Compositions and methods for the detection of human parainfluenza viruses 1-4 (HPIV1-4)

Abstract

Methods are described for rapidly detecting the presence or absence of human parainfluenza viruses (HPIV), including HPIV1-4, in biological or non-biological samples. The methods can include performing amplifying, hybridizing, and detecting steps. Additionally, primers and probes targeted to HPIV1-4, as well as kits designed to detect target regions of HPIV1-4, are provided. Kits, reaction mixtures, and oligonucleotides (e.g., primers and probes) for amplifying and detecting HPIV1-4 are also described.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims priority to U.S. Provisional Patent Application No. US63 / 143,144, filed January 29, 2021, and U.S. Provisional Patent Application No. US63 / 146,158, filed February 5, 2021, both of which are incorporated herein by reference in their entireties.

[0002] FIELD OF THEINVENTION The present disclosure relates to the field of in vitro diagnostics. In this field, the invention relates to the amplification and detection of target nucleic acids that may be present in a sample, in particular the amplification, detection and / or quantification of target nucleic acids, including sequence variants and / or individual variants of human parainfluenza viruses 1-4 (HPIV1-4), using primers and probes. The invention further provides reaction mixtures and kits containing primers and probes for the amplification and detection of HPIV, including HPIV (including HPIV1-4). [Background technology]

[0003] 2. Background of the Invention Human parainfluenza viruses (HPIVs) are viruses that cause human parainfluenza. HPIVs are a group of four distinct single-stranded RNA viruses belonging to the Paramyxoviridae family, known as human parainfluenza virus type 1 (also called HPIV1, HPIV-1, or HPIV serotype 1), human parainfluenza virus type 2 (also called HPIV2, HPIV-2, or HPIV serotype 2), human parainfluenza virus type 3 (also called HPIV3, HPIV-3, or HPIV serotype 3), and human parainfluenza virus type 4 (also called HPIV4, HPIV-4, or HPIV serotype 4) (i.e., collectively HPIV1-4 or HPIV serotypes 1-4). Collectively, these four distinct viruses are sometimes referred to as human parainfluenza virus types 1-4, or HPIV1-4. HPIV1-4 are genetically and antigenically distinct. Most of their structural and biological characteristics are similar, however, HPIV1-4 are each adapted to infect humans of different ages and cause different diseases. Overall, the Paramyxoviridae family of viruses, to which HPIV1-4 belong, are among the most costly viruses in terms of disease burden and economic impact. HPIVs are classified into two genera; (1) the Respiroviruses, which include HPIV1 and HPIV3; and (2) the Rubulaviruses, which include HPIV2 and HPIV4. HPIV4 has also been classified into two distinct antigenic subgroups, 4a and 4b, based on reactivity with monoclonal antibodies. Notably, HPIVs are enveloped viruses that contain nonsegmented negative-stranded genomic RNA. HPIV replication is initiated by entry through attachment, fusion, genome transcription, and replication. Subsequently, the newly synthesized viral components are transported to assembly sites at the cell membrane, where the newly formed virions bud from the cell.

[0004] For all HPIV1-4, the RNA genome is tightly associated with nucleoproteins to form a helical nucleocapsid, to which viral RNA polymerase binds. Replication occurs entirely in the cytoplasm, and progeny virions assemble at the plasma membrane of infected cells and are released by budding. Virions consist of a fibrous nucleocapsid core surrounded by a lipid envelope with virus-specific glycoprotein spikes. In addition to containing the genome, the nucleocapsid of HPIV1-4 also contains two other proteins: the phosphoprotein (P protein) and the large protein (L protein). The outer layer of the lipid membrane contains the spike-like hemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins, which extend from the viral membrane or the surface of the infected cell. Beneath the viral lipid bilayer is the matrix (M) protein, which is the smallest of the major structural proteins and is also the most abundant protein produced by paramyxoviruses. The viral membrane contains the viral RNA, which is encapsidated with the nucleoprotein (NP) to form the nucleocapsid, which binds to a polymerase complex composed of the P and L proteins to form the biologically active ribonucleocapsid. The nucleocapsid protein (or nucleoprotein or NP) is thought to be responsible for the RNA-dependent RNA polymerase activity (along with the P and L proteins). The genomic RNA of all parainfluenza viruses produces six separate, nonoverlapping polyadenylated mRNAs that code for the NP, P, M, F, HN, and L proteins. The mRNA that codes for the P protein contains several additional ORFs that code for C and V (accessory proteins). The genome consists of, in 3' to 5' order, a leader sequence, the NP, P, M, F, HN, L genes, and a trailer sequence.

[0005] Parainfluenza viruses, including HPIV1-4, are respiratory pathogens that act as causative agents of croup, bronchitis, and pneumonia. HPIV infections are responsible for hundreds of thousands of hospitalizations in the United States each year. HPIVs cause severe respiratory infections, especially in children. HPIVs replicate primarily in the respiratory tract and are transmitted by aerosolization. In children, the most common types of illness caused by parainfluenza are rhinitis, pharyngitis, and bronchitis. However, infections in immunocompromised patients are usually prolonged and may be more severe. There is a large diversity in the clinical manifestations of the various types of HPIV. For example, HPIV1 and HPIV2 cause most cases of laryngotracheobronchitis (croup) in children (about 600,000 cases per year in the United States), while HPIV3 is responsible for 3-10% of hospitalizations, usually causing bronchitis, pneumonia, croup, or pneumonia. HPIV4 is less recognized but can cause mild to severe airway disease. HPIV usually infects infants, young children, and people with weakened immune systems, but anyone can become infected with HPIV. Furthermore, a person can have multiple HPIV infections in their lifetime. Reinfection usually causes mild upper respiratory tract illness with cold-like symptoms, but in some people, reinfection can cause severe lower respiratory tract illness, such as pneumonia, bronchitis, and bronchiolitis. Elderly people and people with weakened or compromised immune systems are at higher risk for severe infection. HPIV is usually transmitted by direct contact with infectious droplets or through the air when an infected person breathes, coughs, or sneezes. HPIV can remain infectious in airborne droplets for over an hour, on surfaces for several hours, and up to 10 hours. Unfortunately, there are few, if any, effective treatments for HPIV infections. Ribavirin is one drug that has been shown to have potential efficacy against HPIV3 infections. Furthermore, there is no effective vaccine against HPIV, including any of HPIV1-4.

[0006] Existing methods for laboratory diagnosis of HPIV infection include direct detection of the viral genome by polymerase chain reaction (PCR) assays, direct detection of viral antigens in respiratory secretions (taken within 1 week of symptom onset) by immunofluorescence or enzyme immunoassay methods, isolation and identification of the virus in cell culture, and / or demonstration of a significant rise in HPIV-specific IgG antibodies between appropriately collected paired serum specimens, or specific IgM antibodies in a single serum specimen. Throat swabs, nasopharyngeal swabs, nasal washes, and nasal aspirates have all been used successfully to recover HPIV, although the optimal method for obtaining clinical samples for HPIV has not been well studied and is thought to depend on the detection method used (e.g., PCR or tissue culture), the age of the patient, and the patient's general health (i.e., whether they are immunocompromised or suffer from chronic lung disease) (see, for example, Hendrickson, "Parainfluenza Viruses, Clin. Microbiol. Rev. 16(2):242-264 (2003). The few studies that achieved high virus recovery rates (HPIV-1 and HPIV-3) used nasal washes or nasal aspirates, which are recommended for optimal virus isolation. There are also methods to detect antibodies to HPIV in samples, including enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays, HI, complement fixation, Western blotting, and neutralization assays. However, the development of heterologous antibodies to closely related HPIV serogroups during HPIV infection is a persistent problem when attempting to perform serological diagnosis. ELISAs, radioimmunoassays, and fluoroimmunoassays have also been developed to directly detect HPIV antigens. In addition, although electron microscopy can easily demonstrate the presence of HPIV, the use of electron microscopy is expensive because many paramyxoviruses look alike. Furthermore, immunofluorescence is also widely used as a means of detecting HPIV in samples, but detection of HPIV by direct IF staining of clinical material has been highly variable and sometimes has yielded disappointing results.Shell vial assays, in which tissue cultures are grown on slides and first centrifugation is used to facilitate viral absorption and cell infection, are another method for rapid identification of HPIV, but sensitivity problems have been reported with these techniques. HPIV RNA can be detected directly by Northern hybridization or dot blot analysis using virus-specific probes, but these methods are time-consuming, give inconsistent results, and lack sensitivity. The amount of HPIV RNA in many clinical samples is not sufficient to allow detection without either biological (tissue culture) or molecular amplification. Several studies have demonstrated that PCR is suitable for the detection of HPIV, and multiplex RT-PCR assays have been developed to detect HPIV-1, HPIV-2, and HPIV-3. Another group claims to have developed a multiplex RT-PCR assay for detecting HPIV1-4 in a sample, but the assay has sensitivity problems (see, e.g., Aguilar, et al., "Detection and Identification of Human Parainfluenza Viruses 1, 2, 3, and 4 in Clinical Samples of Pediatric Patients by Multiplex Reverse Transcription-PCR," Journal of Clinical Microbiology 38(3):1191-1195 (2000)). Thus, there remains a need in the art for rapid, reliable, specific and sensitive methods for detecting and / or quantifying the presence of HPIV1-4 in a sample. Summary of the Invention

[0007] In the field of molecular diagnostics, the amplification and detection of nucleic acids is of considerable importance. Such methods can be used to detect any number of microorganisms, such as viruses and bacteria. The most prominent and widely used amplification technique is the polymerase chain reaction (PCR). Other amplification techniques include ligase chain reaction, polymerase ligase chain reaction, Gap-LCR, repair chain reaction, 3SR, NASBA, strand displacement amplification (SDA), transcription-mediated amplification (TMA) and Qβ amplification. Automated systems for PCR-based analysis often utilize real-time detection of product amplification during the PCR process in the same reaction vessel. The key to such methods is the use of modified oligonucleotides bearing reporter groups or labels.

[0008] The present invention is directed to reliable, sensitive, and reproducible amplification and detection of human parainfluenza viruses 1-4 (HPIV1-4). The primers and probes are designed to maximize inclusion for human parainfluenza viruses 1-4 (HPIV1-4) and exclude other viruses of the same family (Paramyxoviridae), thereby preventing cross-reactivity with other templates (e.g., other viral templates). This human parainfluenza viruses 1-4 (HPIV1-4) assay can be used on the cobas® 6800 / 8800 systems. The primers and probes of the present invention can be used as a multi-target assay to detect all four HPIV targets. The design strategy was to select conserved sequence regions from the HPIV1-4 genome and evaluate several primer and probe combinations for each target. These candidates can be used individually to detect single individual HPIV types, for example, one assay to detect HPIV1 in a sample, one assay to detect HPIV2 in a sample, one assay to detect HPIV3 in a sample, and one assay to detect HPIV4 in a sample. Alternatively, these candidates can be used simultaneously in a multiplex assay to detect HPIV1-4 in a single sample. In this way, a single assay can detect the presence of four different types of HPIV (HPIV1-4) in a sample. When used as a multiplex assay targeting four types of HPIV (HPIV1-4), four different sets of primers and probes are used (each set of primers and probe detects HPIV1, HPIV2, HPIV3, and HPIV4). Of course, the candidates can be used in duplex or triplex assays to simultaneously detect two or three targets, respectively. The candidates can be used in any configuration of assays to detect anywhere between one and four HPIV targets of human parainfluenza viruses 1-4 (HPIV1-4).

[0009] Certain embodiments of the present disclosure relate to methods for rapid detection of the presence or absence of human parainfluenza viruses 1-4 (HPIV1-4) in biological or non-biological samples, such as multiplex detection and quantification of HPIV1-4 by real-time polymerase chain reaction (PCR) in a single test tube or container. The embodiments include methods for detection of human parainfluenza viruses 1-4 (HPIV1-4), comprising performing at least one cycling step, which may include an amplification step and a hybridization step. Additionally, the embodiments include primers, probes, and kits designed for detection of human parainfluenza viruses 1-4 (HPIV1-4) in a single tube or container.

[0010] One embodiment of the present disclosure is directed to a method for detecting one or more target nucleic acids of human parainfluenza viruses 1-4 (HPIV1-4). In particular, one embodiment of the present disclosure is directed to a method for detecting one or more target nucleic acids of human parainfluenza viruses 1-4 (HPIV1-4) in a nasopharyngeal sample collected in virus collection medium / universal transport medium (UTM).

[0011] One aspect of the disclosure is a method for detecting human parainfluenza virus (HPIV) in a sample, where HPIV includes human parainfluenza virus type 1 (HPIV-1), human parainfluenza virus type 2 (HPIV-2), human parainfluenza virus type 3 (HPIV-3), and / or human parainfluenza virus type 4 (HPIV-4), comprising: (a) detecting a target nucleic acid of one or more of HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 in a sample; (b) performing an amplification step comprising contacting one or more sets of probes with the amplification products of the HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 target nucleic acids if one or more of the HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 target nucleic acids are present in the sample; and (c) performing a hybridization step comprising contacting one or more probes with the amplification products of the HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 target nucleic acids if the HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 target nucleic acids are present in the sample. and detecting the presence or absence of an amplification product of HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 target nucleic acid, wherein the presence of an amplification product of HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 target nucleic acid indicates the presence of HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4, respectively, in the sample and the absence of an amplification product of HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 target nucleic acid indicates the presence of HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4, respectively, from the sample. detecting the presence or absence of an amplification product of an HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 target nucleic acid, which is indicative of the absence of HPIV-4; the one or more sets of primers and the one or more probes include: (1) a set of primers and a probe specific for an HPIV-1 target nucleic acid, where the set of primers specific for an HPIV-1 target nucleic acid includes a first primer comprising the nucleic acid sequence of SEQ ID NO:1 or a complement thereof, and a second primer comprising the nucleic acid sequence of SEQ ID NO:2 or a complement thereof;(1) a set of primers and probes specific for a target nucleic acid of HPIV-1, the set of primers and probes specific for a target nucleic acid of HPIV-1 comprising the nucleic acid sequence of SEQ ID NO:3 or a complement thereof; and / or (2) a set of primers and probes specific for a target nucleic acid of HPIV-2, the set of primers and probes specific for a target nucleic acid of HPIV-2 comprising a first primer comprising the nucleic acid sequence of SEQ ID NO:4 or a complement thereof, and a second primer comprising the nucleic acid sequence of SEQ ID NO:5 or a complement thereof; the probes specific for a target nucleic acid of HPIV-2 comprising the nucleic acid sequence of SEQ ID NO:6 or SEQ ID NO:7, or a complement thereof; and / or (3) a set of primers and probes specific for a target nucleic acid of HPIV-3, the set of primers and probes specific for a target nucleic acid of HPIV-3, the set of primers and probes specific for a target nucleic acid of HPIV-4, the set of primers and probes specific for a target nucleic acid of HPIV-5, the set of primers and probes specific for a target nucleic acid of HPIV-6, the set of primers and probes specific for a target nucleic acid of HPIV-7, the set of primers and probes specific for a target nucleic acid of HPIV-8, the set of primers and probes specific for a target nucleic acid of HPIV-9, the set of primers and probes specific for a target nucleic acid of HPIV-1 comprising the nucleic acid sequence of SEQ ID NO:3 or a complement thereof; the set of primers specific for an HPIV-3 target nucleic acid comprises a first primer comprising the nucleic acid sequence of SEQ ID NO:8 or SEQ ID NO:15, or a complement thereof, and a second primer comprising the nucleic acid sequence of SEQ ID NO:9 or SEQ ID NO:16, or a complement thereof; the probe specific for an HPIV-3 target nucleic acid comprises a primer set and probe comprising the nucleic acid sequence of SEQ ID NO:10 or SEQ ID NO:17, or a complement thereof; and / or (4) a primer set and probe specific for an HPIV-4 target nucleic acid, wherein the primer set and probe specific for an HPIV-4 target nucleic acid comprises a first primer comprising the nucleic acid sequence of SEQ ID NO:11 or SEQ ID NO:18, or a complement thereof, and a second primer comprising the nucleic acid sequence of SEQ ID NO:12 or SEQ ID NO:19, or a complement thereof;The method includes a set of primers and a probe specific for a target nucleic acid of HPIV-4, the probe comprising the nucleic acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14, or a complement thereof. In another embodiment, the sample is a biological sample. In another embodiment, the biological sample is whole blood, a respiratory specimen, a nasopharyngeal sample, a urine, a fecal specimen, a blood specimen, plasma, a skin swab, a nasal swab, a wound swab, a blood culture, a skin, and a soft tissue infection. In another embodiment, the biological sample is a nasopharyngeal sample. In another embodiment, one or more of the probes are labeled. In another embodiment, one or more of the probes are labeled with a donor fluorescent moiety and a corresponding acceptor moiety. In another embodiment, detecting the presence or absence of an amplification product of an HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 target nucleic acid in step (c) comprises detecting the presence or absence of fluorescence resonance energy transfer (FRET) between a donor fluorescent moiety and an acceptor moiety of a probe specific for an HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 target nucleic acid, where the presence or absence of fluorescence indicates the presence or absence, respectively, of HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 in the sample;

[0012] Another aspect of the disclosure is a method for simultaneously detecting one or more target nucleic acids of HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 in a sample, comprising: (a) performing an amplification step, if one or more target nucleic acids of HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 are present in the sample, comprising contacting the sample with one or more sets of primers to generate amplification products of the target nucleic acids of HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4; (b) detecting one or more target nucleic acids of HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 in the sample; if one or more of the target nucleic acids HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 are present in the sample, performing a hybridization step comprising contacting one or more probes with an amplification product of the HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 target nucleic acid; and (c) detecting the presence or absence of an amplification product of the HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 target nucleic acid, wherein the one or more of the HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 target nucleic acid is present in the sample. detecting the presence or absence of an amplification product of a HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 target nucleic acid, wherein the presence of an amplification product of a HPIV-4 target nucleic acid indicates the presence of HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4, respectively, in the sample, and the absence of an amplification product of a HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 target nucleic acid indicates the absence of HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4, respectively, from the sample; The one or more sets and one or more probes include: (1) a set of primers and a probe specific for a target nucleic acid of HPIV-1, where the set of primers specific for a target nucleic acid of HPIV-1 comprises a first primer comprising a nucleic acid sequence of SEQ ID NO:1 or a complement thereof, and a second primer comprising a nucleic acid sequence of SEQ ID NO:2 or a complement thereof; the probe specific for a target nucleic acid of HPIV-1 comprises a nucleic acid sequence of SEQ ID NO:3 or a complement thereof;and / or (2) a set of primers and probes specific for a target nucleic acid of HPIV-2, wherein the set of primers specific for a target nucleic acid of HPIV-2 comprises a first primer comprising the nucleic acid sequence of SEQ ID NO:4 or a complement thereof, and a second primer comprising the nucleic acid sequence of SEQ ID NO:5 or a complement thereof; the probe specific for a target nucleic acid of HPIV-2 comprises the nucleic acid sequence of SEQ ID NO:6 or SEQ ID NO:7, or a complement thereof; and / or (3) a set of primers and probes specific for a target nucleic acid of HPIV-3, wherein the set of primers specific for a target nucleic acid of HPIV-3 comprises the nucleic acid sequence of SEQ ID NO:8 or SEQ ID NO:15, or a complement thereof, and a second primer comprising the nucleic acid sequence of SEQ ID NO:9 or SEQ ID NO:16, or a complement thereof; the probe specific for a target nucleic acid of HPIV-3 comprises a set of primers and a probe specific for a target nucleic acid of HPIV-3 comprising the nucleic acid sequence of SEQ ID NO:10 or SEQ ID NO:17, or a complement thereof; and / or (4) a set of primers and a probe specific for a target nucleic acid of HPIV-4, wherein the set of primers specific for a target nucleic acid of HPIV-4 comprises a first primer comprising the nucleic acid sequence of SEQ ID NO:11 or SEQ ID NO:18, or a complement thereof, and a second primer comprising the nucleic acid sequence of SEQ ID NO:12 or SEQ ID NO:19, or a complement thereof;The method includes a set of primers and a probe specific for a target nucleic acid of HPIV-4, the probe comprising the nucleic acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14, or a complement thereof. In another embodiment, the sample is a biological sample. In another embodiment, the biological sample is whole blood, a respiratory specimen, a nasopharyngeal sample, a urine, a fecal specimen, a blood specimen, plasma, a skin swab, a nasal swab, a wound swab, a blood culture, a skin, and a soft tissue infection. In another embodiment, the biological sample is a nasopharyngeal sample. In another embodiment, one or more of the probes are labeled. In an embodiment, one or more of the probes are labeled with a donor fluorescent moiety and a corresponding acceptor moiety. In another embodiment, detecting the presence or absence of an amplification product of an HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 target nucleic acid in step (c) comprises detecting the presence or absence of fluorescence resonance energy transfer (FRET) between a donor fluorescent moiety and an acceptor moiety of a probe specific for an HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 target nucleic acid, where the presence or absence of fluorescence indicates the presence or absence, respectively, of HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 in the sample;

