEQ ID NO: 25. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39 and the forward oligomer comprises the sequence of SEQ ID NO: 26. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39 and the forward oligomer comprises the sequence of SEQ ID NO: 27.

[0124] In certain embodiments of the formulation, the target nucleic acid region is SEQ ID NO: 39, the forward amplification primer is selected from the group consisting of SEQ ID NO: 23, 24, 25, 26 and 27, the reverse amplification primer is about 20 to about 22 nucleotides in length and comprises the nucleotide sequence of SEQ ID NO: 34, 35, 36 or 37. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39 and the reverse oligomer comprises the sequence of SEQ ID NO: 34. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39 and the reverse oligomer comprises the sequence of SEQ ID NO: 35. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39 and the reverse oligomer comprises the sequence of SEQ ID NO: 36. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39 and the reverse oligomer comprises the sequence of SEQ ID NO: 37.

[0125] In certain embodiments of the formulation, the target nucleic acid region is SEQ ID NO: 39, the reverse amplification primer is selected from the group consisting of SEQ ID NO: 34, 35, 36, and 37, the forward amplification primer is about 20 to about 23 nucleotides in length, and the forward and reverse amplification primers are configured to generate an amplicon from the target nucleic acid region within SEQ ID NO: 39 that is about 89 to about 143 nucleotides in length.

[0126] In certain embodiments of the formulation, the target nucleic acid region is SEQ ID NO: 39, the forward amplification primer is selected from the group consisting of SEQ ID NO: 23, 24, 25, 26, and 27, the reverse amplification primer is selected from the group consisting of SEQ ID NO: 34, 35, 36, and 37, and the forward and reverse amplification primers are configured to generate an amplicon from the target nucleic acid sequence within SEQ ID NO: 39 selected from the group consisting of lengths of 89, 99, 109, 126, and 143 nucleotides. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39 and the amplicon is 89 nucleotides in length. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39 and the amplicon is 99 nucleotides in length. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39 and the amplicon is 109 nucleotides in length. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39 and the amplicon is 126 nucleotides in length. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39 and the amplicon is 143 nucleotides in length.

[0127] In certain embodiments of the formulation, the target nucleic acid region is SEQ ID NO: 39, the forward amplification primer is selected from the group consisting of SEQ ID NO: 23, 24, 25, 26, and 27, the reverse amplification primer is selected from the group consisting of SEQ ID NO: 34, 35, 36, and 37, and the forward and reverse amplification primers substantially contain the target nucleic acid sequence corresponding to the oligohybridization-forming sequences of (a) SEQ ID NO: 23 and SEQ ID NO: 34, (b) SEQ ID NO: 24 and SEQ ID NO: 34, (c) SEQ ID NO: 25 and SEQ ID NO: 35, (d) SEQ ID NO: 26 and SEQ ID NO: 36, (e) SEQ ID NO: 27 and SEQ ID NO: 37.

[0128] In certain embodiments of the formulation, the target nucleic acid region is SEQ ID NO: 39, and (a) the forward amplification primer and the reverse amplification primer are configured to generate an amplicon from a target nucleic acid sequence having a length of at least about 89 nucleotides adjacent between SEQ ID NO: 25 and SEQ ID NO: 35 within the target nucleic acid region, (b) the forward amplification primer and the reverse amplification primer are configured to generate an amplicon from a target nucleic acid sequence having a length of at least about 99 nucleotides adjacent between SEQ ID NO: 24 and SEQ ID NO: 34 within the target nucleic acid region, (c) the forward amplification primer and the reverse amplification primer are configured to generate an amplicon from a target nucleic acid sequence having a length of at least about 109 nucleotides adjacent between SEQ ID NO: 23 and SEQ ID NO: 34 within the target nucleic acid region, (d) the forward amplification primer and the reverse amplification primer are configured to generate an amplicon from a target nucleic acid sequence having a length of at least about 126 nucleotides adjacent between SEQ ID NO: 27 and SEQ ID NO: 37 within the target nucleic acid region, (e) the forward amplification primer and the reverse amplification primer are configured to generate an amplicon from a target nucleic acid sequence having a length of at least about 143 nucleotides adjacent between SEQ ID NO: 26 and SEQ ID NO: 36 within the target nucleic acid region.

[0129] In certain embodiments of the formulation, at least one amplification primer is configured to anneal to the target nucleic acid sequence in the forward direction, and at least one amplification primer is configured to anneal to the target nucleic acid sequence in the reverse direction. In certain embodiments of the formulation, the forward and reverse amplification primers specifically hybridize to a contiguous nucleotide sequence comprising an oligo-hybridization forming sequence on the target nucleic acid sequence amplified within the target nucleic acid region of SEQ ID NO: 38 or SEQ ID NO: 39 of the VZV nucleic acid sequence (if present) in the sample.

[0130] In some embodiments of the formulation, a composition for determining the presence (or absence) of a target nucleic acid sequence of VZV in a sample comprises (a) at least one forward amplification primer configured to specifically hybridize to an oligo hybridization-forming sequence within the target nucleic acid region of SEQ ID NO: 38 or SEQ ID NO: 39, and (b) at least one reverse amplification primer configured to specifically hybridize to an oligo hybridization-forming sequence within the target nucleic acid region of SEQ ID NO: 38 or SEQ ID NO: 39.

[0131] In certain aspects of the formulation, the forward amplification primer comprises at least one modified nucleobase. In certain aspects, the modified nucleobase is selected from the group consisting of (a) 2'-O-methyl, (b) 5-methyl-cytosine, (c) 2'-fluoro, and (d) combinations of two or more of (a), (b), and (c).

[0132] In certain embodiments of the formulation, the forward amplification primer comprises 2 to 6 modified nucleobases. The 2 to 6 modified nucleobases may be the same or different. In certain embodiments, the forward amplification primer comprises 2 to 6 5-methylcytosine residues. In some embodiments, the forward amplification primer comprises 2 5-methylcytosine residues. In some embodiments, the forward amplification primer comprises 3 5-methylcytosine residues. In certain embodiments, the forward amplification primer comprises 4 5-methylcytosine residues. In certain embodiments, the forward amplification primer comprises 5 5-methylcytosine residues. In certain embodiments, the forward amplification primer comprises 6 5-methylcytosine residues. In certain embodiments, the forward amplification primer comprises 2 to 6 2'-O-methyl residues. In some embodiments, the forward amplification primer comprises 2 2'-O-methyl residues. In some embodiments, the forward amplification primer comprises 3 2'-O-methyl residues. In certain embodiments, the forward amplification primer comprises 4 2'-O-methyl residues. In certain embodiments, the forward amplification primer comprises 5 2'-O-methyl residues. In certain embodiments, the forward amplification primer comprises 6 2'-O-methyl residues.

[0133] In certain embodiments of the formulation, the reverse amplification primer comprises at least one modified nucleobase. In certain embodiments, the modified nucleobase is selected from the group consisting of (a) 2'-O-methyl, (b) 5-methyl-cytosine, (c) 2'-fluoro, and (d) combinations of two or more of (a), (b), and (c).

[0134] In certain embodiments of the formulation, the reverse amplification primer comprises 2 to 6 modified nucleobases. The 2 to 6 modified nucleobases may be the same or different. In certain embodiments, the reverse amplification primer comprises 2 to 6 5-methylcytosine residues. In certain embodiments, the reverse amplification primer comprises 2 5-methylcytosine residues. In some embodiments, the reverse amplification primer comprises 3 5-methylcytosine residues. In some embodiments, the reverse amplification primer comprises 4 5-methyl-cytosine residues. In some embodiments, the reverse amplification primer comprises 5 5-methyl-cytosine residues. In some embodiments, the reverse amplification primer comprises 6 5-methylcytosine residues. In certain embodiments, the reverse amplification primer comprises 2 to 6 2'-O-methyl residues. In certain embodiments, the reverse amplification primer comprises 2 2'-O-methyl residues. In some embodiments, the reverse amplification primer comprises 3 2'-O-methyl residues. In some embodiments, the reverse amplification primer comprises 4 2'-O-methyl residues. In some embodiments, the reverse amplification primer comprises 5 2'-O-methyl residues. In some embodiments, the reverse amplification primer comprises 6 2'-O-methyl residues.

[0135] In certain embodiments of the formulation, the third oligomer is configured to specifically anneal to the target nucleic acid sequence to be amplified within the target nucleic acid regions of SEQ ID NO: 38 and SEQ ID NO: 39 of the VZV nucleic acid sequence (if present) in the sample. In certain embodiments, the third oligomer hybridizes to the oligohybridization-forming sequence within SEQ ID NO: 38. In some embodiments, the third oligomer hybridizes to the oligohybridization-forming sequence within SEQ ID NO: 39. In certain embodiments of the formulation, the third oligomer is a detection probe.

[0136] In certain embodiments of the formulation, the target nucleic acid region is SEQ ID NO: 38 and the detection probe is about 23 to about 27 nucleotides in length.

[0137] In certain embodiments of the formulation, the target nucleic acid region is SEQ ID NO: 38, and the detection probe is selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, and 15. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38, and the detection probe comprises the sequence of SEQ ID NO: 8. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38, and the detection probe comprises the sequence of SEQ ID NO: 9. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38, and the detection probe comprises the sequence of SEQ ID NO: 10. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38, and the detection probe comprises the sequence of SEQ ID NO: 11. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38, and the detection probe comprises the sequence of SEQ ID NO: 12. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38, and the detection probe comprises the sequence of SEQ ID NO: 13. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38, and the detection probe comprises the sequence of SEQ ID NO: 14. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38, and the detection probe comprises the sequence of SEQ ID NO: 15.

[0138] In certain embodiments of the formulation, the target nucleic acid region is SEQ ID NO: 38, and the detection probe comprises a target nucleic acid sequence that substantially corresponds to the following oligohybridization-forming sequences. When the forward and reverse amplification primers are (I) SEQ ID NO: 1 and SEQ ID NO: 16 or (II) SEQ ID NO: 1 and SEQ ID NO: 17, the detection probe is SEQ ID NO: 8; when the forward and reverse amplification primers are (I) SEQ ID NO: 1 and SEQ ID NO: 16 or (II) SEQ ID NO: 1 and SEQ ID NO: 17 or (III) SEQ ID NO: 2 and SEQ ID NO: 17, the detection probe is SEQ ID NO: 9; when the forward and reverse amplification primers are SEQ ID NO: 3 and SEQ ID NO: 18, the detection probe is SEQ ID NO: 10; when the forward and reverse amplification primers are SEQ ID NO: 4 and SEQ ID NO: 19, the detection probe is SEQ ID NO: 11; when the forward and reverse amplification primers are SEQ ID NO: 4 and SEQ ID NO: 19, the detection probe is SEQ ID NO: 12; when the forward and reverse amplification primers are SEQ ID NO: 5 and SEQ ID NO: 20, the detection probe is SEQ ID NO: 13; when the forward and reverse amplification primers are SEQ ID NO: 6 and SEQ ID NO: 21, the detection probe is SEQ ID NO: 14; when the forward and reverse amplification primers are SEQ ID NO: 7 and SEQ ID NO: 22, the detection probe is SEQ ID NO: 15.

[0139] In certain embodiments of the formulation, the target nucleic acid region is SEQ ID NO: 38, and (a) when the forward amplification primer and the reverse amplification primer are configured to generate an amplicon of a target nucleic acid sequence having a length of at least about 89 nucleotides from SEQ ID NO: 3 and SEQ ID NO: 18 on the target nucleic acid region, the detection probe comprises the sequence of SEQ ID NO: 10, (b) when the forward amplification primer and the reverse amplification primer are configured to generate an amplicon of a target nucleic acid sequence having a length of at least about 93 nucleotides from SEQ ID NO: 4 and SEQ ID NO: 19 on the target nucleic acid region, the detection probe comprises the sequence of SEQ ID NO: 11 or SEQ ID NO: 12, (c) when the forward amplification primer and the reverse amplification primer are configured to generate an amplicon of a target nucleic acid sequence having a length of at least about 100 nucleotides from SEQ ID NO: 2 and SEQ ID NO: 17 on the target nucleic acid region, the detection probe comprises the sequence of SEQ ID NO: 9, (d) when the forward amplification primer and the reverse amplification primer are configured to generate an amplicon of a target nucleic acid sequence having a length of at least about 102 nucleotides from SEQ ID NO: 7 and SEQ ID NO: 22 on the target nucleic acid region, the detection probe comprises the sequence of SEQ ID NO: 15, (e) when the forward amplification primer and the reverse amplification primer are configured to generate an amplicon of a target nucleic acid sequence having a length of at least about 119 nucleotides from SEQ ID NO: 6 and SEQ ID NO: 21 on the target nucleic acid region, the detection probe comprises the sequence of SEQ ID NO: 14, (f) when the forward amplification primer and the reverse amplification primer are configured to generate an amplicon of a target nucleic acid sequence having a length of at least about 123 nucleotides from SEQ ID NO: 1 and SEQ ID NO: 17 on the target nucleic acid region, the detection probe comprises the sequence of SEQ ID NO: 8 or SEQ ID NO: 9, (g) when the forward amplification primer and the reverse amplification primer are configured to generate an amplicon of a target nucleic acid sequence having a length of at least about 127 nucleotides from SEQ ID NO: 1 and SEQ ID NO: 16 on the target nucleic acid region, the detection probe comprises the sequence of SEQ ID NO: 8 or SEQ ID NO: 9, or when the forward amplification primer and the reverse amplification primer are configured to generate an amplicon of a target nucleic acid sequence having a length of at least about 127 nucleotides from SEQ ID NO: 5 and SEQ ID NO: 20 on the target nucleic acid region,The detection probe contains the sequence of SEQ ID NO: 13.,

[0140] In certain embodiments of the formulation, the target nucleic acid region is SEQ ID NO: 39 and the detection probe is about 22 to about 27 nucleotides in length.

[0141] In certain embodiments of the formulation, the target nucleic acid region is SEQ ID NO: 39 and the detection probe is selected from the group consisting of SEQ ID NOs: 28, 29, 30, 31, 32, and 33. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39 and the detection probe contains the sequence of SEQ ID NO: 28. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39 and the detection probe contains the sequence of SEQ ID NO: 29. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39 and the detection probe contains the sequence of SEQ ID NO: 30. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39 and the detection probe contains the sequence of SEQ ID NO: 31. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39 and the detection probe contains the sequence of SEQ ID NO: 32. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39 and the detection probe contains the sequence of SEQ ID NO: 33.

[0142] In certain embodiments of the formulation, the target nucleic acid region is SEQ ID NO: 39 and the detection probe contains a target nucleic acid sequence that substantially corresponds to the following oligohybridization-forming sequences. When the forward and reverse amplification primers are (I) SEQ ID NO: 23 and SEQ ID NO: 34 or (II) SEQ ID NO: 24 and SEQ ID NO: 34, the detection probe is SEQ ID NO: 28; when the forward and reverse amplification primers are SEQ ID NO: 25 and SEQ ID NO: 35, the detection probe is SEQ ID NO: 29; when the forward and reverse amplification primers are SEQ ID NO: 25 and SEQ ID NO: 35, the detection probe is SEQ ID NO: 30; when the forward and reverse amplification primers are SEQ ID NO: 26 and SEQ ID NO: 36, the detection probe is SEQ ID NO: 31; when the forward and reverse amplification primers are SEQ ID NO: 27 and SEQ ID NO: 37, the detection probe is SEQ ID NO: 32; when the forward and reverse amplification primers are SEQ ID NO: 27 and SEQ ID NO: 37, the detection probe is SEQ ID NO: 33.

[0143] In certain embodiments of the formulation, the target nucleic acid region is SEQ ID NO: 39, and (a) when the forward amplification primer and the reverse amplification primer are configured to generate an amplicon of a target nucleic acid sequence having a length of at least about 89 nucleotides from SEQ ID NO: 25 and SEQ ID NO: 35 on the target nucleic acid region, the third oligomer comprises the sequence of SEQ ID NO: 29 or SEQ ID NO: 30; (b) when the forward amplification primer and the reverse amplification primer are configured to generate an amplicon of a target nucleic acid sequence having a length of at least about 99 nucleotides from SEQ ID NO: 24 and SEQ ID NO: 34 on the target nucleic acid region, the third oligomer comprises the sequence of SEQ ID NO: 28; (c) when the forward amplification primer and the reverse amplification primer are configured to generate an amplicon of a target nucleic acid sequence having a length of at least about 109 nucleotides from SEQ ID NO: 23 and SEQ ID NO: 34 on the target nucleic acid region, the third oligomer comprises the sequence of SEQ ID NO: 28; (d) when the forward amplification primer and the reverse amplification primer are configured to generate an amplicon of a target nucleic acid sequence having a length of at least about 126 nucleotides from SEQ ID NO: 27 and SEQ ID NO: 37 on the target nucleic acid region, the third oligomer comprises the sequence of SEQ ID NO: 32 or SEQ ID NO: 33; (e) when the forward amplification primer and the reverse amplification primer are configured to generate an amplicon of a target nucleic acid sequence having a length of at least about 143 nucleotides from SEQ ID NO: 26 and SEQ ID NO: 36 on the target nucleic acid region, the third oligomer comprises the sequence of SEQ ID NO: 31.

[0144] In certain embodiments, the formulation for determining the presence (or absence) of VZV in a sample as described herein further comprises at least one detection probe configured to specifically anneal to an oligo-hybridization forming sequence within the target nucleic acid region of SEQ ID NO: 38 or SEQ ID NO: 39, and the detection probe is adjacent between the forward amplification primer and the reverse amplification primer.

[0145] In certain embodiments of the formulation, the detection probe comprises at least one detectable label. In certain embodiments, the detection probe further comprises a second label that interacts with the first label, such as a quencher. In certain embodiments of the formulation, the label is selected from the group consisting of (a) a chemiluminescent label, (b) a fluorescent label, (c) a quencher, and (d) combinations of two or more of (a), (b), and (c). In certain embodiments, the label comprises a fluorescent label. In certain embodiments, the label comprises a quencher. In certain embodiments, the formulation comprises a detection probe having both a fluorescent label and a quencher.

[0146] In certain embodiments of the formulation, the detection probe is linear and does not exhibit any degree of self-complementarity held by intramolecular bonds. In such embodiments, the linear detection probe comprises a fluorophore as a label. In some embodiments, the linear detection probe comprises both a fluorophore and a quenching moiety (e.g., TaqMan™ probe).

[0147] In certain embodiments of the formulation, the detection probe exhibits at least some degree of self-complementarity and is used to facilitate the detection of probe:target duplexes in a sample without the need to first hybridize and remove unhybridized probes prior to detection. In certain embodiments of the formulation, the hairpin detection probe that exhibits at least some degree of self-complementarity is a molecular beacon or a molecular torch.

