Compositions and methods for detecting and analyzing herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), and varicella zoster virus (VZV)

Abstract

Provided herein are compositions and methods useful for the detection of herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), and varicella zoster virus (VZV). In particular, provided herein are compositions, methods, systems, kits, reagents and reaction mixtures related thereto for nucleic acid amplification and detection procedures for detecting HSV-1, HSV-2, and VZV in a sample.

Description

[0001] Sequence Listing

[0002] Having a file size of 19,615 bytes, created on November 3, 2023, and entitled "40733-601_SEQUENCE_LISTING," the text of the computer-readable sequence listing submitted herewith is hereby incorporated by reference in its entirety. Field of the Invention

[0003] Provided herein are compositions and methods useful for detecting herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), and varicella-zoster virus (VZV). Specifically, provided herein are compositions, methods, systems, kits, reagents, and reaction mixtures related thereto for nucleic acid amplification and detection procedures for detecting HSV-1, HSV-2, and VZV in a sample. Background of the Invention

[0005] Human herpesvirus is divided into α, β and γ subtypes. Alpha herpesviridae (Herpesviridae) includes herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2) and varicella zoster virus (VZV). HSV-1 and HSV-2 are large double-stranded DNA viruses and share similar genomes, with about 80% homology of their protein coding regions. HSV-1, HSV-2 and VZV cause various diseases, depending on the anatomical site where the infection begins, the immune status of the host and whether the infection is primary or recurrent. When primary infection occurs, HSV enters through the skin or mucous membrane, infects epithelial cells and begins to replicate. After infection, the virus may become latent in local sensory ganglia, periodically reactivating to cause symptomatic lesions. HSV-1 is the causative agent of " herpes labialis" (i.e., vesicular lesions of the oral mucosa), and can also be transmitted to the genitals by oral-genital contact and cause clinical diseases in the genitals. HSV-2 is sexually transmitted, with lesions primarily confined to the anogenital anatomical region. Primary VZV infection most commonly occurs during childhood and causes varicella (chickenpox), as well as a latent infection in sensory neurons along the neural axis in the dorsal root ganglia (DRG). When virus-specific cellular immunity weakens during aging or due to immunosuppression, reactivation of the latent infection causes herpes zoster (HZ) due to replication of VZV in one or more DRGs.

[0006] Accordingly, there is a need for rapid and accurate diagnostic tests that can detect, identify, and differentiate HSV-1, HSV-2, and VZV from one another. SUMMARY OF THE INVENTION

[0008] Provided herein are compositions and methods useful for detecting herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), and varicella-zoster virus (VZV). Specifically, provided herein are compositions, methods, systems, kits, reagents, and reaction mixtures related thereto for nucleic acid amplification and detection procedures for detecting HSV-1, HSV-2, and VZV in a sample.

[0009] Such compositions and methods include primers, probes, primer sets, primer and probe sets, and methods for detecting HSV-1, HSV-2, and VZV complex in various human samples, including, for example, skin and mucocutaneous lesion swab samples, cerebrospinal fluid, plasma, and serum.

[0010] In some embodiments, two or more polynucleotide reagents provided herein as SEQ ID NOs: 1-21 are combined in a composition (e.g., a reagent set, a kit, a reaction mixture, a system, etc.). In some embodiments, one or more or all nucleic acid reagents comprise a detectable portion (e.g., a synthetic label). In some embodiments, the composition comprises one or more primer pairs of SEQ ID NOs: 1 and 2, 4 and 5, 7 and 8, 10 and 11, 13 and 14, 16 and 17, and 19 and 20. In some embodiments, the composition comprises one or more probes (e.g., labeled probes) of SEQ ID NOs: 3, 6, 9, 12, 15, 18, and 21. In some embodiments, the composition comprises a primer and probe set: SEQ ID NOs: 1-2 and 3, 4-5 and 6, or 7-8 and 9, 10-11 and 12, 13-14 and 15, 16-17 and 18, and / or 19-20 and 21. In some embodiments, the composition comprises an internal control reagent, e.g., SEQ ID NOs: 19-21. In some embodiments, the composition includes a multi-probe system comprising SEQ ID NOs: 3, 6, 9, 12, 15, 18, and 21.

[0011] In some embodiments, the compositions and methods of the present invention employ a reagent set comprising a polynucleotide component having a primer, a probe, a primer set, and / or a probe set. In some embodiments, the polynucleotide component of the composition is composed of the above-mentioned primers, probes, primer sets, or probe set combinations. As a reaction mixture, the composition may be composed of such polynucleotides as well as any polynucleotides included in the sample (i.e., the only non-sample nucleic acid molecule is a polynucleotide represented by SEQ ID NO: 1-21, individually or in combination (e.g., the above-mentioned combination)).

[0012] The primer sets provided herein comprise two primers and can be used for target sequence amplification in, for example, polymerase chain reaction (PCR) amplification. In some embodiments, the compositions comprise at least two primers and one or more (e.g., two or more) probes that detect the amplicon generated by the primers.

[0013] Also provided herein are methods for detecting HSV-1, HSV-2, and VZV in a sample. In some embodiments, the method comprises: (a) forming a reaction mixture comprising a nucleic acid amplification reagent, at least one polynucleotide primer or probe described herein, and a test sample that may contain at least one target sequence; and (b) subjecting the mixture to amplification conditions to generate at least one copy of a nucleic acid sequence that is complementary to the target sequence. In some embodiments, the method further comprises detecting the generated amplicon. In some embodiments, the detection comprises (c) hybridizing the probe to a nucleic acid sequence that is complementary to the target sequence to form a hybrid comprising the probe and the nucleic acid sequence that is complementary to the target sequence; and (d) detecting the hybrid directly or indirectly as an indication of the presence of HSV-1, HSV-2, and VZV in the test sample.

[0014] Furthermore, when the amplification is PCR or a similar thermal cycling amplification process, step (b) can be repeated multiple times to increase the copy number of the target sequence.

[0015] According to another embodiment, HSV-1, HSV-2 and VZV and one or more additional infectious agents (e.g., HIV) or other nucleic acid molecules (e.g., human sequences) are detected. Accordingly, in some embodiments, the composition comprises reagents for detecting such other agents or nucleic acid molecules.

[0016] In some embodiments, the compositions and methods further employ control reagents and / or kit components (e.g., positive controls and / or negative controls). In some embodiments, the control reagents comprise synthetic target nucleic acids. In some embodiments, the control reagents comprise reagents for detecting HSV-1, HSV-2, and VZV, human, or other sequences expected to be present in the sample. In some embodiments, the control target nucleic acids (whether synthetic or endogenous to the sample) are selected so that the amplification primers that amplify the HSV-1, HSV-2, and VZV target nucleic acids also amplify the control target nucleic acids. In some such embodiments, the probes that detect the HSV-1, HSV-2, and VZV target nucleic acids, or the amplicons generated thereby, do not detect the control targets or the amplicons generated thereby. In some embodiments, control probes are provided that detect the control target nucleic acids or the amplicons generated thereby, but do not detect the HSV-1, HSV-2, and VZV target nucleic acids, or the amplicons generated thereby. In some embodiments, internal standards and calibrators for quantification are provided.

[0017] In some embodiments, in addition to the reagents discussed above, the test kit also includes one or more suitable containers, instructions for use, software (e.g., data analysis software), instructions, etc. In some embodiments, the test kit includes a reagent for labeling polynucleotides. In some embodiments, one or more components in the test kit are in lyophilized form. In other embodiments, one or more or all reagents of the present invention are in liquid form.

[0018] Embodiments of the present disclosure provide compositions, kits, systems, and methods for identifying HSV-1, HSV-2, and VZV in complex biological samples, such as skin samples, mucosal samples, surface swab samples, cerebrospinal fluid samples, plasma samples, oral samples, lesion samples, biopsy samples, anogenital samples, and / or blood samples. In some embodiments, the compositions and methods provide one or more single probes for real-time detection methods that can specifically and accurately separate and identify HSV-1, HSV-2, and VZV.