[0013] Another aspect of the disclosure is a method for detecting a first target nucleic acid, a second target nucleic acid, a third target nucleic acid, and / or a fourth target nucleic acid in a sample, if the first target nucleic acid, the second target nucleic acid, the third target nucleic acid, and / or the fourth target nucleic acid are present in the sample, comprising: (a) contacting the sample with one or more sets of primers to detect the first target nucleic acid, the second target nucleic acid, the third target nucleic acid, and / or the fourth target nucleic acid, if the first target nucleic acid, the second target nucleic acid, the third target nucleic acid, and / or the fourth target nucleic acid are present in the sample; (b) performing an amplification step comprising contacting one or more probes with the amplification products of the first target nucleic acid, the second target nucleic acid, the third target nucleic acid, and / or the fourth target nucleic acid if the first target nucleic acid, the second target nucleic acid, the third target nucleic acid, and / or the fourth target nucleic acid are present in the sample; and (c) detecting the presence or absence of the amplification products of the first target nucleic acid, the second target nucleic acid, the third target nucleic acid, and / or the fourth target nucleic acid, wherein the first target nucleic acid, the second target nucleic acid, the third target nucleic acid, and / or the fourth target nucleic acid are present in the sample. detecting the presence or absence of an amplification product of the first target nucleic acid, the second target nucleic acid, the third target nucleic acid, and / or the fourth target nucleic acid, where the presence of the amplification product of the first target nucleic acid, the second target nucleic acid, the third target nucleic acid, and / or the fourth target nucleic acid indicates the presence of the first target nucleic acid, the second target nucleic acid, the third target nucleic acid, and / or the fourth target nucleic acid, respectively, in the sample, and the absence of an amplification product of the first target nucleic acid, the second target nucleic acid, the third target nucleic acid, and / or the fourth target nucleic acid indicates the absence of the first target nucleic acid, the second target nucleic acid, the third target nucleic acid, and / or the fourth target nucleic acid, respectively, in the sample. and wherein the one or more sets of primers and the one or more probes are: (1) a set of primers and a probe for a first target nucleic acid, wherein the set of primers for the first target nucleic acid comprises a first primer comprising a nucleic acid sequence of SEQ ID NO:1 or a complement thereof, and a second primer comprising a nucleic acid sequence of SEQ ID NO:2 or a complement thereof; and the probe for the first target nucleic acid comprises a nucleic acid sequence of SEQ ID NO:3, or a complement thereof;and / or (2) a set of primers and a probe for a second target nucleic acid, wherein the set of primers for the second target nucleic acid comprises a first primer comprising the nucleic acid sequence of SEQ ID NO:4 or a complement thereof, and a second primer comprising the nucleic acid sequence of SEQ ID NO:5 or a complement thereof; the probe for the second target nucleic acid comprises the nucleic acid sequence of SEQ ID NO:6 or SEQ ID NO:7, or a complement thereof; and / or (3) a set of primers and a probe for a third target nucleic acid, wherein the set of primers for the third target nucleic acid comprises a first primer comprising the nucleic acid sequence of SEQ ID NO:8 or SEQ ID NO:15, or a complement thereof, and a second primer comprising the nucleic acid sequence of SEQ ID NO:9 or SEQ ID NO:16, or a complement thereof. (3) a first primer and a second primer comprising a nucleic acid sequence of SEQ ID NO:10 or SEQ ID NO:17, or a complement thereof; and / or (4) a set of primers and a probe for a fourth target nucleic acid, wherein the set of primers for the fourth target nucleic acid comprises a first primer comprising a nucleic acid sequence of SEQ ID NO:11 or SEQ ID NO:18, or a complement thereof, and a second primer comprising a nucleic acid sequence of SEQ ID NO:12 or SEQ ID NO:19, or a complement thereof; and the probe for the fourth target nucleic acid comprises a set of primers and a probe for the fourth target nucleic acid comprising a nucleic acid sequence of SEQ ID NO:13 or SEQ ID NO:14, or a complement thereof;The method is directed to a method, wherein the first target nucleic acid is a target nucleic acid of HPIV-1, the second target nucleic acid is a target nucleic acid of HPIV-2, the third target nucleic acid is a target nucleic acid of HPIV-3, and the fourth target nucleic acid is a target nucleic acid of HPIV-4. In another embodiment, the sample is a biological sample. In another embodiment, the biological sample is whole blood, a respiratory specimen, a nasopharyngeal sample, a urine, a fecal specimen, a blood specimen, plasma, a skin swab, a nasal swab, a wound swab, a blood culture, skin, and a soft tissue infection. In another embodiment, the biological sample is a nasopharyngeal sample. In another embodiment, one or more of the probes are labeled. In another embodiment, one or more of the probes are labeled with a donor fluorescent moiety and a corresponding acceptor moiety. In another embodiment, in step (c), detecting the presence or absence of an amplification product of the first target nucleic acid, the second target nucleic acid, the third target nucleic acid, and / or the fourth target nucleic acid comprises detecting the presence or absence of fluorescence resonance energy transfer (FRET) between a donor fluorescent moiety and an acceptor moiety of the probe for the first target nucleic acid, the second target nucleic acid, the third target nucleic acid, and / or the fourth target nucleic acid, where the presence of fluorescence indicates the presence of HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 in the sample and the absence of fluorescence indicates the absence of HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 in the sample;

[0014] Another aspect is a method for simultaneously detecting a first target nucleic acid, a second target nucleic acid, a third target nucleic acid, and / or a fourth target nucleic acid in a sample, when the first target nucleic acid, the second target nucleic acid, the third target nucleic acid, and / or the fourth target nucleic acid are present in the sample, comprising: (a) contacting the sample with one or more sets of primers to generate amplification products of the first target nucleic acid, the second target nucleic acid, the third target nucleic acid, and / or the fourth target nucleic acid, when the first target nucleic acid, the second target nucleic acid, the third target nucleic acid, and / or the fourth target nucleic acid are present in the sample; (b) if the first target nucleic acid, the second target nucleic acid, the third target nucleic acid, and / or the fourth target nucleic acid are present in the sample, performing a hybridization step comprising contacting one or more probes with the amplification products of the first target nucleic acid, the second target nucleic acid, the third target nucleic acid, and / or the fourth target nucleic acid; and (c) detecting the presence or absence of the amplification products of the first target nucleic acid, the second target nucleic acid, the third target nucleic acid, and / or the fourth target nucleic acid, wherein the presence of an amplification product of the first target nucleic acid, the second target nucleic acid, the third target nucleic acid, and / or the fourth target nucleic acid indicates the presence of the first target nucleic acid, the second target nucleic acid, the third target nucleic acid, and / or the fourth target nucleic acid, respectively, in the sample, and the absence of an amplification product of the first target nucleic acid, the second target nucleic acid, the third target nucleic acid, and / or the fourth target nucleic acid indicates the absence of the first target nucleic acid, the second target nucleic acid, the third target nucleic acid, and / or the fourth target nucleic acid, respectively, in the sample. detecting the presence or absence; the one or more sets of primers and one or more probes include: (1) a set of primers and a probe for a first target nucleic acid, where the set of primers for the first target nucleic acid comprises a first primer comprising a nucleic acid sequence of SEQ ID NO:1 or a complement thereof, and a second primer comprising a nucleic acid sequence of SEQ ID NO:2 or a complement thereof; the set of primers and a probe for the first target nucleic acid comprises a nucleic acid sequence of SEQ ID NO:3, or a complement thereof;(2) a set of primers and a probe for a second target nucleic acid, wherein the set of primers for the second target nucleic acid comprises a first primer comprising a nucleic acid sequence of SEQ ID NO: 4 or a complement thereof, and a second primer comprising a nucleic acid sequence of SEQ ID NO: 5 or a complement thereof; the probe for the second target nucleic acid comprises a nucleic acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7, or a complement thereof; (3) a set of primers and a probe for a third target nucleic acid, wherein the set of primers for the third target nucleic acid comprises a first primer comprising a nucleic acid sequence of SEQ ID NO: 8 or SEQ ID NO: 15, or a complement thereof, and a nucleic acid sequence of SEQ ID NO: 9 or SEQ ID NO: 16, or a complement thereof. (3) a set of primers and a probe for a third target nucleic acid, wherein the set of primers for the fourth target nucleic acid comprises a first primer comprising a nucleic acid sequence of SEQ ID NO:11 or SEQ ID NO:18, or a complement thereof, and a second primer comprising a nucleic acid sequence of SEQ ID NO:12 or SEQ ID NO:19, or a complement thereof; and (4) a set of primers and a probe for a fourth target nucleic acid, wherein the set of primers for the fourth target nucleic acid comprises a first primer comprising a nucleic acid sequence of SEQ ID NO:11 or SEQ ID NO:18, or a complement thereof, and a second primer comprising a nucleic acid sequence of SEQ ID NO:12 or SEQ ID NO:19, or a complement thereof; and the probe for the fourth target nucleic acid comprises a set of primers and a probe for a fourth target nucleic acid comprising a nucleic acid sequence of SEQ ID NO:13 or SEQ ID NO:14, or a complement thereof;The method is directed to a method, wherein the first target nucleic acid is a target nucleic acid of HPIV-1, the second target nucleic acid is a target nucleic acid of HPIV-2, the third target nucleic acid is a target nucleic acid of HPIV-3, and the fourth target nucleic acid is a target nucleic acid of HPIV-4. In another embodiment, the sample is a biological sample. In another embodiment, the biological sample is whole blood, a respiratory specimen, a nasopharyngeal sample, a urine, a fecal specimen, a blood specimen, plasma, a skin swab, a nasal swab, a wound swab, a blood culture, skin, and a soft tissue infection. In another embodiment, the biological sample is a nasopharyngeal sample. In another embodiment, one or more of the probes are labeled. In another embodiment, one or more of the probes are labeled with a donor fluorescent moiety and a corresponding acceptor moiety. In another embodiment, in step (c), detecting the presence or absence of an amplification product of the first target nucleic acid, the second target nucleic acid, the third target nucleic acid, and / or the fourth target nucleic acid comprises detecting the presence or absence of fluorescence resonance energy transfer (FRET) between a donor fluorescent moiety and an acceptor moiety of the probe for the first target nucleic acid, the second target nucleic acid, the third target nucleic acid, and / or the fourth target nucleic acid, where the presence of fluorescence indicates the presence of HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 in the sample and the absence of fluorescence indicates the absence of HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 in the sample;

[0015] Another aspect is directed to a kit for detecting HPIV that may be present in a sample, wherein the HPIV comprises HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4, the kit comprising amplification reagents and detection reagents, wherein the amplification reagents and detection reagents comprise: (i) a DNA polymerase; (ii) nucleotide monomers; and (iii) one or more sets of primers and one or more probes, wherein the one or more sets of primers and one or more probes are selected from the group consisting of: (1) a set of primers and probes specific for a target nucleic acid of HPIV-1, wherein the set of primers specific for a target nucleic acid of HPIV-1 comprises a first primer comprising a nucleic acid sequence of SEQ ID NO:1 or a complement thereof, and a second primer comprising a nucleic acid sequence of SEQ ID NO:2 or a complement thereof; the probe specific for a target nucleic acid of HPIV-1 comprises a nucleic acid sequence of SEQ ID NO:3 or a complement thereof; and / or (2) a set of primers and probes specific for a target nucleic acid of HPIV-2. (2) a set of primers and a probe specific for a target nucleic acid of HPIV-2, wherein the set of primers specific for a target nucleic acid of HPIV-2 comprises a first primer comprising a nucleic acid sequence of SEQ ID NO:4 or a complement thereof, and a second primer comprising a nucleic acid sequence of SEQ ID NO:5 or a complement thereof; the probe specific for a target nucleic acid of HPIV-2 comprises a nucleic acid sequence of SEQ ID NO:6 or SEQ ID NO:7, or a complement thereof; and / or (3) a set of primers and a probe specific for a target nucleic acid of HPIV-3, wherein the set of primers specific for a target nucleic acid of HPIV-3 comprises a first primer comprising a nucleic acid sequence of SEQ ID NO:8 or SEQ ID NO:15, or a complement thereof, and a second primer comprising a nucleic acid sequence of SEQ ID NO:9 or SEQ ID NO:16, or a complement thereof; the probe specific for a target nucleic acid of HPIV-3 comprises a nucleic acid sequence of SEQ ID NO:10 or SEQ ID NO:17, or a complement thereof;and / or (4) a set of primers and a probe specific for a target nucleic acid of HPIV-4, wherein the set of primers specific for a target nucleic acid of HPIV-4 comprises a first primer comprising the nucleic acid sequence of SEQ ID NO:11 or SEQ ID NO:18, or a complement thereof, and a second primer comprising the nucleic acid sequence of SEQ ID NO:12 or SEQ ID NO:19, or a complement thereof; and the probe specific for a target nucleic acid of HPIV-4 comprises the set of primers and a probe specific for a target nucleic acid of HPIV-4 comprising the nucleic acid sequence of SEQ ID NO:13 or SEQ ID NO:14, or a complement thereof. In another embodiment, the sample is a biological sample. In another embodiment, the biological sample is whole blood, a respiratory specimen, a nasopharyngeal sample, urine, a fecal specimen, a blood specimen, plasma, a skin swab, a nasal swab, a wound swab, a blood culture, a skin, and a soft tissue infection. In another embodiment, the biological sample is a nasopharyngeal sample. In another embodiment, one or more of the probes are labeled. In another embodiment, one or more of the probes are labeled with a donor fluorescent moiety and a corresponding acceptor moiety;

[0016] Another aspect is directed to a kit for simultaneous detection of HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 target nucleic acids in a sample, when HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 target nucleic acids are present in the sample, the kit comprising amplification reagents and detection reagents, wherein the amplification reagents and detection reagents comprise: (i) a DNA polymerase; (ii) nucleotide monomers; and (iii) one or more sets of primers and one or more probes, wherein the one or more sets of primers and The one or more probes include: (1) a primer set and probe specific for a target nucleic acid of HPIV-1, where the primer set includes a first primer comprising a nucleic acid sequence of SEQ ID NO:1 or a complement thereof, and a second primer comprising a nucleic acid sequence of SEQ ID NO:2 or a complement thereof; the probe includes a nucleic acid sequence of SEQ ID NO:3 or a complement thereof; (2) a primer set and probe specific for a target nucleic acid of HPIV-2, where the primer set includes a first primer comprising a nucleic acid sequence of SEQ ID NO:1 or a complement thereof, and a second primer comprising a nucleic acid sequence of SEQ ID NO:2 or a complement thereof; (2) a set of primers and probes specific for a target nucleic acid of HPIV-2, the probe comprising a nucleic acid sequence of SEQ ID NO:6 or SEQ ID NO:7, or a complement thereof; (3) a set of primers and probes specific for a target nucleic acid of HPIV-3, the set of primers comprising a first primer comprising a nucleic acid sequence of SEQ ID NO:8 or SEQ ID NO:15, or a complement thereof, and a second primer comprising a nucleic acid sequence of SEQ ID NO:9 or SEQ ID NO:16, or a complement thereof; a set of primers and probes specific for a target nucleic acid of HPIV-3, the probe comprising a nucleic acid sequence of SEQ ID NO:10 or SEQ ID NO:17, or a complement thereof; and (4) a set of primers and probes specific for a target nucleic acid of HPIV-4, the set of primers comprising a first primer comprising a nucleic acid sequence of SEQ ID NO:11 or SEQ ID NO:18, or a complement thereof, and a second primer comprising a nucleic acid sequence of SEQ ID NO:12 or SEQ ID NO:19, or a complement thereof;The probe comprises a set of primers and a probe specific for a target nucleic acid of HPIV-4 comprising the nucleic acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14, or a complement thereof. In another embodiment, the sample is a biological sample. In another embodiment, the biological sample is whole blood, a respiratory specimen, a nasopharyngeal sample, a urine, a fecal specimen, a blood specimen, plasma, a skin swab, a nasal swab, a wound swab, a blood culture, skin, and a soft tissue infection. In another embodiment, the biological sample is a nasopharyngeal sample. In another embodiment, one or more of the probes are labeled. In another embodiment, one or more of the probes are labeled with a donor fluorescent moiety and a corresponding acceptor moiety;