[0148] In certain embodiments of the formulation, the labeled detection probe is non-extendable. For example, the labeled detection probe can have a 3'-terminal 3'-deoxynucleotide (e.g., a terminal 2',3'-dideoxynucleotide), or a 3'-terminal-inverted nucleotide (e.g., the last nucleotide is inverted such that it is attached to the second-to-last nucleotide by a 3' to 3' phosphodiester bond or an analog thereof (such as phosphorothioate)), or a conjugated fluorophore, quencher, or other label that interferes with extension (which may be attached via the 3'-position of the terminal nucleotide, but not necessarily), and can be made non-extendable by 3'-phosphorylation. In certain embodiments, the 3'-terminal nucleotide is not methylated.

[0149] In certain embodiments of the formulation, the detection probe contains at least one modified nucleobase. In certain embodiments, the modified nucleobase is selected from the group consisting of (a) 2'-O-methyl, (b) 5-methyl-cytosine, (c) 2'-fluoro, and (d) combinations of two or more of (a), (b), and (c).

[0150] In certain embodiments, the formulation can further contain additional reagents suitable for performing in vitro amplification, such as, for example, buffers, salts, various dNTPs, and / or enzymes.

[0151] In certain embodiments, the formulation can be packaged in a variety of different embodiments, and one of ordinary skill in the art will understand that the present disclosure encompasses many different kit configurations.

[0152] In certain embodiments, the formulations disclosed herein are aqueous, or can be frozen or lyophilized.

[0153] The presence or absence of the VZV nucleic acid sequence in a sample is determined, and when the target nucleic acid sequence of VZV is present, a reaction mixture for amplifying it is also provided. The amplification primer formulation and the detection probe formulation can be provided as separate formulations or compositions, or as a single formulation of a composition. The reaction mixture further includes other reagents necessary for in vitro amplification, including but not limited to buffers, salts, various dNTPs, enzymes (e.g., thermostable DNA polymerase), and the test sample.

[0154] In certain embodiments, a reaction mixture for amplifying a target nucleic acid sequence within a target nucleic acid region of VZV, or for amplifying an amplicon generated from a target nucleic acid sequence within a target nucleic acid region, includes a first amplification primer and a detection probe.

[0155] In certain embodiments, the reaction mixture includes a set of amplification primers for determining the presence or absence of the VZV nucleic acid sequence in a sample, the first amplification primer includes a forward amplification primer, and the second amplification primer includes a reverse amplification primer.

[0156] In certain embodiments, the reaction mixture is configured to specifically anneal to the oligohybridization-forming sequences within the target nucleic acid regions of SEQ ID NO: 38 and SEQ ID NO: 39 of the VZV nucleic acid sequence (if present) in the sample.

[0157] In certain embodiments, the target nucleic acid region is SEQ ID NO: 38, and the reaction mixture includes forward and reverse amplification primers that are each independently about 19 to about 23 nucleotides in length, and the forward and reverse amplification primers are configured to generate an amplicon that is about 89 to about 127 nucleotides in length from the target nucleic acid region of SEQ ID NO: 38.

[0158] In certain embodiments, the target nucleic acid region is SEQ ID NO: 38, and the reaction mixture includes a forward amplification primer selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7 and a reverse amplification primer of about 19 to about 23 nucleotides in length, wherein the forward and reverse amplification primers are configured to generate an amplicon from the target nucleic acid region within SEQ ID NO: 38 that is about 89 to about 127 nucleotides in length. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the forward amplification primer includes the sequence of SEQ ID NO: 1. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the forward oligomer includes the sequence of SEQ ID NO: 2. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the forward oligomer includes the sequence of SEQ ID NO: 3. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the forward oligomer includes the sequence of SEQ ID NO: 4. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the forward oligomer includes the sequence of SEQ ID NO: 5. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the forward oligomer includes the sequence of SEQ ID NO: 6. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the forward oligomer includes the sequence of SEQ ID NO: 7.

[0159] In certain embodiments, the target nucleic acid region is SEQ ID NO: 38, and the reaction mixture comprises a forward amplification primer selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7, and a reverse amplification primer selected from the group consisting of SEQ ID NOs: 16, 17, 18, 19, 20, 21, and 22 and having a length of about 19 to about 23 nucleotides. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the reverse oligomer comprises the sequence of SEQ ID NO: 16. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the reverse oligomer comprises the sequence of SEQ ID NO: 17. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the reverse oligomer comprises the sequence of SEQ ID NO: 18. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the reverse oligomer comprises the sequence of SEQ ID NO: 19. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the reverse oligomer comprises the sequence of SEQ ID NO: 20. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the reverse oligomer comprises the sequence of SEQ ID NO: 21. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the reverse oligomer comprises the sequence of SEQ ID NO: 22.

[0160] In certain embodiments, the target nucleic acid region is SEQ ID NO: 38, and the reaction mixture comprises a reverse amplification primer selected from the group consisting of SEQ ID NOs: 16, 17, 18, 19, 20, 21, and 22, and a forward amplification primer having a length of about 20 to about 23 nucleotides, wherein the amplification oligomer is configured to generate an amplicon from a target nucleic acid sequence within SEQ ID NO: 38 having a length of about 89 to about 127 nucleotides.

[0161] In certain embodiments, the target nucleic acid region is SEQ ID NO: 38, and the reaction mixture comprises a forward amplification primer selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7, and a reverse amplification primer selected from the group consisting of SEQ ID NOs: 16, 17, 18, 19, 20, 21, and 22, wherein the forward and reverse amplification primers are configured to generate an amplicon from the target nucleic acid sequence within SEQ ID NO: 38 that is 89, 93, 100, 102, 119, 123, or 127 nucleotides in length. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the amplicon is 89 nucleotides in length. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the amplicon is 93 nucleotides in length. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the amplicon is 100 nucleotides in length. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the amplicon is 102 nucleotides in length. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the amplicon is 119 nucleotides in length. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the amplicon is 123 nucleotides in length. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the amplicon is 127 nucleotides in length.

[0162] In certain embodiments, the target nucleic acid region is SEQ ID NO: 38, and the reaction mixture comprises a forward amplification primer selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7, and a reverse amplification primer selected from the group consisting of SEQ ID NOs: 16, 17, 18, 19, 20, 21, and 22, wherein the forward and reverse amplification primers substantially comprise the target nucleic acid sequence corresponding to the oligohybridization formation sequences of (a) SEQ ID NO: 1 and SEQ ID NO: 16, (b) SEQ ID NO: 1 and SEQ ID NO: 17, (c) SEQ ID NO: 2 and SEQ ID NO: 17, (d) SEQ ID NO: 3 and SEQ ID NO: 18, (e) SEQ ID NO: 4 and SEQ ID NO: 19, (f) SEQ ID NO: 5 and SEQ ID NO: 20, (g) SEQ ID NO: 6 and SEQ ID NO: 21, (h) SEQ ID NO: 7 and SEQ ID NO: 22.

[0163] In certain embodiments, the target nucleic acid region is SEQ ID NO: 38, and the reaction mixture comprises one or more of the following: (a) a forward amplification primer and a reverse amplification primer configured to generate an amplicon from a target nucleic acid sequence at least about 89 nucleotides in length adjacent between SEQ ID NO: 3 and SEQ ID NO: 18 within the target nucleic acid region; (b) a forward amplification primer and a reverse amplification primer configured to generate an amplicon from a target nucleic acid sequence at least about 93 nucleotides in length adjacent between SEQ ID NO: 4 and SEQ ID NO: 19 within the target nucleic acid region; (c) a forward amplification primer and a reverse amplification primer configured to generate an amplicon from a target nucleic acid sequence at least about 100 nucleotides in length adjacent between SEQ ID NO: 2 and SEQ ID NO: 17 within the target nucleic acid region; (d) a forward amplification primer and a reverse amplification primer configured to generate an amplicon from a target nucleic acid sequence at least about 102 nucleotides in length adjacent between SEQ ID NO: 7 and SEQ ID NO: 22 within the target nucleic acid region; (e) a forward amplification primer and a reverse amplification primer configured to generate an amplicon from a target nucleic acid sequence at least about 119 nucleotides in length adjacent between SEQ ID NO: 6 and SEQ ID NO: 21 within the target nucleic acid region; (f) a forward amplification primer and a reverse amplification primer configured to generate an amplicon from a target nucleic acid sequence at least about 123 nucleotides in length adjacent between SEQ ID NO: 1 and SEQ ID NO: 17 within the target nucleic acid region; (g) a forward amplification primer and a reverse amplification primer configured to generate an amplicon from a target nucleic acid sequence at least about 127 nucleotides in length adjacent between SEQ ID NO: 1 and SEQ ID NO: 16 or between SEQ ID NO: 5 and SEQ ID NO: 20 within the target nucleic acid region.

[0164] In certain embodiments, the target nucleic acid region is SEQ ID NO: 39, and the reaction mixture comprises a forward amplification primer and a reverse amplification primer, each independently about 20 to about 23 nucleotides in length, wherein the forward and reverse amplification primers are configured to generate an amplicon from a target nucleic acid sequence in SEQ ID NO: 39 that is about 89 to about 143 nucleotides in length.

[0165] In certain embodiments, the target nucleic acid region is SEQ ID NO: 39, and the reaction mixture comprises a forward amplification primer selected from the group consisting of SEQ ID NOs: 23, 24, 25, 26, and 27, and a reverse amplification primer having a length of about 20 to about 22 nucleotides. The forward and reverse amplification primers are configured to generate an amplicon from the target nucleic acid sequence within SEQ ID NO: 39 having a length of about 89 to about 143 nucleotides. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39, and the forward oligomer comprises the sequence of SEQ ID NO: 23. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39, and the forward oligomer comprises the sequence of SEQ ID NO: 24. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39, and the forward oligomer comprises the sequence of SEQ ID NO: 25. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39, and the forward oligomer comprises the sequence of SEQ ID NO: 26. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39, and the forward oligomer comprises the sequence of SEQ ID NO: 27.

[0166] In certain embodiments, the target nucleic acid region is SEQ ID NO: 39, and the reaction mixture comprises a forward amplification primer selected from the group consisting of SEQ ID NOs: 23, 24, 25, 26, and 27, and a reverse amplification primer selected from the group consisting of SEQ ID NOs: 34, 35, 36, and 37 and having a length of about 20 to about 22 nucleotides. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39, and the reverse oligomer comprises the sequence of SEQ ID NO: 34. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39, and the reverse oligomer comprises the sequence of SEQ ID NO: 35. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39, and the reverse oligomer comprises the sequence of SEQ ID NO: 36. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39, and the reverse oligomer comprises the sequence of SEQ ID NO: 37.

[0167] In certain embodiments, the target nucleic acid region is SEQ ID NO: 39, and the reaction mixture comprises a reverse amplification primer selected from the group consisting of SEQ ID NO: 34, 35, 36, and 37, and a forward amplification primer of about 20 to about 23 nucleotides in length, wherein the forward and reverse amplification primers are configured to generate an amplicon from the target nucleic acid sequence within SEQ ID NO: 39 that is about 89 to about 143 nucleotides in length.

[0168] In certain embodiments, the target nucleic acid region is SEQ ID NO: 39, and the reaction mixture comprises a forward amplification primer selected from the group consisting of SEQ ID NO: 23, 24, 25, 26, and 27, and a reverse amplification primer selected from the group consisting of SEQ ID NO: 34, 35, 36, and 37, wherein the forward and reverse amplification primers are configured to generate an amplicon from the target nucleic acid sequence within SEQ ID NO: 39 that is 89, 99, 109, 126, or 143 nucleotides in length. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39 and the amplicon is 89 nucleotides in length. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39 and the amplicon is 99 nucleotides in length. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39 and the amplicon is 109 nucleotides in length. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39 and the amplicon is 126 nucleotides in length. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39 and the amplicon is 143 nucleotides in length.

[0169] In certain embodiments, the target nucleic acid region is SEQ ID NO: 39, and the reaction mixture comprises a forward amplification primer selected from the group consisting of SEQ ID NO: 23, 24, 25, 26, and 27, and a reverse amplification primer selected from the group consisting of SEQ ID NO: 34, 35, 36, and 37, wherein the forward and reverse amplification primers substantially contain the target nucleic acid sequence corresponding to the oligohybridization formation sequences of (a) SEQ ID NO: 23 and SEQ ID NO: 34, (b) SEQ ID NO: 24 and SEQ ID NO: 34, (c) SEQ ID NO: 25 and SEQ ID NO: 35, (d) SEQ ID NO: 26 and SEQ ID NO: 36, (e) SEQ ID NO: 27 and SEQ ID NO: 37.

[0170] In certain embodiments, the target nucleic acid region is SEQ ID NO: 39, and the reaction mixture comprises one or more of the following: (a) a forward amplification primer and a reverse amplification primer configured to generate an amplicon from a target nucleic acid sequence at least about 89 nucleotides in length adjacent between SEQ ID NO: 25 and SEQ ID NO: 35 within the target nucleic acid region; (b) a forward amplification primer and a reverse amplification primer configured to generate an amplicon from a target nucleic acid sequence at least about 99 nucleotides in length adjacent between SEQ ID NO: 24 and SEQ ID NO: 34 within the target nucleic acid region; (c) a forward amplification primer and a reverse amplification primer configured to generate an amplicon from a target nucleic acid sequence at least about 109 nucleotides in length adjacent between SEQ ID NO: 23 and SEQ ID NO: 34 within the target nucleic acid region; (d) a forward amplification primer and a reverse amplification primer configured to generate an amplicon from a target nucleic acid sequence at least about 126 nucleotides in length adjacent between SEQ ID NO: 27 and SEQ ID NO: 37 within the target nucleic acid region; (e) a forward amplification primer and a reverse amplification primer configured to generate an amplicon from a target nucleic acid sequence at least about 143 nucleotides in length adjacent between SEQ ID NO: 26 and SEQ ID NO: 36 within the target nucleic acid region.

[0171] In certain embodiments, the reaction mixture comprises at least one amplification primer configured to anneal to the target nucleic acid sequence in the forward direction and at least one amplification primer configured to anneal to the target nucleic acid sequence in the reverse direction, wherein the amplification primer specifically hybridizes to a contiguous nucleotide sequence comprising an oligo-hybridization forming sequence on the target nucleic acid sequence that is amplified within the target nucleic acid region of SEQ ID NO: 38 or SEQ ID NO: 39 of the VZV nucleic acid sequence (if present) in the sample.

[0172] In some embodiments of the reaction mixture, a composition for determining the presence (or absence) of a target nucleic acid sequence of VZV in a sample comprises (a) at least one forward amplification primer configured to specifically hybridize to an oligo-hybridization forming sequence within the target nucleic acid region of SEQ ID NO: 38 or SEQ ID NO: 39, and (b) at least one reverse amplification primer configured to specifically hybridize to an oligo-hybridization forming sequence within the target nucleic acid region of SEQ ID NO: 38 or SEQ ID NO: 39.

[0173] In certain embodiments of the reaction mixture, the forward amplification primer comprises at least one modified nucleobase. In certain embodiments, the modified nucleobase is selected from the group consisting of (a) 2'-O-methyl, (b) 5-methylcytosine, (c) 2'-fluoro, and (d) combinations of two or more of (a), (b), and (c).

[0174] In certain embodiments of the reaction mixture, the forward amplification primer comprises 2 to 6 modified nucleobases. The 2 to 6 modified nucleobases may be the same or different. In certain embodiments, the forward amplification primer comprises 2 to 6 5-methylcytosine residues. In some embodiments, the forward amplification primer comprises 2 5-methylcytosine residues. In some embodiments, the forward amplification primer comprises 3 5-methylcytosine residues. In some embodiments, the forward amplification primer comprises 4 5-methylcytosine residues. In some embodiments, the forward amplification primer comprises 5 5-methylcytosine residues. In some embodiments, the forward amplification primer comprises 6 5-methylcytosine residues. In certain embodiments, the forward amplification primer comprises 2 to 6 2'-O-methyl residues. In some embodiments, the forward amplification primer comprises 2 2'-O-methyl residues. In some embodiments, the forward amplification primer comprises 3 2'-O-methyl residues. In some embodiments, the forward amplification primer comprises 4 2'-O-methyl residues. In some embodiments, the forward amplification primer comprises 5 2'-O-methyl residues. In some embodiments, the forward amplification primer comprises 6 2'-O-methyl residues.

[0175] In certain embodiments of the reaction mixture, the reverse amplification primer comprises at least one modified nucleobase. In certain embodiments, the modified nucleobase is selected from the group consisting of (a) 2'-O-methyl, (b) 5'-methylcytosine, (c) 2'-fluorine, and (d) combinations of two or more of (a), (b), and (c).

[0176] In certain embodiments, the reverse amplification primer comprises 2 to 6 modified nucleic acid residues. The 2 to 6 modified nucleobases may be the same or different. In certain embodiments, the reverse amplification primer comprises 2 to 6 5-methylcytosine residues. In some embodiments, the reverse amplification primer comprises 2 5-methylcytosine residues. In some embodiments, the reverse amplification primer comprises 3 5-methylcytosine residues. In some embodiments, the reverse amplification primer comprises 4 5-methylcytosine residues. In some embodiments, the reverse amplification primer comprises 5 5-methylcytosine residues. In some embodiments, the reverse amplification primer comprises 6 5-methylcytosine residues. In certain embodiments, the reverse amplification primer comprises 2 to 6 2'-O-methyl residues. In some embodiments, the reverse amplification primer comprises 2 2'-O-methyl residues. In some embodiments, the reverse amplification primer comprises 3 2'-O-methyl residues. In some embodiments, the reverse amplification primer comprises 4 2'-O-methyl residues. In some embodiments, the reverse amplification primer comprises 5 2'-O-methyl residues. In some embodiments, the reverse amplification primer comprises 6 2'-O-methyl residues.

[0177] In certain embodiments, the reaction mixture comprises a third oligomer configured to specifically anneal to a target nucleic acid sequence that is amplified within the target nucleic acid regions of SEQ ID NO: 38 and SEQ ID NO: 39 of the VZV nucleic acid sequence (if present) in the sample. In certain embodiments, the third oligomer hybridizes to the oligohybridization sequence within SEQ ID NO: 38. In some embodiments, the third oligomer hybridizes to the oligohybridization sequence within SEQ ID NO: 39. In certain embodiments, the third oligomer is a detection probe.

[0178] In certain embodiments, the target nucleic acid region is SEQ ID NO: 38 and the reaction mixture comprises a detection probe that is about 23 to about 27 nucleotides in length.