[0019] For example, in some embodiments, the present disclosure provides a composition comprising at least one (e.g., one, two, or three) primer pair selected from SEQ ID NOs: 1 and 2, 4 and 5, 7 and 8, 10 and 11, 13 and 14, 16 and 17, and 19 and 20. In some embodiments, the composition further comprises at least one probe selected from SEQ ID NOs: 3, 6, 9, 12, 15, 18, and 21.

[0020] Additional embodiments provide compositions comprising each of the nucleic acids in SEQ ID NOs: 1-21.

[0021] Embodiments of the present disclosure provide kits comprising: a) any one of the compositions described above, and b) at least one reagent for performing a nucleic acid amplification reaction (eg, a nucleic acid polymerase; a plurality of dNTPs and / or a buffer).

[0022] In other embodiments, the present disclosure provides a reaction mixture comprising any of the above compositions or a nucleic acid that hybridizes to HSV-1, HSV-2, and VZV nucleic acids. In some embodiments, the HSV-1, HSV-2, and VZV target nucleic acid is one or more of HSV-1 US-6, HSV-1 UL-1, HSV-2 UL-1, HSV-2 UL-18, VZV ORF10, and / or VZV ORF21.

[0023] In further embodiments, the present disclosure provides methods for identifying HSV-1, HSV-2, and VZV nucleic acids in a biological sample, comprising: a) contacting a biological sample from a subject with any of the nucleic acid primers or probes described above, and b) directly or indirectly detecting binding of the nucleic acid primers or probes to HSV-1, HSV-2, and VZV nucleic acids. In some embodiments, the method further comprises the step of c) determining the presence of HSV-1, HSV-2, and VZV in the sample when binding is detected. In some embodiments, detection is via real-time PCR, also known as quantitative PCR (qPCR).

[0024] Still other embodiments provide methods for detecting HSV-1, HSV-2, and VZV nucleic acids in a biological sample, comprising: a) extracting DNA from the sample; b) contacting the DNA with one or more primer pairs and one or more nucleic acid probes; c) performing an amplification assay to amplify one or more HSV-1, HSV-2, and VZV nucleic acid targets; and d) identifying the presence of the target in the sample.

[0025] Further embodiments provide methods for detecting HSV-1, HSV-2 and / or VZV nucleic acids in a biological sample, comprising: a) extracting DNA from the sample; b) contacting the DNA with one or more primer pairs selected from SEQ ID NOs: 1 and 2, 4 and 5, 7 and 8, 10 and 11, 13 and 14, 16 and 17, and 19 and 20; and one or more nucleic acid probes selected from SEQ ID NOs: 3, 6, 9, 12, 15, 18 and 21; c) performing an amplification assay to amplify one or more HSV-1, HSV-2 and VZV nucleic acid targets; and d) identifying the presence of the targets in the sample.

[0026] Further embodiments provide methods for detecting HSV-1, HSV-2 and / or VZV nucleic acids in a biological sample, comprising: a) extracting DNA from the sample; b) contacting the DNA with one or more primer pairs selected from SEQ ID NOs: 1 and 2, 4 and 5, 7 and 8, 10 and 11, 13 and 14, 16 and 17, and 19 and 20; and one or more nucleic acid probes selected from SEQ ID NOs: 3, 6, 9, 12, 15, 18 and 21; c) performing a real-time polymerase chain reaction assay to amplify one or more HSV-1, HSV-2 and VZV nucleic acid targets; and d) identifying the presence of the targets in the sample.

[0027] In some embodiments, the methods, compositions, kits, reaction mixtures, and systems of the present invention provide automated multiplex assays for the qualitative detection and differentiation of HSV-1, HSV-2, and VZV from skin and mucocutaneous lesion specimens. In some embodiments, the present invention aids in the diagnosis and care of individuals with HSV-1, HSV-2, and VZV infections.

[0028] Additional embodiments are described herein. Detailed Description of the Invention

[0030] Provided herein are compositions and methods useful for detecting herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), and varicella-zoster virus (VZV). Specifically, provided herein are compositions, methods, systems, kits, reagents, and reaction mixtures related thereto for nucleic acid amplification and detection procedures for detecting HSV-1, HSV-2, and VZV in a sample.

[0031] In some embodiments, the present invention provides two sets of primers and probes for detecting HSV-1, two sets of primers and probes for detecting HSV-2, and two sets of primers and probes for detecting VZV. In addition to the primers and probes, the present invention also includes diagnostic methods and assay formats that use the primers and probes mentioned above to detect VZV using, for example, Alinity The real-time PCR technology on the system enables specific and sensitive detection of HSV-1, HSV-2, and VZV.In some embodiments, the present invention further provides internal controls that serve as validity controls for sample processing and amplification efficiency.

[0032] Multiple strains of HSV-1, HSV-2, and VZV are prevalent in the population, and the genomic sequences of different strains may exhibit genetic variability. Such natural polymorphisms within the primer-probe binding site can result in invalid hybridization and a lack of detection for nucleic acid single-plex testing methods based on PCR technology. In order to ensure assay robustness, the HSV 1&2 and VZV assays of the present invention are configured to target two conserved sequences within the respective viral genomes, i.e., dual target testing for each target virus. In some embodiments, for HSV-1, the gene target is glycoprotein D (US6) + unique long region (Unique Long Region) (UL1), for HSV-2, the gene target is UL1 + UL18, and for VZV, the gene target is open reading frame (ORF) 10 + ORF 21.

[0033] In some embodiments, provided herein are polynucleotides that specifically hybridize to nucleic acid sequences of HSV-1, HSV-2, and VZV, or their complements. These polynucleotides can be used to amplify HSV-1, HSV-2, and VZV (if present) in a sample and specifically detect the presence of HSV-1, HSV-2, and VZV. Exemplary polynucleotides are described, for example, by SEQ ID NOs: 1-18.

[0034] In some embodiments, the assays described herein utilize multiple (e.g., two) different sets of primers / probes specific for HSV-1, HSV-2, and VZV. For example, in some embodiments, a first set is designed to detect one gene, while a second set is designed to detect another gene. Experiments described herein demonstrate that the dual-target strategy of the present invention results in the detection of HSV-1, HSV-2, and VZV genomic DNA with high reliability.

[0035] Embodiments of the technology described herein provide high-throughput, automated detection of HSV-1, HSV-2, and VZV with high sensitivity and specificity.

[0036] As used herein, the term "specific hybridization" refers to the ability of a nucleic acid to detectably and specifically bind to a second nucleic acid. Under hybridization and wash conditions, a polynucleotide specifically hybridizes to a target nucleic acid strand while minimizing detectable binding to nonspecific nucleic acids. Stringent conditions that can be used to achieve specific hybridization are known in the art.

[0037] As used herein, "target sequence" or "target nucleic acid sequence" means a nucleic acid sequence of HSV-1, HSV-2, and VZV, or other sequences to be detected (e.g., HIV), or their complements, which are amplified, detected, or both amplified and detected using one or more polynucleotides provided herein. In addition, although the term target sequence sometimes refers to a double-stranded nucleic acid sequence, those skilled in the art will recognize that the target sequence can also be single-stranded. In cases where the target is double-stranded, the polynucleotide primer sequences preferably amplify both strands of the target sequence. It is possible to select a target sequence that is more or less specific for a particular organism. For example, the target sequence may be specific for an entire genus, more than one genus, species or subspecies, serogroup, auxotype, serotype, strain, isolate, or other subset of organisms.