[0017] Another embodiment is a method for detecting HPIV-1 in a sample, comprising: (a) performing an amplification step, if one or more target nucleic acids of HPIV-1 are present in the sample, comprising contacting the sample with one or more sets of primers to generate amplification products of the HPIV-1 target nucleic acids; (b) performing a hybridization step, if one or more target nucleic acids of HPIV-1 are present in the sample, comprising contacting one or more probes with the amplification products of the HPIV-1 target nucleic acids; and (c) detecting the presence or absence of the amplification products of the HPIV-1 target nucleic acids, wherein the presence of the amplification products of the HPIV-1 target nucleic acids indicates the presence or absence of an HPIV-1 target nucleic acid in the sample. detecting the presence or absence of an amplification product of the HPIV-1 target nucleic acid, wherein the absence of an amplification product of the HPIV-1 target nucleic acid indicates the presence of HPIV-1 from the sample; wherein the one or more sets of primers and the one or more probes comprise a set of primers and a probe specific for the HPIV-1 target nucleic acid, wherein the set of primers specific for the HPIV-1 target nucleic acid comprises a first primer comprising the nucleic acid sequence of SEQ ID NO:1 or a complement thereof, and a second primer comprising the nucleic acid sequence of SEQ ID NO:2 or a complement thereof; and the probe specific for the HPIV-1 target nucleic acid comprises the nucleic acid sequence of SEQ ID NO:3 or a complement thereof.Another embodiment is a method for detecting HPIV-2 in a sample comprising: (a) performing an amplification step, if one or more target nucleic acids of HPIV-2 are present in the sample, comprising contacting the sample with one or more sets of primers to generate amplification products of the HPIV-2 target nucleic acids; (b) performing a hybridization step, if one or more target nucleic acids of HPIV-2 are present in the sample, comprising contacting one or more probes with the amplification products of the HPIV-2 target nucleic acids; and (c) detecting the presence or absence of the amplification products of the HPIV-2 target nucleic acids, wherein the presence of the amplification products of the HPIV-2 target nucleic acids indicates the presence or absence of HPIV-2 in the sample. and detecting the presence or absence of an amplification product of the HPIV-2 target nucleic acid, wherein the absence of an amplification product of the HPIV-2 target nucleic acid indicates the absence of HPIV-2 from the sample; the one or more sets of primers and the one or more probes comprise a set of primers and a probe specific for the HPIV-2 target nucleic acid, wherein the set of primers specific for the HPIV-2 target nucleic acid comprises a first primer comprising the nucleic acid sequence of SEQ ID NO:4 or a complement thereof, and a second primer comprising the nucleic acid sequence of SEQ ID NO:5 or a complement thereof; and the probe specific for the HPIV-2 target nucleic acid comprises the nucleic acid sequence of SEQ ID NO:6 or SEQ ID NO:7 or a complement thereof.Another embodiment is a method for detecting HPIV-3 in a sample comprising: (a) performing an amplification step, if one or more target nucleic acids of HPIV-3 are present in the sample, comprising contacting the sample with one or more sets of primers to generate amplification products of the HPIV-3 target nucleic acids; (b) performing a hybridization step, if one or more target nucleic acids of HPIV-3 are present in the sample, comprising contacting one or more probes with the amplification products of the HPIV-3 target nucleic acids; and (c) detecting the presence or absence of the amplification products of the HPIV-3 target nucleic acids, wherein the presence of the amplification products of the HPIV-3 target nucleic acids indicates the presence of HPIV-3 in the sample, and detecting the presence or absence of an amplification product of the HPIV-3 target nucleic acid, wherein the absence of an amplification product of the HPIV-3 target nucleic acid indicates the absence of HPIV-3 from the sample; the one or more sets of primers and the one or more probes comprise a set of primers and a probe specific for the HPIV-3 target nucleic acid, wherein the set of primers specific for the HPIV-3 target nucleic acid comprises a first primer comprising the nucleic acid sequence of SEQ ID NO:8 or SEQ ID NO:15, or a complement thereof, and a second primer comprising the nucleic acid sequence of SEQ ID NO:9 or SEQ ID NO:16, or a complement thereof; and the probe specific for the HPIV-3 target nucleic acid comprises the nucleic acid sequence of SEQ ID NO:10 or SEQ ID NO:17, or a complement thereof.Another embodiment is a method for detecting HPIV-4 in a sample, comprising: (a) performing an amplification step, if one or more target nucleic acids of HPIV-4 are present in the sample, comprising contacting the sample with one or more sets of primers to generate amplification products of the HPIV-4 target nucleic acids; (b) performing a hybridization step, if one or more target nucleic acids of HPIV-4 are present in the sample, comprising contacting one or more probes with the amplification products of the HPIV-4 target nucleic acids; and (c) detecting the presence or absence of the amplification products of the HPIV-4 target nucleic acids, wherein the presence of the amplification products of the HPIV-4 target nucleic acids indicates the presence of HPIV-4 in the sample, and detecting the presence or absence of an amplification product of the HPIV-4 target nucleic acid, where the absence of an amplification product of the HPIV-4 target nucleic acid indicates the absence of HPIV-4 from the sample; wherein the one or more sets of primers and the one or more probes comprise a set of primers and a probe specific for the HPIV-4 target nucleic acid, where the set of primers specific for the HPIV-4 target nucleic acid comprises a first primer comprising the nucleic acid sequence of SEQ ID NO: 11 or SEQ ID NO: 18, or a complement thereof, and a second primer comprising the nucleic acid sequence of SEQ ID NO: 12 or SEQ ID NO: 19, or a complement thereof; and the probe specific for the HPIV-3 target nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14, or a complement thereof. Another related embodiment is directed to methods of detecting HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 in a sample, comprising performing these methods. In another embodiment, the sample is a biological sample. In another embodiment, the biological sample is whole blood, a respiratory specimen, a nasopharyngeal sample, a urine, a fecal specimen, a blood specimen, plasma, a skin swab, a nasal swab, a wound swab, a blood culture, skin, and a soft tissue infection. In another embodiment, the biological sample is a nasopharyngeal sample. In another embodiment, one or more probes are labeled. In another embodiment, one or more probes are labeled with a donor fluorescent moiety and a corresponding acceptor moiety.

[0018] Other aspects provide oligonucleotides comprising or consisting of a sequence of nucleotides selected from SEQ ID NOs: 1-19 or a complement thereof, having 100 or fewer nucleotides. In another aspect, the disclosure provides oligonucleotides comprising a nucleic acid having at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90% or 95%, etc.) to one of SEQ ID NOs: 1-19 or a complement thereof, the oligonucleotide having 100 or fewer nucleotides. Generally, these oligonucleotides can be primer nucleic acids, probe nucleic acids, etc. in these embodiments. In certain of these embodiments, the oligonucleotides have 40 or fewer nucleotides (e.g., 35 or fewer nucleotides, 30 or fewer nucleotides, 25 or fewer nucleotides, 20 or fewer nucleotides, 15 or fewer nucleotides, etc.). In some embodiments, the oligonucleotides comprise at least one modified nucleotide, e.g., to alter nucleic acid hybridization stability compared to unmodified nucleotides. The oligonucleotides optionally comprise at least one label and / or optionally at least one quencher moiety. In some embodiments, the oligonucleotides comprise at least one conservatively modified mutation. "Conservatively modified variations" or simply "conservative variations" of a particular nucleic acid sequence refer to nucleic acids that encode identical or essentially identical amino acid sequences, or, where the nucleic acid does not encode an amino acid sequence, essentially identical sequences. Those of skill in the art will recognize that individual substitutions, deletions, or additions that alter, add, or delete a single nucleotide or a small percentage of nucleotides (typically less than 5%, more typically less than 4%, 2%, or 1%) in an encoded sequence are "conservatively modified variations" where the alteration results in the deletion of an amino acid, the addition of an amino acid, or the substitution of an amino acid with a chemically similar amino acid.

[0019] In one aspect, the amplification can use a polymerase enzyme with 5' to 3' nuclease activity. Thus, the donor fluorescent moiety and the acceptor moiety, e.g., a quencher, can be within 5 to 20 nucleotides (e.g., 7 or 10 nucleotides) of each other along the length of the probe. In other aspects, the probe includes a nucleic acid sequence that allows for the formation of a secondary structure. The formation of such a secondary structure can result in spatial proximity between the first fluorescent moiety and the second fluorescent moiety. According to this method, the second fluorescent moiety on the probe can be a quencher.

[0020] The present disclosure also provides methods for detecting the presence or absence of HPIV (including HPIV1-4) or HPIV (including HPIV1-4) nucleic acids in a biological sample from an individual. These methods can be used, for example, to detect the presence or absence of HPIV (including HPIV1-4) nucleic acids in plasma for use in blood screening and diagnostic tests. Moreover, one skilled in the art can use the same tests to evaluate urine and other types of samples to detect and / or quantify HPIV (including HPIV1-4) nucleic acids. Such methods generally include performing at least one cycling step including an amplification step and a dye-binding step. Typically, the amplification step includes contacting the sample with multiple pairs of oligonucleotide primers to generate one or more amplification products if nucleic acid molecules are present in the sample, and the dye-binding step includes contacting the amplification products with a double-stranded DNA-binding dye. Such methods also include detecting the presence or absence of binding of a double-stranded DNA binding dye to the amplification product, the presence of binding indicating the presence of HPIV (including HPIV1-4) nucleic acid in the sample, and the absence of binding indicating the absence of HPIV (including HPIV1-4) nucleic acid in the sample. An exemplary double-stranded DNA binding dye is ethidium bromide. Other nucleic acid binding dyes include DAPI, Hoechst dyes, PicoGreen®, RiboGreen®, OliGreen®, and cyanine dyes such as YO-YO® and SYBR® Green. Additionally, such methods can include determining the melting temperature between the amplification product and the double-stranded DNA binding dye, where the melting temperature confirms the presence or absence of HPIV (including HPIV1-4) nucleic acid.

[0021] In a further aspect, a kit is provided for detecting and / or quantifying one or more nucleic acids of HPIV (including HPIV1-4). The kit may include one or more sets of primers specific for amplifying a gene target; and one or more detectable oligonucleotide probes specific for detecting the amplification product.

[0022] In one aspect, the kit can include probes that are already labeled with donor and corresponding acceptor moieties, such as other fluorescent moieties or dark quenchers, or can include fluorophore moieties for labeling the probes. The kit can also include nucleoside triphosphates, a nucleic acid polymerase, and buffers necessary for the function of the nucleic acid polymerase. The kits can also include inserts and instructions for using the primers, probes, and fluorophore moieties to detect the presence or absence of HPIV (including HPIV1-4) nucleic acid in a sample.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present subject matter, suitable methods and materials are described below. Furthermore, the materials, methods, and examples are merely illustrative and are not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0024] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will become apparent from the drawings and detailed description, as well as the claims.