[0179] In certain embodiments, the target nucleic acid region is SEQ ID NO: 38, and the reaction mixture comprises a detection probe selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, and 15. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38, and the detection probe comprises the sequence of SEQ ID NO: 8. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38, and the detection probe comprises the sequence of SEQ ID NO: 9. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38, and the detection probe comprises the sequence of SEQ ID NO: 10. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38, and the detection probe comprises the sequence of SEQ ID NO: 11. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38, and the detection probe comprises the sequence of SEQ ID NO: 12. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38, and the detection probe comprises the sequence of SEQ ID NO: 13. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38, and the detection probe comprises the sequence of SEQ ID NO: 14. In certain embodiments, the target nucleic acid region is SEQ ID NO: 38, and the detection probe oligomer comprises the sequence of SEQ ID NO: 15.

[0180] In certain embodiments, the target nucleic acid region is SEQ ID NO: 38, and the reaction mixture comprises a detection probe that comprises a target nucleic acid sequence that substantially corresponds to the following oligohybridization-forming sequences. SEQ ID NO: 8 when the forward and reverse amplification primers are (I) SEQ ID NO: 1 and SEQ ID NO: 16 or (II) SEQ ID NO: 1 and SEQ ID NO: 17; SEQ ID NO: 9 when the forward and reverse amplification primers are (I) SEQ ID NO: 1 and SEQ ID NO: 16 or (II) SEQ ID NO: 1 and SEQ ID NO: 17 or (III) SEQ ID NO: 2 and SEQ ID NO: 17; SEQ ID NO: 10 when the forward and reverse amplification primers are SEQ ID NO: 3 and SEQ ID NO: 18; SEQ ID NO: 11 when the forward and reverse amplification primers are SEQ ID NO: 4 and SEQ ID NO: 19; SEQ ID NO: 12 when the forward and reverse amplification primers are SEQ ID NO: 4 and SEQ ID NO: 19; SEQ ID NO: 13 when the forward and reverse amplification primers are SEQ ID NO: 5 and SEQ ID NO: 20; SEQ ID NO: 14 when the forward and reverse amplification primers are SEQ ID NO: 6 and SEQ ID NO: 21; SEQ ID NO: 15 when the forward and reverse amplification primers are SEQ ID NO: 7 and SEQ ID NO: 22.

[0181] In certain embodiments, the target nucleic acid region is SEQ ID NO: 38, and the reaction mixture comprises one or more of the following.(a) When the forward amplification primer and the reverse amplification primer are configured to generate an amplicon of a target nucleic acid sequence with a length of at least about 89 nucleotides from SEQ ID NO: 3 and SEQ ID NO: 18 on the target nucleic acid region, a detection probe containing the sequence of SEQ ID NO: 10; (b) When the forward amplification primer and the reverse amplification primer are configured to generate an amplicon of a target nucleic acid sequence with a length of at least about 93 nucleotides from SEQ ID NO: 4 and SEQ ID NO: 19 on the target nucleic acid region, a detection probe containing the sequence of SEQ ID NO: 11 or SEQ ID NO: 12; (c) When the forward amplification primer and the reverse amplification primer are configured to generate an amplicon of a target nucleic acid sequence with a length of at least about 100 nucleotides from SEQ ID NO: 2 and SEQ ID NO: 17 on the target nucleic acid region, a detection probe containing the sequence of SEQ ID NO: 9; (d) When the forward amplification primer and the reverse amplification primer are configured to generate an amplicon of a target nucleic acid sequence with a length of at least about 102 nucleotides from SEQ ID NO: 7 and SEQ ID NO: 22 on the target nucleic acid region, a detection probe containing the sequence of SEQ ID NO: 15; (e) When the forward amplification primer and the reverse amplification primer are configured to generate an amplicon of a target nucleic acid sequence with a length of at least about 119 nucleotides from SEQ ID NO: 6 and SEQ ID NO: 21 on the target nucleic acid region, a detection probe containing the sequence of SEQ ID NO: 14; (f) When the forward amplification primer and the reverse amplification primer are configured to generate an amplicon of a target nucleic acid sequence with a length of at least about 123 nucleotides from SEQ ID NO: 1 and SEQ ID NO: 17 on the target nucleic acid region, a detection probe containing the sequence of SEQ ID NO: 8 or SEQ ID NO: 9; (g) When the forward amplification primer and the reverse amplification primer are configured to generate an amplicon of a target nucleic acid sequence with a length of at least about 127 nucleotides from SEQ ID NO: 1 and SEQ ID NO: 16 on the target nucleic acid region, a detection probe containing the sequence of SEQ ID NO: 8 or SEQ ID NO: 9, or when the forward amplification primer and the reverse amplification primer are configured to generate an amplicon of a target nucleic acid sequence with a length of at least about 127 nucleotides from SEQ ID NO: 5 and SEQ ID NO: 20 on the target nucleic acid region, a detection probe containing the sequence of SEQ ID NO: 13.

[0182] In certain embodiments, the target nucleic acid region is SEQ ID NO: 39, and the reaction mixture comprises a detection probe that is about 22 to about 27 nucleotides in length.

[0183] In certain embodiments, the target nucleic acid region is SEQ ID NO: 39, and the reaction mixture comprises a detection probe selected from the group consisting of SEQ ID NOs: 28, 29, 30, 31, 32, and 33. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39, and the detection probe comprises the sequence of SEQ ID NO: 28. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39, and the detection probe comprises the sequence of SEQ ID NO: 29. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39, and the detection probe comprises the sequence of SEQ ID NO: 30. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39, and the detection probe comprises the sequence of SEQ ID NO: 31. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39, and the detection probe comprises the sequence of SEQ ID NO: 32. In certain embodiments, the target nucleic acid region is SEQ ID NO: 39, and the detection probe comprises the sequence of SEQ ID NO: 33.

[0184] In certain embodiments, the target nucleic acid region is SEQ ID NO: 39, and the reaction mixture comprises a detection probe that comprises a target nucleic acid sequence that substantially corresponds to the following oligohybridization forming sequences. When the forward and reverse amplification primers are (I) SEQ ID NO: 23 and SEQ ID NO: 34 or (II) SEQ ID NO: 24 and SEQ ID NO: 34, SEQ ID NO: 28; when the forward and reverse amplification primers are SEQ ID NO: 25 and SEQ ID NO: 35, SEQ ID NO: 29; when the forward and reverse amplification primers are SEQ ID NO: 25 and SEQ ID NO: 35, SEQ ID NO: 30; when the forward and reverse amplification primers are SEQ ID NO: 26 and SEQ ID NO: 36, SEQ ID NO: 31; when the forward and reverse amplification primers are SEQ ID NO: 27 and SEQ ID NO: 37, SEQ ID NO: 32; when the forward and reverse amplification primers are SEQ ID NO: 27 and SEQ ID NO: 37, SEQ ID NO: 33.

[0185] In certain embodiments, the target nucleic acid region is SEQ ID NO: 39, and the reaction mixture comprises one or more of the following: (a) a third oligomer comprising the sequence of SEQ ID NO: 29 or SEQ ID NO: 30, when the forward amplification primer and the reverse amplification primer are configured to generate an amplicon of a target nucleic acid sequence at least about 89 nucleotides in length from SEQ ID NO: 25 and SEQ ID NO: 35 on the target nucleic acid region; (b) a third oligomer comprising the sequence of SEQ ID NO: 28, when the forward amplification primer and the reverse amplification primer are configured to generate an amplicon of a target nucleic acid sequence at least about 99 nucleotides in length from SEQ ID NO: 24 and SEQ ID NO: 34 on the target nucleic acid region; (c) a third oligomer comprising the sequence of SEQ ID NO: 28, when the forward amplification primer and the reverse amplification primer are configured to generate an amplicon of a target nucleic acid sequence at least about 109 nucleotides in length from SEQ ID NO: 23 and SEQ ID NO: 34 on the target nucleic acid region; (d) a third oligomer comprising the sequence of SEQ ID NO: 32 or SEQ ID NO: 33, when the forward amplification primer and the reverse amplification primer are configured to generate an amplicon of a target nucleic acid sequence at least about 126 nucleotides in length from SEQ ID NO: 27 and SEQ ID NO: 37 on the target nucleic acid region; (e) a third oligomer comprising the sequence of SEQ ID NO: 31, when the forward amplification primer and the reverse amplification primer are configured to generate an amplicon of a target nucleic acid sequence at least about 143 nucleotides in length from SEQ ID NO: 26 and SEQ ID NO: 36 on the target nucleic acid region.

[0186] In certain embodiments, the reaction mixture for determining the presence (or absence) of VZV in a sample comprises at least one detection probe configured to specifically anneal to an oligohybridization formation sequence within the target nucleic acid region of SEQ ID NO: 38 or SEQ ID NO: 39, and the detection probe is adjacent between the forward amplification primer and the reverse amplification primer.

[0187] In certain embodiments of the reaction mixture, the detection probe comprises at least one detectable label. In some embodiments, the detection probe further comprises a second label that interacts with the first label. In some embodiments, the second label is a quencher.

[0188] In certain embodiments of the reaction mixture, the label is selected from the group consisting of (a) a chemiluminescent label, (b) a fluorescent label, (c) a quencher, and (d) combinations of two or more of (a), (b), and (c). In certain embodiments, the reaction mixture comprises a fluorescent label. In certain embodiments, the reaction mixture comprises a quencher. In certain embodiments, the reaction mixture comprises both a fluorescent dye and a quencher.

[0189] In certain embodiments of the reaction mixture, the detection probe is linear and does not exhibit any degree of self-complementarity held by intramolecular bonds. In some embodiments, the linear detection probe comprises a fluorophore as a label. In some embodiments, the linear detection probe comprises both a fluorophore and a quenching moiety (e.g., TaqMan™ probe).

[0190] In certain embodiments of the reaction mixture, the detection probe exhibits at least a degree of self-complementarity and is used to facilitate the detection of probe:target duplexes in a sample without the need to first hybridize and remove unhybridized probes prior to detection. In certain embodiments of the reaction mixture, the hairpin detection probe that exhibits at least a degree of self-complementarity is a molecular beacon or a molecular torch.

[0191] In certain embodiments of the reaction mixture, the labeled detection probe is non-extendable. For example, the labeled detection probe can have a 3'-terminal 3'-deoxynucleotide (e.g., a terminal 2',3'-dideoxy-nucleotide), or a 3'-terminal inverted nucleotide (e.g., the last nucleotide is inverted such that it is linked to the penultimate nucleotide by a 3' to 3' phosphodiester bond or an analog thereof (such as phosphorothioate)), or a bound fluorophore, quencher, or other label that interferes with extension (possibly bound via the 3'-position of the terminal nucleotide, but not necessarily so), and can be made non-extendable by 3'-phosphorylation. In certain embodiments, the 3'-terminal nucleotide is not methylated.

[0192] In certain embodiments of the reaction mixture, the detection probe comprises at least one modified nucleobase. In certain embodiments, the modified nucleobase is selected from the group consisting of (a) 2'-O-methyl, (b) 5-methylcytosine, (c) 2'-fluorine, and (d) combinations of two or more of (a), (b), and (c).

[0193] In certain embodiments, the reaction mixture comprises at least one amplification primer or detection probe as described herein. In certain embodiments, the reaction mixture comprises a plurality of amplification primers, and / or detection probes. In certain embodiments, the reaction mixture comprises a single set of forward and reverse amplification primers that generate a single amplicon of the target nucleic acid sequence from the target nucleic acid region. In certain embodiments, the reaction mixture comprises a plurality of sets of amplification primers that generate a plurality of amplicons from various target nucleic acid sequences within various target nucleic acid regions. In certain embodiments, the reaction mixture comprises a plurality of sets of amplification primers that generate a plurality of amplicons from various target nucleic acid sequences within a single target nucleic acid region.

[0194] In certain embodiments, the reaction mixture includes additional reagents for determining the presence of VZV in the sample and, if present, amplification of the target nucleic acid sequence of the VZV nucleic acid sequence in the sample. In certain embodiments, the reaction mixture may include reagents suitable for performing in vitro amplification, such as various dNTPs, enzymes, buffers, and / or salts.

[0195] In certain embodiments, the reaction mixture may include various nucleotide subunits of DNA, such as dATP, dCTP, dGTP, and dTTP, and / or ATP, CTP, GTP, and UTP. In certain embodiments, the reaction mixture may include a DNA polymerase enzyme or a reverse transcriptase. In certain embodiments, the reaction mixture may include an organic buffer. In certain embodiments, the reaction mixture may include one or more surfactants.

[0196] In certain embodiments, the reaction mixture may include one or more inorganic salts selected from the group consisting of magnesium chloride, sodium chloride, potassium chloride, and sodium citrate. In certain embodiments, the reaction mixture may include magnesium chloride. In certain embodiments, the reaction mixture may include magnesium chloride at a concentration of 3 mM to 6 mM. In certain embodiments, the concentration of magnesium chloride is 2 mM. In certain embodiments, the concentration of magnesium chloride is 4 mM. In certain embodiments, the concentration of magnesium chloride is 6 mM.

[0197] In certain embodiments, the reaction mixture may be an aqueous reaction mixture. In certain embodiments, the reaction mixture may be frozen. In certain embodiments, the reaction mixture may be lyophilized. In certain embodiments, the lyophilized reaction mixture may appear as a powder or a solid or a sphere. In certain embodiments, the lyophilized reaction mixture may include a filler, such as trehalose, raffinose, or a combination thereof.

[0198] Exemplary compositions, kits, reaction mixtures, formulations, and methods are further illustrated by the following non-limiting examples.

[0199] Exemplary compositions, kits, reaction mixtures, formulations, and methods are further illustrated by the following non-limiting examples.

[0200] List of Embodiments 1. An oligonucleotide composition for amplifying a target nucleic acid sequence within a target nucleic acid region of VZV, or an oligonucleotide composition for amplifying an amplicon generated from a target nucleic acid sequence within a target nucleic acid region, comprising at least two amplification primers, wherein the first amplification primer is a forward amplification primer and the second amplification primer is a reverse amplification primer.

[0201] 2. The oligonucleotide composition of embodiment 1, wherein the target nucleic acid region is SEQ ID NO: 38 or SEQ ID NO: 39.

[0202] 3. The oligonucleotide composition of embodiment 1 or 2, wherein the target nucleic acid region is SEQ ID NO: 38, the forward and reverse amplification primers are each independently about 19 to about 23 nucleotides in length, and the forward and reverse amplification primers are configured to generate an amplicon about 89 to about 127 nucleotides in length from the target nucleic acid region of SEQ ID NO: 38.

[0203] 4. The oligonucleotide composition according to any one of embodiments 1 to 3, wherein the target nucleic acid region is SEQ ID NO: 38, the forward amplification primer is selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7, the reverse amplification primer is about 19 to about 23 nucleotides in length, and the two amplification primers are configured to generate an amplicon about 89 to about 127 nucleotides in length from the target nucleic acid region of SEQ ID NO: 38.

[0204] 5. The oligonucleotide composition according to any one of embodiments 1 to 4, wherein the target nucleic acid region is SEQ ID NO: 38 and the reverse amplification primer is selected from the group consisting of SEQ ID NOs: 16, 17, 18, 19, 20, 21, and 22.

[0205] 6. The target nucleic acid region is SEQ ID NO: 38, the reverse amplification primer is selected from the group consisting of SEQ ID NOs: 16, 17, 18, 19, 20, 21, and 22, the forward amplification primer has a length of about 20 to about 23 nucleotides, and the reverse and forward amplification primers are configured to generate an amplicon having a length of about 89 to about 127 nucleotides from the target nucleic acid region of SEQ ID NO: 38. The oligonucleotide composition according to any one of Embodiments 1 to 3.

[0206] 7. The forward amplification primer is configured to hybridize to the oligohybridization-forming region within SEQ ID NO: 38 or its complement, the reverse amplification primer is configured to hybridize to the oligohybridization-forming region within SEQ ID NO: 38 or its complement, and the distance between the oligohybridization-forming region of the first amplification primer and the oligohybridization-forming region of the second amplification region is 89, 93, 100, 102, 119, 123, or 127 nucleotides along SEQ ID NO: 38 when measured from the most distant nucleotides of the two oligohybridization-forming regions. The oligonucleotide composition according to Embodiment 4 or Embodiment 6.

[0207] 8. The target nucleic acid region is SEQ ID NO: 38, and the forward and reverse amplification primers include target nucleic acid sequences corresponding to (a) SEQ ID NO: 1 and SEQ ID NO: 16, (b) SEQ ID NO: 1 and SEQ ID NO: 17, (c) SEQ ID NO: 2 and SEQ ID NO: 17, (d) SEQ ID NO: 3 and SEQ ID NO: 18, (e) SEQ ID NO: 4 and SEQ ID NO: 19, (f) SEQ ID NO: 5 and SEQ ID NO: 20, (g) SEQ ID NO: 6 and SEQ ID NO: 21, or (h) SEQ ID NO: 7 and SEQ ID NO: 22. The oligonucleotide composition according to any one of Embodiments 1 to 7.

[0208] 9. The target nucleic acid region is SEQ ID NO: 39, the forward and reverse amplification primers are, independently of each other, about 20 to about 23 nucleotides in length, and the forward and reverse amplification primers are configured to generate an amplicon having a length of about 89 to about 143 nucleotides from the target nucleic acid region of SEQ ID NO: 39. The oligonucleotide composition of Embodiment 1 or 2.

[0209] 10. The target nucleic acid region is SEQ ID NO: 39, the forward amplification primer is selected from the group consisting of SEQ ID NOs: 23, 24, 25, 26, and 27, the reverse amplification primer is about 20 to about 23 nucleotides in length, and the two amplification primers are configured to generate an amplicon having a length of about 89 to about 143 nucleotides from the target nucleic acid region of SEQ ID NO: 39. The oligonucleotide composition of Embodiment 1 or 2 or 9.

[0210] 11. The target nucleic acid region is SEQ ID NO: 39, and the reverse amplification primer is selected from the group consisting of SEQ ID NOs: 34, 35, 36, and 37. The oligonucleotide composition of Embodiment 10.

[0211] 12. The target nucleic acid region is SEQ ID NO: 39, the reverse amplification primer is selected from the group consisting of SEQ ID NOs: 34, 35, 36, and 37, the forward amplification primer is about 20 to about 23 nucleotides in length, and the reverse and forward amplification primers are configured to generate an amplicon having a length of about 89 to about 143 nucleotides from the target nucleic acid region of SEQ ID NO: 39. The oligonucleotide composition of Embodiment 1 or 2 or 9.