[0038] As used herein, the term "test sample" means a sample obtained from an organism, a biological fluid, an environmental sample, or other sample suspected of containing or potentially containing HSV-1, HSV-2, and VZV target sequences. The test sample can be obtained from any biological source, such as tissue, blood, saliva, sputum, mucus, bronchial sweat, urine, urethral swab, cervical swab, urogenital or anal swab, lesion swab, conjunctival swab, lens fluid, cerebrospinal fluid, milk, ascites, synovial fluid, peritoneal fluid, amniotic fluid, fermentation broth, cell culture, tissue biopsy, chemical reaction mixture, and the like. The test sample can be used (i) directly as obtained from the source, or (ii) after pretreatment to modify the properties of the sample. Thus, the test sample can be pretreated prior to use by, for example, preparing plasma or serum from blood, disrupting cells or viral particles, preparing a liquid from a solid material, diluting a viscous liquid, filtering a liquid, distilling a liquid, concentrating a liquid, inactivating interfering components, adding reagents, purifying nucleic acids, and the like.

[0039] As used herein, the term "label" means a molecule or part having a characteristic or property that allows detection and optionally quantification. The label can be directly detectable, such as using, for example, but not limited to, radioisotopes, fluorophores, chemiluminophores, enzymes, colloidal particles, fluorescent microparticles, etc.; or the label may be indirectly detectable, such as using, for example, specific binding members. It should be understood that directly detectable labels may require additional components, such as substrates, triggering reagents, quenching moieties, light, etc., to allow detection and / or quantification of the label. When using indirectly detectable labels, they are generally used in combination with "conjugates". Conjugates are typically specific binding members that have been attached or coupled to a directly detectable label. The coupling chemistry used to synthesize conjugates is well known in the art and can include, for example, any chemical means and / or physical means that do not destroy the specific binding properties of the specific binding members or the detectable properties of the label. As used herein, "specific binding members" means a member of a pair of binding pairs, such as two different molecules in which one of the molecules specifically binds to another molecule by, for example, chemical or physical means. In addition to antigen and antibody specific binding pairs, other specific binding pairs include, but are not limited to, avidin and biotin; a hapten and an antibody specific for the hapten; complementary nucleotide sequences; an enzyme cofactor or substrate and an enzyme; and the like.

[0040] Polynucleotides are nucleic acid polymers of ribonucleic acids (RNA), deoxyribonucleic acids (DNA), modified RNA or DNA, RNA or DNA mimics (such as, but not limited to, PNA), and derivatives thereof and their homologs. Thus, polynucleotides include polymers composed of naturally occurring nucleobases, sugars, and covalent internucleoside (backbone) linkages, as well as polymers with non-naturally occurring portions that function similarly. Such modified or substituted nucleic acid polymers are well known in the art and, for purposes of the present invention, are referred to as "analogs." For ease of preparation and familiarity to the skilled artisan, polynucleotides are preferably modified or unmodified polymers of deoxyribonucleic acids or ribonucleic acids.

[0041] Available polynucleotide analogs include the polymer of the main chain or non-natural internucleoside bonding with modification.The main chain of modification is included in those main chains retaining phosphorus atoms in the main chain, such as phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methylphosphonate and other alkylphosphonates, and those main chains no longer having phosphorus atoms, such as by bonding between short chain alkyl or cycloalkyl nucleosides, mixing heteroatoms and alkyl or cycloalkyl nucleosides bonding or one or more short chain heteroatoms or heterocyclic nucleosides bonding forming main chain.The example of this type of main chain without phosphorus is morpholino bonding (see, for example, U.S. Patent number 5,185,444,5,034,506 and 5,142,047, described patents are all incorporated herein by reference).The nucleic acid polymer (analog) of modification may contain the sugar moiety of one or more modifications. For example, it is possible to modify the sugar moiety by substitution at the 2' position with a 2-methoxyethoxy (2-MOE) group (see, eg, Martin et al., (1995) Helv. Chim. Acta, 78:486-504).

[0042] Embodiment also contemplates analogs that are RNA or DNA mimics, in which the sugar and internucleoside linkages of the nucleotide units are replaced by novel groups. In these mimetics, the base unit is maintained for hybridization with the target sequence. The example of such mimetics that has been shown to have excellent hybridization properties is peptide nucleic acid (PNA) (Nielsen et al., (1991) Science, 254:1497-1500, incorporated herein by reference; International Patent Application WO 92 / 20702). In PNA compounds, the sugar backbone of the oligonucleotide is replaced by a main chain containing amides, such as an aminoethylglycine main chain. The core base is retained and is directly or indirectly combined with the aza-nitrogen atom of the amide portion of the main chain.

[0043] Contemplated polynucleotides may further include derivatives in which the nucleic acid molecule has been covalently modified by substitution, chemical, enzymatic or other appropriate means with moieties other than naturally occurring nucleotides, for example, moieties that serve as labels as described herein.

[0044] The present invention further comprises homologs of the polynucleotides having the nucleic acid sequences shown in SEQ ID NOs: 1-21. Homologs are nucleic acids having at least one alteration in the primary sequence shown in any one of SEQ ID NOs: 1-21, wherein the at least one alteration does not destroy the ability of the polynucleotide to specifically hybridize to the target sequence, as described above. Accordingly, the primary sequence can be altered, for example, by insertion, addition, deletion, or substitution of one or more nucleotides, such as in SEQ ID NOs: 1-21. Thus, homologs that are fragments of the sequences disclosed in SEQ ID NOs: 1-21 may have a continuous sequence of at least about 7, 10, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or more nucleotides of the nucleic acid sequences of SEQ ID NOs: 1-21 and will retain the ability to specifically hybridize to the target sequence, as described above. Generally, homologues will have a nucleotide sequence with at least about 50%, 60%, 70%, 80%, 85%, 90% or 95% nucleotide sequence identity to the nucleotide sequence shown in SEQ ID NO:1-21. Identity about this type of sequence is defined herein as the percentage of the nucleotides identical to known polynucleotides in the candidate sequence after comparing sequences and, if necessary, introducing room to reach maximum percentage identity. The end (5' or 3') or internal deletion, extension or insertion of the nucleotide sequence should not be construed as affecting identity.

[0045] In some embodiments, the polynucleotides comprise primers and probes that specifically hybridize to HSV-1, HSV-2, and VZV target sequences, for example, nucleic acid molecules having any one of the nucleic acid sequences set forth in SEQ ID NOs: 1-21, including analogs and / or derivatives of such nucleic acid sequences and homologs thereof, which can specifically hybridize to HSV-1, HSV-2, and VZV target sequences. As described below, the polynucleotides can be used as primers and / or probes for amplifying or detecting HSV-1, HSV-2, and VZV.

[0046] Polynucleotides can be prepared by various techniques. For example, solid phase synthesis can be used using commercially available equipment, such as those available from Applied Biosystems USA Inc. (Foster City, Calif.), DuPont (Wilmington, Del.), or Milligen (Bedford, Mass.). Modified polynucleotides, such as phosphorothioate and alkylated derivatives (see, for example, U.S. Patent Nos. 5,464,746; 5,424,414; and 4,948,882), can also be readily prepared.

[0047] Polynucleotides can be used directly as probes for the detection or quantification of HSV-1, HSV-2, and VZV nucleic acids in a test sample, or both. Under suitable hybridization conditions, the test sample is contacted with at least one polynucleotide, and hybridization between the target sequence and the at least one polynucleotide is detected. Detection can be direct or indirect. In some embodiments, hybrids are detected directly between the probe and the target. In some embodiments, hybrids are detected indirectly, for example, by detecting reaction byproducts generated by an enzymatic reaction that occurs in the presence of a duplex between the probe and the HSV-1, HSV-2, and VZV target.

[0048] The polynucleotide may incorporate one or more detectable labels. A detectable label is a molecule or moiety having a characteristic or property that can be detected directly or indirectly and is selected so that the ability of the polynucleotide to hybridize to its target sequence is not adversely affected.

[0049] Detection label has the same definition as previously defined "label", and "capture label" is generally used to separate extension products and probes associated with such products from other amplification reactions. Specific binding members (as previously defined) are very suitable for this purpose. In addition, the probes used according to this method may be blocked at their 3' ends so that they do not extend under hybridization conditions. Methods for preventing probe extension are well known and are a matter of choice for those skilled in the art.