[0025] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0026] [Figure 1]Figure 1 shows the different probe dye labels for the various HPIV1-4 targets used in the assay, e.g., HEX dye is dedicated to probe HPIV1, COU dye is dedicated to probe HPIV2, JA270 dye is dedicated to probe HPIV3, FAM dye is dedicated to probe HPIV4, and Cy5.5 dye is dedicated as an internal standard (GIC). [Diagram 2] FIG. 2 shows the primer sets and probe sequences for each assay for each of the four HPIV targets (HPIV1-4). [Diagram 3] FIG. 3 shows the alignment of a set of primers (SEQ ID NOs: 1 and 2) and a probe (SEQ ID NO: 3) for the amplification and detection of an HPIV1 target. [Figure 4] FIG. 4 shows the alignment of a set of primers (SEQ ID NOs: 4 and 5) and a probe (SEQ ID NOs: 6 and 7) for the amplification and detection of an HPIV2 target. [Diagram 5] FIG. 5 shows the alignment of a set of primers (SEQ ID NOs: 8 and 9) and a probe (SEQ ID NO: 10) for amplification and detection of an HPIV3 target. [Figure 6] FIG. 6 shows the alignment of a set of primers (SEQ ID NOs: 11 and 12) and probes (SEQ ID NOs: 13 and 14) for amplification and detection of HPIV4 targets. [Figure 7A] FIG. 7A shows PCR growth curves of a dilution series of HPIV1 IVT transcripts described in Example 2 (HPIV1 oligonucleotides including SEQ ID NOs: 1 to 3 were used). [Figure 7B] FIG. 7B shows the efficiency of the HPIV1 assay described in Example 2 (using HPIV1 oligonucleotides including SEQ ID NOs: 1-3). [Figure 8A] FIG. 8A shows a dilution series PCR growth curve of the performance of the HPIV2 assay described in Example 3 (using HPIV2 oligonucleotides including SEQ ID NOs: 4, 5, and 7). [Figure 8B] FIG. 8B shows the efficiency of the HPIV2 assay described in Example 3 (using HPIV2 oligonucleotides including SEQ ID NOs: 4, 5, and 7). [Figure 8C] FIG. 8C shows primer and probe concentrations under three different test conditions described in Example 3 (using HPIV2 oligonucleotides including SEQ ID NOs: 4, 5, and 7). [Figure 8D] FIG. 8D shows the growth curves of the performance of the HPIV2 assay under three different conditions for various primer and probe concentrations, demonstrating that increasing primer and probe concentrations improve the signal. [Figure 9A] FIG. 9A shows PCR growth curves of a dilution series of the performance of the HPIV3 assay described in Example 4 (using HPIV3 oligonucleotides including SEQ ID NOs: 8-10). [Figure 9B] FIG. 9B shows the efficiency of the HPIV3 assay described in Example 4 (using HPIV3 oligonucleotides including SEQ ID NOs:8-10). [Figure 10A] FIG. 10A shows PCR growth curves of a dilution series of the performance of the HPIV4 assay described in Example 5 (using HPIV4 oligonucleotides including SEQ ID NOs: 11-13). [Figure 10B] FIG. 10B shows the efficiency of the HPIV4 assay described in Example 5 (using HPIV4 oligonucleotides including SEQ ID NOs: 11-13). [Figure 11A] Figures 11A and 11B show growth curves and data, respectively, of the ability of HPIV1-4 primers and probes to simultaneously amplify and detect HPIV1-4 target nucleic acid from viral eluates in a multiplexed manner, as described in Example 6, using the following oligonucleotides: HPIV1 (SEQ ID NOs: 1-3), HPIV2 (SEQ ID NOs: 4, 5, and 7), HPIV3 (SEQ ID NOs: 8-10), and HPIV4 (SEQ ID NOs: 11-13). [Figure 11B]Figures 11A and 11B show growth curves and data, respectively, of the ability of HPIV1-4 primers and probes to simultaneously amplify and detect HPIV1-4 target nucleic acid from viral eluates in a multiplexed manner, as described in Example 6, using the following oligonucleotides: HPIV1 (SEQ ID NOs: 1-3), HPIV2 (SEQ ID NOs: 4, 5, and 7), HPIV3 (SEQ ID NOs: 8-10), and HPIV4 (SEQ ID NOs: 11-13). [Figure 12A] FIG. 12A shows the composition of the artificial nasopharyngeal simulated clinical samples used in Example 7, which were used to test HPIV1-4 oligonucleotides for detecting HPIV1 (sequence numbers 1-3), HPIV2 (sequence numbers 4, 5, and 7), HPIV3 (sequence numbers 8-10), and HPIV4 (sequence numbers 11-13). [Figure 12B] Figures 12B (HPIV1), 12C (HPIV2), 12D (HPIV3), and 12E (HPIV4) show PCR growth curves of dilution series of HPIV1-4 multiplex assay performance on artificial nasopharyngeal simulated clinical samples. Figures 12B-12D demonstrate that HPIV1-4 oligonucleotides can simultaneously detect target HPIV1-4 nucleic acids from artificial nasopharyngeal simulated clinical samples in a multiplex setting. [Figure 12C] Figures 12B (HPIV1), 12C (HPIV2), 12D (HPIV3), and 12E (HPIV4) show PCR growth curves of dilution series of HPIV1-4 multiplex assay performance on artificial nasopharyngeal simulated clinical samples. Figures 12B-12D demonstrate that HPIV1-4 oligonucleotides can simultaneously detect target HPIV1-4 nucleic acids from artificial nasopharyngeal simulated clinical samples in a multiplex setting. [Figure 12D] Figures 12B (HPIV1), 12C (HPIV2), 12D (HPIV3), and 12E (HPIV4) show PCR growth curves of dilution series of HPIV1-4 multiplex assay performance on artificial nasopharyngeal simulated clinical samples. Figures 12B-12D demonstrate that HPIV1-4 oligonucleotides can simultaneously detect target HPIV1-4 nucleic acids from artificial nasopharyngeal simulated clinical samples in a multiplex setting. [Figure 12E] Figures 12B (HPIV1), 12C (HPIV2), 12D (HPIV3), and 12E (HPIV4) show PCR growth curves of dilution series of HPIV1-4 multiplex assay performance on artificial nasopharyngeal simulated clinical samples. Figures 12B-12D demonstrate that HPIV1-4 oligonucleotides can simultaneously detect target HPIV1-4 nucleic acids from artificial nasopharyngeal simulated clinical samples in a multiplex setting. [Figure 13A] Figures 13A-D show that HPIV1-4 primers and probes simultaneously and specifically detect target HPIV1-4 nucleic acids from viral eluates in an HPIV1-4 multiplex real-time PCR assay as described in Example 8. HPIV1-4 oligonucleotides were tested using primers / probes to detect HPIV1 (SEQ ID NOs: 1-3), HPIV2 (SEQ ID NOs: 4, 5, and 7), HPIV3 (SEQ ID NOs: 8-10), and HPIV4 (SEQ ID NOs: 11-13). HPIV1-4 oligonucleotides were tested under multiplex conditions such that samples were exposed to all oligonucleotides for HPIV1-4 simultaneously. Results are shown in Figures 13A (HPIV1), 13B (HPIV2), 13C (HPIV3), and 13D (HPIV4), which show dilution series PCR growth curves of the performance of the HPIV1-4 assay. These results demonstrate that the HPIV1-4 oligonucleotides are specific for their respective intended targets. As can be seen in Figures 13A-13D, no cross-reactivity was observed when these viral eluates were used in unintended channels for specific HPIV types. [Figure 13B]Figures 13A-D show that HPIV1-4 primers and probes simultaneously and specifically detect target HPIV1-4 nucleic acids from viral eluates in an HPIV1-4 multiplex real-time PCR assay as described in Example 8. HPIV1-4 oligonucleotides were tested using primers / probes to detect HPIV1 (SEQ ID NOs: 1-3), HPIV2 (SEQ ID NOs: 4, 5, and 7), HPIV3 (SEQ ID NOs: 8-10), and HPIV4 (SEQ ID NOs: 11-13). HPIV1-4 oligonucleotides were tested under multiplex conditions such that samples were exposed to all oligonucleotides for HPIV1-4 simultaneously. Results are shown in Figures 13A (HPIV1), 13B (HPIV2), 13C (HPIV3), and 13D (HPIV4), which show dilution series PCR growth curves of the performance of the HPIV1-4 assay. These results demonstrate that the HPIV1-4 oligonucleotides are specific for their respective intended targets. As can be seen in Figures 13A-13D, no cross-reactivity was observed when these viral eluates were used in unintended channels for specific HPIV types. [Figure 13C]Figures 13A-D show that HPIV1-4 primers and probes simultaneously and specifically detect target HPIV1-4 nucleic acids from viral eluates in an HPIV1-4 multiplex real-time PCR assay as described in Example 8. HPIV1-4 oligonucleotides were tested using primers / probes to detect HPIV1 (SEQ ID NOs: 1-3), HPIV2 (SEQ ID NOs: 4, 5, and 7), HPIV3 (SEQ ID NOs: 8-10), and HPIV4 (SEQ ID NOs: 11-13). HPIV1-4 oligonucleotides were tested under multiplex conditions such that samples were exposed to all oligonucleotides for HPIV1-4 simultaneously. Results are shown in Figures 13A (HPIV1), 13B (HPIV2), 13C (HPIV3), and 13D (HPIV4), which show dilution series PCR growth curves of the performance of the HPIV1-4 assay. These results demonstrate that the HPIV1-4 oligonucleotides are specific for their respective intended targets. As can be seen in Figures 13A-13D, no cross-reactivity was observed when these viral eluates were used in unintended channels for specific HPIV types. [Figure 13D]Figures 13A-D show that HPIV1-4 primers and probes simultaneously and specifically detect target HPIV1-4 nucleic acids from viral eluates in an HPIV1-4 multiplex real-time PCR assay as described in Example 8. HPIV1-4 oligonucleotides were tested using primers / probes to detect HPIV1 (SEQ ID NOs: 1-3), HPIV2 (SEQ ID NOs: 4, 5, and 7), HPIV3 (SEQ ID NOs: 8-10), and HPIV4 (SEQ ID NOs: 11-13). HPIV1-4 oligonucleotides were tested under multiplex conditions such that samples were exposed to all oligonucleotides for HPIV1-4 simultaneously. Results are shown in Figures 13A (HPIV1), 13B (HPIV2), 13C (HPIV3), and 13D (HPIV4), which show dilution series PCR growth curves of the performance of the HPIV1-4 assay. These results demonstrate that the HPIV1-4 oligonucleotides are specific for their respective intended targets. As can be seen in Figures 13A-13D, no cross-reactivity was observed when these viral eluates were used in unintended channels for specific HPIV types. [Figure 14A] 14A and 14B show PCR growth curves of the performance of the HPIV1-4 multiplex assay on samples known to be negative for any and all of HPIV1-4, as described in Example 9. HPIV1-4 oligonucleotides were tested using primers / probes to detect HPIV1 (SEQ ID NOs: 1-3), HPIV2 (SEQ ID NOs: 4, 5, and 7), HPIV3 (SEQ ID NOs: 8-10), and HPIV4 (SEQ ID NOs: 11-13). HPIV1-4 oligonucleotides were tested under multiplex conditions such that samples were exposed to all oligonucleotides for HPIV1-4 simultaneously. These studies were designed to test the specificity of HPIV1-4 oligonucleotides for HPIV1-4 negative eluates. The results, as shown in FIG. 14A and FIG. 14B, show no amplification in any channel using HPIV1-4 oligonucleotides on nasopharyngeal eluates known to be negative for HPIV1-4. [Figure 14B] 14A and 14B show PCR growth curves of the performance of the HPIV1-4 multiplex assay on samples known to be negative for any and all of HPIV1-4, as described in Example 9. HPIV1-4 oligonucleotides were tested using primers / probes to detect HPIV1 (SEQ ID NOs: 1-3), HPIV2 (SEQ ID NOs: 4, 5, and 7), HPIV3 (SEQ ID NOs: 8-10), and HPIV4 (SEQ ID NOs: 11-13). HPIV1-4 oligonucleotides were tested under multiplex conditions such that samples were exposed to all oligonucleotides for HPIV1-4 simultaneously. These studies were designed to test the specificity of HPIV1-4 oligonucleotides for HPIV1-4 negative eluates. The results, as shown in FIG. 14A and FIG. 14B, show no amplification in any channel using HPIV1-4 oligonucleotides on nasopharyngeal eluates known to be negative for HPIV1-4. [Figure 15] FIG. 15 shows the primer sets and probe sequences for each assay for each of the four HPIV targets (HPIV1-4). [Figure 16A] FIG. 16A shows PCR growth curves of a dilution series of HPIV1 IVT transcripts described in Example 10 (using HPIV1 oligonucleotides including SEQ ID NOs: 1-3). [Figure 16B] FIG. 16B shows the efficiency of the HPIV1 assay described in Example 10 (using HPIV1 oligonucleotides comprising SEQ ID NOs: 1-3). [Figure 16C] FIG. 16C shows PCR growth curves of a dilution series of HPIV2 IVT transcripts described in Example 10 (using HPIV2 oligonucleotides including SEQ ID NOs: 4-6). [Figure 16D] FIG. 16D shows the efficiency of the HPIV2 assay described in Example 10 (using HPIV2 oligonucleotides containing SEQ ID NOs: 4-6). [Figure 16E]FIG. 16E shows PCR growth curves of a dilution series of HPIV3 IVT transcripts described in Example 10 (using HPIV3 oligonucleotides including SEQ ID NOs: 15-17). [Figure 16F] FIG. 16F shows the efficiency of the HPIV3 assay described in Example 10 (using HPIV3 oligonucleotides including SEQ ID NOs: 15-17). [Figure 16G] FIG. 16G shows PCR growth curves of a dilution series of HPIV4 IVT transcripts described in Example 10 (using HPIV4 oligonucleotides including SEQ ID NOs: 11, 13, 18 and 19). [Figure 16H] FIG. 16H shows the efficiency of the HPIV4 assay described in Example 10 (using HPIV4 oligonucleotides including SEQ ID NOs: 11, 13, 18, and 19). [Figure 17A] FIG. 17A shows growth curves of the ability of HPIV1-4 primers and probes to simultaneously amplify and detect HPIV1-4 target nucleic acids in a single-stranded format from viral eluates using the following oligonucleotides: HPIV1 (SEQ ID NOs: 1-3), HPIV2 (SEQ ID NOs: 4-6), HPIV3 (SEQ ID NOs: 15-17), and HPIV4 (SEQ ID NOs: 11, 13, 18, and 19), as described in Example 11. [Figure 17B] FIG. 17B shows growth curves of the ability of HPIV1-4 primers and probes to simultaneously amplify and detect HPIV1-4 target nucleic acids in the presence of other respiratory virus targets to test for exclusivity, using the following oligonucleotides: HPIV1 (SEQ ID NOs: 1-3), HPIV2 (SEQ ID NOs: 4-6), HPIV3 (SEQ ID NOs: 15-17), and HPIV4 (SEQ ID NOs: 11, 13, 18, and 19), as described in Example 11. [Figure 18A] Figure 18A shows the composition of the artificial nasopharynx matrix eluate used in Example 12. Example 12 describes HPIV1-4 oligonucleotides for detecting HPIV1 (SEQ ID NOs: 1-3), HPIV2 (SEQ ID NOs: 4-6), HPIV3 (SEQ ID NOs: 15-17), and HPIV4 (SEQ ID NOs: 11, 13, 18, and 19) that were tested in artificial nasopharynx simulated clinical samples. [Figure 18B] Figures 18B (HPIV1), 18C (HPIV2), 18D (HPIV3), and 18E (HPIV4) show PCR growth curves of dilution series of HPIV1-4 multiplex assay performance in artificial nasopharyngeal simulated clinical samples. Figures 18B, 18C, 18D, and 18E demonstrate that HPIV1-4 oligonucleotides can simultaneously detect target HPIV1-4 nucleic acids from artificial nasopharyngeal simulated clinical samples in a multiplex setting. [Figure 18C] Figures 18B (HPIV1), 18C (HPIV2), 18D (HPIV3), and 18E (HPIV4) show PCR growth curves of dilution series of HPIV1-4 multiplex assay performance in artificial nasopharyngeal simulated clinical samples. Figures 18B, 18C, 18D, and 18E demonstrate that HPIV1-4 oligonucleotides can simultaneously detect target HPIV1-4 nucleic acids from artificial nasopharyngeal simulated clinical samples in a multiplex setting. [Figure 18D] Figures 18B (HPIV1), 18C (HPIV2), 18D (HPIV3), and 18E (HPIV4) show PCR growth curves of dilution series of HPIV1-4 multiplex assay performance in artificial nasopharyngeal simulated clinical samples. Figures 18B, 18C, 18D, and 18E demonstrate that HPIV1-4 oligonucleotides can simultaneously detect target HPIV1-4 nucleic acids from artificial nasopharyngeal simulated clinical samples in a multiplex setting. [Figure 18E] Figures 18B (HPIV1), 18C (HPIV2), 18D (HPIV3), and 18E (HPIV4) show PCR growth curves of dilution series of HPIV1-4 multiplex assay performance in artificial nasopharyngeal simulated clinical samples. Figures 18B, 18C, 18D, and 18E demonstrate that HPIV1-4 oligonucleotides can simultaneously detect target HPIV1-4 nucleic acids from artificial nasopharyngeal simulated clinical samples in a multiplex setting. [Figure 19A]Figures 19A, 19B, 19C, and 19D show that HPIV1-4 primers and probes simultaneously and specifically detect target HPIV1-4 nucleic acids from viral eluates in an HPIV1-4 multiplex real-time PCR assay as described in Example 13. HPIV1-4 oligonucleotides were tested using primers / probes to detect HPIV1 (SEQ ID NOs: 1-3), HPIV2 (SEQ ID NOs: 4-6), HPIV3 (SEQ ID NOs: 15-17), and HPIV4 (SEQ ID NOs: 11, 13, 18, and 19). HPIV1-4 oligonucleotides were tested under multiplex conditions such that samples were exposed to all oligonucleotides for HPIV1-4 simultaneously. Results are shown in Figures 19A (HPIV1), 19B (HPIV2), 19C (HPIV3), and 19D (HPIV4), which show PCR growth curves of a dilution series of the performance of the HPIV1-4 assay. These results demonstrate that the HPIV1-4 oligonucleotides are specific for their intended targets, and no cross-reactivity was observed when these viral eluates were used in unintended channels for specific HPIV types, as seen in Figures 19A, 19B, 19C, and 19D. [Figure 19B]Figures 19A, 19B, 19C, and 19D show that HPIV1-4 primers and probes simultaneously and specifically detect target HPIV1-4 nucleic acids from viral eluates in an HPIV1-4 multiplex real-time PCR assay as described in Example 13. HPIV1-4 oligonucleotides were tested using primers / probes to detect HPIV1 (SEQ ID NOs: 1-3), HPIV2 (SEQ ID NOs: 4-6), HPIV3 (SEQ ID NOs: 15-17), and HPIV4 (SEQ ID NOs: 11, 13, 18, and 19). HPIV1-4 oligonucleotides were tested under multiplex conditions such that samples were exposed to all oligonucleotides for HPIV1-4 simultaneously. Results are shown in Figures 19A (HPIV1), 19B (HPIV2), 19C (HPIV3), and 19D (HPIV4), which show PCR growth curves of a dilution series of the performance of the HPIV1-4 assay. These results demonstrate that the HPIV1-4 oligonucleotides are specific for their intended targets, and no cross-reactivity was observed when these viral eluates were used in unintended channels for specific HPIV types, as seen in Figures 19A, 19B, 19C, and 19D. [Figure 19C]Figures 19A, 19B, 19C, and 19D show that HPIV1-4 primers and probes simultaneously and specifically detect target HPIV1-4 nucleic acids from viral eluates in an HPIV1-4 multiplex real-time PCR assay as described in Example 13. HPIV1-4 oligonucleotides were tested using primers / probes to detect HPIV1 (SEQ ID NOs: 1-3), HPIV2 (SEQ ID NOs: 4-6), HPIV3 (SEQ ID NOs: 15-17), and HPIV4 (SEQ ID NOs: 11, 13, 18, and 19). HPIV1-4 oligonucleotides were tested under multiplex conditions such that samples were exposed to all oligonucleotides for HPIV1-4 simultaneously. Results are shown in Figures 19A (HPIV1), 19B (HPIV2), 19C (HPIV3), and 19D (HPIV4), which show PCR growth curves of a dilution series of the performance of the HPIV1-4 assay. These results demonstrate that the HPIV1-4 oligonucleotides are specific for their intended targets, and no cross-reactivity was observed when these viral eluates were used in unintended channels for specific HPIV types, as seen in Figures 19A, 19B, 19C, and 19D. [Figure 19D]Figures 19A, 19B, 19C, and 19D show that HPIV1-4 primers and probes simultaneously and specifically detect target HPIV1-4 nucleic acids from viral eluates in an HPIV1-4 multiplex real-time PCR assay as described in Example 13. HPIV1-4 oligonucleotides were tested using primers / probes to detect HPIV1 (SEQ ID NOs: 1-3), HPIV2 (SEQ ID NOs: 4-6), HPIV3 (SEQ ID NOs: 15-17), and HPIV4 (SEQ ID NOs: 11, 13, 18, and 19). HPIV1-4 oligonucleotides were tested under multiplex conditions such that samples were exposed to all oligonucleotides for HPIV1-4 simultaneously. Results are shown in Figures 19A (HPIV1), 19B (HPIV2), 19C (HPIV3), and 19D (HPIV4), which show PCR growth curves of a dilution series of the performance of the HPIV1-4 assay. These results demonstrate that the HPIV1-4 oligonucleotides are specific for their intended targets, and no cross-reactivity was observed when these viral eluates were used in unintended channels for specific HPIV types, as seen in Figures 19A, 19B, 19C, and 19D. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Diagnosis of HPIV (including HPIV1-4) infections by nucleic acid amplification provides a method for rapid, accurate, reliable, specific and sensitive detection and / or quantification of HPIV (including HPIV1-4) infections. Described herein are real-time PCR assays for detection and / or quantification of HPIV (including HPIV1-4) nucleic acids, including DNA and / or RNA, in non-biological or biological samples. Primers and probes for detection and / or quantification of HPIV (including HPIV1-4) are provided, as are articles of manufacture or kits containing such primers and probes. The increased specificity and sensitivity of real-time PCR for detection of HPIV (including HPIV1-4) compared to other methods, and improved features of real-time PCR, including sample containment, real-time detection and quantification of amplification products, make the implementation of this technology feasible for routine diagnosis of HPIV (including HPIV1-4) infections in clinical laboratories. Additionally, this technology can be used for blood screening as well as prognosis. This HPIV (including HPIV1-4) detection assay can also be multiplexed such that all of the oligonucleotides for detection and amplification of HPIV1-4 are added to a sample and the oligonucleotides for HPIV1-4, if present in the sample, can simultaneously amplify and detect each target nucleic acid in parallel. In this way, a single sample can be assayed for the presence of four different types of HPIV (HPIV1-4) in a single reaction and reaction vessel. Such multiplex assays are advantageous from a cost-saving standpoint and in situations where sample is limited.

[0028] The disclosure includes, for example, oligonucleotide primers and fluorescently labeled hydrolysis probes that hybridize to the HPIV (including HPIV1-4) genome to specifically identify HPIV (including HPIV1-4) using TaqMan® amplification and detection technology.

[0029] The disclosed methods may include performing at least one cycling step that includes amplifying one or more portions of a nucleic acid molecule genetic target from a sample using one or more primer pairs. As used herein, "HPIV primer" or "HPIV1-4 primer" refers to an oligonucleotide primer that specifically anneals to a nucleic acid sequence found in the HPIV (including HPIV1-4) genome and initiates DNA synthesis therefrom under appropriate conditions to generate a respective amplification product. Each of the HPIV (including HPIV1-4) primers discussed anneals to a target such that at least a portion of each amplification product contains a nucleic acid sequence corresponding to the target. If one or more nucleic acids are present in the sample, one or more amplification products are generated, and thus the presence of one or more amplification products indicates the presence of HPIV (including HPIV1-4) in the sample. The amplification product must contain a nucleic acid sequence complementary to one or more detectable probes for HPIV (including HPIV1-4). As used herein, "HPIV probe(s)" or "HPIV1-4 probe(s)" refer to oligonucleotide probes that specifically anneal to nucleic acid sequences found in the HPIV (including HPIV1-4) genome. Each cycling step includes an amplification step, a hybridization step, and a detection step, in which a sample is contacted with one or more detectable HPIV (including HPIV1-4) probes to detect the presence or absence of HPIV (including HPIV1-4) in the sample.

[0030] As used herein, the term "amplifying" refers to the process of synthesizing a nucleic acid molecule complementary to one or both strands of a template nucleic acid molecule (e.g., a nucleic acid molecule from the HPIV (including HPIV1-4) genome). Amplifying a nucleic acid molecule typically involves denaturing the template nucleic acid, annealing a primer to the template nucleic acid at a temperature below the melting temperature of the primer, and enzymatically extending from the primer to generate an amplification product. Amplification typically requires the presence of deoxyribonucleoside triphosphates, a DNA polymerase enzyme (e.g., Platinum® Taq), and appropriate buffers and / or cofactors (e.g., MgCl2 and / or KCl) for optimal activity of the polymerase enzyme.

[0031] The term "primer" as used herein is known to those skilled in the art and refers to oligomeric compounds, primarily oligonucleotides, but also modified oligonucleotides that are capable of "priming" DNA synthesis by a template-dependent DNA polymerase, i.e., for example, the 3' end of the oligonucleotide provides a free 3'-OH group to which a further "nucleotide" can be attached by a template-dependent DNA polymerase that establishes a 3' to 5' phosphodiester bond, thereby using a deoxynucleoside triphosphate, thereby releasing pyrophosphate.

[0032] The term "hybridizing" refers to the annealing of one or more probes to an amplification product. "Hybridization conditions" typically include a temperature below the melting temperature of the probe, but which avoids non-specific hybridization of the probe.

[0033] The term "5' to 3' nuclease activity" refers to the activity of a nucleic acid polymerase typically associated with nucleic acid chain synthesis whereby nucleotides are removed from the 5' end of a nucleic acid chain.

[0034] The term "thermostable polymerase" refers to a polymerase enzyme that is thermostable, i.e., that catalyzes the formation of primer extension products complementary to a template and does not irreversibly denature when exposed to high temperatures for the time required to effect denaturation of the double-stranded template nucleic acid. Generally, synthesis is initiated at the 3' end of each primer and proceeds in the 5' to 3' direction along the template strand. Thermostable polymerases have been isolated, for example, from Thermus flavus, T. ruber, T. thermophilus, T. aquaticus, T. lacteus, T. rubens, Bacillus stearothermophilus, and Methanothermus fervidus. Nonetheless, non-thermostable polymerases can also be used in PCR assays, provided the enzyme is replenished as needed.

[0035] The term "complement thereof" refers to a nucleic acid that is the same length as and exactly complementary to a given nucleic acid.

[0036] The term "extension" or "lengthening" when used in reference to a nucleic acid refers to when additional nucleotides (or other similar molecules) are incorporated into a nucleic acid. For example, a nucleic acid is optionally extended by a biocatalyst that incorporates a nucleotide, e.g., a polymerase that typically adds a nucleotide to the 3' end of the nucleic acid.

[0037] The terms "identical" or "percent identity" in the context of two or more nucleic acid sequences refer to two or more sequences or subsequences that are identical or have a certain percentage of identical nucleotides when compared and aligned for maximum correspondence, e.g., as determined using one of the sequence comparison algorithms available to those of skill in the art or by visual inspection. Exemplary algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST programs, see, e.g., Altschul et al. (1990) "Basic local alignment search tool" J. Mol. Biol. 215:403-410, Gish et al. (1993) "Identification of protein coding regions by database similarity search" Nature Genet. 3:266-272, Madden et al. (1996) "Applications of network BLAST server" Meth. Enzymol. 266:131-141, Altschul et al. (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs" Nucleic Acids Res. 25:3389-3402, and Zhang et al. (1997) "PowerBLAST: A new network BLAST application for interactive or automated sequence analysis and annotation" Genome Res. 7:649-656, each of which is incorporated herein by reference.

[0038] "Modified nucleotide" in the context of oligonucleotide refers to a change in which at least one nucleotide of an oligonucleotide sequence is replaced by a different nucleotide that provides the oligonucleotide with desired properties.Exemplary modified nucleotides that can be substituted in the oligonucleotides described herein include, for example, t-butylbenzyl, C5-methyl-dC, C5-ethyl-dC, C5-methyl-dU, C5-ethyl-dU, 2,6-diaminopurine, C5-propynyl-dC, C5-propynyl-dU, C7-propynyl-dA, C7-propynyl-dG, C5-propargylamino-dC, C5-propargylamino-dU ... Examples of modified nucleotides include 7-propargylamino-dA, C7-propargylamino-dG, 7-deaza-2-deoxy-xanthosine, pyrazolopyrimidine analogs, pseudo-dU, nitropyrrole, nitroindole, 2'-0-methylribo-U, 2'-0-methylribo-C, N4-ethyl-dC, N6-methyl-dA, 5-propynyl-dU, 5-propynyl-dC, 7-deaza-deoxyguanosine (deazaG(u-deaza)), and the like. Many other modified nucleotides that can be substituted in an oligonucleotide are mentioned herein or known in the art. In certain embodiments, the modified nucleotide substitution modifies the melting temperature (Tm) of the oligonucleotide compared to the melting temperature of the corresponding unmodified oligonucleotide. To further illustrate, in some embodiments, certain modified nucleotide substitutions can reduce non-specific nucleic acid amplification (e.g., minimize primer dimer formation, etc.), increase the yield of the intended target amplicon, and the like. Examples of these types of nucleic acid modifications are described, for example, in U.S. Patent No. 6,001,611, which is incorporated herein by reference. Other modified nucleotide substitutions may alter the stability of the oligonucleotide or provide other desirable characteristics.

[0039] Detection / quantification of HPIV (including HPIV1-4) target nucleic acid The present disclosure provides methods for detecting HPIV (including HPIV1-4), for example, by amplifying a portion of an HPIV (including HPIV1-4) nucleic acid sequence. Specifically, primers and probes for amplifying, detecting and / or quantitating HPIV (including HPIV1-4) nucleic acid molecular targets are provided by embodiments of the present disclosure.

[0040] For detection and / or quantification of HPIV (including HPIV1-4), primers and probes are provided for amplifying and detecting / quantifying HPIV (including HPIV1-4). HPIV (including HPIV1-4) nucleic acids other than those exemplified herein can also be used to detect HPIV (including HPIV1-4) in a sample. For example, functional variants can be assessed for specificity and / or sensitivity by those of skill in the art using routine methods. Exemplary functional variants can include, for example, one or more deletions, insertions, and / or substitutions in the HPIV (including HPIV1-4) nucleic acids disclosed herein.