[0212] 13. The forward amplification primer is configured to hybridize to the oligo hybridization-forming region within SEQ ID NO: 39 or its complement, the reverse amplification primer is configured to hybridize to the oligo hybridization-forming region within SEQ ID NO: 39 or its complement, and the distance between the oligo hybridization-forming region of the first amplification primer and the oligo hybridization-forming region of the second amplification region, when measured from the most distant nucleotides of the two oligo hybridization-forming regions, is 89, 99, 109, 126, and 143 nucleotides in length along SEQ ID NO: 39. The oligonucleotide composition of Embodiment 10 or 12.

[0213] 14. The target nucleic acid region is SEQ ID NO: 39, and the forward and reverse amplification primers contain the target nucleic acid sequences corresponding to (a) SEQ ID NO: 23 and SEQ ID NO: 34, (b) SEQ ID NO: 24 and SEQ ID NO: 34, (c) SEQ ID NO: 25 and SEQ ID NO: 35, (d) SEQ ID NO: 26 and SEQ ID NO: 36, or (e) SEQ ID NO: 27 and SEQ ID NO: 37. The oligonucleotide composition according to any one of Embodiment 1 or 2 or Embodiments 9 to 13.

[0214] 15. The oligonucleotide composition according to any one of Embodiments 1 to 14, further comprising a third oligonucleotide.

[0215] 16. The oligonucleotide composition of Embodiment 15, wherein the third oligonucleotide is a detection probe.

[0216] 17. The target nucleic acid region is SEQ ID NO: 38, and the detection probe is about 23 to about 27 nucleotides in length. The oligonucleotide composition according to any one of Embodiments 2 to 8.

[0217] 18. The target nucleic acid region is SEQ ID NO: 38, and the detection probe is selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, and 15. The oligonucleotide composition of Embodiment 17.

[0218] 19. When the detection probe contains the following target hybridization-forming sequences: (a) If the sequence number is 8, the forward and reverse amplification primers carefully contain (I) sequence number 1 and sequence number 16 or (II) sequence number 1 and sequence number 17; (b) If the sequence number is 9, the forward and reverse amplification primers carefully contain (I) sequence number 1 and sequence number 16 or (II) sequence number 1 and sequence number 17 or (III) sequence number 2 and sequence number 17; (c) If the sequence number is 10, the forward and reverse amplification primers carefully contain sequence number 3 and sequence number 18; (d) If the sequence number is 11, the forward and reverse amplification primers carefully contain sequence number 4 and sequence number 19; (e) If the sequence number is 12, the forward and reverse amplification primers carefully contain sequence number 4 and sequence number 19; (f) If the sequence number is 13, the forward and reverse amplification primers carefully contain sequence number 5 and sequence number 20; (g) If the sequence number is 14, the forward and reverse amplification primers carefully contain sequence number 6 and sequence number 21; or (h) If the sequence number is 15, the forward and reverse amplification primers carefully contain sequence number 7 and sequence number 22. The oligonucleotide composition of embodiment 17.

[0219] 20. The oligonucleotide composition according to any one of embodiments 2 or 9 to 14, wherein the target nucleic acid region is sequence number 39 and the detection probe has a length of about 22 to about 27 nucleotides.

[0220] 21. The oligonucleotide composition of embodiment 20, wherein the target nucleic acid region is sequence number 39 and the detection probe is selected from the group consisting of sequence numbers 28, 29, 30, 31, 32, and 33.

[0221] 22. When the detection probe comprises the following target hybridization-forming sequences: (a) if SEQ ID NO: 28, the forward and reverse amplification primers carefully comprise (I) SEQ ID NO: 23 and SEQ ID NO: 34 or (II) SEQ ID NO: 24 and SEQ ID NO: 34; (b) if SEQ ID NO: 29, the forward and reverse amplification primers carefully comprise SEQ ID NO: 25 and SEQ ID NO: 35; (c) if SEQ ID NO: 30, the forward and reverse amplification primers carefully comprise SEQ ID NO: 25 and SEQ ID NO: 35; (d) if SEQ ID NO: 31, the forward and reverse amplification primers carefully comprise SEQ ID NO: 26 and SEQ ID NO: 36; (e) if SEQ ID NO: 32, the forward and reverse amplification primers carefully comprise SEQ ID NO: 27 and SEQ ID NO: 37; or (f) if SEQ ID NO: 33, the forward and reverse amplification primers carefully comprise SEQ ID NO: 27 and SEQ ID NO: 37, the oligonucleotide composition of embodiment 20.

[0222] 23. The oligonucleotide composition according to any one of embodiments 15 to 22, wherein the detection probe further comprises at least one detectable label.

[0223] 24. The oligonucleotide composition of embodiment 23, wherein one or more of the detectable labels are selected from the group consisting of (a) a chemiluminescent label, (b) a fluorescent label, (c) a quencher, or (d) a combination of two or more of (a), (b), and (c).

[0224] 25. The oligonucleotide composition of embodiment 24, wherein one or more of the detectable labels comprise a fluorescent label, or one or more of the detectable labels comprise a quencher, or one or more of the detectable labels comprise both a fluorescent label and a quencher.

[0225] 26. The oligonucleotide composition according to any one of embodiments 15 to 25, wherein the detection probe is a TaqMan™ detection probe.

[0226] 27. The oligonucleotide composition according to any one of Embodiments 15 to 25, wherein the detection probe further comprises a non-target hybridization-forming array, or the detection probe comprising a non-target hybridization-forming array is a hairpin detection probe, or the hairpin detection probe is a molecular beacon or a molecular torch.

[0227] 28. The oligonucleotide composition according to any one of Embodiments 15 to 27, wherein the detection probe further comprises at least one modified nucleobase.

[0228] 29. The oligonucleotide composition according to Embodiment 28, wherein one or more of the modified nucleobases are selected from the group consisting of (a) 2'-O-methyl, (b) 5-methylcytosine, (c) 2'-fluoro, or (d) a combination of two or more of (a), (b), and (c).

[0229] 30. The oligonucleotide composition according to Embodiment 28 or 29, wherein the detection probe comprises 3 to 10 modified nucleobases, or the detection probe comprises 3 to 10 5-methylcytosine-modified nucleobases, or the detection probe comprises 3 5-methylcytosine-modified nucleobases, or the detection probe comprises 6 5-methylcytosine-modified nucleobases, or the detection probe comprises 7 5-methylcytosine-modified nucleobases, or the detection probe comprises 10 5-methylcytosine-modified nucleobases, or at least one modification is a 5-methylcytosine-modified nucleobase, or the detection probe comprises 3 to 10 2'-O-methyl-modified nucleobases, or the detection probe comprises 3 2'-O-methyl-modified nucleobases, or the detection probe comprises 6 2'-O-methyl-modified nucleobases, or the detection probe comprises 7 2'-O-methyl-modified nucleobases, or the detection probe comprises 10 2'-O-methyl-modified nucleobases, or at least one modification is a 2'-O-methyl-modified nucleobase.

[0230] 31. The oligonucleotide composition according to any one of Embodiments 1 to 30, wherein the forward amplification primer further comprises at least one modified nucleobase.

[0231] 32. An oligonucleotide composition according to embodiment 31, wherein one or more of the modified nucleobases are selected from the group consisting of (a) 2'-O-methyl, (b) 5-methylcytosine, (c) 2'-fluoro, or (d) a combination of two or more of (a), (b), and (c).

[0232] 33. An oligonucleotide composition according to embodiment 31 or 32, wherein the forward amplification primer comprises 2 to 6 modified nucleobases, or the forward amplification primer comprises 2 to 6 5-methylcytosine-modified nucleobases, or the forward amplification primer comprises 2 5-methylcytosine-modified nucleobases, or the forward amplification primer comprises 3 5-methylcytosine-modified nucleobases, or the forward amplification primer comprises 4 5-methylcytosine-modified nucleobases, or the forward amplification primer comprises 6 5-methylcytosine-modified nucleobases, or at least one modification is a 5-methylcytosine-modified nucleobase, or the forward amplification primer comprises 2 to 6 2'-O-methyl-modified nucleobases, or the forward amplification primer comprises 2 2'-O-methyl-modified nucleobases, or the forward amplification primer comprises 3 2'-O-methyl-modified nucleobases, or the forward amplification primer comprises 4 2'-O-methyl-modified nucleobases, or the forward amplification primer comprises 6 2'-O-methyl-modified nucleobases, or at least one modification is a 2'-O-methyl-modified nucleobase.

[0233] 34. An oligonucleotide composition according to any one of embodiments 1 to 33, wherein the reverse amplification primer further comprises at least one modified nucleobase.

[0234] 35. An oligonucleotide composition according to embodiment 34, wherein one or more of the modified nucleobases are selected from the group consisting of (a) 2'-O-methyl, (b) 5-methylcytosine, (c) 2'-fluoro, or (d) a combination of two or more of (a), (b), and (c).

[0235] 36. The reverse amplification primer contains 2 to 6 modified nucleobases, or the reverse amplification primer contains 2 to 6 2'-fluorine modified nucleobases, or the reverse amplification primer contains 1 2'-fluorine modified nucleobase, or the reverse amplification primer contains 3 2'-fluorine modified nucleobases, or the reverse amplification primer contains 4 2'-fluorine modified nucleobases, or the reverse amplification primer contains 6 2'-fluorine modified nucleobases, or at least 1 modification is a 2'-fluorine modified nucleobase, or the reverse amplification primer contains 2 to 6 5-methylcytosine modified nucleobases, or the reverse amplification primer contains 1 5-methylcytosine modified nucleobase, or the reverse amplification primer contains 3 5-methylcytosine modified nucleobases, or the reverse amplification primer contains 4 5-methylcytosine modified nucleobases, or the reverse amplification primer contains 5 5-methylcytosine modified nucleobases, or the reverse amplification primer contains 6 5-methylcytosine modified nucleobases, or at least 1 modification is a 5-methylcytosine modified nucleobase, the oligonucleotide composition of embodiment 34 or 35.

[0236] 37. An oligonucleotide composition for detecting a target nucleic acid sequence within a target nucleic acid region of VZV, or an oligonucleotide composition for detecting an amplicon generated from a target nucleic acid sequence within a target nucleic acid region, the oligonucleotide composition comprising at least one oligonucleotide for detecting the target nucleic acid sequence.

[0237] 38. The oligonucleotide composition of embodiment 37, wherein the target nucleic acid region is SEQ ID NO: 38 or SEQ ID NO: 39.

[0238] 39. The oligonucleotide composition of embodiment 38, wherein the target nucleic acid region is SEQ ID NO: 38 and the detection probe is about 23 to about 27 nucleotides in length.

[0239] 40. The oligonucleotide composition of embodiment 39, wherein the target nucleic acid region is SEQ ID NO: 38 and the detection probe is selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, and 15.

[0240] 41. The oligonucleotide composition of embodiment 39 or 40, further comprising at least one set of amplification primers, wherein one amplification primer is a forward amplification primer and one amplification primer is a reverse amplification primer.

[0241] 42. The oligonucleotide composition of embodiment 41, wherein the target nucleic acid region is SEQ ID NO: 38, the forward and reverse amplification primers are each individually about 19 to about 23 nucleotides in length, and the forward and reverse amplification primers are configured to generate an amplicon about 89 to about 127 nucleotides in length from the target nucleic acid region of SEQ ID NO: 38.

[0242] 43. The oligonucleotide composition of embodiment 42, wherein the target nucleic acid region is SEQ ID NO: 38 and the forward amplification primer is selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7.

[0243] 44. The oligonucleotide composition of embodiment 42 or 43, wherein the target nucleic acid region is SEQ ID NO: 38 and the reverse amplification primer is selected from the group consisting of SEQ ID NOs: 16, 17, 18, 19, 20, 21, and 22.

[0244] 45. The oligonucleotide composition of embodiment 41, wherein the target nucleic acid region is SEQ ID NO: 38, the reverse amplification primer is selected from the group consisting of SEQ ID NOs: 16, 17, 18, 19, 20, 21, and 22, the forward amplification primer is about 20 to about 23 nucleotides in length, and the forward and reverse amplification primers are configured to generate an amplicon about 89 to about 127 nucleotides in length from the target nucleic acid region of SEQ ID NO: 38.

[0245] 46. The forward amplification primer is configured to hybridize to the oligo hybridization-forming region within SEQ ID NO: 38 or its complement, the reverse amplification primer is configured to hybridize to the oligo hybridization-forming region within SEQ ID NO: 38 or its complement, and the distance between the oligo hybridization-forming region of the first amplification primer and the oligo hybridization-forming region of the second amplification region, when measured from the most distant nucleotides of the two oligo hybridization-forming regions, is 89, 93, 100, 102, 119, 123, or 127 nucleotides in length along SEQ ID NO: 38. The oligonucleotide composition of embodiment 43 or 45.

[0246] 47. The target nucleic acid region is SEQ ID NO: 38, and the forward and reverse amplification primers contain target nucleic acid sequences corresponding to (a) SEQ ID NO: 1 and SEQ ID NO: 16, (b) SEQ ID NO: 1 and SEQ ID NO: 17, (c) SEQ ID NO: 2 and SEQ ID NO: 17, (d) SEQ ID NO: 3 and SEQ ID NO: 18, (e) SEQ ID NO: 4 and SEQ ID NO: 19, (f) SEQ ID NO: 5 and SEQ ID NO: 20, (g) SEQ ID NO: 6 and SEQ ID NO: 21, or (h) SEQ ID NO: 7 and SEQ ID NO: 22. The oligonucleotide composition according to any one of embodiments 41 to 46.

[0247] 48. When the detection probe comprises the following target hybridization-forming sequences: (a) if the sequence number is 8, the forward and reverse amplification primers exactly contain (I) sequence number 1 and sequence number 16 or (II) sequence number 1 and sequence number 17; (b) if the sequence number is 9, the forward and reverse amplification primers exactly contain (I) sequence number 1 and sequence number 16 or (II) sequence number 1 and sequence number 17 or (III) sequence number 2 and sequence number 17; (c) if the sequence number is 10, the forward and reverse amplification primers exactly contain sequence number 3 and sequence number 18; (d) if the sequence number is 11, the forward and reverse amplification primers exactly contain sequence number 4 and sequence number 19; (e) if the sequence number is 12, the forward and reverse amplification primers exactly contain sequence number 4 and sequence number 19; (f) if the sequence number is 13, the forward and reverse amplification primers exactly contain sequence number 5 and sequence number 20; (g) if the sequence number is 14, the forward and reverse amplification primers exactly contain sequence number 6 and sequence number 21; (h) if the sequence number is 15, the forward and reverse amplification primers exactly contain sequence number 7 and sequence number 22. An oligonucleotide composition of Embodiment 39.

[0248] 49. An oligonucleotide composition of Embodiment 38, wherein the target nucleic acid region is sequence number 39 and the detection probe has a length of about 22 to about 27 nucleotides.

[0249] 50. An oligonucleotide composition of Embodiment 49, wherein the target nucleic acid region is sequence number 39 and the detection probe is selected from the group consisting of sequence numbers 28, 29, 30, 31, 32 and 33.

[0250] 51. An oligonucleotide composition of Embodiment 49 or 50, further comprising at least one set of amplification primers, wherein one amplification primer is a forward amplification primer and one amplification primer is a reverse amplification primer.

[0251] 52. The target nucleic acid region is SEQ ID NO: 39, the forward and reverse amplification primers are each individually about 20 to about 23 nucleotides in length, and the forward and reverse amplification primers are configured to generate an amplicon having a length of about 89 to about 143 nucleotides from the target nucleic acid region of SEQ ID NO: 39. The oligonucleotide composition of embodiment 51.

[0252] 53. The target nucleic acid region is SEQ ID NO: 39, and the forward amplification primer is selected from the group consisting of SEQ ID NOs: 23, 24, 25, 26, and 27. The oligonucleotide composition of embodiment 52.

[0253] 54. The target nucleic acid region is SEQ ID NO: 39, and the reverse amplification primer is selected from the group consisting of SEQ ID NOs: 34, 35, 36, and 37. The oligonucleotide composition of embodiment 52 or 53.

[0254] 55. The target nucleic acid region is SEQ ID NO: 39, the reverse amplification primer is selected from the group consisting of SEQ ID NOs: 34, 35, 36, and 37, the forward amplification primer is about 20 to about 23 nucleotides in length, and the reverse and forward amplification primers are configured to generate an amplicon having a length of about 89 to about 143 nucleotides from the target nucleic acid region of SEQ ID NO: 39. The oligonucleotide composition of embodiment 51.

[0255] 56. The forward amplification primer is configured to hybridize to the oligohybridization region within SEQ ID NO: 39 or its complement, the reverse amplification primer is configured to hybridize to the oligohybridization region within SEQ ID NO: 39 or its complement, and the distance between the oligohybridization region of the first amplification primer and the oligohybridization region of the second amplification region is 89, 99, 109, 126, or 143 nucleotides along SEQ ID NO: 39 when measured from the most distant nucleotides of the two oligohybridization regions. The oligonucleotide composition of embodiment 53 or 55.

[0256] 57. The target nucleic acid region is SEQ ID NO: 39, and the forward and reverse amplification primers contain target nucleic acid sequences corresponding to (a) SEQ ID NO: 23 and SEQ ID NO: 34, (b) SEQ ID NO: 24 and SEQ ID NO: 34, (c) SEQ ID NO: 25 and SEQ ID NO: 35, (d) SEQ ID NO: 26 and SEQ ID NO: 36, or (e) SEQ ID NO: 27 and SEQ ID NO: 37. The oligonucleotide composition according to any one of Embodiments 51 to 56.

[0257] 58. When the detection probe contains the following target hybridization-forming sequences: (a) If it is SEQ ID NO: 28, the forward and reverse amplification primers carefully contain (I) SEQ ID NO: 23 and SEQ ID NO: 34 or (II) SEQ ID NO: 24 and SEQ ID NO: 34; (b) If it is SEQ ID NO: 29, the forward and reverse amplification primers carefully contain SEQ ID NO: 25 and SEQ ID NO: 35; (c) If it is SEQ ID NO: 30, the forward and reverse amplification primers carefully contain SEQ ID NO: 25 and SEQ ID NO: 35; (d) If it is SEQ ID NO: 31, the forward and reverse amplification primers carefully contain SEQ ID NO: 26 and SEQ ID NO: 36; (e) If it is SEQ ID NO: 32, the forward and reverse amplification primers carefully contain SEQ ID NO: 27 and SEQ ID NO: 37; (f) If it is SEQ ID NO: 33, the forward and reverse amplification primers carefully contain SEQ ID NO: 27 and SEQ ID NO: 37. The oligonucleotide composition of Embodiment 49.