[0050] In cases where labeling is used to detect primer amplification products, the primer sequence can optionally be labeled with a capture label or a detection label. In some embodiments, the primer comprises a 3' portion that hybridizes to HSV-1, HSV-2, and VZV target nucleic acids, and a 5' portion that introduces non-HSV-1, HSV-2, and VZV sequences into the extension products generated thereby. Such a 5' portion may include a synthetic tag sequence for use, for example, in next-generation sequencing technologies such as Illumina, PacBio, ONT, and the like.

[0051] In some embodiments, the probe is used to hybridize with the extension product or amplicon generated by the primer sequence, and is typically hybridized with a sequence that does not include the primer sequence. Similar to the primer sequence, the probe sequence can also be marked with a capture label or a detection label, but it should be noted that in some embodiments, when the primer is marked with a capture label, the probe is marked with a detection label, and vice versa. After forming a copy sequence / probe hybrid, the differential labeling on the copy sequence and the probe sequence (i.e., capture label and detection label) can be used to separate and detect such hybrids.

[0052] Polynucleotides are also suitable for use as capture probes in sandwich assays. The polynucleotide capture probe is attached to a solid support and contacted with a test sample under suitable hybridization conditions such that a probe: target hybrid is formed between the capture probe and any target nucleic acid present in the test sample. After one or more appropriate washing steps, the probe: target hybrid is typically detected with the aid of a second "disclosure" probe or a specific antibody that recognizes the hybrid molecule.

[0053] Embodiment also contemplates the use of polynucleotides in the nucleic acid hybridization assay of modification. For example, U.S. Patent number 5,627,030 discloses a method for amplifying detection signals in nucleic acid hybridization assays. In the disclosed assay, a first polynucleotide probe sequence is hybridized with a target sequence under suitable conditions, and the probe: target hybrid is then immunocaptured and fixed. A second polynucleotide probe containing a plurality of repeat units is then hybridized with the probe: target hybrid component. Detection is achieved by hybridization of a plurality of labeled nucleic acid sequence probes, and each probe hybridizes with each repeat unit present in the second probe. The attachment of the multi-labeled probe to the second probe therefore amplifies the detection signal and increases the sensitivity of the assay.

[0054] Amplification and detection of HSV-1, HSV-2, and VZV nucleotide sequences

[0055] Polynucleotides can be used as primers or probes to amplify and / or detect HSV-1, HSV-2, and VZV in a test sample. The primer / probe sets provided herein comprise at least two primers and at least one probe. These primer / probe sets can be employed according to nucleic acid amplification techniques. Thus, the primers in any particular primer / probe set can be used to amplify a target sequence. In most cases, the probe hybridizes to a copy of the target sequence generated by one or more primers and generally facilitates the detection of any copy of the target sequence generated during the course of the amplification reaction. All primer / probe sets can be employed according to nucleic acid amplification procedures to specifically and sensitively detect HSV-1, HSV-2, and VZV when appropriate primers and probes are combined. It is contemplated that the individual primers and probes in the primer / probe sets provided herein may be used alternatively in combination with primers and / or probes other than those described in the primer / probe sets provided herein. In some embodiments, two primer and probe sets are used to detect two different HSV-1, HSV-2, and VZV target sequences.

[0056] Amplification procedures include, but are not limited to, polymerase chain reaction (PCR), TMA, rolling circle amplification, nucleic acid sequence-based amplification (NASBA), and strand displacement amplification (SDA). Those skilled in the art will appreciate that primers may need to be modified for use in certain amplification techniques, e.g., for SDA, the primer includes additional nucleotides near its 5' end that constitute a recognition site for a restriction endonuclease. Similarly, for NASBA, the primer includes additional nucleotides near its 5' end that constitute an RNA polymerase promoter.

[0057] In some embodiments, certain criteria are considered when selecting primers for an amplification reaction. For example, when a primer pair is required for an amplification reaction, the primers should be selected so that the probability of forming a 3' duplex is minimized and the melting temperature (T M ) are sufficiently similar to optimize annealing to the target sequence and minimize the amount of nonspecific annealing.

[0058] In some embodiments, the amplification method comprises: (a) forming a reaction mixture comprising a nucleic acid amplification reagent, at least one primer / probe set, and a test sample suspected of containing at least one target sequence, and (b) subjecting the mixture to amplification conditions to generate at least one copy of a nucleic acid sequence complementary to the target sequence. Step (b) of the above method can be repeated any suitable number of times (before step (c) in the detection method), for example, by thermally cycling the reaction mixture 10 to 100 times, typically about 20 to 60 times, more typically about 25 to 45 times.

[0059] Nucleic acid amplification reagents include, but are not limited to, an enzyme having at least polymerase activity, an enzyme cofactor such as magnesium or manganese; a salt; nicotinamide adenine dinucleotide (NAD); and deoxynucleotide triphosphates (dNTPs), such as deoxyadenine triphosphate, deoxyguanine triphosphate, deoxycytosine triphosphate, and deoxythymine triphosphate.

[0060] Amplification conditions are conditions that generally promote annealing and extension of one or more nucleic acid sequences.

[0061] As mentioned above, can detect by the whole bag of tricks the specific amplicon that uses the polynucleotide amplification target nucleic acid sequence to produce.For example, one or more primers that use in the amplified reaction may carry out mark like this, make and can directly detect amplicon by conventional technique after the amplified reaction.Alternately, can add the probe that forms by the mark of one of the primer that uses in the amplified reaction or the 3rd polynucleotide different from primer sequence after amplified reaction is finished, said 3rd polynucleotide has carried out mark and with the regional complementation of amplified sequence.Then mixture is submitted to suitable hybridization and washing conditions, and detects mark by conventional method.

[0062] The amplification product produced as above can be detected during or after the amplification process of the target sequence. Methods for detecting the amplification of the target sequence in amplification (e.g., real-time PCR) processes are outlined above and are, for example, described in U.S. Patent number 5,210,015. Alternatively, the amplification product is hybridized with a probe, then separated from the other reaction components and detected using microparticles and labeled probes.

[0063] It is easy to understand that it will be advantageous to allow the amplification and detection of target nucleic acid sequences to occur simultaneously in a single, unopened reaction vessel. Such programs avoid the risk of "carry-over" contamination in the post-amplification processing step, and also promote the adaptation of high-throughput screening or determination and program automation. In addition, such programs allow "real-time" monitoring and "end-point" monitoring of amplified reactions. Examples of probe molecules particularly well suited for such programs include molecular beacon probes and TAQMAN probes. TAQMAN probes are dual-labeled fluorescent nucleic acid probes generally consisting of polynucleotides complementary to the target sequence, which are labeled with a fluorophore at the 5' end and labeled with a quencher at the 3' end. In free probes, the close proximity of fluorophore and quencher ensures that the fluorophore is internally quenched. During the extension phase of the amplified reaction, the probe is cut by the 5' nuclease activity of the polymerase and the fluorophore is released. The released fluorophore can then emit fluorescence and therefore produce a detectable signal.

[0064] In some embodiments, a "molecular beacon" probe is employed. Molecular beacon probes are described, for example, in U.S. Patent Nos. 6,150,097; 5,925,517 and 6,103,476 (incorporated herein by reference in their entirety). A molecular beacon is a polynucleotide probe capable of forming a stem-loop (hairpin) structure. The loop is a single-stranded structure containing a sequence complementary to the target sequence, while the stem is generally unrelated to the target sequence and self-hybridizes to form a double-stranded region. Nucleotides that are both complementary to the target sequence and can self-hybridize may also constitute part of the stem region. Attached to one arm of the stem is a fluorophore portion and attached to the other arm is a quencher portion. When the polynucleotide takes a hairpin shape, the fluorophore and quencher are in close proximity, and the energy emitted by the fluorophore is therefore absorbed by the quencher and released as heat, resulting in internal quenching of the fluorophore. After the polynucleotide binds to its target sequence, the fluorophore and quencher become spatially separated and the fluorophore can emit fluorescence, generating a detectable signal.