[0041] More specifically, each of the oligonucleotide embodiments includes a nucleic acid having a sequence selected from SEQ ID NOs: 1-19, a substantially identical variant thereof having at least, e.g., 80%, 90%, or 95% sequence identity to one of SEQ ID NOs: 1-19, or the complement of SEQ ID NOs: 1-19 and variants thereof.

[0042] In one embodiment, HPIV (including HPIV1-4) primer sets and probes are used to provide for detection of HPIV (including HPIV1-4) in a biological sample suspected of containing HPIV (Table 1). The primer sets and probes can comprise or consist of primers and probes specific for HPIV (including HPIV1-4) nucleic acid sequences comprising or consisting of the nucleic acid sequences of SEQ ID NOs: 1-19. In another embodiment, the primers and probes for HPIV (including HPIV1-4) targets comprise or consist of functionally active variants of any of the primers and probes of SEQ ID NOs: 1-19.

[0043] Functionally active variants of any of the primers and / or probes of SEQ ID NOs: 1-19 can be identified by using the primers and / or probes in the disclosed methods. Functionally active variants of any of the primers and / or probes of SEQ ID NOs: 1-19 relate to primers and / or probes that provide similar or higher specificity and sensitivity compared to the respective sequences of SEQ ID NOs: 1-19 in the described methods or kits.

[0044] [Table 1]

[0045] A variant may vary from the sequence of SEQ ID NO: 1-19 by the addition, deletion or substitution of one or more nucleotides, for example, the addition, deletion or substitution of one or more nucleotides at the 5'-end and / or 3'-end of each of the sequences of SEQ ID NO: 1-19. As detailed above, the primer and / or probe may be chemically modified, i.e., the primer and / or probe may comprise modified nucleotides or non-nucleotide compounds. Thus, the probe (or primer) is a modified oligonucleotide. A "modified nucleotide" (or "nucleotide analog") differs from a natural "nucleotide" by some modification, but still consists of a base or base-like compound, a pentofuranosyl sugar or pentofuranosyl sugar-like compound, a phosphate moiety or a phosphate-like moiety, or a combination thereof. For example, a "label" may be attached to the base portion of a "nucleotide" to obtain a "modified nucleotide". A natural base in a "nucleotide" may be replaced, for example, with a 7-deazapurine, thereby also obtaining a "modified nucleotide". The terms "modified nucleotide" or "nucleotide analog" are used interchangeably in this application. A "modified nucleoside" (or "nucleoside analogue") differs from a naturally occurring nucleoside by some modification, as outlined above for "modified nucleotides" (or "nucleotide analogues").

[0046] Oligonucleotides, including modified oligonucleotides and oligonucleotide analogs, that amplify nucleic acid molecules encoding HPIV (including HPIV1-4) targets, e.g., nucleic acids encoding other portions of HPIV (including HPIV1-4), can be designed using computer programs such as OOLIGO (Molecular Biology Insights Inc. Cascade, Colo.). Important features in designing oligonucleotides to be used as amplification primers include, but are not limited to, an appropriate size of the amplification product to facilitate detection (e.g., by electrophoresis), similar melting temperatures for the members of the pair of primers, and the length of each primer (i.e., the primers must be long enough to anneal and initiate synthesis with sequence specificity, but not so long that fidelity is compromised during oligonucleotide synthesis). Typically, oligonucleotide primers are 8-50 nucleotides in length (e.g., 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50 nucleotides in length).

[0047] In addition to a set of primers, the method may use one or more probes to detect the presence or absence of HPIV (including HPIV1-4). The term "probe" refers to a synthetic or biologically produced nucleic acid (DNA or RNA) that contains a specific nucleotide sequence that allows it to specifically (i.e., preferentially) hybridize, by design or selection, to a "target nucleic acid," in this case HPIV (including HPIV1-4) (target) nucleic acid, under a defined, predetermined stringency. A "probe" can also be referred to as a "detection probe," which means that it detects a target nucleic acid.

[0048] In some embodiments, the described HPIV (including HPIV1-4) probes can be labeled with at least one fluorescent label. In one embodiment, the HPIV (including HPIV1-4) probes can be labeled with a donor fluorescent moiety, e.g., a fluorescent dye, and a corresponding acceptor moiety, e.g., a quencher. In one embodiment, the probe comprises or consists of a fluorescent moiety and the nucleic acid sequence comprises or consists of SEQ ID NOs: 3, 6, 7, 10, 13, 14, and / or 17.

[0049] The design of oligonucleotides used as probes can be performed in a similar manner to the design of primers. Embodiments can use a single probe or a pair of probes for detection of the amplification product. Depending on the embodiment, the probes used can include at least one label and / or at least one quencher moiety. Like primers, the probes usually have similar melting temperatures, and the length of each probe must be sufficient for sequence-specific hybridization to occur, but not so long that fidelity is reduced during synthesis. Oligonucleotide probes are generally 15-40 (e.g., 16, 18, 20, 21, 22, 23, 24, or 25) nucleotides in length.

[0050] The constructs can include vectors that include one of the HPIV (including HPIV1-4) primer and probe nucleic acid molecules (e.g., SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19), respectively. The constructs can be used, for example, as control template nucleic acid molecules. Vectors suitable for use are commercially available and / or produced by recombinant nucleic acid technology methods routine in the art. HPIV (including HPIV1-4) nucleic acid molecules can be obtained, for example, by chemical synthesis, by direct cloning from HPIV (including HPIV1-4), or by nucleic acid amplification.

[0051] Constructs suitable for use in the present methods typically include, in addition to an HPIV (including HPIV1-4) nucleic acid molecule (e.g., a nucleic acid molecule comprising one or more of the sequences of SEQ ID NOs: 1-19), a sequence encoding a selection marker (e.g., an antibiotic resistance gene) for selection of the desired construct and / or transformant, and an origin of replication. The choice of vector system will usually depend on several factors, including, but not limited to, the choice of host cell, replication efficiency, selectability, inducibility, and ease of recovery.

[0052] Constructs containing HPIV (including HPIV1-4) nucleic acid molecules can be propagated in host cells. The term host cell as used herein is meant to include prokaryotic and eukaryotic organisms, such as yeast, plant and animal cells. Prokaryotic hosts include E. coli, Salmonella typhimurium, Serratia marcescens and Bacillus subtilis. Eukaryotic hosts include yeasts such as S. cerevisiae, S. pombe, Pichia pastoris, mammalian cells such as COS cells or Chinese hamster ovary (CHO) cells, insect cells, and plant cells such as Arabidopsis thaliana and Nicotiana tabacum. The construct can be introduced into the host cell using any of the techniques commonly known to those skilled in the art. For example, calcium phosphate precipitation, electroporation, heat shock, lipofection, microinjection and viral-mediated nucleic acid transfer are common methods for introducing nucleic acid into the host cell. In addition, naked DNA can be delivered directly into the cell (see, for example, U.S. Patent Nos. 5,580,859 and 5,589,466).

[0053] Polymerase chain reaction (PCR) U.S. Patent Nos. 4,683,202, 4,683,195, 4,800,159 and 4,965,188 disclose conventional PCR techniques. PCR typically uses two oligonucleotide primers that bind to a selected nucleic acid template (e.g., DNA or RNA). Primers useful in some embodiments include oligonucleotides that can act as initiation points for nucleic acid synthesis within the described HPIV (including HPIV1-4) nucleic acid sequences (e.g., SEQ ID NOs: 1, 2, 4, 5, 8, 9, 11, 12, 15, 16, 18 and / or 19). Primers can be purified from restriction digests by conventional methods or can be produced synthetically. Primers are preferably single-stranded to maximize amplification efficiency, although primers may be double-stranded. Double-stranded primers are first denatured, i.e., treated to separate the strands. One method of denaturing double-stranded nucleic acids is by heating.

[0054] If the template nucleic acid is double-stranded, it is necessary to separate the two strands before it can be used as a template in PCR. Strand separation can be achieved by any suitable denaturing method, including physical, chemical, or enzymatic means. One method of separating the nucleic acid strands involves heating until the nucleic acid is predominantly denatured (e.g., greater than 50%, 60%, 70%, 80%, 90%, or 95% denatured). The heating conditions required to denature the template nucleic acid depend, for example, on the buffer salt concentration, as well as the length and nucleotide composition of the nucleic acid to be denatured, but typically range from about 90°C to about 105°C for a period of time, depending on the characteristics of the reaction, such as temperature and nucleic acid length. Denaturation is typically performed for about 30 seconds to 4 minutes (e.g., 1 minute to 2 minutes and 30 seconds, or 1.5 minutes).

[0055] When the double-stranded template nucleic acid has been denatured by heat, the reaction mixture is cooled to a temperature that promotes annealing of each primer to its target sequence. The annealing temperature is typically about 35°C to about 65°C, (e.g., about 40°C to about 60°C, about 45°C to about 50°C). The annealing time can be about 10 seconds to about 1 minute (e.g., about 20 seconds to about 50 seconds, about 30 seconds to about 40 seconds). The reaction mixture is then adjusted to a temperature that promotes or optimizes the activity of the polymerase, i.e., a temperature sufficient for extension to occur from the annealed primers to generate products complementary to the template nucleic acid. The temperature should be sufficient to synthesize an extension product from each primer annealed to the nucleic acid template, but not so high as to denature the extension product from its complementary template (e.g., the temperature for extension is generally in the range of about 40°C to about 80°C (e.g., about 50°C to about 70°C, about 60°C)). The extension time can be from about 10 seconds to about 5 minutes (eg, from about 30 seconds to about 4 minutes, from about 1 minute to about 3 minutes, from about 1 minute 30 seconds to about 2 minutes).

[0056] The genome of retroviruses or RNA viruses, or the mRNA produced by DNA viruses such as HPIV (including HPIV1-4), is composed of ribonucleic acid, or RNA. In such cases, the template nucleic acid, RNA, must first be transcribed into complementary DNA (cDNA) through the action of the enzyme reverse transcriptase. Using the RNA template and a short primer complementary to the 3' end of the RNA, reverse transcriptase directs the synthesis of first strand cDNA, which can then be used directly as a template for the polymerase chain reaction.

[0057] PCR assays can use HPIV (including HPIV1-4) nucleic acids, such as RNA or DNA (cDNA). The template nucleic acid need not be purified and can be a minor fraction of a complex mixture, such as HPIV (including HPIV1-4) nucleic acids contained in human cells. HPIV (including HPIV1-4) nucleic acid molecules can be extracted from biological samples by routine techniques, such as those described in Diagnostic Molecular Microbiology: Principles and Applications (Persing et al. (eds), 1993, American Society for Microbiology, Washington DC). Nucleic acids can be obtained from any number of sources, including plasmids or natural sources, including bacteria, yeast, viruses, organelles, or higher organisms such as plants or animals.

[0058] Oligonucleotide primers (e.g., SEQ ID NOs: 1, 2, 4, and 5) are combined with PCR reagents under reaction conditions conducive to primer extension. For example, a chain extension reaction typically contains 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 15 mM MgCl2, 0.001% (w / v) gelatin, 0.5-1.0 μg of denatured template DNA, 50 pmol of each oligonucleotide primer, 2.5 U of Taq polymerase, and 10% DMSO. Reactions typically contain 150-320 μM each of dATP, dCTP, dTTP, dGTP, or one or more analogs thereof.

[0059] The newly synthesized strands form double-stranded molecules that can be used in subsequent steps of the reaction. The steps of strand separation, annealing, and extension can be repeated as many times as necessary to generate a desired amount of amplification product corresponding to the target HPIV (including HPIV1-4) nucleic acid molecule. The limiting factors of the reaction are the amount of primers, thermostable enzyme, and nucleoside triphosphates present in the reaction. The cycling steps (i.e., denaturation, annealing, and extension) are preferably repeated at least once. For detection use, the number of cycling steps depends, for example, on the nature of the sample. If the sample is a complex mixture of nucleic acids, more cycling steps will be required to amplify the target sequence enough for detection. Generally, the cycling steps are repeated at least about 20 times, but may be repeated 40, 60, or 100 times.

[0060] Fluorescence Resonance Energy Transfer (FRET) FRET technology (see, e.g., U.S. Pat. Nos. 4,996,143, 5,565,322, 5,849,489, and 6,162,603) is based on the concept that when a donor fluorescent moiety and a corresponding acceptor fluorescent moiety are positioned within a certain distance of each other, energy transfer occurs between the two fluorescent moieties, which can be visualized or otherwise detected and / or quantified. Typically, the donor transfers energy to the acceptor when excited by light irradiation of a suitable wavelength. Typically, the acceptor re-emits the transferred energy in the form of light irradiation of a different wavelength. In certain systems, non-fluorescent energy can be transferred between the donor and acceptor moieties via a biomolecule that includes a substantially non-fluorescent donor moiety (see, e.g., U.S. Pat. No. 7,741,467).

[0061] In one example, an oligonucleotide probe can include a donor fluorescent moiety or dye (e.g., HEX or FAM) and a corresponding quencher (e.g., BlackHole Quencher™ (BHQ) (e.g., BHQ-2)) that may or may not be fluorescent and dissipates the transferred energy in a form other than light. When the probe is intact, energy transfer typically occurs between the donor and acceptor moieties such that the fluorescent emission from the donor fluorescent moiety is quenched by the acceptor moiety. During the extension step of the polymerase chain reaction, the probe bound to the amplification product is cleaved, for example, by the 5' to 3' nuclease activity of Taq polymerase such that the fluorescent emission of the donor fluorescent moiety is no longer quenched. Exemplary probes for this purpose are described, for example, in U.S. Patent Nos. 5,210,015, 5,994,056, and 6,171,785. Commonly used donor-acceptor pairs include the FAM-TAMRA pair. Commonly used quenchers are DABCYL and TAMRA. Commonly used dark quenchers include BlackHole Quencher™ (BHQ) (such as BHQ2), (Biosearch Technologies, Inc., Novato, Calif.), Iowa Black™ (Integrated DNA Tech., Inc., Coralville, Iowa), BlackBerry® Quencher 650 (BBQ-650) (Berry & Assoc, Dexta, Mich.).

[0062] In another example, two oligonucleotide probes, each containing a fluorescent moiety, can hybridize to the amplification product at specific positions determined by the complementarity of the oligonucleotide probes to the HPIV (including HPIV1-4) target nucleic acid sequence. When the oligonucleotide probes hybridize to the amplification product nucleic acid at the appropriate positions, a FRET signal is generated. Hybridization temperatures can range from about 35° C. to about 65° C. for about 10 seconds to about 1 minute.

[0063] Fluorescence analysis can be performed, for example, using a photon-counting epifluorescence microscope system (equipped with appropriate dichroic mirrors and filters to monitor a particular range of fluorescence emission), a photon-counting photomultiplier system, or a fluorometer. Excitation to initiate energy transfer or to allow direct detection of the fluorophore can be performed using an argon ion laser, a high intensity mercury (Hg) arc lamp, a xenon lamp, a fiber optic light source, or other high intensity light source appropriately filtered for excitation of the desired range.

[0064] As used herein with respect to a donor moiety and a corresponding acceptor moiety, "corresponding" refers to an acceptor fluorescent moiety or dark quencher that has an absorbance spectrum that overlaps with the emission spectrum of the donor fluorescent moiety. The wavelength maximum of the emission spectrum of the acceptor fluorescent moiety must be at least 100 nm greater than the wavelength maximum of the excitation spectrum of the donor fluorescent moiety. Thus, efficient non-irradiation energy transfer can occur between them.

[0065] Fluorescent donor moieties and corresponding acceptor moieties are generally selected for (a) efficient Förster energy transfer, (b) a large final Stokes shift (>100 nm), (c) a shift in emission as far as possible into the red portion of the visible spectrum (>600 nm); and (d) a shift in emission to a wavelength higher than the Raman water fluorescence emission produced by excitation at the donor excitation wavelength. For example, a donor fluorescent moiety can be selected that has its maximum excitation wavelength near a laser line (e.g., helium-cadmium 442 nm or argon 488 nm), a high extinction coefficient, a high quantum yield, and good overlap of its fluorescence emission with the excitation spectrum of the corresponding acceptor fluorescent moiety. A corresponding acceptor fluorescent moiety can be selected that has a high extinction coefficient, a high quantum yield, good overlap of its excitation with the emission of the donor fluorescent moiety, and emission in the red portion of the visible spectrum (>600 nm).

[0066] Representative donor fluorescent moieties that can be used with various acceptor fluorescent moieties in FRET technology include fluorescein, lucifer yellow, B-phycoerythrin, 9-acridine isothiocyanate, lucifer yellow VS, 4-acetamido-4'-isothio-cyanatostilbene-2,2'-disulfonic acid, 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin, succinyl 1-pyrenebutyrate, and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid derivatives. Representative acceptor fluorescent moieties include LC Red 640, LC Red 705, Cy5, Cy5.5, Lissamine rhodamine B sulfonyl chloride, tetramethylrhodamine isothiocyanate, rhodamine x isothiocyanate, erythrosine isothiocyanate, fluorescein, diethylenetriaminepentaacetate, or other chelates of lanthanide ions (e.g., europium, or terbium), depending on the donor fluorescent moiety used. Donor and acceptor fluorescent moieties can be obtained, for example, from Molecular Probes (Junction City, Oreg.) or Sigma Chemical Co. (St. Louis, Mo.).

[0067] The donor and acceptor fluorescent moieties can be attached to the appropriate probe oligonucleotide via a linker arm. The length of each linker arm is important since it affects the distance between the donor and acceptor fluorescent moieties. The length of the linker arm can be the distance in angstroms (Å) from the nucleotide base to the fluorescent moiety. Generally, the linker arm is from about 10 Å to about 25 Å. The linker arm can be of the type described in WO 84 / 03285. WO 84 / 03285 also discloses methods of attaching the linker arm to a particular nucleotide base and attaching the fluorescent moiety to the linker arm.

[0068] Acceptor fluorescent moieties such as LC Red 640 can be combined with oligonucleotides containing amino linkers (e.g., C6-amino phosphoramidites available from ABI (Foster City, Calif.) or Glen Research (Sterling, Va.) to generate, for example, LC Red 640-labeled oligonucleotides. Linkers commonly used to attach donor fluorescent moieties such as fluorescein to oligonucleotides include thiourea linkers (derived from FITC, e.g., Fluorescein-CPG from Glen Research or ChemGene (Ashland, Mass.)), amide-linkers (derived from Fluorescein-NHS-esters, such as CX-Fluorescein-CPG from BioGenex (San Ramon, Calif.)), or 3'-amino-CPG, which requires attachment of a Fluorescein-NHS-ester after oligonucleotide synthesis.

[0069] Detection of HPIV (including HPIV1-4) amplification products (amplicons) The present disclosure provides a method for detecting the presence or absence of HPIV (including HPIV1-4) in a biological or non-biological sample. The provided method avoids problems of sample contamination, false negatives, and false positives. The method includes performing at least one cycling step including amplifying a portion of a HPIV (including HPIV1-4) target nucleic acid molecule from a sample using one or more pairs of HPIV (including HPIV1-4) primers, and a FRET detection step. The multiple cycling steps are preferably performed in a thermocycler. The method can be performed using HPIV (including HPIV1-4) primers and probes to detect the presence of HPIV (including HPIV1-4), where detection of HPIV (including HPIV1-4) indicates the presence of HPIV (including HPIV1-4) in the sample.