[0258] 59. The oligonucleotide composition according to any one of Embodiments 37 or 38, wherein the detection probe further contains at least one detectable label.

[0259] 60. One or more of the detectable labels are selected from the group consisting of (a) chemiluminescent labels, (b) fluorescent labels, (c) quenchers, or (d) combinations of two or more of (a), (b), and (c). The oligonucleotide composition of Embodiment 59.

[0260] 61. The oligonucleotide composition of embodiment 60, wherein one or more detectable labels comprise a fluorescent label, or one or more detectable labels comprise a quencher, or one or more detectable labels comprise both a fluorescent label and a quencher.

[0261] 62. The oligonucleotide composition according to any one of embodiments 37 to 61, wherein the detection probe is a TaqMan™ detection probe.

[0262] 63. The oligonucleotide composition according to any one of embodiments 37 to 61, wherein the detection probe further comprises a non-target hybridizing sequence, or the detection probe comprising a non-target hybridizing sequence is a hairpin detection probe, or the hairpin detection probe is a molecular beacon or a molecular torch.

[0263] 64. The oligonucleotide composition according to any one of embodiments 37 to 63, wherein the detection probe further comprises at least one modified nucleobase.

[0264] 65. The oligonucleotide composition of embodiment 64, wherein one or more of the modified nucleobases are selected from the group consisting of (a) 2'-O-methyl, (b) 5-methylcytosine, (c) 2'-fluoro, or (d) a combination of two or more of (a), (b), and (c).

[0265] 66. The detection probe contains 3 to 10 modified nucleobases, or the detection probe contains 3 to 10 5-methylcytosine modified nucleobases, or the detection probe contains 3 5-methylcytosine modified nucleobases, or the detection probe contains 6 5-methylcytosine modified nucleobases, or the detection probe contains 7 5-methylcytosine modified nucleobases, or the detection probe contains 10 5-methylcytosine modified nucleobases, or at least one modification is a 5-methylcytosine modified nucleobase, or the detection probe contains 3 to 10 2'-O-methyl modified nucleobases, or the detection probe contains 3 2'-O-methyl modified nucleobases, or the detection probe contains 6 2'-O-methyl modified nucleobases, or the detection probe contains 7 2'-O-methyl modified nucleobases, or the detection probe contains 10 2'-O-methyl modified nucleobases, or at least one modification is a 2'-O-methyl modified nucleobase, the oligonucleotide composition of embodiment 64 or 65.

[0266] 67. The oligonucleotide composition according to any one of embodiments 41 to 66, wherein the forward amplification primer further contains at least one modified nucleobase.

[0267] 68. The oligonucleotide composition of embodiment 67, wherein one or more of the modified nucleobases are selected from the group consisting of (a) 2'-O-methyl, (b) 5-methylcytosine, (c) 2'-fluorine, or (d) a combination of two or more of (a), (b), and (c).

[0268] 69. The forward amplification primer contains 2 to 6 modified nucleobases, or the forward amplification primer contains 2 to 6 5-methylcytosine modified nucleobases, or the forward amplification primer contains 2 5-methylcytosine modified nucleobases, or the forward amplification primer contains 3 5-methylcytosine modified nucleobases, or the forward amplification primer contains 4 5-methylcytosine modified nucleobases, or the forward amplification primer contains 6 5-methylcytosine modified nucleobases, or at least one modification is a 5-methylcytosine modified nucleobase, or the forward amplification primer contains 2 to 6 2'-O-methyl modified nucleobases, or the forward amplification primer contains 2 2'-O-methyl modified nucleobases, or the forward amplification primer contains 3 2'-O-methyl modified nucleobases, or the forward amplification primer contains 4 2'-O-methyl modified nucleobases, or the forward amplification primer contains 6 2'-O-methyl modified nucleobases, or at least one modification is a 2'-O-methyl modified nucleobase, the oligonucleotide composition of embodiment 67 or 68.

[0269] 70. The oligonucleotide composition according to any one of embodiments 41 to 69, wherein the reverse amplification primer further contains at least one modified nucleobase.

[0270] 71. The oligonucleotide composition of embodiment 70, wherein one or more of the modified nucleobases are selected from the group consisting of (a) 2'-O-methyl, (b) 5-methylcytosine, (c) 2'-fluorine, or (d) a combination of two or more of (a), (b), and (c).

[0271] 72. The reverse amplification primer contains 2 to 6 modified nucleobases, or the reverse amplification primer contains 2 to 6 2'-fluoro modified nucleobases, or the reverse amplification primer contains 2 2'-fluoro modified nucleobases, or the reverse amplification primer contains 3 2'-fluoro modified nucleobases, or the reverse amplification primer contains 4 2'-fluoro modified nucleobases, or the reverse amplification primer contains 5 2'-fluoro modified nucleobases, or the reverse amplification primer contains 6 2'-fluoro modified nucleobases, or at least 2 of the modifications are 2'-fluoro modified nucleobases, or the reverse amplification primer contains 2 to 6 5-methylcytosine modified nucleobases, or the reverse amplification primer contains 2 5-methylcytosine modified nucleobases, or the reverse amplification primer contains 3 5-methylcytosine modified nucleobases, or the reverse amplification primer contains 4 5-methylcytosine modified nucleobases, or the reverse amplification primer contains 5 5-methylcytosine modified nucleobases, or the reverse amplification primer contains 6 5-methylcytosine modified nucleobases, or at least 1 of the modifications is a 5-methylcytosine modified nucleobase, the oligonucleotide composition of embodiment 70 or 71.

[0272] 73. A kit comprising a composition for detecting the presence of VZV in a sample and, if present, amplifying the target nucleic acid sequence of VZV, generally comprising one or more oligonucleotides for detecting the target nucleic acid sequence, or one or more oligonucleotides for detecting an amplicon generated from the target nucleic acid sequence, and one or more oligonucleotides for amplifying the target nucleic acid sequence, or one or more oligonucleotides for amplifying an amplicon generated from the target nucleic acid sequence.

[0273] 74. The kit of embodiment 73, further comprising at least two amplification primers as in any one of embodiments 1 to 36, wherein the first amplification primer is a forward amplification primer and the second amplification primer is a reverse amplification primer.

[0274] 75. The kit of embodiment 74 further includes guidance for determining whether a probe:target hybrid is formed under hybridization conditions in a test sample, using any of a variety of reagents for performing in vitro amplification from a target nucleic acid sequence and generating amplicons if a VZV nucleic acid sequence is present in the sample, as well as any of a variety of known techniques for amplifying a target nucleic acid sequence.

[0275] 76. The kit of embodiment 75 can include a variety of reagents suitable for performing in vitro amplification, such as buffers, salts, various dNTPs, or enzymes.

[0276] 77. The kit of embodiment 76 can include various salts, such as magnesium chloride, sodium chloride, potassium chloride, or sodium citrate.

[0277] 78. The kit of embodiment 76 can include various dNTPs, such as deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), deoxythymidine triphosphate (dTTP), or adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), and uridine triphosphate (UTP).

[0278] 79. The kit of embodiment 76 can include various enzymes, such as thermostable DNA polymerase, reverse transcriptase, or RNA polymerase.

[0279] 80. The amplification primers of the kit of embodiment 76 can be aqueous, or can be frozen or lyophilized.

[0280] 81. The various reagents as described herein can be packaged in a variety of different embodiments in the kit of embodiment 76.

[0281] The amplification primers included in the kit include a single set of forward and reverse amplification primers that generate a single amplicon of the target nucleic acid sequence, or the kit includes multiple sets of amplification primers that generate multiple amplicons from various target nucleic acid sequences across various target nucleic acid regions, or the kit may include multiple sets of amplification primers that generate multiple amplicons from various target nucleic acid sequences within a single target nucleic acid region, the kit of embodiment 74.

[0282] 83. The kit of embodiment 74, wherein the kit includes guidance instructions for amplifying the target nucleic acid sequence of the target region using conventional endpoint PCR amplification and generating additional dsDNA molecules using DNA polymerase.

[0283] 84. The kit of embodiment 83, wherein the kit includes various reagents suitable for performing conventional endpoint PCR amplification, or the kit includes various reagents suitable for performing real-time PCR amplification, or the kit includes various reagents suitable for performing LCR amplification, or the kit includes various reagents suitable for performing SDA amplification, or the kit includes various reagents suitable for performing TMA amplification, or the kit includes various reagents suitable for performing NASBA amplification.

[0284] 85. The kit of embodiment 73, further comprising at least one oligonucleotide similar to any of embodiments 37-72 for detecting the target nucleic acid sequence or for detecting an amplicon generated from the target nucleic acid sequence within the target nucleic acid region.

[0285] 86. The kit of embodiment 85, wherein the kit further includes guidance instructions for determining whether a probe:target hybrid is formed under hybridization conditions in a test sample using various reagents for in vitro detection of the target nucleic acid sequence or detection of an amplicon generated from the target nucleic acid sequence when the VZV nucleic acid sequence is present in the sample, and using any of various known techniques for amplifying the target nucleic acid sequence.

[0286] Embodiment 86 kit, wherein the kit may contain various reagents suitable for performing in vitro amplification, such as buffer, salts, various dNTPs, or enzymes.

[0287] Embodiment 87 kit, wherein the kit may contain various salts, such as magnesium chloride, sodium chloride, potassium chloride, or sodium citrate.

[0288] Embodiment 87 kit, wherein the kit may contain various dNTPs, such as deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), deoxythymidine triphosphate (dTTP), or adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), and uridine triphosphate (UTP).

[0289] Embodiment 87 kit, wherein the kit may contain various enzymes, such as thermostable DNA polymerase, reverse transcriptase, or RNA polymerase.

[0290] Embodiment 87 kit, wherein the detection probe is aqueous or can be frozen or lyophilized.

[0291] Embodiment 87 kit, wherein the various reagents described herein can be packaged in various different embodiments.

[0292] Embodiment 87 kit, wherein the oligonucleotides contained in the kit are intended to form base pairs with various amplification oligonucleotides according to the specific requirements of tests developed in the end - user's laboratory.

[0293] Embodiment 86 kit, wherein the kit contains various reagents suitable for performing real - time PCR.

[0294] 95. The kit of embodiment 94, which includes instructions for detecting a target nucleic acid sequence with one or more detection probes using real-time PCR, wherein the detection probes hybridize to the amplification product to generate a signal.

[0295] 96. The kit of embodiment 94, which includes instructions for detecting a target nucleic acid sequence with one or more detection probes using real-time PCR, wherein the detection probes are labeled with a detectable label.

[0296] 97. The kit of embodiment 94, which includes instructions for detecting a target nucleic acid sequence with one or more detection probes using real-time PCR, wherein the detection probes are unlabeled and can be detected indirectly by binding another binding partner to a moiety on the probe.

[0297] 98. The kit of embodiment 96, which includes instructions for detecting a target nucleic acid sequence with one or more detection probes using real-time PCR, wherein the detection probes are labeled with a detectable label and the labeled probes include a second moiety such as a quencher.

[0298] 99. The kit of embodiment 86, which includes instructions for detecting a target nucleic acid sequence with one or more detection probes using conventional endpoint PCR, wherein the detection probes hybridize to the amplification product to generate a signal.

[0299] 100. The kit of embodiment 99, wherein the endpoint detection is performed using agarose gel electrophoresis.

[0300] 101. The kit of embodiment 73, which may optionally include a non-VZV internal standard nucleic acid that is amplified and detected in the same assay reaction mixture by using amplification and detection probes specific for the IC sequence.

[0301] 102. The kit of embodiment 73 may include additional guidance regarding sample preparation before amplification, or the use of capture oligomers for hybridizing to the target nucleic acid sequence, and probes: conventional methods for washing non-target materials found in the target duplex, etc.

[0302] 103. The kit of embodiment 102 may include additional instructions regarding conventional methods of target capture, which may include guidance for lysing the sample to release intracellular contents containing the target nucleic acid sequence if the VZV nucleic acid sequence is present in the sample.

[0303] 104. The kit of embodiment 103 may include additional instructions regarding conventional methods of target capture, which may include guidance for specific or non-specific target capture of the target nucleic acid sequence found in the sample.

[0304] 105. The kit of embodiment 104 may have guidance that may recommend non-specific capture probes that preferentially hybridize to the target nucleic acid sequence or its complement under stringent hybridization conditions that allow detection by forming a probe:target duplex.

[0305] 106. The kit according to any one of embodiments 102 to 105 may have guidance that may prefer non-specific capture probes for a substantially aqueous mixture.

[0306] 107. The kit according to any one of embodiments 102 to 106 may have guidance that may recommend washing the probe:target duplex to remove any non-target nucleic acid components that may have bound to the non-specific capture probes.

[0307] 108. The kit of embodiment 107 may have guidance that may recommend washing the probe:target duplex multiple times.

[0308] 109. The kit according to any one of embodiments 102 to 108 may have guidance that may recommend other means of physically separating the target nucleic acid sequence from the sample.

[0309] Kit of Embodiment 109, which can recover the bound target nucleic acid sequence using paramagnetic beads.

[0310] 111. A method for amplifying or detecting a target nucleic acid sequence of VZV, generally comprising using one or more oligonucleotides for detecting the target nucleic acid sequence, or detecting an amplicon generated from the target nucleic acid sequence, and using one or more oligonucleotides for amplifying the target nucleic acid sequence or amplifying an amplicon generated from the target nucleic acid sequence.

[0311] 112. The method of Embodiment 111 for amplifying a target nucleic acid sequence or amplifying an amplicon generated from the target nucleic acid sequence, comprising the steps of obtaining a sample, contacting the sample with at least two amplification primers (the first amplification primer being a forward amplification primer and the second amplification primer being a reverse amplification primer), providing conditions for generating an amplicon from the target nucleic acid sequence, and determining whether VZV is present in the sample.

[0312] 113. The method of Embodiment 112, wherein the target nucleic acid region is SEQ ID NO: 38 or SEQ ID NO: 39.

[0313] 114. The method of Embodiment 112 or 113, wherein the target nucleic acid region is SEQ ID NO: 38, the forward and reverse amplification primers are independently of each other about 19 to about 23 nucleotides in length, and the forward and reverse amplification primers are configured to generate an amplicon about 89 to about 127 nucleotides in length from the target nucleic acid region of SEQ ID NO: 38.

[0314] 115. The method according to any one of embodiments 112 to 114, wherein the target nucleic acid region is SEQ ID NO: 38, the forward amplification primer is selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7, the reverse amplification primer is about 19 to about 23 nucleotides in length, and the two amplification primers are configured to generate an amplicon having a length of about 89 to about 127 nucleotides from the target nucleic acid region of SEQ ID NO: 38.

[0315] 116. The method according to any one of embodiments 112 to 115, wherein the target nucleic acid region is SEQ ID NO: 38, and the reverse amplification primer is selected from the group consisting of SEQ ID NOs: 16, 17, 18, 19, 20, 21, and 22.

[0316] 117. The method according to any one of embodiments 112 to 114, wherein the target nucleic acid region is SEQ ID NO: 38, the reverse amplification primer is selected from the group consisting of SEQ ID NOs: 16, 17, 18, 19, 20, 21, and 22, the forward amplification primer is about 20 to about 23 nucleotides in length, and the reverse and forward amplification primers are configured to generate an amplicon having a length of about 89 to about 127 nucleotides from the target nucleic acid region of SEQ ID NO: 38.

[0317] 118. The forward amplification primer is configured to hybridize to an oligohybridization-forming region within SEQ ID NO: 38 or its complement, the reverse amplification primer is configured to hybridize to an oligohybridization-forming region within SEQ ID NO: 38 or its complement, and the distance between the oligohybridization-forming region of the first amplification primer and the oligohybridization-forming region of the second amplification region is 89, 93, 100, 102, 119, 123, or 127 nucleotides along SEQ ID NO: 38 when measured from the most distant nucleotides of the two oligohybridization-forming regions. The method according to any one of embodiments 115 to 117.

[0318] 119. The method according to any one of embodiments 112 to 118, wherein the target nucleic acid region is SEQ ID NO: 38, and the forward and reverse amplification primers comprise target nucleic acid sequences corresponding to (a) SEQ ID NO: 1 and SEQ ID NO: 16, (b) SEQ ID NO: 1 and SEQ ID NO: 17, (c) SEQ ID NO: 2 and SEQ ID NO: 17, (d) SEQ ID NO: 3 and SEQ ID NO: 18, (e) SEQ ID NO: 4 and SEQ ID NO: 19, (f) SEQ ID NO: 5 and SEQ ID NO: 20, (g) SEQ ID NO: 6 and SEQ ID NO: 21, or (h) SEQ ID NO: 7 and SEQ ID NO: 22.

[0319] 120. The method according to embodiment 112 or 113, wherein the target nucleic acid region is SEQ ID NO: 39, the forward and reverse amplification primers are each independently about 20 to about 23 nucleotides in length, and the forward and reverse amplification primers are configured to generate an amplicon about 89 to about 143 nucleotides in length from the target nucleic acid region of SEQ ID NO: 39.

[0320] 121. The method according to embodiment 112 or 113 or 120, wherein the target nucleic acid region is SEQ ID NO: 39, the forward amplification primer is selected from the group consisting of SEQ ID NOs: 23, 24, 25, 26, and 27, the reverse amplification primer is about 20 to about 22 nucleotides in length, and the two amplification primers are configured to generate an amplicon about 89 to about 143 nucleotides in length from the target nucleic acid region of SEQ ID NO: 39.

[0321] 122. The method according to embodiment 121, wherein the target nucleic acid region is SEQ ID NO: 39 and the reverse amplification primer is selected from the group consisting of SEQ ID NOs: 34, 35, 36, and 37.

[0322] 123. The method according to embodiment 112 or 113 or 120, wherein the target nucleic acid region is SEQ ID NO: 39, the reverse amplification primer is selected from the group consisting of SEQ ID NOs: 34, 35, 36, and 37, the forward amplification primer is about 20 to about 23 nucleotides in length, and the reverse and forward amplification primers are configured to generate an amplicon about 89 to about 143 nucleotides in length from the target nucleic acid region of SEQ ID NO: 39.

[0323] 124. The method according to embodiment 121 or 123, wherein the forward amplification primer is configured to hybridize to the oligohybridization region within SEQ ID NO: 39 or its complement, the reverse amplification primer is configured to hybridize to the oligohybridization region within SEQ ID NO: 39 or its complement, and the distance between the oligohybridization region of the first amplification primer and the oligohybridization region of the second amplification region is 89, 99, 109, 126 or 143 nucleotides along SEQ ID NO: 39 when measured from the most distant nucleotides of the two oligohybridization regions.