[0065] Examples of fluorophores that can be used in the present invention include, but are not limited to, fluorescein and fluorescein derivatives such as dihalo-(C1 to C8) dialkoxycarboxyfluorescein, 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), coumarin and coumarin derivatives, Lucifer yellow, Texas Red, tetramethylrhodamine, tetrachloro-6-carboxyfluorescein, 5-carboxyrhodamine, cyanine dyes, etc. Quenchers include, but are not limited to, DABCYL, 4'-(4-dimethylaminophenylazo)benzoic acid (DABSYL), 4-dimethylaminophenylazophenyl-4'-maleimide (DABMI), tetramethylrhodamine, carboxytetramethylrhodamine (TAMRA), Black Hole Quencher (BHQ) dyes, and the like.

[0066] In some embodiments, a quantitative assay is employed. In some such embodiments, an internal standard is employed in the reaction. Such internal standards generally comprise a control target nucleic acid sequence and a control polynucleotide probe. The internal standard may optionally further comprise another pair of primers. The primary sequences of these control primers may be unrelated to HSV-1, HSV-2, and VZV polynucleotides and are specific for the control target nucleic acid sequence. Alternatively, if the control target sequence is designed so that it binds to HSV-1, HSV-2, and VZV primers, no additional primers are required. The amount of target nucleic acid in the test sample can be quantified using an "endpoint" method or a "real-time" method.

[0067] In some embodiments, HSV-1, HSV-2 and VZV detection assays are provided as high-throughput assays. For high-throughput assays, reaction components are typically contained in multi-container carriers or platforms, such as multi-well microtiter plates, which allow monitoring of multiple assay reactions containing different test samples in the same assay. In some embodiments, a highly automated high-throughput assay is employed to increase the efficiency of the screening or assay process. Many high-throughput screening or assay systems are currently commercially available, as are the automation capabilities of many programs, such as sample and reagent pipetting, liquid distribution, timed incubation, sample formatting into microarrays, microplate thermal cycling, and microplate readings in appropriate detectors, resulting in much faster throughput times. In some embodiments, the reaction is performed in a microfluidic device (e.g., card).

[0068] The polynucleotides, methods, and kits can be used in clinical or research settings for detecting and / or quantifying HSV-1, HSV-2, and VZV nucleic acids. Thus, in these settings, the polynucleotides can be used in assays to diagnose HSV-1, HSV-2, and VZV infection in a subject, or to monitor the amount of HSV-1, HSV-2, and VZV target nucleic acid sequences in a subject infected with HSV-1, HSV-2, and VZV. Monitoring the amount of virus in a subject is particularly important in identifying or monitoring the response to antiviral therapy.

[0069] In some embodiments, the assay is compatible with automated real-time PCR detection systems, such as the Abbott m2000 system or the Abbott Alinity System. Therefore, in some embodiments, before measuring, prepare the sample for use with this type of system. For example, in some embodiments, the preparation of target DNA is performed using a technology based on magnetic particles (AbbottmSample Preparation SystemDNA). This can be performed using the Abbott m2000sp for automated sample preparation or using a manual sample preparation protocol. In some embodiments, internal controls (IC), positive controls, and negative controls are processed from sample preparation to confirm that the process has been correctly performed.

[0070] For amplification, in some embodiments, purified sample DNA and master mix are added to a 96-well PCR plate using an Abbott m2000sp instrument or manually. After addition, each plate is sealed and transferred to an Abbott m2000rt, where PCR amplification is performed using a DNA polymerase.

[0071] In some embodiments, the presence of HSV-1, HSV-2 and VZV amplification products is detected during the annealing / extension step by measuring the real-time fluorescence signal of the HSV-1, HSV-2 and VZV probes. The presence of IC amplification products is detected by measuring the real-time fluorescence signal of the IC probe. In some embodiments, the HSV-1, HSV-2 and VZV and IC probes are single-stranded DNA oligonucleotides that consist of a target-specific binding sequence, a fluorescent moiety covalently linked to the 5' end of the probe, and a quenching moiety covalently linked to the 3' end of the probe. In the absence of the HSV-1, HSV-2 and VZV or IC target sequence, the probe fluorescence is quenched. In the presence of the HSV-1, HSV-2 and VZV or IC target sequence, the HSV-1, HSV-2 and VZV or IC probes specifically bind to their complementary sequences in the target during the annealing / extension step, allowing fluorescence emission and detection. In some embodiments, the HSV-1, HSV-2, and VZV probes are labeled with different fluorescent dyes (e.g., C560 for HSV-1, FAM for TM For HSV-2, 705 for VZV target probe, and 670 for IC), thus allowing the simultaneous detection of amplification products of HSV-1, HSV-2, and VZV and IC in the same reaction.

[0072] In some embodiments, steps are taken to avoid nucleic acid contamination. For example, in some embodiments, contamination is minimized because: PCR amplification and oligonucleotide hybridization occur in sealed reaction vessels; detection is performed automatically without opening the reaction vessel (e.g., plate well); aerosol barrier pipette tips are used for all pipetting; pipette tips are discarded after use; and separate dedicated areas are used to perform HSV-1, HSV-2, and VZV assays.

[0073] In some embodiments, the above reagents are provided in the form of a kit and / or system (e.g., a system comprising automated sample processing and assay instruments as described herein). For example, in some embodiments, the kit and / or system comprises, consists essentially of, or consists of:

[0074] 1. Alinity m HSV 1&2 / VZV AMP Kit: The Alinity m HSV 1&2 / VZV AMP Kit consists of two types of multiwell trays: Alinity m HSV 1&2 / VZV AMP TRAY 1 and Alinity m HSV 1&2 / VZV ACT TRAY 2.

[0075] i. Each Alinity m HSV 1&2 / VZV AMP TRAY 1 (individually packaged in foil pouches) contains 48 unit doses of liquid amplification reagent wells and 48 unit doses of liquid IC wells. One well of each is used for each test. The amplification reagent wells consist of synthetic oligonucleotides, DNA polymerase, dNTPs, and 0.15% 950. Internal control (IC) wells consisted of 0.15% The PCR product consisted of linearized plasmid DNA with irrelevant IC sequence and poly dA:dT in TE buffer with 950 μL as preservative.

[0076] ii. Each Alinity m HSV 1&2 / VZV ACT TRAY 2 (individually packaged in foil pouches) contains 48 unit doses of liquid activation reagent wells. Use one reagent well per test. The Alinity m HSV 1&2 / VZV ACT TRAY 2 contains unit doses of activation reagents (KCl, MgCl2, TMAC, and 950).

[0077] 2. Alinity m HSV 1&2 / VZV CTRL Kit: The Alinity m HSV 1&2 / VZV CTRL Kit consists of a negative control and a positive control, each supplied as a liquid in a single-use tube.

[0078] i. Alinity m HSV 1&2 / VZV Negative CTRL (Catalog No. 9N61Z) contains a buffer solution.

[0079] ii. Alinity m HSV 1&2 / VZV Positive CTRL (Catalog No. 9N61W) contains a mixture of linearized plasmid DNA containing HSV-1, HSV-2, and VZV DNA sequences in a buffered solution.

[0080] In some embodiments, Alinity HSV 1&2 / VZV AMP kits and / or systems comprise, consist essentially of, or consist of Type 2 multiwell trays, i.e., Alinity HSV 1&2 / VZVAMP TRAY 1 and Alinity HSV 1&2 / VZV ACT TRAY 2. Alinity HSV 1&2 / VZV AMP TRAY1 is individually packaged in foil pouches and contains 48 unit doses of liquid amplification reagent wells and 48 unit doses of liquid internal control (IC) wells. One well of each is used for each test. The amplification reagent wells contain synthetic oligonucleotides, DNA polymerase, dNTPs, and 0.15% 950. Internal control (IC) wells contain 0.15% Linearized plasmid DNA with irrelevant IC sequence and poly dA:dT in TE buffer with 950 μL as preservative. Alinity HSV 1&2 / VZV ACT TRAY 2 is individually packaged in foil pouches and contains 48 unit dose liquid activated reagent wells. One reagent well is used per test. In some embodiments, Alinity HSV 1&2 / VZV ACT TRAY 2 contains unit doses of activation reagents such as KCl, MgCl2, TMAC and 950. In some embodiments, Alinity The HSV 1&2 / VZV CTRL Kit contains negative and positive controls supplied as liquid in single-use tubes. The HSV 1&2 / VZVNegative CTRL kit contains buffer solution. The HSV 1&2 / VZV Positive CTRL Kit contains a mixture of linearized plasmid DNA containing HSV-1, HSV-2, and VZV DNA sequences in a buffered solution.