[0070] As described herein, the amplification products can be detected using labeled hybridization probes that utilize FRET technology. One FRET format utilizes TaqMan® technology to detect the presence or absence of amplification products and thus the presence or absence of HPIV (including HPIV1-4). TaqMan® technology utilizes one single-stranded hybridization probe labeled with, for example, one fluorescent moiety or dye (e.g., HEX or FAM) and one quencher (e.g., BHQ-2), which may or may not be fluorescent. When the first fluorescent moiety is excited with light of an appropriate wavelength, the absorbed energy is transferred to a second fluorescent moiety or dark quencher according to the principles of FRET. The second fluorescent moiety is typically a quencher molecule. During the annealing step of the PCR reaction, the labeled hybridization probe binds to the target DNA (i.e., the amplification products) and is degraded during the subsequent extension step, for example, by the 5' to 3' nuclease activity of Taq polymerase. As a result, the fluorescent moiety and the quencher moiety are spatially separated from one another. As a result, upon excitation of the first fluorescent moiety in the absence of the quencher, the fluorescent emission from the first fluorescent moiety can be detected. By way of example, the ABI PRISM® 7700 Sequence Detection System (Applied Biosystems) uses TaqMan® technology and is suitable for carrying out the methods described herein for detecting the presence or absence of HPIV (including HPIV1-4) in a sample.

[0071] Molecular beacons in combination with FRET can also be used to detect the presence of amplification products using real-time PCR. Molecular beacon technology uses a hybridization probe labeled with a first fluorescent moiety and a second fluorescent moiety. The second fluorescent moiety is generally a quencher, and a fluorescent label is typically placed at each end of the probe. Molecular beacon technology uses a probe oligonucleotide with a sequence that allows for secondary structure formation (e.g., a hairpin). The secondary structure formed within the probe results in both fluorescent sites being in spatial proximity when the probe is in solution. After hybridization to the target nucleic acid (i.e., the amplification product), the secondary structure of the probe is disrupted and the fluorescent moieties are separated from each other, so that the emission of the first fluorescent moiety can be detected after excitation with light of an appropriate wavelength.

[0072] Another common form of FRET technology utilizes two hybridization probes. Each probe can be labeled with a different fluorescent moiety and is generally designed to hybridize in close proximity to one another within a target DNA molecule (e.g., an amplification product). A donor fluorescent moiety, e.g., fluorescein, is excited at 470 nm by the light source of the LightCycler® Instrument. During FRET, fluorescein transfers its energy to an acceptor fluorescent moiety, e.g., LightCycler®-Red 640 (LC Red 640) or LightCycler®-Red 705 (LC Red 705). The acceptor fluorescent moiety then emits light of a longer wavelength, which is detected by the optical detection system of the LightCycler® instrument. Efficient FRET can only occur when the fluorescent moieties are in direct local proximity and the emission spectrum of the donor fluorescent moiety overlaps with the absorption spectrum of the acceptor fluorescent moiety. The intensity of the emitted signal can be correlated with the number of original target DNA molecules (e.g., the number of HPIV (including HPIV1-4) genomes). When amplification of the HPIV (including HPIV1-4) target nucleic acid occurs and an amplification product is produced, the hybridizing step results in a detectable signal based on FRET between the members of the probe pair.

[0073] Generally, the presence of FRET indicates the presence of HPIV (including HPIV1-4) in the sample, and the absence of FRET indicates the absence of HPIV (including HPIV1-4) in the sample. However, inadequate specimen collection, delayed transportation, improper transportation conditions, or use of certain collection swabs (calcium alginate or aluminum shafts) are all conditions that can affect the success and / or accuracy of the test results.

[0074] Representative biological samples that can be used to practice the method include, but are not limited to, whole blood, respiratory specimens, urine, fecal specimens, blood specimens, plasma, skin swabs, nasal swabs, nasopharyngeal samples, wound swabs, blood cultures, skin and soft tissue infections. Methods for collecting and storing biological samples are known to those skilled in the art. The biological sample may be processed (e.g., by nucleic acid extraction methods and / or kits known in the art) to release HPIV (including HPIV1-4) nucleic acids, or in some cases, the biological sample may be directly contacted with PCR reaction components and appropriate oligonucleotides. In some examples, the biological sample is whole blood. When whole blood is typically collected, it is often collected in a container that contains an anticoagulant, such as heparin, citrate, or EDTA, which allows the whole blood to be stored at an appropriate temperature. However, under such conditions, the nucleic acids in whole blood undergo a significant amount of degradation. Therefore, it may be advantageous to collect the blood in a reagent that dissolves, denatures, and stabilizes the whole blood components, including nucleic acids, such as a nucleic acid stabilizing solution. In such cases, the nucleic acids may be better preserved and stabilized for subsequent isolation and analysis by nucleic acid tests such as PCR, etc. Such nucleic acid stabilizing solutions are well known in the art and include, but are not limited to, cobas PCR medium containing 4.2 M guanidinium salt (GuHCl) and 50 mM Tris at pH 7.5.

[0075] The sample can be collected by any method or device designed to adequately hold and preserve the sample before analysis. Such methods and devices are well known in the art. When the sample is a biological sample such as whole blood, the method or device can include a blood collection tube. Such blood collection tubes are well known in the art and can include, for example, a blood collection tube. In many cases, it may be advantageous to use a blood collection tube, where the blood collection tube is under pressure in the space intended for sample intake, such as a blood tube with a vacuum chamber, such as a vacutainer blood collection tube. Such blood collection tubes with vacuum chambers, such as vacutainer blood collection tubes, are well known in the art. It may be further advantageous to collect blood into a blood collection tube with or without a vacuum chamber, such that the whole blood drawn is immediately in contact with the nucleic acid stabilizing solution in the blood collection tube, with or without a vacuum chamber, containing therein a solution that dissolves, denatures, and stabilizes whole blood components, including nucleic acids, such as a nucleic acid stabilizing solution.

[0076] Melting curve analysis is an additional step that can be included in the cycle profile. Melting curve analysis is based on the fact that DNA melts at a characteristic temperature called the melting temperature (Tm), which is defined as the temperature at which one half of a DNA double strand separates into single strands. The melting temperature of DNA depends mainly on its nucleotide composition. Thus, DNA molecules rich in G and C nucleotides have a higher Tm than DNA molecules rich in A and T nucleotides. By detecting the temperature at which the signal is lost, the melting temperature of the probe can be determined. Similarly, by detecting the temperature at which the signal is generated, the annealing temperature of the probe can be determined. The melting temperature(s) of the HPIV (including HPIV1-4) probe from the HPIV (including HPIV1-4) amplification product can confirm the presence or absence of HPIV (including HPIV1-4) in the sample.

[0077] During each thermocycler run, control samples can be cycled as well. A positive control sample can amplify a target nucleic acid control template (other than the amplification product of the described target gene), for example, using control primers and a control probe. A positive control sample can also amplify, for example, a plasmid construct containing a target nucleic acid molecule. Such a plasmid control can be amplified internally (e.g., in the sample) or in a separate sample run alongside the patient sample, using the same primers and probes used to detect the intended target. Such controls are indicators of the success or failure of the amplification, hybridization and / or FRET reaction. Each thermocycler run can also include a negative control, for example, lacking target template DNA. The negative control can measure contamination. This ensures that the system and reagents do not produce false positive signals. Thus, the control reaction can easily determine, for example, the ability of primers to anneal with sequence specificity and initiate extension, and the ability of probes to hybridize with sequence specificity and FRET to occur.

[0078] In one embodiment, the method includes steps to avoid contamination, for example, the enzymatic method utilizing uracil-DNA glycosylase described in U.S. Patent Nos. 5,035,996, 5,683,896, and 5,945,313 to reduce or eliminate contamination between one thermocycling run and the next.

[0079] This method can be carried out using conventional PCR combined with FRET technology. In one embodiment, the LightCycler® instrument is used. The following patent applications describe real-time PCR used with LightCycler® technology: WO 97 / 46707, WO 97 / 46714, and WO 97 / 46712.

[0080] The LightCycler® can be operated using a PC workstation. Signals from samples are obtained as the machine sequentially positions the capillaries on the optical unit. The software can display the fluorescence signal in real time immediately after each measurement. Fluorescence acquisition times range from 10 to 100 milliseconds (msec). After each cycling step, a quantitative display of fluorescence versus cycle number can be continuously updated for all samples. The generated data can be stored for further analysis.

[0081] The LightCycler® 480 II Real-Time PCR System can also be operated using a PC workstation. The instrument has a thermal block cycler, and heating and cooling are achieved using Peltier elements. Fluorescent signals from samples are obtained from 96-well plates using high-intensity xenon lamps that emit light over a broad spectrum. Flexible combinations of built-in filters for specific excitation and emission allow the use of a variety of fluorescent dyes and detection formats. The software can display the fluorescent signal and calculate CT values, and the data generated can be stored for further analysis.

[0082] As an alternative to FRET, double-stranded DNA binding dyes such as fluorescent DNA binding dyes (e.g., SYBR® Green or SYBR® Gold (Molecular Probes)) can be used to detect the amplification products. Upon interaction with double-stranded nucleic acids, such fluorescent DNA binding dyes emit a fluorescent signal after excitation with light of an appropriate wavelength. Also, double-stranded DNA binding dyes such as nucleic acid intercalating dyes can be used. When using double-stranded DNA binding dyes, a melting curve analysis is usually performed to confirm the presence of the amplification product.

[0083] Those skilled in the art will appreciate that other nucleic acid or signal amplification methods may also be used, including, but not limited to, branched DNA signal amplification, loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), self-sustained sequence replication (3 SR), strand displacement amplification (SDA), or smart amplification process version 2 (SMAP 2).

[0084] It is understood that embodiments of the present disclosure are not limited by the configuration of one or more commercially available devices.

[0085] Manufactured products / kits Embodiments of the present disclosure further provide articles of manufacture or kits for detecting HPIV (including HPIV1-4). The articles of manufacture may include primers and probes used to detect target HPIV (including HPIV1-4) genes along with suitable packaging materials. Representative primers and probes for detecting HPIV (including HPIV1-4) may hybridize to HPIV (including HPIV1-4) target nucleic acid molecules. In addition, the kits may also include appropriately packaged reagents and materials necessary for DNA immobilization, hybridization and detection, such as solid supports, buffers, enzymes and DNA standards. Methods for designing primers and probes are disclosed herein, and representative examples of primers and probes that amplify and hybridize to HPIV (including HPIV1-4) target nucleic acid molecules are provided.

[0086] The article of manufacture can also include one or more fluorescent moieties for labeling the probe or can label the probes provided with the kit. For example, the article of manufacture can include donor and / or acceptor fluorescent moieties for labeling HPIV (including HPIV1-4) probes. Examples of suitable FRET donor fluorescent moieties and corresponding acceptor fluorescent moieties are provided above.

[0087] The article of manufacture may also include a package insert or packaging label with instructions for using the HPIV (including HPIV1-4) primers and probes to detect HPIV (including HPIV1-4) in a sample. The article of manufacture may further include reagents (e.g., buffers, polymerase enzymes, cofactors, or agents to prevent contamination) for carrying out the methods disclosed herein. Such reagents may be specific for one of the commercially available instruments described herein.

[0088] Embodiments of the present disclosure also provide a set of primers and one or more detectable probes for preferentially and / or selectively detecting and / or quantifying HPIV (including HPIV1-4) in a sample.

[0089] Embodiments of the present invention are further described in the following examples, which do not limit the scope of the invention described in the claims. EXAMPLES

[0090] The following examples and figures are provided to aid in the understanding of the subject matter, the scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

[0091] In this study, fully automated sample preparation (nucleic acid extraction and purification) followed by PCR amplification and detection was performed. The system used was a cobas® 6800 / 8800 system, consisting of a sample delivery module, a transfer module, a processing module, and an analysis module. Automated data management was performed by the cobas® 6800 / 8800 system.

[0092] The master mix contained detection probes specific for human parainfluenza virus 1-4 (HPIV1-4) and control nucleic acids. Each specific human parainfluenza virus 1-4 (HPIV1-4) and control detection probe was labeled with a unique fluorescent dye that served as a reporter. Each probe also had a second dye that acted as a quencher. The reporter dye was measured at a defined wavelength, thus allowing detection and discrimination of the amplified human parainfluenza virus 1-4 (HPIV1-4) targets and controls. The fluorescent signal of the intact probe was suppressed by the quencher dye. During the PCR amplification step, hybridization of the probe to the specific single-stranded DNA template resulted in cleavage by the 5' to 3' nuclease activity of the DNA polymerase, separating the reporter and quencher dyes and producing a fluorescent signal. With each PCR cycle, the amount of cleaved probe increased, and the cumulative signal of the reporter dye increased concomitantly. Two specific reporter dyes were measured at defined wavelengths, allowing simultaneous detection and discrimination of amplified human parainfluenza virus 1-4 (HPIV1-4) targets and controls.

[0093] Primers and probes for human parainfluenza virus 1-4 (HPIV1-4) testing were designed by seeding the primers and probes according to the genome in the most conserved regions based on the alignment. One set of oligonucleotides (SEQ ID NOs: 1-3) was designed to detect and amplify HPIV1 target nucleic acid. Another set of oligonucleotides (SEQ ID NOs: 4-7) was designed to detect and amplify HPIV2 target nucleic acid. Another set of oligonucleotides (SEQ ID NOs: 8-10) was designed to detect and amplify HPIV3 target nucleic acid. Another set of oligonucleotides (SEQ ID NOs: 11-14) was designed to detect and amplify HPIV4 target nucleic acid. Another set of oligonucleotides (SEQ ID NOs: 15-17) was designed to detect and amplify HPIV3 target nucleic acid. Another set of oligonucleotides (SEQ ID NOs: 18-19 and 13-14) was designed to detect and amplify HPIV4 target nucleic acid. Another set of oligonucleotides (SEQ ID NOs: 11, 18-19 and 13-14) was designed to detect and amplify HPIV4 target nucleic acid.

[0094] Each set of oligonucleotides (or primers / probes) can be used singleplex in its own reaction to amplify and detect a particular target region of interest (i.e., HPIV1, HPIV2, HPIV3, or HPIV4). However, oligonucleotide sets can also be combined in a multiplex target assay, whereby the reaction mixture includes one or more sets of oligonucleotides (SEQ ID NOs: 1-3 for HPIV1, SEQ ID NOs: 3-7 for HPIV2, SEQ ID NOs: 8-10 or SEQ ID NOs: 15-17 for HPIV3; and SEQ ID NOs: 11-14, or SEQ ID NOs: 18-19 and 13-14, or SEQ ID NOs: 11, 18-19 and 13-14 for HPIV4) such that in a single real-time PCR reaction, any or all of the HPIV1-4 target nucleic acids are amplified and detected in the sample (if the targets are present in the sample). For detection of HPIV1 target nucleic acids, the forward primer corresponds to the nucleic acid sequence of SEQ ID NO: 1, the reverse primer corresponds to the nucleic acid sequence of SEQ ID NO: 2, and the probe corresponds to the nucleic acid sequence of SEQ ID NO: 3. For detection of HPIV2 target nucleic acids, the forward primer corresponds to the nucleic acid sequence of SEQ ID NO: 4, the reverse primer corresponds to the nucleic acid sequence of SEQ ID NO: 5, and the probe corresponds to the nucleic acid sequence of SEQ ID NO: 6 and / or 7. For detection of HPIV3 target nucleic acids, the forward primer corresponds to the nucleic acid sequence of SEQ ID NO: 8, the reverse primer corresponds to the nucleic acid sequence of SEQ ID NO: 9, and the probe corresponds to the nucleic acid sequence of SEQ ID NO: 10. Alternatively, for detection of HPIV3 target nucleic acids, the forward primer corresponds to the nucleic acid sequence of SEQ ID NO: 15, the reverse primer corresponds to the nucleic acid sequence of SEQ ID NO: 16, and the probe corresponds to the nucleic acid sequence of SEQ ID NO: 17. For detection of HPIV4 target nucleic acids, the forward primer corresponds to the nucleic acid sequence of SEQ ID NO: 11, the reverse primer corresponds to the nucleic acid sequence of SEQ ID NO: 12, and the probe corresponds to the nucleic acid sequence of SEQ ID NO: 13 and / or 14. Alternatively, for detection of HPIV4 target nucleic acids, the forward primer corresponds to the nucleic acid sequence of SEQ ID NO: 18, the reverse primer corresponds to the nucleic acid sequence of SEQ ID NO: 19, and the probe corresponds to the nucleic acid sequence of SEQ ID NO: 13 and / or 14.In some cases, the set for detection of HPIV4 target nucleic acid further comprises a second forward primer corresponding to the nucleic acid sequence of SEQ ID NO: 11. These oligonucleotides can be used in individual assays for detection and amplification of HPIV1 target nucleic acid (using oligonucleotides corresponding to SEQ ID NOs: 1-3), HPIV2 target nucleic acid (using oligonucleotides corresponding to SEQ ID NOs: 4-7), HPIV3 target nucleic acid (using oligonucleotides corresponding to SEQ ID NOs: 8-10 or SEQ ID NOs: 15-17), and HPIV4 target nucleic acid (using oligonucleotides corresponding to SEQ ID NOs: 11-14 or SEQ ID NOs: 18-19 and 13-14 or SEQ ID NOs: 11, 18-19 and 13-14). Alternatively, the oligonucleotides can be used in multiplex target assays designed to simultaneously detect and amplify multiple nucleic acid targets from different types of HPIV (e.g., HPIV1-4). For example, oligonucleotides for detection and amplification of HPIV1, HPIV2, HPIV3, and / or HPIV4 can be added to the sample simultaneously. Multiplex assays have the advantage that any or all of the four types of HPIV (HPIV1-4) can be detected in a single sample without using additional resources. In this way, multiplex assays save resources (e.g., PCR reagents, samples), costs, and time. Multiplex assays allow for the simultaneous detection of multiple HPIV types in a single sample in an efficient, rapid, reliable, and inexpensive manner.

[0095] Example 1: Primer and probe design for detection of HPIV1-4 by real-time PCR HPIV1-4 nucleic acid tests were designed to detect all four types of HPIV (HPIV1-4). Viral pathogen-specific assays were designed by proprietary software using optimal oligonucleotide sequences found within the genome and based on specific inclusiveness and exclusivity requirements. Specific target regions (i.e., four target regions in total) were selected for each of the HPIV1-4 assays. The HPIV1 assay targets the L polymerase protein gene (see Figure 3), the HPIV2 assay targets the large protein (see Figure 4), the HPIV3 assay is designed in the nucleocapsid protein (see Figure 5), and the HPIV4 assay targets the large protein (see Figure 6). Each assay has one forward primer, one reverse primer, and one probe for each HPIV type (HPIV1-4).

[0096] Example 2: HPIV1 primers and probes amplify and detect the L polymerase protein gene of HPIV1 in a real-time PCR assay HPIV1 oligonucleotides were tested using primers / probes for detection of HPIV1 (SEQ ID NO:1-3). The final concentrations of forward primer (SEQ ID NO:1) were 200 nM, reverse primer (SEQ ID NO:2) were 150 nM, and probe (SEQ ID NO:3) were 75 nM. HPIV1 assays were tested in the HEX channel. In vitro generated transcripts were used at the following concentrations: 10, 10 per reaction. 2 , 10 3 , 10 4 , and 10 5 Reagents used include cobas® 6800 / 8800 Universal PCR Master Mix with profiles and conditions for use on the cobas® 6800 / 8800, using TaqMan® amplification and detection technology. The cobas® 6800 / 8800 PCR profile used is shown in Table 2 below:

[0097] [Table 2]

[0098] The results are shown in FIG. 7A, which shows the PCR growth curves of a dilution series of the HPIV1 assay performance. FIG. 7A shows that the HPIV1 primers and probes used (SEQ ID NOs: 1-3) can amplify and detect HPIV1. FIG. 7B shows the efficiency of the HPIV1 assay, demonstrating that the HPIV1 real-time PCR assay is efficient and exhibits excellent sensitivity at low target concentrations. These data show that the HPIV1 real-time PCR assay is linear over the range tested and can detect up to 10 copies of target per reaction. Taken together, these data demonstrate that the HPIV1 real-time assay (comprising the oligonucleotide sequences of SEQ ID NOs: 1-3) can detect and amplify HPIV1.