[0324] 125. The method according to any one of embodiments 112 or 113 or any one of embodiments 120 - 124, wherein the target nucleic acid region is SEQ ID NO: 39, and the forward and reverse amplification primers comprise target nucleic acid sequences corresponding to (a) SEQ ID NO: 23 and SEQ ID NO: 34, (b) SEQ ID NO: 24 and SEQ ID NO: 34, (c) SEQ ID NO: 25 and SEQ ID NO: 35, (d) SEQ ID NO: 26 and SEQ ID NO: 36, or (e) SEQ ID NO: 27 and SEQ ID NO: 37.

[0325] 126. The method according to any one of embodiments 112 - 125, further comprising a third oligonucleotide.

[0326] 127. The method according to embodiment 126, wherein the third oligonucleotide is a detection probe.

[0327] 128. The method according to any one of embodiments 113 - 119, wherein the target nucleic acid region is SEQ ID NO: 38, and the detection probe is about 23 to about 27 nucleotides in length.

[0328] 129. The method according to embodiment 128, wherein the target nucleic acid region is SEQ ID NO: 38, and the detection probe is selected from the group consisting of SEQ ID NO: 8, 9, 10, 11, 12, 13, 14 and 15.

[0329] 130. When the detection probe comprises the following target hybridization-forming sequences: (a) if the sequence number is 8, the forward and reverse amplification primers carefully include (I) sequence number 1 and sequence number 16 or (II) sequence number 1 and sequence number 17; (b) if the sequence number is 9, the forward and reverse amplification primers carefully include (I) sequence number 1 and sequence number 16 or (II) sequence number 1 and sequence number 17 or (III) sequence number 2 and sequence number 17; (c) if the sequence number is 10, the forward and reverse amplification primers carefully include sequence number 3 and sequence number 18; (d) if the sequence number is 11, the forward and reverse amplification primers carefully include sequence number 4 and sequence number 19; (e) if the sequence number is 12, the forward and reverse amplification primers carefully include sequence number 4 and sequence number 19; (f) if the sequence number is 13, the forward and reverse amplification primers carefully include sequence number 5 and sequence number 20; (g) if the sequence number is 14, the forward and reverse amplification primers carefully include sequence number 6 and sequence number 21; or (h) if the sequence number is 15, the forward and reverse amplification primers carefully include sequence number 7 and sequence number 22. The method of embodiment 128.

[0330] 131. The method according to any one of embodiments 113 or 120 - 125, wherein the target nucleic acid region is sequence number 39 and the detection probe has a length of about 22 to about 27 nucleotides.

[0331] 132. The method of embodiment 131, wherein the target nucleic acid region is sequence number 39 and the detection probe is selected from the group consisting of sequence numbers 28, 29, 30, 31, 32, and 33.

[0332] 133. When the detection probe includes the following target hybridization-forming sequences: (a) if the sequence number is 28, the forward and reverse amplification primers carefully include (I) sequence number 23 and sequence number 34 or (II) sequence number 24 and sequence number 34; (b) if the sequence number is 29, the forward and reverse amplification primers carefully include sequence number 25 and sequence number 35; (c) if the sequence number is 30, the forward and reverse amplification primers carefully include sequence number 25 and sequence number 35; (d) if the sequence number is 31, the forward and reverse amplification primers carefully include sequence number 26 and sequence number 36; (e) if the sequence number is 32, the forward and reverse amplification primers carefully include sequence number 27 and sequence number 37; or (f) if the sequence number is 33, the forward and reverse amplification primers carefully include sequence number 27 and sequence number 37. The method of embodiment 131.

[0333] 134. The method according to any one of embodiments 126 to 133, wherein the detection probe further includes at least one detectable label.

[0334] 135. The method of embodiment 134, wherein one or more of the detectable labels are selected from the group consisting of (a) chemiluminescent labels, (b) fluorescent labels, (c) quenchers, or (d) combinations of two or more of (a), (b), and (c).

[0335] 136. The method of embodiment 135, wherein one or more detectable labels include a fluorescent label, or one or more detectable labels include a quencher, or one or more detectable labels include both a fluorescent label and a quencher.

[0336] 137. The method according to any one of embodiments 126 to 136, wherein the detection probe is a TaqMan (trademark) detection probe.

[0337] 138. The method according to any one of embodiments 126 to 136, wherein the detection probe further comprises a non-target hybridization-forming array, or the detection probe comprising a non-target hybridization-forming array is a hairpin detection probe, or the hairpin detection probe is a molecular beacon or a molecular torch.

[0338] 139. The method according to any one of embodiments 126 to 138, wherein the detection probe further comprises at least one modified nucleobase.

[0339] 140. The method of embodiment 139, wherein one or more of the modified nucleobases are selected from the group consisting of (a) 2'-O-methyl, (b) 5-methylcytosine, (c) 2'-fluoro, or (d) a combination of two or more of (a), (b), and (c).

[0340] 141. The detection probe comprises 3 to 10 modified nucleobases, or the detection probe comprises 3 to 10 5-methylcytosine-modified nucleobases, or the detection probe comprises 3 5-methylcytosine-modified nucleobases, or the detection probe comprises 6 5-methylcytosine-modified nucleobases, or the detection probe comprises 7 5-methylcytosine-modified nucleobases, or the detection probe comprises 10 5-methylcytosine-modified nucleobases, or at least one modification is a 5-methylcytosine-modified nucleobase, or the detection probe comprises 3 to 10 2'-O-methyl-modified nucleobases, or the detection probe comprises 3 2'-O-methyl-modified nucleobases, or the detection probe comprises 6 2'-O-methyl-modified nucleobases, or the detection probe comprises 7 2'-O-methyl-modified nucleobases, or the detection probe comprises 10 2'-O-methyl-modified nucleobases, or at least one modification is a 2'-O-methyl-modified nucleobase, the method of embodiment 139 or 140.

[0341] 142. The method according to any one of embodiments 112 to 141, wherein the forward amplification primer further comprises at least one modified nucleobase.

[0342] 143. The method of embodiment 142, wherein one or more of the modified nucleobases are selected from the group consisting of (a) 2'-O-methyl, (b) 5-methylcytosine, (c) 2'-fluoro, or (d) a combination of two or more of (a), (b), and (c).

[0343] 144. The method of embodiment 142 or 143, wherein the forward amplification primer comprises 1 to 6 modified nucleobases, or the forward amplification primer comprises 1 to 6 5-methylcytosine modified nucleobases, or the forward amplification primer comprises 1 5-methylcytosine modified nucleobase, or the forward amplification primer comprises 2 5-methylcytosine modified nucleobases, or the forward amplification primer comprises 3 5-methylcytosine modified nucleobases, or the forward amplification primer comprises 4 5-methylcytosine modified nucleobases, or the forward amplification primer comprises 5 5-methylcytosine modified nucleobases, or the forward amplification primer comprises 6 5-methylcytosine modified nucleobases, or at least one modification is a 5-methylcytosine modified nucleobase, or the forward amplification primer comprises 1 to 6 2'-O-methyl modified nucleobases, or the forward amplification primer comprises 1 2'-O-methyl modified nucleobase, or the forward amplification primer comprises 2 2'-O-methyl modified nucleobases, or the forward amplification primer comprises 3 2'-O-methyl modified nucleobases, or the forward amplification primer comprises 4 2'-O-methyl modified nucleobases, or the forward amplification primer comprises 5 2'-O-methyl modified nucleobases, or the forward amplification primer comprises 6 2'-O-methyl modified nucleobases, or at least one modification is a 2'-O-methyl modified nucleobase.

[0344] 145. The method according to any one of embodiments 112 to 144, wherein the reverse amplification primer further comprises at least one modified nucleobase.

[0345] 146. The method of embodiment 145, wherein one or more of the modified nucleobases are selected from the group consisting of (a) 2'-O-methyl, (b) 5'-methylcytosine, (c) 2'-fluoro, or (d) a combination of two or more of (a), (b), and (c).

[0346] 147. The method of embodiment 145 or 146, wherein the reverse amplification primer comprises 2 to 6 modified nucleobases, or the reverse amplification primer comprises 2 to 6 2'-fluoro modified nucleobases, or the reverse amplification primer comprises 2 2'-fluoro modified nucleobases, or the reverse amplification primer comprises 3 2'-fluoro modified nucleobases, or the reverse amplification primer comprises 4 2'-fluoro modified nucleobases, or the reverse amplification primer comprises 5 2'-fluoro modified nucleobases, or the reverse amplification primer comprises 6 2'-fluoro modified nucleobases, or at least 2 of the modifications are 2'-fluoro modified nucleobases, or the reverse amplification primer comprises 2 to 6 5-methylcytosine modified nucleobases, or the reverse amplification primer comprises 2 5-methylcytosine modified nucleobases, or the reverse amplification primer comprises 3 5-methylcytosine modified nucleobases, or the reverse amplification primer comprises 4 5-methylcytosine modified nucleobases, or the reverse amplification primer comprises 5 5-methylcytosine modified nucleobases, or the reverse amplification primer comprises 6 5-methylcytosine modified nucleobases, or at least 1 of the modifications is a 5-methylcytosine modified nucleobase.

[0347] 148. A method according to embodiment 111 for detecting a target nucleic acid sequence or for detecting an amplicon generated from a target nucleic acid sequence, the method comprising the steps of obtaining a sample, contacting the sample with at least one oligonucleotide for detecting the target nucleic acid sequence, providing conditions for detecting the presence of the target nucleic acid sequence, and determining whether VZV is present in the sample.

[0348] 149. The method of embodiment 148, wherein the target nucleic acid region is SEQ ID NO: 38 or SEQ ID NO: 39.

[0349] 150. The method of embodiment 149, wherein the target nucleic acid region is SEQ ID NO: 38 and the detection probe is about 23 to about 27 nucleotides in length.

[0350] 151. The method of embodiment 150, wherein the target nucleic acid region is SEQ ID NO: 38 and the detection probe is selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, and 15.

[0351] 152. The method of embodiment 150 or 151, further comprising at least one set of amplification primers, wherein one amplification primer is a forward amplification primer and one amplification primer is a reverse amplification primer.

[0352] 153. The method of embodiment 152, wherein the target nucleic acid region is SEQ ID NO: 38, the forward and reverse amplification primers are each individually about 19 to about 23 nucleotides in length, and the forward and reverse amplification primers are configured to generate an amplicon about 89 to about 127 nucleotides in length from the target nucleic acid region of SEQ ID NO: 38.

[0353] 154. The method of embodiment 153, wherein the target nucleic acid region is SEQ ID NO: 38 and the forward amplification primer is selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7.

[0354] 155. The method of embodiment 153 or 154, wherein the target nucleic acid region is SEQ ID NO: 38 and the reverse amplification primer is selected from the group consisting of SEQ ID NOs: 16, 17, 18, 19, 20, 21, and 22.

[0355] 156. The method of embodiment 155, wherein the target nucleic acid region is SEQ ID NO: 38, the reverse amplification primer is selected from the group consisting of SEQ ID NO: 16, 17, 18, 19, 20, 21, and 22, the forward amplification primer is about 20 to about 23 nucleotides in length, and the reverse and forward amplification primers are configured to generate an amplicon having a length of about 89 to about 127 nucleotides from the target nucleic acid region of SEQ ID NO: 38.

[0356] 157. The method of embodiment 154 or 156, wherein the forward amplification primer is configured to hybridize to the oligohybridization-forming region within SEQ ID NO: 38 or its complement, the reverse amplification primer is configured to hybridize to the oligohybridization-forming region within SEQ ID NO: 38 or its complement, and the distance between the oligohybridization-forming region of the first amplification primer and the oligohybridization-forming region of the second amplification region is 89, 93, 100, 102, 119, 123, or 127 nucleotides in length along SEQ ID NO: 38 when measured from the most distant nucleotides of the two oligohybridization-forming regions.

[0357] 158. The method according to any one of embodiments 152 to 157, wherein the target nucleic acid region is SEQ ID NO: 38, and the forward and reverse amplification primers comprise target nucleic acid sequences corresponding to (a) SEQ ID NO: 1 and SEQ ID NO: 16, (b) SEQ ID NO: 1 and SEQ ID NO: 17, (c) SEQ ID NO: 2 and SEQ ID NO: 17, (d) SEQ ID NO: 3 and SEQ ID NO: 18, (e) SEQ ID NO: 4 and SEQ ID NO: 19, (f) SEQ ID NO: 5 and SEQ ID NO: 20, (g) SEQ ID NO: 6 and SEQ ID NO: 21, or (h) SEQ ID NO: 7 and SEQ ID NO: 22.

[0358] 159. When the detection probe comprises the following target hybridization-forming sequences: (a) if the sequence number is 8, the forward and reverse amplification primers exactly contain (I) sequence number 1 and sequence number 16 or (II) sequence number 1 and sequence number 17; (b) if the sequence number is 9, the forward and reverse amplification primers exactly contain (I) sequence number 1 and sequence number 16 or (II) sequence number 1 and sequence number 17 or (III) sequence number 2 and sequence number 17; (c) if the sequence number is 10, the forward and reverse amplification primers exactly contain sequence number 3 and sequence number 18; (d) if the sequence number is 11, the forward and reverse amplification primers exactly contain sequence number 4 and sequence number 19; (e) if the sequence number is 12, the forward and reverse amplification primers exactly contain sequence number 4 and sequence number 19; (f) if the sequence number is 13, the forward and reverse amplification primers exactly contain sequence number 5 and sequence number 20; (g) if the sequence number is 14, the forward and reverse amplification primers exactly contain sequence number 6 and sequence number 21; (h) if the sequence number is 15, the forward and reverse amplification primers exactly contain sequence number 7 and sequence number 22. The method of embodiment 150.

[0359] 160. The method of embodiment 149, wherein the target nucleic acid region is sequence number 39 and the detection probe is about 22 to about 27 nucleotides in length.

[0360] 161. The method of embodiment 160 or 161, wherein the target nucleic acid region is sequence number 39 and the detection probe is selected from the group consisting of sequence numbers 28, 29, 30, 31, 32 and 33.

[0361] 162. The method of embodiment 161, further comprising at least one set of amplification primers, wherein one amplification primer is a forward amplification primer and one amplification primer is a reverse amplification primer.

[0362] 163. The method of embodiment 162, wherein the target nucleic acid region is SEQ ID NO: 39, the forward and reverse amplification primers are each individually about 20 to about 23 nucleotides in length, and the forward and reverse amplification primers are configured to generate an amplicon having a length of about 89 to about 143 nucleotides from the target nucleic acid region of SEQ ID NO: 39.

[0363] 164. The method of embodiment 163, wherein the target nucleic acid region is SEQ ID NO: 39 and the forward amplification primer is selected from the group consisting of SEQ ID NOs: 23, 24, 25, 26, and 27.

[0364] 165. The method of embodiment 163 or 164, wherein the target nucleic acid region is SEQ ID NO: 39 and the reverse amplification primer is selected from the group consisting of SEQ ID NOs: 34, 35, 36, and 37.

[0365] 166. The method of embodiment 162, wherein the target nucleic acid region is SEQ ID NO: 39, the reverse amplification primer is selected from the group consisting of SEQ ID NOs: 34, 35, 36, and 37, the forward amplification primer is about 20 to about 23 nucleotides in length, and the reverse and forward amplification primers are configured to generate an amplicon having a length of about 89 to about 143 nucleotides from the target nucleic acid region of SEQ ID NO: 39.

[0366] 167. The method of embodiment 164 or 166, wherein the forward amplification primer is configured to hybridize to an oligohybridization-forming region within SEQ ID NO: 39 or its complement, the reverse amplification primer is configured to hybridize to an oligohybridization-forming region within SEQ ID NO: 39 or its complement, and the distance between the oligohybridization-forming region of the first amplification primer and the oligohybridization-forming region of the second amplification region is 89, 99, 109, 126, or 143 nucleotides along SEQ ID NO: 39 as measured from the most distant nucleotides of the two oligohybridization-forming regions.

[0367] 168. The method according to any one of embodiments 162 to 167, wherein the target nucleic acid region is SEQ ID NO: 39, and the forward and reverse amplification primers contain target nucleic acid sequences corresponding to (a) SEQ ID NO: 23 and SEQ ID NO: 34, (b) SEQ ID NO: 24 and SEQ ID NO: 34, (c) SEQ ID NO: 25 and SEQ ID NO: 35, (d) SEQ ID NO: 26 and SEQ ID NO: 36, or (e) SEQ ID NO: 27 and SEQ ID NO: 37.

[0368] 169. When the detection probe contains the following target hybridization-forming sequences: (a) If it is SEQ ID NO: 28, the forward and reverse amplification primers strictly contain (I) SEQ ID NO: 23 and SEQ ID NO: 34 or (II) SEQ ID NO: 24 and SEQ ID NO: 34; (b) If it is SEQ ID NO: 29, the forward and reverse amplification primers strictly contain SEQ ID NO: 25 and SEQ ID NO: 35; (c) If it is SEQ ID NO: 30, the forward and reverse amplification primers strictly contain SEQ ID NO: 25 and SEQ ID NO: 35; (d) If it is SEQ ID NO: 31, the forward and reverse amplification primers strictly contain SEQ ID NO: 26 and SEQ ID NO: 36; (e) If it is SEQ ID NO: 32, the forward and reverse amplification primers strictly contain SEQ ID NO: 27 and SEQ ID NO: 37; (f) If it is SEQ ID NO: 33, the forward and reverse amplification primers strictly contain SEQ ID NO: 27 and SEQ ID NO: 37. The method of embodiment 160.

[0369] 170. The method according to embodiment 148 or 149, wherein the detection probe further contains at least one detectable label.

[0370] 171. The method according to embodiment 170, wherein one or more of the detectable labels are selected from the group consisting of (a) chemiluminescent labels, (b) fluorescent labels, (c) quenchers, or (d) combinations of two or more of (a), (b), and (c).

[0371] 172. The method according to embodiment 171, wherein one or more detectable labels contain a fluorescent label, or one or more detectable labels contain a quencher, or one or more detectable labels contain both a fluorescent label and a quencher.

[0372] 173. The method according to any one of embodiments 148 to 172, wherein the detection probe is a TaqMan (trademark) detection probe.

[0373] 174. The method according to any one of embodiments 148 to 172, wherein the detection probe further comprises a non-target hybridization-forming sequence, or the detection probe comprising a non-target hybridization-forming sequence is a hairpin detection probe, or the hairpin detection probe is a molecular beacon or a molecular torch.