[0081] In some embodiments, all forms of HSV-1, HSV-2, and VZV are detected (e.g., primers and probes are selected to identify all HSV-1, HSV-2, and VZV nucleic acid target sequences that may be present in the sample). In some embodiments, specific HSV-1, HSV-2, and VZV sequences are detected. Example

[0082] The following examples are for illustrative purposes only and are not to be construed as limiting the scope of the present invention in any way.

[0083] Example 1 - Exemplary Assay Workflow

[0084] This example describes a method for performing real-time PCR to detect HSV-1, HSV-2, and VZV in a sample. In some embodiments, the real-time PCR method comprises or consists of the following steps:

[0085] 1. Sample preparation, in which DNA is extracted from the sample using reagents; sample preparation is automated using Abbott Alinity system (Abbott Molecular) or performed manually;

[0086] 2. PCR assembly, using Alinity The purified sample and the assay PCR components are added to the reaction vessel together, either systematically or manually;

[0087] 3. Transfer the reaction vessel to Alinity Amplification detection module on the system.

[0088] 4. Use automated Alinity The system amplifies and detects PCR products. Automatic reporting on system workstations.

[0089] Example 2 - HSV-1, HSV-2 and VZV triple assay using cultured virus testing

[0090] A multiplex (e.g., triplex) real-time polymerase chain reaction (PCR) assay is configured and tested for direct qualitative detection and differentiation of HSV-1, HSV-2, and VZV DNA from cultured viral samples. In some embodiments, the assay can be used as an aid in the diagnosis of HSV-1, HSV-2, and / or VZV infection in symptomatic and / or asymptomatic patients. In some embodiments, the assay is configured for use with an automated Abbott Alinity In some embodiments, the assay can be used with other real-time PCR instruments and can be used with other samples, such as cerebrospinal fluid (CSF) samples. The performance of the system's HSV 1, HSV 2, and VZV assays was tested with viral panel members (PMs) containing HSV-1, HSV-2, or VZV viral particles in comparison to other methods available at other sites, including: the Simplexa HSV 1 & 2 Direct Kit (DIASORIN MOLECULAR, Cypress, CA) at Northshore University Health center, Evanston, IL; the Artus HSV-1 / 2 RG PCR Kit (QIAGEN, Hilden, Germany) at Quest Diagnostics, Lewisville, TX; the Luminex ARIES HSV 1 & 2 (Luminex, Austin, TX) at Tricore Reference Labs, Albuquerque, NM; the Solana HSV / VZV (Quidel, San Diego, CA) at Tricore Reference Labs, Albuquerque, NM; and the Simplexa HSV 1 & 2 Direct Kit (DIASORIN MOLECULAR, Cypress, CA) at Northshore University Health center, Evanston, IL. Center, Evanston, IL; the Simplexa VZV Direct Kit (DIASORIN MOLECULAR, Cypress, CA) at Quest Diagnostics, Lewisville, TX; the Artus VZV QS-RGQ Kit (QIAGEN, Hilden, Germany) at Boca Biolitics, Pompano Beach, FL. HSV 1 & 2 on 4800 (Roche Diagnostics, Indianapolis, IN); on Boca Biolitics, Pompano Beach, FL Aptima HSV 1 & 2 assays were performed on Hologic (Hologic, Santa Clara, CA). Samples were tested on Alinity Biosciences (Abbott, Des Plaines, IL). Further testing was performed with HSV 1&2 / VZV assay.

[0091] Preparation of HSV-1, HSV-2 and VZV target experimental panel

[0092] Panels consisting of varying levels of HSV-1, HSV-2, and VZV DNA were prepared using separate viral cultures diluted in different transport media. (See Table 5 below.) The viruses and strains used in the panel preparation were: HSV-1 MacIntyre (ATCC, Manassas, VA), HSV-1 HF (ATCC, Manassas, VA), HSV-2 MS (ATCC, Manassas, VA), HSV-2 G (ATCC, Manassas, VA), VZV 82 (ZeptoMetrix, Buffalo, NY), and VZV Ellen (ZeptoMetrix, Buffalo, NY). For HSV-1, HSV-1 MacIntyre and HSV-1 HF strains were tested. For HSV-2, HSV-2 MS and HSV-2 G were tested. For VZV, VZV 82 and VZV Ellen were tested. Strains were diluted in BD Universal Viral Transport medium (BD-UVT) (BD, Franklin Lakes, NJ) to generate seven concentrations for each HSV-1 and HSV-2 analyte panel member. For HSV-1, the concentrations were: 1024, 256, 64, 16, 4, 1, and 0.5 TCID 50 / mL. For HSV-2, the concentrations are: 4096, 1024, 256, 64, 16, 4, 1, and 0.5 TCID 50 / mL. For the VZV panel members, the concentrations were 51200, 12800, 3200, 800, 200, 50, 25, 12.5, 6.25, 3.125, 1.56, and copies / mL. The panel members were dispensed into 1 mL aliquots and stored at -70°C. For each analyte, a negative control panel member consisting of BD-UVT alone was also dispensed into 1 mL aliquots and stored at -70°C.

[0093] Primer and probe design

[0094] Primers and probes are designed to amplify genomic regions from HSV-1, HSV-2, and VZV. The assay formulation contains two sets of primer / probe sets for each of the three analytes (i.e., HSV-1, HSV-2, and VZV) (i.e., dual-target primer / probe sets). The genes / targets for the dual-target design for HSV-1 are glycoprotein D (US6) and unique long region (UL) 1. For HSV-2, the genes / targets for the dual-target design are UL1+UL18. Open reading frame (ORF) 10 and ORF 21 are used as dual genes / targets for VZV. Primer and probe sequences for the targets of each virus are provided in Table 1. The forward and reverse primers for each gene and target anneal to the specific target sequence and direct the amplification of the target. The probes then specifically bind to their respective amplicons and facilitate the detection of HSV-1, HSV-2, and / or VZV. The probe has a fluorescent moiety covalently linked to the 5' end and a quencher molecule at the 3' end. In the absence of the target sequence, the probe fluorescence is quenched. In the presence of the target sequence, hybridization with the complementary sequence separates the fluorophore and quencher and allows fluorescence emission and detection. The probe for each analyte is connected to a specific dye, which allows detection of each analyte in a specific laminate channel, resulting in the differentiation of all four targets (HSV-1, HSV-2, VZV, and IC).

[0095] Table 1. Primer pairs and probe sequences for the Alinity m HSV-1, HSV-2, and VZV assays

[0096]

[0097]

[0098] In some embodiments, the probe / dye combinations of Table 1 are reconfigured to achieve various detection outputs. In some embodiments, the primer pairs and probes of Table 1 are configured to achieve detection outputs in a multiplex format, in simultaneous testing, in a single reaction vessel or container, with shared reagents and reagent concentrations, and under shared reaction and amplification conditions. The probe sequences in Table 1 are derived from one strand of the viral genome. In some embodiments, complementary sequences may be used.

[0099] In some embodiments, the label at the opposite end from the dye may be any portion that can effectively quench a fluorescent dye and allow the effective binding of these probes to their target sequence. In some embodiments, the dye is labeled at the 5' end of the probe with a quencher at the 3' end. In some embodiments, the dye is labeled at the 3' end with a quencher at the 5' end. In some embodiments, primers and / or probes are modified to include one or more binding enhancers, such as mgb or pdU / pdC.