[0099] Example 3: HPIV2 primers and probes amplify and detect the large protein gene of HPIV1 in a real-time PCR assay HPIV2 oligonucleotides were tested using primers / probes for detection of HPIV2 (SEQ ID NO: 4, 5 and 7). The final concentrations of the forward primer (SEQ ID NO: 4) were 150 nM, the reverse primer (SEQ ID NO: 5) was 150 nM and the probe (SEQ ID NO: 7) was 75 nM. The HPIV2 assay was tested in the COU channel. In vitro generated transcripts were used at the following concentrations: 10, 10 per reaction. 2 , 10 3 , 10 4 , 10 5 and 10 6Copy. Reagents used include cobas® 6800 / 8800 Universal PCR Master Mix with profile and conditions for use on the cobas® 6800 / 8800, using TaqMan® amplification and detection technology. The cobas® 6800 / 8800 PCR profile used is shown in Table 2 above. The results are shown in Figure 8A, which shows the PCR growth curves of a dilution series of the HPIV2 assay performance. Figure 8A shows that the HPIV2 primers and probes used (SEQ ID NOs: 4, 5 and 7) can amplify and detect HPIV2. Figure 8B shows the efficiency of the HPIV2 assay, demonstrating that the HPIV2 real-time PCR assay is efficient and exhibits excellent sensitivity at low target concentrations. These data show that the HPIV1 real-time PCR assay is linear over the range tested. However, overall, these results indicate that the RFI may be higher and improvements are possible with respect to the performance of the HPIV2 assay, suggesting that assay optimization (e.g., primer / probe oligonucleotide concentrations) is required. For this purpose, primer and probe concentrations were titrated. Three different concentration conditions were tested: (1) primer concentration 150 nM, probe concentration 75 nM; (2) primer concentration 200 nM, probe concentration 100 nM; (3) primer concentration 300 nM and probe concentration 100 nM (shown in Figure 8C). In vitro generated transcripts were used at the following concentrations: 10, 10 per reaction. 2 , 10 3 , and 10 4 copies. The results for each of the three different concentration conditions are shown in FIG. 8D and show that the signal improves with increasing primer and probe concentrations. These studies show that the HPIV2 assay is robust enough to detect up to 10 copies per reaction. Taken together, these data demonstrate that the HPIV1 real-time assay (comprising the oligonucleotide sequences of SEQ ID NOs: 4, 5, and 7) can detect and amplify HPIV2.

[0100] Example 4: HPIV3 primers and probes amplify and detect the nucleocapsid protein gene of HPIV3 in a real-time PCR assay HPIV3 oligonucleotides were tested using primers / probes for detection of HPIV3 (SEQ ID NO:8-10). The final concentrations of the forward primer (SEQ ID NO:8) were 200 nM, the reverse primer (SEQ ID NO:9) was 150 nM, and the probe (SEQ ID NO:10) was 100 nM. The HPIV3 assay was tested in the JA270 channel. In vitro generated transcripts were used at the following concentrations: 10, 10 per reaction. 2 , 10 3 , 10 4 , 10 5 and 10 6 copies. Reagents used include cobas® 6800 / 8800 Universal PCR Master Mix with profile and conditions for use with cobas® 6800 / 8800, using TaqMan® amplification and detection technology. The cobas® 6800 / 8800 PCR profile used is shown in Table 2 above. The results are shown in Figure 9A, which shows the PCR growth curves of a dilution series of the HPIV3 assay performance. Figure 9A shows that the HPIV3 primers and probes used (SEQ ID NOs: 9-10) can amplify and detect HPIV3. Figure 9B shows the efficiency of the HPIV3 assay, demonstrating that the HPIV3 real-time PCR assay is efficient and exhibits excellent sensitivity at low target concentrations. These data show that the HPIV3 real-time PCR assay is linear over the range tested and can detect up to 10 copies per reaction. Taken together, these data demonstrate that the HPIV3 real-time assay (comprising the oligonucleotide sequences of SEQ ID NOs: 8-10) can detect and amplify HPIV3.

[0101] Example 5: HPIV4 primers and probes amplify and detect the large protein gene of HPIV4 in a real-time PCR assay HPIV4 oligonucleotides were tested using primers / probes to detect HPIV3 (SEQ ID NO:11-13). The final concentrations of forward primer (SEQ ID NO:11) were 200 nM, reverse primer (SEQ ID NO:12) were 200 nM, and probe (SEQ ID NO:13) were 100 nM. The HPIV4 assay was tested in the FAM channel. In vitro generated transcripts were used at the following concentrations: 10, 10 per reaction. 2 , 10 3 , 10 4 , 10 5 and 10 6 copies. Reagents used include cobas® 6800 / 8800 Universal PCR Master Mix with profile and conditions for use with cobas® 6800 / 8800, using TaqMan® amplification and detection technology. The cobas® 6800 / 8800 PCR profile used is shown in Table 2 above. The results are shown in Figure 10A, which shows the PCR growth curves of a dilution series of the performance of the HPIV4 assay. Figure 10A shows that the HPIV4 primers and probes used (SEQ ID NOs: 11-13) can amplify and detect HPIV4. Figure 10B shows the efficiency of the HPIV4 assay, demonstrating that the HPIV4 real-time PCR assay is efficient and exhibits excellent sensitivity at low target concentrations. These data show that the HPIV1 real-time PCR assay is linear over the range tested and can detect up to 10 copies per quadruple reaction. Taken together, these data demonstrate that the HPIV4 real-time assay (comprising the oligonucleotide sequences of SEQ ID NOs: 11-13) can detect and amplify HPIV4.

[0102] Example 6: HPIV1-4 primers and probes simultaneously amplify and detect target HPIV1-4 nucleic acids from viral eluates in an HPIV1-4 multiplex real-time PCR assay HPIV1-4 oligonucleotides were tested using primers / probes to detect HPIV1 (SEQ ID NOs: 1-3), HPIV2 (SEQ ID NOs: 4, 5, and 7), HPIV3 (SEQ ID NOs: 8-10), and HPIV4 (SEQ ID NOs: 11-13). The oligonucleotides for HPIV1-4 were tested under multiplex conditions such that samples were exposed to all oligonucleotides for HPIV1-4 simultaneously. Oligonucleotides for the HPIV1 assay were tested in the HEX channel, oligonucleotides for the HPIV2 assay were tested in the COU channel, oligonucleotides for the HPIV3 assay were tested in the JA270 channel, and oligonucleotides for the HPIV4 assay were tested in the FAM channel. Reagents used include cobas® 6800 / 8800 Universal PCR Master Mix with profile and conditions for use on the cobas® 6800 / 8800, using TaqMan® amplification and detection technology. The cobas® 6800 / 8800 PCR profile used is shown in Table 2 above. Viral elution samples were obtained from ZeptoMetrix (catalog number NATRVP-IDI (batch 318302)). The results are shown in Figures 11A and 11B, which show the PCR growth curves for each of the multiplex assays for viral eluates 1-4. Figures 11A and 11B show that the HPIV1-4 primers and probes used (SEQ ID NOs: 1-5 and 7-13) can amplify and detect HPIV1-4 from viral eluates. That is, all of the viral eluates tested were detected in the expected channels. Taken together, these data demonstrate that the HPIV1-4 multiplex real-time assay (comprising the oligonucleotide sequences of SEQ ID NOs: 1-5 and 6-13) can detect and amplify viral eluates containing the target nucleic acid sequence of HPIV1-4. Additionally, the exclusivity of the HPIV1-4 assay was tested to determine whether there was any cross-reactivity between the oligonucleotides for detecting and amplifying HPIV1-4 and other respiratory virus targets.To this end, other respiratory virus targets including adenovirus (AV), enterovirus / rhinovirus (EV / RV), and human metapneumovirus (HMPV) from high titer virus cultures were used. In this study, oligonucleotides for detection and amplification of HPIV1-4 (SEQ ID NOs: 1-5 and 6-13) were tested in PCR assays against HPIV (HPIV1-4) as well as AV (AVB, AVE, AVC, and AVA strains), EV / RV (EVA, EVB, EVD68, and RVA strains), and HMPV targets. Primers specific for AV, EV / RV, and HMPV targets were also used. The results shown in Figure 11B indicate that the HPIV1-4 oligonucleotides do not show cross-reactivity with multiple respiratory virus targets from high titer virus cultures. Thus, these studies demonstrate the exclusivity and specificity of the HPIV1-4 oligonucleotides for only the HPIV1-4 target.

[0103] Example 7: HPIV1-4 primers and probes simultaneously amplify and detect target HPIV1-4 nucleic acids from artificial nasopharyngeal simulated clinical samples in an HPIV1-4 multiplex real-time PCR assay HPIV1-4 oligonucleotides were tested using primers / probes to detect HPIV1 (SEQ ID NOs: 1-3), HPIV2 (SEQ ID NOs: 4, 5, and 7), HPIV3 (SEQ ID NOs: 8-10), and HPIV4 (SEQ ID NOs: 11-13). Oligonucleotides for HPIV1-4 were tested under multiplex conditions such that samples were exposed to all oligonucleotides for HPIV1-4 simultaneously. Oligonucleotides for the HPIV1 assay were tested in the HEX channel, oligonucleotides for the HPIV2 assay were tested in the COU channel, oligonucleotides for the HPIV3 assay were tested in the JA270 channel, and oligonucleotides for the HPIV4 assay were tested in the FAM channel. Test samples were tested in the presence of an artificial nasopharyngeal matrix eluate intended to simulate the background of clinical samples containing cells, albumin, and mucin, and were used at the following concentrations: 10, 10, 10 per reaction. 2 , 10 3 , and 10 4copies. The composition of the matrix is ​​shown in Figure 12A. The reagents used include cobas® 6800 / 8800 Universal PCR Master Mix with profiles and conditions for use with the cobas® 6800 / 8800 and using TaqMan® amplification and detection technology. The cobas® 6800 / 8800 PCR profile used is shown in Table 2 above. The results are shown in Figure 12B (HPIV1), Figure 12C (HPIV2), Figure 12D (HPIV3), and Figure 12E (HPIV4), which show the PCR growth curves of a dilution series of the performance of the HPIV1-4 assay. The results showed that in the presence of the nasopharyngeal matrix, the sensitivity of HPIV1-4 types is down to a minimum of 100 copies / reaction. Figures 12B-12D demonstrate that the HPIV1-4 oligonucleotides can simultaneously amplify and detect target HPIV1-4 nucleic acids from artificial nasopharyngeal simulated clinical samples in a multiplex setting. Because the artificial nasopharyngeal matrix simulates a nasopharyngeal clinical sample, these data suggest that HPIV1-4 oligonucleotides can simultaneously amplify and detect target HPIV1-4 nucleic acid from actual nasopharyngeal samples.

[0104] Example 8: HPIV1-4 primers and probes simultaneously and specifically amplify and detect target HPIV1-4 nucleic acids from viral eluates in an HPIV1-4 multiplex real-time PCR assay HPIV1-4 oligonucleotides were tested using primers / probes to detect HPIV1 (SEQ ID NOs: 1-3), HPIV2 (SEQ ID NOs: 4, 5, and 7), HPIV3 (SEQ ID NOs: 8-10), and HPIV4 (SEQ ID NOs: 11-13). Oligonucleotides for HPIV1-4 were tested under multiplex conditions such that samples were exposed to all oligonucleotides for HPIV1-4 simultaneously. Oligonucleotides for the HPIV1 assay were tested in the HEX channel, oligonucleotides for the HPIV2 assay were tested in the COU channel, oligonucleotides for the HPIV3 assay were tested in the JA270 channel, and oligonucleotides for the HPIV4 assay were tested in the FAM channel. Samples tested were viral eluates (ATCC catalog numbers: HPIV1-VR-94; HPIV2-VR-92; HPIV3-VR-1782; ZeptoMetrix catalog number: HPIV4A-0810060CF) and were tested at two concentrations, neat and at a 1:100,000 dilution. Reagents used include cobas® 6800 / 8800 Universal PCR Master Mix with profiles and conditions for use on the cobas® 6800 / 8800, using TaqMan® amplification and detection technology. The cobas® 6800 / 8800 PCR profile used is shown in Table 2 above. These studies were designed to test the specificity of the HPIV1-4 oligonucleotides for viral eluates. The results are shown in Figures 13A (HPIV1), 13B (HPIV2), 13C (HPIV3), and 13D (HPIV4), which show PCR growth curves of a dilution series of the performance of the HPIV1-4 assay. These results demonstrate that the HPIV1-4 oligonucleotides are specific for their respective intended targets. As seen in Figures 13A-13D, no cross-reactivity was observed when using these viral eluates in unintended channels for specific HPIV types. Taken together, these studies demonstrate that HPIV1-4 oligonucleotides specifically amplify and detect intended targets in a multiplex setting.

[0105] Example 9: HPIV1-4 primers and probes demonstrate specificity for the HPIV1-4 target as they fail to amplify and detect the HPIV1-4 target in clinical samples known to be negative for HPIV1-4 HPIV1-4 oligonucleotides were tested using primers / probes for detecting HPIV1 (SEQ ID NOs: 1-3), HPIV2 (SEQ ID NOs: 4, 5, and 7), HPIV3 (SEQ ID NOs: 8-10), and HPIV4 (SEQ ID NOs: 11-13). Oligonucleotides for HPIV1-4 were tested under multiplex conditions such that samples were simultaneously exposed to all oligonucleotides for HPIV1-4. Oligonucleotides for the HPIV1 assay were tested in the HEX channel, oligonucleotides for the HPIV2 assay were tested in the COU channel, oligonucleotides for the HPIV3 assay were tested in the JA270 channel, and oligonucleotides for the HPIV4 assay were tested in the FAM channel. Samples tested were nasopharyngeal eluates from six individual patients known to be negative for HPIV1-4. Reagents used include the cobas® 6800 / 8800 Universal PCR Master Mix with profiles and conditions for use on the cobas® 6800 / 8800 and using TaqMan® amplification and detection technology. The cobas® 6800 / 8800 PCR profiles used are shown in Table 2 above. These studies were designed to test the specificity of the HPIV1-4 oligonucleotides for HPIV1-4 negative eluates. The results are shown in Figures 14A and 14B, which show the PCR growth curves of the HPIV1-4 assay performance. These results show that there is no amplification in any channel using HPIV1-4 oligonucleotides with nasopharyngeal eluates known to be negative for HPIV1-4. These results indicate that the HPIV1-4 oligonucleotides do not cross-react and / or inadvertently amplify nucleic acids when the intended target is not present in the nasopharyngeal eluate.

[0106] Example 10: HPIV1-4 primers and probes simultaneously specifically amplify and detect target HPIV1-4 nucleic acids from viral eluates in an HPIV1-4 multiplex real-time PCR assay HPIV1 oligonucleotides were tested using primers / probes to detect HPIV1 (SEQ ID NOs: 1-3). HPIV1-4 oligonucleotides were tested using primers / probes to detect HPIV1 (SEQ ID NOs: 1-3), HPIV2 (SEQ ID NOs: 4-6), HPIV3 (SEQ ID NOs: 15-17), and HPIV4 (SEQ ID NOs: 11, 13, 18, and 19). HPIV1-4 oligonucleotides are shown in Figure 15. In vitro generated transcripts were used at the following concentrations: 10, 10 per reaction, 10 2 , 10 3 , 10 4 , 10 5 and 10 6 Copy. Reagents used include cobas® 6800 / 8800 Universal PCR Master Mix with profile and conditions for use on the cobas® 6800 / 8800, using TaqMan® amplification and detection technology. The cobas® 6800 / 8800 PCR profile used is shown in Table 2 above. For HPIV1, the forward primer (SEQ ID NO: 1) is at a concentration of 400 nM, the reverse primer (SEQ ID NO: 2) is at a concentration of 300 nM, and the probe (SEQ ID NO: 3) is at a concentration of 100 nM. For HPIV2, the forward primer (SEQ ID NO: 4) is at a concentration of 200 nM, the reverse primer (SEQ ID NO: 5) is at a concentration of 150 nM, and the probe (SEQ ID NO: 6) is at a concentration of 100 nM. For HPIV3, the forward primer (SEQ ID NO:15) was at a concentration of 500 nM, the reverse primer (SEQ ID NO:16) was at a concentration of 300 nM, and the probe (SEQ ID NO:17) was at a concentration of 100 nM. For HPIV4, the forward primers (SEQ ID NOs:11 and 18) were at concentrations of 100 nM and 400 nM, respectively, the reverse primer (SEQ ID NO:19) was at a concentration of 400 nM, and the probe (SEQ ID NO:13) was at a concentration of 100 nM. The results are shown in Figure 16A (HPIV1), Figure 16B (HPIV2), Figure 16C (HPIV3), and Figure 16D (HPIV4), which show the PCR growth curves of the dilution series of the performance of the HPIV1-4 assay.

[0107] The results are shown in FIG. 16A, which shows the PCR growth curves of the dilution series of the performance of the HPIV1 assay. FIG. 16A shows that the HPIV1 primers and probes (SEQ ID NOs: 1-3) used can amplify and detect HPIV1. FIG. 16B shows the efficiency of the HPIV1 assay, demonstrating that the HPIV1 real-time PCR assay is efficient and exhibits excellent sensitivity at low target concentrations. The results are shown in FIG. 16C, which shows the PCR growth curves of the dilution series of the performance of the HPIV2 assay. FIG. 16C shows that the HPIV2 primers and probes (SEQ ID NOs: 4-6) used can amplify and detect HPIV2. FIG. 16D shows the efficiency of the HPIV2 assay, demonstrating that the HPIV2 real-time PCR assay is efficient and exhibits excellent sensitivity at low target concentrations. The results are shown in FIG. 16E, which shows the PCR growth curves of the dilution series of the performance of the HPIV3 assay. FIG. 16E shows that the HPIV3 primers and probes (SEQ ID NOs: 15-17) used can amplify and detect HPIV3. Figure 16F shows the efficiency of the HPIV3 assay, demonstrating that the HPIV3 real-time PCR assay is efficient and exhibits excellent sensitivity at low target concentrations. The results are shown in Figure 16G, which shows the PCR growth curves of a dilution series of the performance of the HPIV4 assay. Figure 16G shows that the HPIV4 primers and probes used (SEQ ID NOs: 11, 13, 18 and 19) can amplify and detect HPIV4. Figure 16H shows the efficiency of the HPIV4 assay, demonstrating that the HPIV4 real-time PCR assay is efficient and exhibits excellent sensitivity at low target concentrations.

[0108] These data indicate that the HPIV1, HPIV2, HPIV3, and HPIV4 real-time PCR assays are linear over the range tested and can detect up to 10 copies of target per reaction. Collectively, these data demonstrate that the HPIV1 (SEQ ID NOs: 1-3), HPIV2 (SEQ ID NOs: 4-6), HPIV3 (SEQ ID NOs: 15-17), and HPIV4 (SEQ ID NOs: 11, 13, 18, and 19) real-time assays can detect and amplify HPIV1, HPIV2, HPIV3, and HPIV4, respectively.