[0374] 175. The method according to any one of embodiments 148 to 174, wherein the detection probe further comprises at least one modified nucleobase.

[0375] 176. The method of embodiment 175, wherein one or more of the modified nucleobases are selected from the group consisting of (a) 2'-O-methyl, (b) 5-methylcytosine, (c) 2'-fluoro, or (d) a combination of two or more of (a), (b), and (c).

[0376] 177. The method of embodiment 175 or 176, wherein the detection probe comprises 3 to 10 modified nucleobases, or the detection probe comprises 3 to 10 5-methylcytosine-modified nucleobases, or the detection probe comprises 3 5-methylcytosine-modified nucleobases, or the detection probe comprises 6 5-methylcytosine-modified nucleobases, or the detection probe comprises 7 5-methylcytosine-modified nucleobases, or the detection probe comprises 10 5-methylcytosine-modified nucleobases, or at least one modification is a 5-methylcytosine-modified nucleobase, or the detection probe comprises 3 to 10 2'-O-methyl-modified nucleobases, or the detection probe comprises 3 2'-O-methyl-modified nucleobases, or the detection probe comprises 6 2'-O-methyl-modified nucleobases, or the detection probe comprises 7 2'-O-methyl-modified nucleobases, or the detection probe comprises 10 2'-O-methyl-modified nucleobases, or at least one modification is a 2'-O-methyl-modified nucleobase.

[0377] 178. The method according to any one of embodiments 152 to 177, wherein the forward amplification primer further comprises at least one modified nucleobase.

[0378] 179. The method of embodiment 178, wherein one or more of the modified nucleobases are selected from the group consisting of (a) 2'-O-methyl, (b) 5-methylcytosine, (c) 2'-fluorine, or (d) a combination of two or more of (a), (b), and (c).

[0379] 180. The forward amplification primer comprises 2 to 6 modified nucleobases, or the forward amplification primer comprises 2 to 6 5-methylcytosine-modified nucleobases, or the forward amplification primer comprises 2 5-methylcytosine-modified nucleobases, or the forward amplification primer comprises 3 5-methylcytosine-modified nucleobases, or the forward amplification primer comprises 4 5-methylcytosine-modified nucleobases, or the forward amplification primer comprises 6 5-methylcytosine-modified nucleobases, or at least one modification is a 5-methylcytosine-modified nucleobase, or the forward amplification primer comprises 2 to 6 2'-O-methyl-modified nucleobases, or the forward amplification primer comprises 2 2'-O-methyl-modified nucleobases, or the forward amplification primer comprises 3 2'-O-methyl-modified nucleobases, or the forward amplification primer comprises 4 2'-O-methyl-modified nucleobases, or the forward amplification primer comprises 6 2'-O-methyl-modified nucleobases, or at least one modification is a 2'-O-methyl-modified nucleobase, the method of embodiment 178 or 179.

[0380] 181. The method according to any one of embodiments 152 to 180, wherein the reverse amplification primer further comprises at least one modified nucleobase.

[0381] 182. The method of embodiment 181, wherein one or more of the modified nucleobases are selected from the group consisting of (a) 2'-O-methyl, (b) 5-methylcytosine, (c) 2'-fluorine, or (d) a combination of two or more of (a), (b), and (c).

[0382] 183. The reverse amplification primer contains 1 to 6 modified nucleobases, or the reverse amplification primer contains 1 to 6 2'-fluorine modified nucleobases, or the reverse amplification primer contains 1 2'-fluorine modified nucleobase, or the reverse amplification primer contains 2 2'-fluorine modified nucleobases, or the reverse amplification primer contains 3 2'-fluorine modified nucleobases, or the reverse amplification primer contains 4 2'-fluorine modified nucleobases, or the reverse amplification primer contains 5 2'-fluorine modified nucleobases, or the reverse amplification primer contains 6 2'-fluorine modified nucleobases, or at least 1 modification is a 2'-fluorine modified nucleobase, or the reverse amplification primer contains 1 to 6 5-methylcytosine modified nucleobases, or the reverse amplification primer contains 1 5-methylcytosine modified nucleobase, or the reverse amplification primer contains 2 5-methylcytosine modified nucleobases, or the reverse amplification primer contains 3 5-methylcytosine modified nucleobases, or the reverse amplification primer contains 4 5-methylcytosine modified nucleobases, or the reverse amplification primer contains 5 5-methylcytosine modified nucleobases, or the reverse amplification primer contains 6 5-methylcytosine modified nucleobases, or at least 1 modification is a 5-methylcytosine modified nucleobase, the method of Embodiment 181 or 182.

[0383] 184. A preparation for amplifying the target nucleic acid sequence of VZV, generally comprising one or more oligonucleotides for detecting the target nucleic acid sequence or detecting an amplicon generated from the target nucleic acid sequence, or one or more oligonucleotides for amplifying the target nucleic acid sequence or amplifying an amplicon generated from the target nucleic acid sequence.

[0384] 185. The preparation of Embodiment 184, wherein the amplification primer preparation and the detection probe preparation are two separate products.

[0385] 186. The preparation according to embodiment 184, further comprising at least two amplification primers as in any one of embodiments 1 to 36, wherein the first amplification primer is a forward amplification primer and the second amplification primer is a reverse amplification primer.

[0386] 187. The amplification primers included in the kit include a single set of forward and reverse amplification primers that generate a single amplicon of the target nucleic acid sequence, or the kit includes multiple sets of amplification primers that generate multiple amplicons from various target nucleic acid sequences across various target nucleic acid regions, or the kit may include multiple sets of amplification primers that generate multiple amplicons from various target nucleic acid sequences within a single target nucleic acid region, the preparation according to embodiment 186.

[0387] 188. The preparation according to embodiment 184 may further include additional reagents for determining the presence of the VZV nucleic acid sequence in the sample.

[0388] 189. The preparation according to embodiment 188, further comprising at least one oligonucleotide as in any one of embodiments 37 to 72 for detecting the target nucleic acid sequence or for detecting an amplicon generated from the target nucleic acid sequence within the target nucleic acid region.

[0389] 190. The preparation according to any one of embodiments 184 or 189 may further include additional reagents for amplifying the sequence if the target nucleic acid sequence of the VZV nucleic acid sequence is present in the sample.

[0390] 191. The preparation according to embodiment 184 may include reagents suitable for performing in vitro amplification, such as various dNTPs, enzymes, buffers, or salts.

[0391] The formulation of embodiment 191, wherein the formulation can contain each of various nucleotide subunits of DNA, such as deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), deoxythymidine triphosphate (dTTP), or adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), and uridine triphosphate (UTP).

[0392] The formulation of embodiment 191, wherein the formulation can contain a DNA polymerase enzyme, or the formulation can contain a reverse transcriptase, or the formulation can contain an organic buffer, or the formulation can contain a surfactant, or the formulation can contain an inorganic salt.

[0393] The formulation of embodiment 191, wherein the formulation can contain an inorganic salt selected from the group consisting of magnesium chloride, sodium chloride, potassium chloride, and sodium citrate.

[0394] The formulation of embodiment 191, wherein the aqueous formulation can be dropped into liquid nitrogen and lyophilized according to procedures well known to those skilled in the art of molecular biology.

[0395] The formulation of embodiment 195, wherein the lyophilized formulation can appear as a powder or solid or sphere.

[0396] The formulation of embodiment 196, wherein when the formulation is lyophilized, the formulation can further contain a filler such as, for example, trehalose, raffinose, or a combination thereof.

[0397] A reaction mixture for amplifying a target nucleic acid sequence of VZV, generally comprising one or more oligonucleotides for detecting the target nucleic acid sequence or for detecting an amplicon generated from the target nucleic acid sequence, and one or more oligonucleotides for amplifying the target nucleic acid sequence or for amplifying an amplicon generated from the target nucleic acid sequence.

[0398] 199. Similar to any one of Embodiments 1 to 36, further comprising at least two amplification primers, wherein the first amplification primer is a forward amplification primer and the second amplification primer is a reverse amplification primer, the reaction mixture of Embodiment 198.

[0399] 200. The amplification primers included in the kit include a single set of forward and reverse amplification primers that generate a single amplicon of the target nucleic acid sequence, or the kit includes a plurality of sets of amplification primers that generate a plurality of amplicons from various target nucleic acid sequences across various target nucleic acid regions, or the kit may include a plurality of sets of amplification primers that generate a plurality of amplicons from various target nucleic acid sequences within a single target nucleic acid region, the reaction mixture according to any one of Embodiment 199.

[0400] 201. The reaction mixture of Embodiment 198 may further include additional reagents for determining the presence of VZV nucleic acid sequences in the sample.

[0401] 202. The reaction mixture of Embodiment 198 further includes at least one oligonucleotide similar to any one of Embodiments 37 to 72 for detecting the target nucleic acid sequence or for detecting an amplicon generated from the target nucleic acid sequence within the target nucleic acid region.

[0402] 203. When the target nucleic acid sequence of the VZV nucleic acid sequence is present in the sample, the reaction mixture of any one of Embodiments 198 to 202 may further include additional reagents for amplifying the sequence.

[0403] 204. The reaction mixture of Embodiment 198 may include reagents suitable for performing in vitro amplification, such as various dNTPs, enzymes, buffers, or salts.

[0404] The reaction mixture of embodiment 204 may contain each of the various nucleotide subunits of DNA, such as deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), deoxythymidine triphosphate (dTTP), or adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), and uridine triphosphate (UTP).

[0405] The reaction mixture of embodiment 204 may contain a DNA polymerase enzyme, or the reaction mixture may contain a reverse transcriptase, or the reaction mixture may contain an organic buffer, or the reaction mixture may contain a surfactant, or the reaction mixture may contain an inorganic salt.

[0406] The reaction mixture of embodiment 204 may contain an inorganic salt selected from the group consisting of magnesium chloride, sodium chloride, potassium chloride, and sodium citrate.

[0407] The reaction mixture according to embodiment 207 contains magnesium chloride, or the concentration of magnesium chloride is 3 mM to 6 mM, or the concentration of magnesium chloride is 2 mM, or the concentration of magnesium chloride is 4 mM, or the concentration of magnesium chloride is 6 mM.

Examples

[0408] The oligonucleotides provided are useful for the amplification or detection of the target nucleic acid regions of SEQ ID NO: 38 and SEQ ID NO: 39 within the VZV nucleic acid sequence. Specifically, by combining primers and probes, the target nucleic acid sequences within the target nucleic acid regions of VZV can be amplified and detected. In some embodiments, the primers and probes are used in combination with a fluorescently labeled probe. The oligonucleotides function to amplify or detect the target nucleic acid sequences in clinical specimens or in devised clinical specimens, do not cross-react with common organisms potentially present in the samples, and do not interfere with internal standards.

[0409] The following examples illustrate certain specific embodiments disclosed and should not be construed as limiting the scope of the disclosure in any way.

[0410] Example 1 - Considerations in Oligomer Design As shown in Table 1, 18 unique combinations of primers and probes (PPRs) were evaluated for in vitro VZV detection. All oligosets cover the target nucleic acid regions of SEQ ID NO: 38 and SEQ ID NO: 39 within the broader VZV nucleic acid sequence.

Table 1-1

Table 1-2

Table 1-3

Table 1-4

[0411] Eighteen different primer and probe combinations were selected and tested (on the same day as preparation). Samples were stored at 4 °C until just prior to testing. VZV culture, Ellen (Catalog No. 0810171CF, Zeptometrix, Buffalo, NY) was diluted in Specimen Transport Medium (STM) (Catalog No. 5128-1220, QIAGEN (Digene), Germantown, MD). To each of 18 specimen tubes, 1000 ul of STM containing VZV culture (10000 cp / rxn) was added. A negative control consisting of 1000 ul of STM (without VZV) was also run in parallel. Detection probes included the use of non-canonical bases such as 5-methyl-2'-deoxycytosine (5-Me-dC) to raise the melting temperature (Tm). All PPR PCR reactions were run using the thermal cycle conditions listed in Table 2 and tested against an internal standard (Table 3).

Table 2

Table 3

[0412] Table 4 shows the threshold cycle (Ct) and number of positive reactions from each of the 18 different PPR combinations reported in Table 1. All PPR PCR reactions were run using the thermal cycle conditions listed in Table 2 on a Hologic PANTHER FUSION® instrument in the FAM channel. Since the PPR PCR reactions were fully performed on the PANTHER FUSION® instrument for automation, no plates were used. A total of two sample extractions were processed for each PPR. One extraction contained 3 PCR replicates from the eluate. The other extraction contained 1 PCR replicate. Thus, for each PPR, 4 PCR replicates were obtained from two sample extractions. The total reaction volume for each PPR was 400.0 μl.

Table 4

[0413] Results: Ct is the number of cycles at which the relative fluorescence unit signal exceeds the set RFU threshold (associated with the point at which the measured fluorescence signal becomes statistically greater than the baseline signal), thereby distinguishing the amplified signal from background noise. Based on the data, some mixtures showed inadequate results, while other combinations of primers and probes showed good results and were selected for further evaluation of the target nucleic acid regions (SEQ ID NO: 38 and SEQ ID NO: 39). For SEQ ID NO: 38, after analyzing the gradient (logarithmic linear phase measurement of reaction efficiency), the number of cycles at which the fluorescence signal of the reaction exceeded the threshold, the relative fluorescence units, and the known mismatches (via analysis software), it was determined that PPR mixture 8 was the best candidate for advancement. When comparing real-time PCR results from samples containing different amounts of target nucleic acid sequences, a low Ct value indicates a large amount of amplicon (copies of the target nucleic acid sequence), while a high Ct value indicates a small amount of amplicon product. PPR mixture 8 showed a low Ct value and thus showed the highest amount of amplicon. Generally, a Ct value of less than about 29 cycles indicates an abundant PCR product, while a Ct value of greater than about 38 cycles indicates a minimal amount of polynucleotide. PPR mixture 8 also has a high RFU value. (In samples with a large amount of polynucleotide amplicon, the corresponding RFU value is high). For SEQ ID NO: 39, PPR mixture 15 was selected for the same reasons described herein. However, one strain showed a 1-base pair mismatch with the reverse primer. The RFU was not as high as that of PPR mixture 8, but the gradient and Ct values were found to be good. PPR mixture 16 was similarly proven to be ideal (having a low Ct and a 1-base pair mismatch with the reverse primer of one strain), but PPR mixture 16 generated a larger amplicon of 143 base pairs, which was larger than that of PPR mixture 15.

[0414] Example 2: Performance of Oligonucleotides, Probes, and MgCl2 Concentrations under Different Primers To evaluate the flexibility of the oligonucleotide of SEQ ID NO: 38 that functions under different assay conditions, various concentrations of primers, probes, and MgCl2 were combined and tested. Three concentrations of primers (0.4, 0.7, and 1.0 μM), three concentrations of probes (0.2, 0.5, and 0.8 μM), and three concentrations of MgCl2 (2, 4, and 6 mM) were tested against a VZV plasmid (Hologic, Marlborough, Massachusetts) diluted to 1000 cp / rxn, tested against PPR mixtures 1 - 18 using the thermal cycling conditions listed in Table 2, and tested against an internal standard (Table 3). The RFU and Ct data indicate that the PPR is robust and can withstand changes in oligo concentration and salt concentration without causing significant problems with Ct values. Thus, the combination of VZV oligonucleotides can function under a wide range of assay conditions. The Ct values are consistent across all test conditions and the range of MgCl2 concentrations. The baseline fluorescence (and final RFU) is affected by the probe concentration as expected.

[0415] Subsequently, both PPR mixture 8 and PPR mixture 15 were tested with various concentrations of VZV culture to determine which oligo set to advance. The results are shown in Table 5. All PPR PCR reactions were performed using the thermal cycling conditions listed in Table 2.

Table 5

[0416] Result: Based on Ct, reactivity, RFU, and the signal - to - noise ratio, PPR mixture 8 of SEQ ID NO: 38 was selected as the best candidate for further testing.

[0417] To further confirm that PPR mixture 8 and PPR mixture 15 are the best PPR candidates, two oligonucleotide sets were determined for cross-reactivity against HSV-1 and HSV-2 (two strains of herpes simplex virus that similarly share similar nucleic acid sequences) to determine whether they cross-react (e.g., anneal, amplify, and detect) with off-target sequences of HSV-1 or HSV-2.

Table 6

[0418] Results: Both herpes simplex virus type 1 (HSV-1) (MacIntyre strain) and herpes simplex virus type 2 (HSV-2) (MS strain) were diluted with STM (high concentration). The positive control consisted of STM containing VZV culture fluid at 1000 cp / rxn. The negative control consisted of STM (without VZV). The PCR thermal cycle conditions correlated with those in Table 2. Using software that plots the rate of change in relative fluorescence units (RFU) on the Y-axis against the time (number of cycles) on the Y-axis (-ΔF / ΔT) (e.g., melting curve analysis), the data showed no measurable change in fluorescence (RFU) over time (e.g., no increase in PCR product (amplicon)). The measured RFU (e.g., background noise associated with the dissociation of dsDNA to single-stranded DNA (ssDNA) by PCR) was consistent throughout 45 PCR cycles. Based on the data summarized herein, PPR mixture 8 (for SEQ ID NO: 38) was selected for evaluation of sensitivity and specificity.

[0419] Example 3 - Analytical Sensitivity, Virus Sensitivity Generally, those skilled in the art of molecular biology will understand that the viral susceptibility experiment most closely resembles a clinical sample because the presence of host cells in the culture medium emulates in vivo conditions. As described herein, one VZV strain (isolate A) (catalog number 0810172CF, Zeptometrix, Buffalo, NY) diluted with STM was evaluated for reactivity with PPR mixture 8. A negative control (composed of STM without VZV) was run in parallel. A second VZV strain (unknown) (catalog number 23-279-161, Thermo Fisher Scientific (AcroMetrix), Waltham, MA) diluted to 31.6 cp / ml and 10 cp / ml was similarly tested against a negative control consisting of a PBS / PK mixture (final PK concentration of 3 mg / ml) and a positive control (consisting of a VZV plasmid diluted to 100 cp / rxn in STM). VZV in STM was evaluated at 10 - 1000 cp / rxn (278 - 27778 cp / ml). VZV in plasma was tested at 10 - 10,000 cp / ml (0.4 - 360 cp / rxn). For both strains, PPR mixture 8 was run using the thermal cycle conditions listed in Table 2 and tested against an internal standard (Table 3). The results are listed in Tables 7 and 8.