[0100] PCR preparation

[0101] The multiplex assay contains a single reaction mixture for simultaneous detection of DNA from HSV-1, HSV-2, VZV, and IC. The PCR reaction contains sample DNA and a PCR master mix. The PCR master mix contains primers and probes for HSV-1, HSV-2, VZV, and IC, 12x Buffer, dNTPs, DNA polymerase, C610 reference dye, gelatin, Proclin, KCl, TMAC (tetramethylammonium chloride), MgCl2, and water. KCl, TMAC, and MgCl2 are components of the activation reagent. The compositions of the oligonucleotide mixture and the activation reagent are provided in Tables 2 and 3.

[0102] Table 2.- Oligonucleotide Master Mix Composition

[0103]

[0104]

[0105] Table 3.- Activation reagent composition

[0106]

[0107] PCR reaction

[0108] Once the sample preparation process is complete, the PCR reaction vessel is filled with Alinity System combination purified nucleic acid eluate, unit dose Alinity HSV 1, HSV-2, and VZV amplification reagents, and unit dose activation reagents. The Vapor Barrier Solution is then automatically added to the reaction vessel.The reaction vessel is then capped and transferred to the thermocycling position in the Assay Processing Unit (APU) for amplification and detection of the designated target.

[0109] Loop Condition

[0110] During the PCR process, the reaction temperature is raised to a temperature that dissociates the double-stranded DNA. As the reaction temperature subsequently decreases, HSV-1, HSV-2, and VZV primers anneal to their respective DNA chains and are extended by Platinum II Taq DNA polymerase. During several consecutive rounds of thermal cycling, the amplified product dissociates into single strands at high temperatures, followed by primer annealing and extension as the temperature decreases. Exponential amplification is achieved by repeated cycles between high and low temperatures. The amplification of the dual targets of the four assay analytes (i.e., HSV-1, HSV-2, VZV, and IC) occurs simultaneously in the same reaction. During the primer annealing / extension step, a read step is performed to allow real-time fluorescence detection of the amplified product when the probe anneals to its respective targets. The probe has a fluorescent moiety covalently linked to the 5' end, accompanied by a quencher molecule at the 3' end. In the absence of the target sequence, the probe fluorescence is quenched. In the presence of the target sequence, hybridization with the complementary sequence separates the fluorophore and quencher and allows fluorescence emission and detection. The PCR cycling conditions are provided in Table 4.

[0111] Table 4. PCR cycling conditions

[0112]

[0113] result

[0114] Results are reported as cycle number (CN) for each target (HSV-1, HSV-2, VZV, and IC) based on the threshold cycle (Ct), at which the fluorescence signal exceeds the threshold to indicate detection of the target nucleic acid. The Ct is inversely proportional to the concentration of each analyte present.

[0115] Accordingly, in some embodiments, the present invention provides a diagnostic method comprising the steps of:

[0116] 1. Prepare the 21 oligonucleotides provided in Table 1, as well as PCR reagents such as dNTP, 12xAlinity Prepare a master mix by mixing a reaction mixture of buffer, fish gelatin, C610 reference dye, and platinum II polymerase.

[0117] 2. The reaction mixture is mixed with divalent ions as activators.

[0118] 3. DNA extraction from HSV-1, HSV-2 or VZV viruses.

[0119] 4. The eluted DNA was mixed with a master mix and activation reagent to amplify HSV-1 US6, HSV-1 UL1, HSV-2 UL1, HSV-2 UL18, VZV ORF10, and VZV ORF21 sequences from their respective viral genomes.

[0120] 5. Detect each analyte with a separate fluorophore to identify and differentiate HSV-1, HSV-2, and VZV targets.

[0121] Comparison Test

[0122] The cultured samples are also sent to the laboratory for testing determined by the following comparisons:

[0123] 1. At Northshore University Health Center HSV 1&2 DirectKit

[0124] 2. At Quest Diagnostics HSV-1 / 2RG PCR Kit

[0125] 3. In Tricore Reference Labs ARIES HSV 1&2

[0126] 4. In Tricore Reference Labs HSV / VZV

[0127] 5. At Northshore University Health Center VZV Direct Test

[0128] 6. At Quest Diagnostics VZV QS-RGQ Kit

[0129] 7. At BocaBiolistics On the 4800System HSV 1 and 2 Test

[0130] 8. At BocaBiolistics Aptima Herpes Simplex Viruses1&2Assay on

[0131] Using the method described in the previous section, The same panel of HSV-1, HSV-2, and VZV subjects was tested on the system.

[0132] Comparative results in BD-UVT (UTM) medium

[0133] Use the separate assay package insert or instructions for use. and The assay tested the panel members prepared in BD-UVT (UTM). System HSV-1, HSV-2, and VZV assays were tested on panel members. Assay specificity comparison data are provided in Table 5.

[0134] Table 5. Comparative HSV-1, HSV-2, and VZV Assay Results

[0135]

[0136]

[0137] use Panel members prepared in MSwab medium (Roche Diagnostics, Indianapolis, IN) were tested using the HSV 1 and 2 Test. Panel members prepared in the Aptima Multitest Swab Specimen Collection Kit (Hologic, Santa Clara, CA) were tested using the Aptima Herpes Simplex Viruses 1 & 2 Assay. Panel members prepared in the Aptima Multitest Swab Specimen Collection Kit (Hologic, Santa Clara, CA) were also tested using the Alinity The panel members were tested for HSV / VZV. The results for HSV-1 are provided in Table 6, and the results for HSV-2 are provided in Table 7.

[0138] Table 6. Comparative HSV-1 assay results

[0139]

[0140] Table 7. Comparative HSV-2 assay results

[0141]

[0142] Table cells highlighted in green show the assays used for the respective analytes. Cells highlighted in gray were not tested or data was not available from the comparative assays due to invalid replicates. For the HSV-1 MacIntyre and HSV-1 HF targets, Alinity The assay was performed at 1 and 0.5 TCID 50 100% of the replicates were detected at the 400 μg / mL level. For HSV-2MS and HSV-2G targets, Alinity Determined at 0.5 TCID 50100% of the replicates were detected at 100% of the PCR product. For VZV 82 and VZV Ellen, Alinity The assay detected 100% of the replicates at levels of 3.125 and 6.25 copies / mL, respectively.

[0143] These data indicate that using multiple Alinity The performance of the assay is equivalent to or better than conventional assays in terms of precision, with improvements in throughput, cost, sensitivity, and specificity achieved by Alinity Testing formats and platforms offer additional advantages.

[0144] Example 3 - HSV-1, HSV-2 and VZV Triple Assay Testing with Patient Samples

[0145] In some embodiments, Alinity The HSV 1&2 / VZV assay is a multiplex real-time polymerase chain reaction (PCR) assay designed for the direct qualitative detection and differentiation of HSV-1, HSV-2, and VZV DNA from samples of skin or mucocutaneous lesions. To evaluate the assay performance on patient samples, clinical samples of lesions collected from swabs in viral transport medium were assayed with the Alinity HSV 1&2 / VZV assay was performed.

[0146] sample

[0147] Clinical samples were obtained from commercial suppliers (TrinaBioreactives, Switzerland; Discovery LifeSciences, Huntsville, AL). The samples were frozen before testing. The samples were never frozen prior to testing and were tested as "fresh" (ie, never frozen) samples. Fresh samples were collected from patients and were not frozen at any time during storage. An aliquot of each sample was tested.

[0148] Sample preparation

[0149] Loading lesion swab samples into Alinity The sample preparation protocol is performed in a disposable multi-well sample preparation cartridge that is loaded into the Alinity The sample (i.e., specimen and / or control sample) and magnetic particles are pipetted through the instrument into one of the Assay Processing Units (APUs) on the system. The magnetic particles are then transferred sequentially to the wells of the IRU containing a series of wash solutions. After the wash steps are complete, the magnetic particles are captured by the plunger magnet and transferred to the elution wells within the IRU, where the purified nucleic acids are eluted from the particles into the Alinity Elution buffer 2.