[0109] Example 11: HPIV1-4 primers and probes simultaneously amplify and detect target HPIV1-4 nucleic acids from viral eluates in an HPIV1-4 multiplex real-time PCR assay HPIV1-4 oligonucleotides were tested using primers / probes to detect HPIV1 (SEQ ID NOs: 1-3), HPIV2 (SEQ ID NOs: 4-6), HPIV3 (SEQ ID NOs: 15-17), and HPIV4 (SEQ ID NOs: 11, 13, 18, and 19). Oligonucleotides for HPIV1-4 were tested under multiplex conditions such that samples were exposed to all oligonucleotides for HPIV1-4 simultaneously. Oligonucleotides for the HPIV1 assay were tested in the HEX channel, oligonucleotides for the HPIV2 assay were tested in the COU channel, oligonucleotides for the HPIV3 assay were tested in the JA270 channel, and oligonucleotides for the HPIV4 assay were tested in the FAM channel. HPIV1-4 oligonucleotides were tested using primers / probes to detect HPIV1 (SEQ ID NOs: 1-3), HPIV2 (SEQ ID NOs: 4-6), HPIV3 (SEQ ID NOs: 15-17), and HPIV4 (SEQ ID NOs: 11, 13, 18, and 19). Reagents used include cobas® 6800 / 8800 Universal PCR Master Mix with profile and conditions for use with the cobas® 6800 / 8800, using TaqMan® amplification and detection technology. The cobas® 6800 / 8800 PCR profile used is shown in Table 2 above. Viral cultures were obtained from ZeptoMetrix (catalog number NATRVP-IDI (batch 318302)) and ATCC. Results are shown in FIG. 17A, which shows the PCR growth curves for each of the viral eluate HPIV1-4 singleplex assays. FIG. 17A shows that the HPIV1-4 primers and probes used (HPIV1 (SEQ ID NOs: 1-3), HPIV2 (SEQ ID NOs: 4-6), HPIV3 (SEQ ID NOs: 15-17), and HPIV4 (SEQ ID NOs: 11, 13, 18, and 19)) can detect and amplify HPIV1-4 from viral eluate. That is, all viral eluates tested were detected in the expected channels.Taken together, these data demonstrate that the HPIV1-4 multiplex real-time assays (HPIV1 (SEQ ID NOs: 1-3), HPIV2 (SEQ ID NOs: 4-6), HPIV3 (SEQ ID NOs: 15-17), and HPIV4 (SEQ ID NOs: 11, 13, 18, and 19)) can detect and amplify viral eluates containing target nucleic acid sequences for HPIV1-4.

[0110] Additionally, the exclusivity of the HPIV1-4 assay was tested to determine whether there was cross-reactivity between the oligonucleotides for detecting and amplifying HPIV1-4 and other respiratory viral targets, including adenovirus (AdV), human coronavirus (CoV), enterovirus (EV), influenza, human metapneumovirus (HMPV), respiratory syncytial virus (RSV), rhinovirus (RV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from high-titer virus cultures. In this study, oligonucleotides for detection and amplification of HPIV1-4 (HPIV1 (SEQ ID NOs: 1-3), HPIV2 (SEQ ID NOs: 4-6), HPIV3 (SEQ ID NOs: 15-17), and HPIV4 (SEQ ID NOs: 11, 13, 18, and 19)) were tested in PCR assays against HPIV (HPIV1-4), AdV (AdV-B, AdV-E, AdV-C strains), CoV (CoV229E, CoV HKU1 + HPIV3 (clinical samples), CoV NL63, CoV OC43), EV (EV D68 and EV C), influenza (FluA H1N1, FluA H3N2, and FluB), HMPV (HMPV A1 and HMPV B1), RSV (RSV A and RSVB), rhinovirus (RV A and RV B), and SARS-CoV-2 eluates. The results shown in Figure 17B indicate that the HPIV1-4 oligonucleotides in a multiplex setting do not exhibit cross-reactivity with multiple respiratory virus targets from multiple high titer respiratory virus cultures. Thus, these studies demonstrate the exclusivity and specificity of the HPIV1-4 oligonucleotides in a multiplex setting for only the HPIV1-4 target.

[0111] Example 12: HPIV1-4 primers and probes simultaneously amplify and detect target HPIV1-4 nucleic acids from artificial nasopharyngeal simulated clinical samples in an HPIV1-4 multiplex real-time PCR assay HPIV1-4 oligonucleotides were tested using primers / probes to detect HPIV1 (SEQ ID NOs: 1-3), HPIV2 (SEQ ID NOs: 4-6), HPIV3 (SEQ ID NOs: 15-17), and HPIV4 (SEQ ID NOs: 11, 13, 18, and 19). Oligonucleotides for HPIV1-4 were tested under multiplex conditions such that samples were exposed to all oligonucleotides for HPIV1-4 simultaneously. Oligonucleotides for the HPIV1 assay were tested in the HEX channel, oligonucleotides for the HPIV2 assay were tested in the COU channel, oligonucleotides for the HPIV3 assay were tested in the JA270 channel, and oligonucleotides for the HPIV4 assay were tested in the FAM channel. Test samples were tested in the presence of an artificial nasopharyngeal matrix eluate intended to simulate the background of clinical samples containing cells and mucin, and were used at the following concentrations: 5x10 per reaction. 0 , 5x10 1 , 5x10 2 , 5x10 3 , and 5x10 4copies. The composition of the matrix is ​​shown in Figure 18A. The reagents used include cobas® 6800 / 8800 Universal PCR Master Mix with profiles and conditions for use with the cobas® 6800 / 8800, using TaqMan® amplification and detection technology. The cobas® 6800 / 8800 PCR profile used is shown in Table 2 above. The results are shown in Figure 18B (HPIV1), Figure 18C (HPIV2), Figure 18D (HPIV3), and Figure 18E (HPIV4), which show PCR growth curves of a dilution series of the performance of the HPIV1-4 assay. The results showed that in the presence of nasopharyngeal matrix, the sensitivity of HPIV1-4 types is down to 50 copies / reaction. Figures 18B, 18C, 18D, and 18E show that HPIV1-4 oligonucleotides can simultaneously amplify and detect target HPIV1-4 nucleic acids from artificial nasopharyngeal simulated clinical samples in a multiplex setting. The data show that the HPIV1-4 multiplex assay exhibits sensitivity of four HPIV types down to 50 copies / reaction in the presence of a nasopharyngeal background. Because the artificial nasopharyngeal matrix simulates a nasopharyngeal clinical sample, these data suggest that HPIV1-4 oligonucleotides can simultaneously amplify and detect target HPIV1-4 nucleic acids from actual nasopharyngeal samples.

[0112] Example 13: HPIV1-4 primers and probes simultaneously specifically amplify and detect target HPIV1-4 nucleic acids from viral eluates in an HPIV1-4 multiplex real-time PCR assay HPIV1-4 oligonucleotides were tested using primers / probes to detect HPIV1 (SEQ ID NOs: 1-3), HPIV2 (SEQ ID NOs: 4-6), HPIV3 (SEQ ID NOs: 15-17), and HPIV4 (SEQ ID NOs: 11, 13, 18, and 19). Oligonucleotides for HPIV1-4 were tested under multiplex conditions such that samples were exposed to all oligonucleotides for HPIV1-4 simultaneously. Oligonucleotides for the HPIV1 assay were tested in the HEX channel, oligonucleotides for the HPIV2 assay were tested in the COU channel, oligonucleotides for the HPIV3 assay were tested in the JA270 channel, and oligonucleotides for the HPIV4 assay were tested in the FAM channel. The samples tested were viral eluates (ATCC Catalog Numbers: HPIV1-VR-94; HPIV2-VR-92; HPIV3-VR-1782; HPIV-4B-VR1377; ZeptoMetrix Catalog Number: HPIV4A-0810060CF) and were tested at two concentrations, neat and at a 1:100,000 dilution. Reagents used included the cobas® 6800 / 8800 Universal PCR Master Mix, with profiles and conditions for use on the cobas® 6800 / 8800, using TaqMan® amplification and detection technology. The cobas® 6800 / 8800 PCR profiles used are shown in Table 2 above. These studies were designed to test the specificity of the HPIV1-4 oligonucleotides for viral eluates. The results are shown in Figure 19A (HPIV1), Figure 19B (HPIV2), Figure 19C (HPIV3), and Figure 19D (HPIV4), which show the PCR growth curves of a dilution series of the performance of the HPIV1-4 assay. These results demonstrate that the HPIV1-4 oligonucleotides are specific for their intended targets. As seen in Figures 19A, 19B, 19C, and 19D, no cross-reactivity was observed when these viral eluates were used in unintended channels for a particular HPIV type.Taken together, these studies demonstrate that HPIV1-4 oligonucleotides specifically amplify and detect intended targets in a multiplex setting.

[0113] Although the foregoing invention has been described in some detail for clarity and understanding, it will be apparent to those skilled in the art upon reading this disclosure that various changes in form and detail may be made without departing from the true scope of the invention. For example, all of the techniques and devices described above may be used in various combinations. All publications, patents, patent applications, and / or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and / or other document was individually indicated to be incorporated by reference for all purposes.

Claims

1. 1. A method for detecting human parainfluenza virus (HPIV) in a sample, wherein the HPIV comprises human parainfluenza virus type 1 (HPIV-1), human parainfluenza virus type 2 (HPIV-2), human parainfluenza virus type 3 (HPIV-3), and / or human parainfluenza virus type 4 (HPIV-4), the method comprising: (a) if one or more target nucleic acids of HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 are present in the sample, performing an amplification step comprising contacting the sample with one or more sets of primers to generate amplification products of the HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 target nucleic acids; (b) if one or more target nucleic acids of HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 are present in the sample, performing a hybridization step comprising contacting one or more probes with amplification products of the HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 target nucleic acids; and (c) detecting the presence or absence of an amplification product of a HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 target nucleic acid, wherein the presence of an amplification product of a HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 target nucleic acid indicates the presence of HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4, respectively, in the sample. detecting the presence or absence of an amplification product of a HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 target nucleic acid, wherein the absence of an amplification product of a HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 target nucleic acid indicates the absence of HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4, respectively, from the sample; Including; wherein the one or more sets of primers and the one or more probes are (1) a set of primers and a probe specific for a target nucleic acid of HPIV-1, wherein the set of primers specific for a target nucleic acid of HPIV-1 comprises a first primer comprising the nucleic acid sequence of SEQ ID NO:1 and a second primer comprising the nucleic acid sequence of SEQ ID NO:2; the probe specific for a target nucleic acid of HPIV-1 comprises the nucleic acid sequence of SEQ ID NO:3 or a complement thereof; and / or (2) a set of primers and a probe specific for a target nucleic acid of HPIV-2, wherein the set of primers specific for a target nucleic acid of HPIV-2 comprises a first primer comprising the nucleic acid sequence of SEQ ID NO:4 and a second primer comprising the nucleic acid sequence of SEQ ID NO:5; the probe specific for a target nucleic acid of HPIV-2 comprises the nucleic acid sequence of SEQ ID NO:6 or SEQ ID NO:7, or a complement thereof; and / or (3) A set of primers and a probe specific for a target nucleic acid of HPIV-3, wherein the set of primers specific for a target nucleic acid of HPIV-3 comprises a first primer comprising the nucleic acid sequence of SEQ ID NO:8 or SEQ ID NO:15, and a second primer comprising the nucleic acid sequence of SEQ ID NO:9 or SEQ ID NO:16; the probe specific for a target nucleic acid of HPIV-3 comprises the nucleic acid sequence of SEQ ID NO:10 or SEQ ID NO:17, or a complement thereof; and / or (4) A set of primers and a probe specific for a target nucleic acid of HPIV-4, wherein the set of primers specific for a target nucleic acid of HPIV-4 comprises a first primer comprising the nucleic acid sequence of SEQ ID NO:11 or SEQ ID NO:18, and a second primer comprising the nucleic acid sequence of SEQ ID NO:12 or SEQ ID NO:19; and the probe specific for a target nucleic acid of HPIV-4 comprises the nucleic acid sequence of SEQ ID NO:13 or SEQ ID NO:14, or a complement thereof. A method comprising:

2. 1. A method for simultaneously detecting one or more target nucleic acids of HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 in a sample, the method comprising: (a) if one or more target nucleic acids of HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 are present in the sample, performing an amplification step comprising contacting the sample with one or more sets of primers to generate amplification products of the HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 target nucleic acids; (b) if one or more target nucleic acids of HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 are present in the sample, performing a hybridization step comprising contacting one or more probes with amplification products of the HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 target nucleic acids; and (c) detecting the presence or absence of an amplification product of a HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 target nucleic acid, wherein the presence of an amplification product of a HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 target nucleic acid indicates the presence of HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4, respectively, in the sample. detecting the presence or absence of an amplification product of a HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 target nucleic acid, wherein the absence of an amplification product of a HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 target nucleic acid indicates the absence of HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4, respectively, from the sample; Including; wherein the one or more sets of primers and the one or more probes are (1) a set of primers and a probe specific for a target nucleic acid of HPIV-1, wherein the set of primers specific for a target nucleic acid of HPIV-1 comprises a first primer comprising the nucleic acid sequence of SEQ ID NO:1 and a second primer comprising the nucleic acid sequence of SEQ ID NO:2; the probe specific for a target nucleic acid of HPIV-1 comprises the nucleic acid sequence of SEQ ID NO:3 or a complement thereof; and / or (2) a set of primers and a probe specific for a target nucleic acid of HPIV-2, wherein the set of primers specific for a target nucleic acid of HPIV-2 comprises a first primer comprising the nucleic acid sequence of SEQ ID NO:4 and a second primer comprising the nucleic acid sequence of SEQ ID NO:5; the probe specific for a target nucleic acid of HPIV-2 comprises the nucleic acid sequence of SEQ ID NO:6 or SEQ ID NO:7, or a complement thereof; and / or (3) A set of primers and a probe specific for a target nucleic acid of HPIV-3, wherein the set of primers specific for a target nucleic acid of HPIV-3 comprises a first primer comprising the nucleic acid sequence of SEQ ID NO:8 or SEQ ID NO:15, and a second primer comprising the nucleic acid sequence of SEQ ID NO:9 or SEQ ID NO:16; the probe specific for a target nucleic acid of HPIV-3 comprises the nucleic acid sequence of SEQ ID NO:10 or SEQ ID NO:17, or a complement thereof; and / or (4) A set of primers and a probe specific for a target nucleic acid of HPIV-4, wherein the set of primers specific for a target nucleic acid of HPIV-4 comprises a first primer comprising the nucleic acid sequence of SEQ ID NO:11 or SEQ ID NO:18, and a second primer comprising the nucleic acid sequence of SEQ ID NO:12 or SEQ ID NO:19; and the probe specific for a target nucleic acid of HPIV-4 comprises the nucleic acid sequence of SEQ ID NO:13 or SEQ ID NO:14, or a complement thereof. A method comprising:

3. The method according to any one of claims 1 to 2, wherein the sample is a biological sample.

4. 4. The method of claim 3, wherein the biological sample is whole blood, a respiratory specimen, a nasopharyngeal sample, a urine, a fecal specimen, a blood specimen, plasma, a skin swab, a nasal swab, a wound swab, a blood culture, a skin, and a soft tissue infection.

5. The method of claim 4 , wherein the biological sample is a nasopharyngeal sample.

6. The method of any one of claims 1 to 5, wherein the one or more probes are labeled.

7. The method of claim 6 , wherein the one or more probes are labeled with a donor fluorescent moiety and a corresponding acceptor moiety.

8. 8. The method of claim 7, wherein detecting the presence or absence of an amplification product of an HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 target nucleic acid in step (c) comprises detecting the presence or absence of fluorescence resonance energy transfer (FRET) between a donor fluorescent moiety and an acceptor moiety of a probe specific for an HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 target nucleic acid, wherein the presence or absence of fluorescence indicates the presence or absence, respectively, of HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 in the sample.

9. 1. A kit for detecting HPIV that may be present in a sample, wherein the HPIV comprises HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4, the kit comprising amplification and detection reagents, wherein the amplification and detection reagents comprise: (i) a DNA polymerase; (ii) nucleotide monomers; and (iii) one or more sets of primers and one or more probes, wherein the one or more sets of primers and one or more probes are: (1) a set of primers and a probe specific for a target nucleic acid of HPIV-1, wherein the set of primers specific for a target nucleic acid of HPIV-1 comprises a first primer comprising the nucleic acid sequence of SEQ ID NO:1 and a second primer comprising the nucleic acid sequence of SEQ ID NO:2; the probe specific for a target nucleic acid of HPIV-1 comprises the nucleic acid sequence of SEQ ID NO:3 or a complement thereof; and / or (2) a set of primers and a probe specific for a target nucleic acid of HPIV-2, wherein the set of primers specific for a target nucleic acid of HPIV-2 comprises a first primer comprising the nucleic acid sequence of SEQ ID NO:4 and a second primer comprising the nucleic acid sequence of SEQ ID NO:5; the probe specific for a target nucleic acid of HPIV-2 comprises the nucleic acid sequence of SEQ ID NO:6 or SEQ ID NO:7, or a complement thereof; and / or (3) A set of primers and a probe specific for a target nucleic acid of HPIV-3, wherein the set of primers specific for a target nucleic acid of HPIV-3 comprises a first primer comprising the nucleic acid sequence of SEQ ID NO:8 or SEQ ID NO:15, and a second primer comprising the nucleic acid sequence of SEQ ID NO:9 or SEQ ID NO:16; the probe specific for a target nucleic acid of HPIV-3 comprises the nucleic acid sequence of SEQ ID NO:10 or SEQ ID NO:17, or a complement thereof; and / or (4) A set of primers and a probe specific for a target nucleic acid of HPIV-4, wherein the set of primers specific for a target nucleic acid of HPIV-4 comprises a first primer comprising the nucleic acid sequence of SEQ ID NO:11 or SEQ ID NO:18, and a second primer comprising the nucleic acid sequence of SEQ ID NO:12 or SEQ ID NO:19; and the probe specific for a target nucleic acid of HPIV-4 comprises the nucleic acid sequence of SEQ ID NO:13 or SEQ ID NO:14, or a complement thereof. Including the kit.

10. The kit of claim 9 , wherein the sample is a biological sample.

11. 11. The kit of claim 10, wherein the biological sample is whole blood, a respiratory specimen, a nasopharyngeal sample, urine, a fecal specimen, a blood specimen, plasma, a skin swab, a nasal swab, a wound swab, a blood culture, skin, and a soft tissue infection.

12. The kit of claim 11 , wherein the biological sample is a nasopharyngeal sample.

13. The kit according to any one of claims 9 to 12, wherein the one or more probes are labeled.

14. The kit of claim 13 , wherein the one or more probes are labeled with a donor fluorescent moiety and a corresponding acceptor moiety.

15. The kit according to any one of claims 9 to 14, wherein the one or more sets of primers and the one or more probes are (1) A primer set and a probe specific for a target nucleic acid of HPIV-1, wherein the primer set comprises a first primer comprising the nucleic acid sequence of SEQ ID NO:1 and a second primer comprising the nucleic acid sequence of SEQ ID NO:2; and the probe comprises the nucleic acid sequence of SEQ ID NO:3 or a complement thereof; (2) A primer set and a probe specific for a target nucleic acid of HPIV-2, wherein the primer set comprises a first primer comprising the nucleic acid sequence of SEQ ID NO:4 and a second primer comprising the nucleic acid sequence of SEQ ID NO:5; and the probe comprises the nucleic acid sequence of SEQ ID NO:6 or SEQ ID NO:7, or a complement thereof; (3) A set of primers and a probe specific for a target nucleic acid of HPIV-3, wherein the set of primers comprises a first primer comprising a nucleic acid sequence of SEQ ID NO:8 or SEQ ID NO:15, and a second primer comprising a nucleic acid sequence of SEQ ID NO:9 or SEQ ID NO:16; the probe comprises a nucleic acid sequence of SEQ ID NO:10 or SEQ ID NO:17, or a complement thereof; and (4) A primer set and a probe specific for a target nucleic acid of HPIV-4, wherein the primer set comprises a first primer comprising the nucleic acid sequence of SEQ ID NO:11 or SEQ ID NO:18, and a second primer comprising the nucleic acid sequence of SEQ ID NO:12 or SEQ ID NO:19; and the probe comprises the nucleic acid sequence of SEQ ID NO:13 or SEQ ID NO:14, or a complement thereof. Including the kit.

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