Table 7

Table 8

[0420] Results: 100% detection was seen with VZV added to STM at 100 cp / rxn and 20% at 10 cp / rxn. VZV in plasma was 100% detected at 36 cp / rxn and 66% at 3.6 cp / rxn. The predicted limit of detection (LoD) for VZV in plasma is 31.6 cp / ml based on the Ct value at 100 cp / ml. The AcroMetrix VZV panel displays the exact LoD for VZV below 1000 cp / ml (36 cp / rxn). The internal standard was 100% detected in both studies.

[0421] Plasmid Sensitivity Unlike viral samples, plasmid DNA is readily accessible in solution and does not require cell lysis for testing. Testing plasmid DNA eliminates problems associated with the DNA extraction process because the detection limit is lowered and the performance of the LDT appears poor due to challenges in nucleic acid extraction. Here, plasmid sensitivity was evaluated by testing VZV plasmid in STM at six concentrations (10 to 1,000,000 copies / reaction). The limit of detection (LoD) of the VZV plasmid was determined using an oligo set (PPR Mix 8) to ensure compatibility. The VZV plasmid (Hologic, Marlborough, Massachusetts) was diluted in STM to 1,000,000, 10,000, 1,000, 100, 10, 1 cp / rxn and tested against PPR Mix 8 (specific to SEQ ID NO: 38). The negative control was composed of STM (without VZV). PPR Mix 8 was run using the thermal cycling conditions listed in Table 2 and tested against an internal standard (Table 3). The results are shown in Table 9. [Table 9]

[0422] Results: Generally, the slope of the curve is used to determine the reaction efficiency, which should be about 90% to about 110% corresponding to a slope of about -3.6 to about -3.10. Here, the PPR mixture 8 shows a linear slope of 3.44. The correlation coefficient (R2) value is a measure of the reproducibility of replicates (corresponding to a measure of how well the data fits the standard curve, e.g., the linearity of the standard curve), ideally equal to 1, but generally 0.999 is the maximum value. Here, it is 0.9982 for R2. A slope of 3.44 and an R2 of 0.9982 mean high PCR efficiency. The limit of detection (LoD) of the plasmid is 1 to 10 cp / rxn (27 to 277 cp / ml). Generally, the theoretical limit of detection for 2 to 10 reactions is considered the minimum number of target nucleic acid sequences that can be reliably quantified. The suitability was confirmed by evaluating the VZV plasmid LoD using the selected oligo set (PPR mixture 8). 100% detection of the VZV plasmid (Hologic, Marlborough, Massachusetts) was measured at 10 cp / rxn in town.

[0423] Sensitivity of viral genomic DNA and concentration comparison of plasmid, gDNA, and virus In the genomic DNA test, it is necessary to lyse the cells to access the virus. The sensitivity of genomic DNA was evaluated by testing STM containing VZV gDNA (Ellen) (catalog number VR-1367, ATCC, Manassas, Virginia), VZV plasmid (Hologic, Marlborough, Massachusetts), and VZV culture (Ellen) at six concentrations (10 to 1,000,000 copies / reaction). The PCR formulation was prepared according to PPR mixture 8 in Table 1. Plasmid, gDNA, and virus culture were added to STM at the specified concentrations separately. The results are shown in Table 10.

Table 10

[0424] Results: Using an IC without reverse transcriptase, contamination of gDNA was detected. If the Ct of the IC is higher than the Ct generated by the most dilute target, the Ct indicates that gDNA did not contribute to signal generation. Here, 100% detection of gDNA and plasmid was observed even when it was lowered to 31.6 cp / rxn, and the Ct values were similar (difference 0.3). In the case of the virus culture solution, 100% detection was measured only at 1000 cp / rxn, showing a logarithmic difference of 1.3 - 2.2 in concentration from gDNA.

[0425] Example 4: Specificity For the specificity test, 38 organisms commonly found in blood, tissue, or lesions were prepared in 9 panels by adding them to STM as close as possible to 1E6 cp / ml (depending on availability). The specificity of each panel was evaluated using a PCR formulation according to PPR mixture 8 in Table 1. The composition of the panels and the results of the reactivity are listed in Table 11. The positive control was composed of STM containing VZV culture solution, while the negative control was composed of STM (without VZV). PPR mixture 8 was run using the thermal cycle conditions listed in Table 2 and tested against the internal standard (Table 3).

Table 11-1

Table 11-2

[0426] Results: Of the 38 organisms tested, 0% were positive for VZV and 100% were positive for the internal standard. The positive control consisting of STM containing VZV culture solution was reported to be positive for VZV and IC, while the negative control (STM without VZV) was positive only for IC.

[0427] Example 5: Interference To measure interference, VZV reactivity was evaluated in the presence of 38 organisms from the specificity study. Panels 2 - 8 were diluted with STM to 1:10 VZV strain (Isolate A) at 27,778 cp / ml. Isolate A is a culture of a specific strain of VZV and is "alive" until the cells lyse. Panels 1 and 9 were similarly diluted with STM to 1:10 VZV strain (Isolate A) culture at 27,778 cp / ml. Panels 1 and 9 were newly prepared as they were not obtained from the specificity study. To evaluate CMV interference, CMV culture was added to each panel at 27,778 cp / ml. Each of the 9 panels was tested with PPR mixture 8. The positive control was composed of STM containing CMV and VZV at 27,778 cp / ml. The negative control was composed of STM (without VZV). PPR mixture 8 was run using the thermal cycling conditions listed in Table 2 and tested against the internal standard (Table 3). The results are shown in Table 12.

Table 12 - 1

Table 12 - 2

[0428] Results: VZV and CMV were detected in 100% of the specificity panels tested. For VZV, out of the 38 organisms tested, 0 organisms interfered with the detection of VZV. For CMV, out of the 34 organisms tested, 0 organisms interfered with the detection of CMV. The Ct values varied, but the maximum Ct difference for CMV was 1.6 (when compared to the positive control). Since all samples contained concentrations higher than those expected for clinical specimens, Ct was not considered significant unless it was greater than 3 Ct. The internal standard was detected in 100% of the panels. The positive control (detected in 100% of the panels) was positive for VZV and the internal standard. The negative control was positive only for the internal standard.

[0429] Example 6: Reactivity In the reactivity test, it is confirmed that the selected oligo combination (PPR mixture 8) functions similarly with all virus strains available in the market. This is because testing only one strain of VZV does not suggest that PPR mixture 8 functions equivalently with similar strains, and isolate A is not representative of all VZV strains. The eight isolates tested characterize all the quantified VZV strains available in the market at the time of testing. Strains that have not been quantified do not contribute to the sensitivity test, so like the strains available from ATCC, unquantified VZV strains were not tested. Here, PPR mixture 8 was tested against eight different VZV strains in both virus transport medium (VTM) containing 2E4 cells / ml HeLa and STM containing 2E4 cells / ml. All eight strains were tested at 100 cp / rxn and 1000 cp / rxn. The threshold cycle (Ct) and relative fluorescence unit (RFU) data are shown in Table 14. The positive control consisted of STM containing VZV at 100 cp / rxn and 2E4 cells / ml HeLa. The negative controls consisted of STM and HeLa (without VZV), and VTM and HeLa (without VZV). PPR mixture 8 was run using the thermal cycle conditions listed in Table 2 and tested against an internal standard (Table 3). The results are shown in Table 13.

Table 13

[0430] Results: All strains reacted with the VZV oligo. 100% positivity was seen in all eight VZV strains at 1000 cp / rxn. Isolate A, Isolate B, Isolate D, 82, 275, and 9939 were also 100% positive at 100 cp / rxn. The positive control of the VZV plasmid in STM at 100 cp / rxn was positive for VZV. The negative controls consisting of both simulated clinical matrices were negative for VZV. The internal standard was detected in all samples tested. All strains reacted with the VZV oligo.

[0431] Example 7: Clinical performance study of reagents specific for VZV analytes. The VZV clinical reactivity with 20 positive and 20 negative clinical specimens was examined. VZV in the stored clinical specimens was detected using a PCR preparation with the described primers and probes for VZV. 37.5 μM of VZV primers (SEQ ID NOs: 4 and 19) and 25 μM of VZV probe (SEQ ID NO: 11, 5'-Fluorescein, 3'BHQ1, all C modified with 5-Me-dC) were used in the reaction (PPR mixture 8). The test samples included 20 known VZV-positive and 20 known VZV-negative lesion swab specimens. A VZV plasmid of 50 cp / reaction was used as a positive control. The samples were processed with 300 μL of specimen and 468 μL of STM (1:1.56) using the cycles described in Table 7-1 and the PPR mixture described in Table 7-2.

Table 14

Table 15

Table 16-1

Table 16-2

Table 17-1

Table 17-2

Table 18-1

Table 18-2

Table 19

[0432] Conclusion: Primers and probes specific for VZV showed more than 90% clinical concordance with the comparative VZV assay. The negative concordance for 20 VZV-negative clinical specimens was 100.0%. The positive concordance for 20 VZV-positive clinical specimens was 100.0%. The VZV-specific oligomers detected VZV in all samples known to contain VZV and did not detect VZV in any samples known to lack VZV.

[0433] Example 8: Analysis of oligomer reactivity specific for VZV. The ability to amplify and detect different strains or isolates of VZV and VZV control plasmids was evaluated. 37.5 μM VZV primers (SEQ ID NOs: 4 and 19) and 25 μM VZV probe (SEQ ID NO: 11, 5'-Fluorescein, 3'BHQ1, all C modified with 5-Me-dC) were used in the reaction (PPR mixture 8). VZV virus was present in the reaction at 500 cp / reaction. VZV plasmid was present in the reaction at 158, 50, or 15.8 cp / reaction. The positive control plasmid was present in the reaction at 50 cp / reaction.

[0434] Samples were processed using the cycles described in Table 8-1 and the PPR mixtures described in Table 8-2.

Table 20

Table 21

Table 22

Table 23

Table 24

[0435] Conclusion: The VZV-specific oligomers can detect VZV plasmid DNA of less than 50 cp / rxn and VZV genomic DNA (VZV isolates and / or strains) of 500 cp / rxn. The detection rate was over 95%. The VZV-specific oligomers in multiplex reactions with control primers and probes can amplify and detect both VZV DNA and the control plasmid even in the presence of a large number of VZV-positive samples.

[0436] Example 9. Specificity and interference test of VZV-specific oligomers. The specificity of the VZV-specific oligomers was evaluated against 35 organisms commonly found in plasma and serum (specificity analysis). The ability of the VZV-specific oligomers to amplify and detect VZV in the presence of cross-reactants was also evaluated (interference analysis). 37.5 μM of VZV primers (SEQ ID NOs: 4 and 19) and 25 μM of VZV probe (SEQ ID NO: 11, 5'-Fluorescein, 3'-BHQ1, all C modified with 5-me-dC) were used in the reaction (PPR mixture 8).

[0437] Samples were processed using the cycles described in Table 9-1 and the PPR mixture described in Table 9-2.

Table 25

Table 26

Table 27

[0438] The specificity reaction contained 60 μL of panel stock and 540 μL of diluent.

[0439] The interference reaction contained 60 μL of panel stock, 60 μL of VZV, and 480 μL of diluent.

Table 28

Table 29

Table 30

Table 31

[0440] Conclusion: Primers and probes specific for VZV showed 100% specificity when tested against a panel of microorganisms commonly found in plasma, serum, and lesion swabs. When VZV was present at a concentration of 1.5 × 103 copies / mL or less in the presence of microorganisms commonly found in plasma, serum, and lesion swabs, the primers and probes specific for VZV also had a 100% VZV detection rate. The primers and probes specific for VZV are robust and specific for VZV EBNA1. The primers and probes specific for VZV can detect VZV in the presence of potential interfering organisms at 1500 cp / rxn without significantly affecting Ct or RFU.

[0441] Sequence In the following table, degenerate (mixed) positions (Y = C or T, R = A or G, W = A or T, S = G or C, K = G or T, M = A or C, etc.) are identified using the IUPAC nucleotide codes, and individual molecules in the composition or kit can have any of the nucleotides corresponding to the IUPAC code.

Table 32 - 1

Table 32 - 2

Table 32 - 3

Claims

1. 1. A composition for amplifying a Varicella Zoster Virus (VZV) target nucleic acid sequence, comprising: (a) a forward amplification primer, and (b) Reverse amplification primer Including, (i) the forward amplification primer comprises the nucleobase sequence of SEQ ID NO:6 and the reverse amplification primer comprises the nucleobase sequence of SEQ ID NO:21; or (ii) the forward amplification primer comprises the nucleobase sequence of SEQ ID NO:25, and the reverse amplification primer comprises the nucleobase sequence of SEQ ID NO:

35.

2. 10. The composition of claim 1, further comprising a detection probe for detecting the amplified Varicella-Zoster Virus (VZV) target nucleic acid sequence, said detection probe comprising at least one detectable label.

3. said detection probe comprises the nucleobase sequence of SEQ ID NO:14, said forward amplification primer comprises the nucleobase sequence of SEQ ID NO:6, and said reverse amplification primer comprises the nucleobase sequence of SEQ ID NO:21; or 3. The composition of claim 2, wherein the detection probe comprises the nucleobase sequence of SEQ ID NO:29 or 30, the forward amplification primer comprises the nucleobase sequence of SEQ ID NO:25, and the reverse amplification primer comprises the nucleobase sequence of SEQ ID NO:

35.

4. The composition of any one of claims 1 to 3, wherein the forward amplification primer, the reverse amplification primer, and / or the detection probe comprise at least one modified nucleotide.

5. 5. The composition of claim 4, wherein the modified nucleotide comprises a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, or a 5-methylcytosine.

6. The at least one detectable label is (a) a chemiluminescent label; (b) a fluorescent label; (c) a quencher, or (d) a combination of two or more of (a), (b), and (c).

7. The composition of claim 6 , wherein the at least one detectable label comprises the fluorescent label, the quencher, or both the fluorescent label and the quencher.

8. The composition of any one of claims 2 to 7, wherein the detection probe comprises a 5' non-target hybridizing sequence that base pairs with the 3' end of the detection probe, or a 3' non-target hybridizing sequence that base pairs with the 5' end of the detection probe.

9. The composition of claim 8 , wherein the detection probe comprises a molecular beacon or a molecular torch.

10. The composition of any one of claims 1 to 9, further comprising one or more of a buffer, a salt, a dNTP, a detergent, and an enzyme.

11. 11. The composition of claim 10, wherein the enzyme comprises a thermostable DNA polymerase, a reverse transcriptase, an RNA polymerase, or a combination of any two or more of a thermostable DNA polymerase, a reverse transcriptase, and an RNA polymerase.

12. The composition of any one of claims 1 to 11, wherein the amplification primers are in aqueous solution, frozen or lyophilized.

13. 13. The composition of claim 12, wherein the composition comprises two or more pairs of amplification primers and / or two or more detection probes, each pair of amplification primers consisting of a forward amplification primer and a reverse amplification primer.

14. The composition of claim 13, wherein the two or more pairs of amplification primers and / or the two or more detection probes amplify target nucleic acid sequences of the same or different organisms.

15. The composition of any one of claims 1 to 14, further comprising an internal standard target nucleic acid sequence, an oligomer for amplifying and / or detecting the internal standard target nucleic acid sequence, or a combination thereof.

16. A detection probe for detecting a VZV target nucleic acid sequence, comprising: (a) the detection probe is for use in combination with a forward amplification primer comprising the nucleobase sequence of SEQ ID NO:6 and a reverse amplification primer comprising the nucleobase sequence of SEQ ID NO:21, wherein the detection probe comprises an oligonucleotide comprising the nucleobase sequence of SEQ ID NO:14, comprises one or more detectable labels, and hybridizes to a VZV target nucleic acid sequence amplified by the amplification primers of SEQ ID NO:6 and SEQ ID NO:21; or (b) the detection probe is for use in combination with a forward amplification primer comprising the nucleobase sequence of SEQ ID NO:25 and a reverse amplification primer comprising the nucleobase sequence of SEQ ID NO:35, the detection probe comprising an oligonucleotide comprising the nucleobase sequence of SEQ ID NO:29 or 30, comprising one or more detectable labels, and hybridizing to a VZV target nucleic acid sequence amplified by the amplification primers of SEQ ID NO:25 and SEQ ID NO:

35.

17. 17. The detection probe of claim 16, wherein the detection probe comprises at least one modified nucleotide.

18. 18. The detection probe of claim 17, wherein the modified nucleotides comprise 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, or 5-methylcytosine.

19. One or more of the detectable labels is (a) a chemiluminescent label; (b) a fluorescent label; (c) a quencher, or (d) a combination of two or more of (a), (b), and (c).

20. 20. The detection probe of claim 19, wherein one or more of the detectable labels comprises the fluorescent label, the quencher, or both the fluorescent label and the quencher.

21. A detection probe according to any one of claims 16 to 20, wherein the detection probe comprises a 5' non-target hybridizing sequence that is base-paired to the 3' end of the detection probe, or a 3' non-target hybridizing sequence that is base-paired to the 5' end of the detection probe.

22. 22. The detection probe of claim 21, wherein the detection probe comprises a molecular beacon or a molecular torch.

23. 1. A method for amplifying a VZV target nucleic acid sequence, comprising: (a) obtaining a sample containing or suspected of containing a VZV target nucleic acid sequence; (b) contacting said sample with a composition according to any one of claims 1 to 15; (c) providing conditions sufficient to amplify the target nucleic acid sequence, thereby producing an amplification product of the VZV target nucleic acid sequence, if the VZV target nucleic acid sequence is present in the sample; The method includes:

24. The method according to claim 23, wherein the method further comprises the step of contacting the sample with a detection probe according to any one of claims 16 to 21 to determine the presence or absence of the amplification product.

25. 1. A method for determining the presence or absence of VZV in a sample, comprising: (a) obtaining a sample containing or suspected of containing a VZV target nucleic acid sequence; (b) contacting said sample with a composition according to any one of claims 1 to 15; (c) generating an amplification product by providing conditions sufficient to amplify the target nucleic acid sequence; (d) detecting the presence or absence of the amplification product; The method includes:

26. A kit for amplifying and detecting a Varicella-Zoster Virus (VZV) target nucleic acid sequence, comprising: (a) a forward amplification primer comprising the nucleobase sequence of SEQ ID NO:6, a reverse amplification primer comprising the nucleobase sequence of SEQ ID NO:21, and a detection probe comprising the nucleobase sequence of SEQ ID NO:14 and one or more detectable labels; or (b) a forward amplification primer comprising the nucleobase sequence of SEQ ID NO:25, a reverse amplification primer comprising the nucleobase sequence of SEQ ID NO:35, and a detection probe comprising the nucleobase sequence of SEQ ID NO:29 or 30 and one or more detectable labels. Including the kit.

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