[0150] Sample testing

[0151] Testing using Alinity HSV 1&2 / VZV Master Mix with Alinity Alinity for HSV 1&2 / VZV Application Specification Documents The application specification file contains the software for sample processing and amplification of targets (HSV-1, HSV-2, VZV, IC) executed on the instrument.

[0152] Data Analysis

[0153] Results were analyzed for cycle number (CN) for each target (HSV-1, HSV-2, VZV, and IC).CN values are based on the threshold cycle (Ct) at which the fluorescent signal exceeds the threshold to indicate detection of the target nucleic acid.

[0154] Experimental results - frozen samples

[0155] Data from 91 frozen positive samples are shown in Table 8. The data includes results from 30 HSV-1 positive samples that were obtained from both the supplier and Alinity The assay results were both positive for HSV-1. Results from 29 HSV-2 positive samples were also tabulated, and the samples were obtained from the supplier results and Alinity The data also includes results from 32 VZV-positive samples that were obtained through vendor results and Alinity The assays were positive for both. Two VZV-positive samples were doubly positive for HSV. One was doubly positive for VZV and HSV-1, while the other was doubly positive for VZV and HSV-2.

[0156] Table 8. Frozen sample data

[0157]

[0158]

[0159]

[0160] Experimental results - fresh samples

[0161] Data from 48 fresh samples are shown in Table 9. The data include results from 22 HSV-1 positive samples that were obtained from both the supplier and Alinity The assay results were both positive for HSV-1. Results from 26 HSV-2 positive samples were also tabulated, and the samples were obtained from the supplier results and Alinity The assays were positive for both. Samples were tested as "fresh" and also after one freeze / thaw cycle, in which the samples were frozen at -70°C or lower and then thawed at room temperature or 2-8°C before testing. A single freeze / thaw cycle had no effect on the assay results.

[0162] Table 9. Fresh sample data

[0163]

[0164]

[0165] These data show that the use of Alinity The performance of the assay was equivalent for fresh and frozen sample HSV-1, HSV-2, or VZV testing.

Claims

1. A method for detecting herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), and varicella zoster virus (VZV) nucleic acids in a biological sample from a subject, comprising: a) extracting nucleic acid from the biological sample; b) contacting the nucleic acid from the biological sample of the subject with at least two nucleic acid primer pairs selected from the group consisting of SEQ ID NOs: 1 and 2, SEQ ID NOs: 4 and 5, SEQ ID NOs: 7 and 8, SEQ ID NOs: 10 and 11, SEQ ID NOs: 13 and 14, SEQ ID NOs: 16 and 17, and SEQ ID NOs: 19 and 20, and at least two probes selected from the group consisting of SEQ ID NOs: 3, 6, 9, 12, 15, 18, and 21; d) performing an amplification assay to amplify one or more HSV-1, HSV-2, and VZV nucleic acid targets; and e) detecting the presence of said HSV-1, HSV-2 and VZV nucleic acid targets in said sample.

2. The method of claim 1, wherein the detection is binding of at least one of the nucleic acid primers or probes to the HSV-1, HSV-2, and VZV nucleic acids.

3. The method of claim 1, wherein the detecting comprises real-time polymerase chain reaction (RT-PCR).

4. The method of claim 1, wherein the biological sample is a skin sample, a mucosal sample, an anogenital sample, an oral sample, a cerebrospinal fluid sample and / or a blood sample.

5. The method of claim 1, wherein one or more of the SEQ ID NOs comprises a tag. The method of claim 5 , wherein the label comprises a fluorophore. The method of claim 5 , wherein the label comprises a fluorophore / quencher pair.

8. The method of claim 1, further comprising contacting the biological sample from the subject with a reaction mixture.

9. The method of claim 8, wherein the reaction mixture comprises a reagent selected from the group consisting of a nucleic acid polymerase, a plurality of dNTPs, and a buffer.

10. The method of claim 8, wherein the reaction mixture comprises magnetic particles.

11. A composition comprising at least two nucleic acid primer pairs selected from SEQ ID NOs: 1 and 2, SEQ ID NOs: 4 and 5, SEQ ID NOs: 7 and 8, SEQ ID NOs: 10 and 11, SEQ ID NOs: 13 and 14, SEQ ID NOs: 16 and 17, and SEQ ID NOs: 19 and 20, and at least two probes selected from SEQ ID NOs: 3, 6, 9, 12, 15, 18, and 21.

12. The composition of claim 11, wherein one or more of the SEQ ID NOs comprises a tag.

13. The composition of claim 11, wherein the label comprises a fluorophore.

14. The composition of claim 12, wherein the label comprises a fluorophore / quencher pair.

15. The composition of claim 11, wherein the composition is a reaction mixture.

16. A reaction mixture comprising: a) at least two nucleic acid primer pairs selected from SEQ ID NOs: 1 and 2, SEQ ID NOs: 4 and 5, SEQ ID NOs: 7 and 8, SEQ ID NOs: 10 and 11, SEQ ID NOs: 13 and 14, SEQ ID NOs: 16 and 17, and SEQ ID NOs: 19 and 20, and at least two probes selected from SEQ ID NOs: 3, 6, 9, 12, 15, 18, and 21; and b) at least one reagent for performing a nucleic acid amplification reaction. The reaction mixture of claim 16 , further comprising a sample.

18. The reaction mixture of claim 17, wherein the sample comprises HSV-1, HSV-2 and / or VZV target nucleic acid.

19. The reaction mixture of claim 16, wherein the reagents are selected from the group consisting of a nucleic acid polymerase, a plurality of dNTPs, and a buffer.

20. The reaction mixture of claim 16, further comprising magnetic particles.

21. The reaction mixture of claim 16, further comprising one or more reagents specific for RT-PCR.

22. A system comprising: a) at least two nucleic acid primer pairs selected from SEQ ID NOs: 1 and 2, SEQ ID NOs: 4 and 5, SEQ ID NOs: 7 and 8, SEQ ID NOs: 10 and 11, SEQ ID NOs: 13 and 14, SEQ ID NOs: 16 and 17, and SEQ ID NOs: 19 and 20, and at least two probes selected from SEQ ID NOs: 3, 6, 9, 12, 15, 18, and 21; and b) Automated sample processing equipment.

23. The system of claim 22, further comprising an assay instrument.

24. The system of claim 23, wherein the assay instrument is a high-throughput assay instrument.

25. The system of claim 22, further comprising reaction components in a multi-container carrier.

26. The system of claim 22, further comprising a microplate thermal cycler and / or a microplate detector.

27. The system of claim 22, further comprising a microfluidic device.

28. A kit comprising: a) at least two nucleic acid primer pairs selected from SEQ ID NOs: 1 and 2, SEQ ID NOs: 4 and 5, SEQ ID NOs: 7 and 8, SEQ ID NOs: 10 and 11, SEQ ID NOs: 13 and 14, SEQ ID NOs: 16 and 17, and SEQ ID NOs: 19 and 20, and at least two probes selected from SEQ ID NOs: 3, 6, 9, 12, 15, 18, and 21; and b) at least one reagent for performing a nucleic acid amplification reaction.

29. The kit of claim 28, wherein the reagents are selected from the group consisting of a nucleic acid polymerase, a plurality of dNTPs, and a buffer.

30. The kit of claim 28, wherein one or more of the primers or probes comprises a label.

31. The kit of claim 30, wherein the label comprises a fluorophore.

32. The kit of claim 31 , wherein the label comprises a fluorophore / quencher pair.

33. The kit of claim 28, further comprising magnetic particles.

34. The kit of claim 28, further comprising one or more reagents specific for RT-PCR.

35. The kit according to claim 28, further comprising a positive control nucleic acid, a negative control nucleic acid, a calibrator nucleic acid and / or instructions for use of the kit.

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