Real-time PCR method for detecting bovine parvovirus 3

Abstract

The present invention provides a primer-probe combination for detecting DNA encoding bovine parvovirus 3 (BPV-3) genomic DNA in DNA extracted from a test sample. An endogenous positive control is also provided.

Description

Technical Field

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 126,939, filed December 17, 2020, and U.S. Provisional Patent Application No. 63 / 211,607, filed June 17, 2021, which are hereby incorporated by reference in their entirety for all purposes as if fully set forth herein.

[0002] This application is being filed with a Sequence Listing in electronic format. The Sequence Listing is provided as a file named A-2741-WO-PCT_ST25.txt, created on December 9, 2021, and having a size of 6 kb. The information in the electronic format of this Sequence Listing is hereby incorporated by reference in its entirety.

[0003]

Background Art

[0004] Manufacturing therapeutic biological products using cell culture processes carries an inherent risk of transmitting viral contaminants. Such contaminants can arise from numerous sources, including contamination of the manufacturing system due to materials and equipment, the use of animal-derived reagents during manufacturing, and failures in GMP processes. Animal-derived raw materials, such as bovine serum (FBS), are sometimes used as components of cell culture-based manufacturing processes and are one of the main sources of bovine-derived viral contamination in biomanufacturing processes. To meet regulatory health requirements ensuring the absence of exogenous substances in biological therapeutics derived from mammalian cells, biomanufacturers must take measures to detect, remove, and / or inactivate viral contamination. Raw materials can be tested for viral contamination before use, and corrective actions can be taken. Depending on the biological molecule being manufactured, dedicated viral inactivation and removal processes can be added to downstream purification processes to ensure the viral safety of the biological therapeutic.

[0005] The Parvoviridae family includes single-stranded DNA (ssDNA) viruses, collectively known as parvoviruses, which have a small, non-enveloped capsid with T=1 icosahedral symmetry. Their diameters range from 18 to 26 nm. The viral capsid contains a genome of approximately 5 kb that encodes two major proteins: a non-structural (NS) protein and a structural capsid (VP) protein. Viruses of the Parvoviridae family infect a wide range of hosts and are divided into two subfamilies: Parvovirinae and Densovirinae. These infect vertebrate and arthropod hosts, respectively.

[0006] Bovine parvovirus 3 (BPV-3) belongs to the subfamily Parvovirinae and the genus Erythroparvovirus (a species of ungulate erythroparvovirus 1). BPV-3 is known to infect cattle, but its pathogenesis and clinical symptoms remain unclear.

[0007] In accordance with the testing requirements of the Code of Federal Regulations (9 CFR113), the presence or absence of specific viruses in animal-derived components used in the manufacture of biologics must be assessed by incubating the animal-derived raw materials on specific indicator cells, and then observing for virus-induced cytopathic effects, which are tested by hemostatic adsorption or antibody fluorescence. Among bovine parvoviruses, BPV-1 can replicate in at least one of the selected indicator cell lines and can be detected by the 9 CFR 113 test. [Overview of the project] [Problems that the invention aims to solve]

[0008] However, tolerant cell lines that support BPV-3 replication have not yet been identified and therefore cannot be detected by routine 9 CFR113 testing. Although BPV-3 was detected by next-generation sequencing (NGS) several years ago, it remains a newly emerging virus with limited available information. Conventional cell culture-based viral tests cannot support BPV-3 replication, and therefore molecular biology assays are an essential alternative method for detecting BPV-3 genomic DNA. The impact of BPV-3 on cell culture-based manufacturing remains unclear. There is a need for a rapid and specific method to detect the presence or absence of BPV-3 genomic DNA in raw materials from cell culture-based manufacturing, such as fetal bovine serum (FBS) and / or untreated non-GMP bulk samples containing FBS. The present invention described herein addresses this need. [Means for solving the problem]

[0009] The present invention provides a composition comprising oligonucleotides selected from the group consisting of a) oligonucleotides having the nucleic acid sequences of SEQ ID NOs: 1, 2, and 3; b) oligonucleotides having the nucleic acid sequences of SEQ ID NOs: 4, 5, and 6; c) oligonucleotides having the nucleic acid sequences of SEQ ID NOs: 7, 8, and 9; d) oligonucleotides having the nucleic acid sequences of SEQ ID NOs: 10, 11, and 12; e) oligonucleotides having the nucleic acid sequences of SEQ ID NOs: 13, 14, and 15; f) oligonucleotides having the nucleic acid sequences of SEQ ID NOs: 20, 21, and 22; g) oligonucleotides having the nucleic acid sequences of SEQ ID NOs: 23, 24, and 25; and h) oligonucleotides having the nucleic acid sequences of SEQ ID NOs: 26, 27, and 28. In one embodiment, the composition optionally comprises a second composition comprising oligonucleotides having the sequences of SEQ ID NOs: 16, 17, and 18. In one embodiment, the composition comprises oligonucleotides having the nucleic acid sequences of SEQ ID NOs: 7, 8, and 9. In one embodiment, the composition comprises a first composition containing an oligonucleotide having the nucleic acid sequences of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, and a second composition containing an oligonucleotide having the sequences of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18.

[0010] The present invention provides a reagent for detecting DNA encoding bovine parvovirus 3 (BPV-3) in extracted DNA of a test sample, selected from the group consisting of a) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; b) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6; c) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9; d) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12; e) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15; f) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22; g) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25; and h) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28.

[0011] In one embodiment, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 22, SEQ ID NO: 25, or SEQ ID NO: 28 has a fluorescent reporter dye and / or a non-fluorescent quencher. In related embodiments, one or more of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 22, SEQ ID NO: 25, or SEQ ID NO: 28 have the fluorescent reporter dye 6-carboxyfluorescein (FAM) at the 5' end and / or either a sub-groove binder non-fluorescent quencher (MGB-NFQ) or ZEN-IB and an Iowa Black fluorescent quencher (IBFQ) at the 3' end. In one embodiment, the primer-probe combination detects DNA encoding the structural capsid (VP) protein and / or non-structural (NS) protein of bovine parvovirus 3 in the test sample. In one embodiment, the reagent is in combination with a combination of endogenous positive control primers. In one embodiment, the reagent includes SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9.

[0012] The present invention provides reagents for use as an endogenous positive control primer-probe combination in an assay for detecting bovine parvovirus 3 (BPV-3) genomic DNA in extracted DNA of a test sample, including a primer-probe combination. In relevant embodiments, the endogenous positive control primer-probe combination has the nucleic acid sequences of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18. In one embodiment, SEQ ID NO: 18 has a fluorescent reporter dye and / or a non-fluorescent quencher. In relevant embodiments, SEQ ID NO: 18 has the fluorescent reporter dye 2'-chloro-7'phenyl-1,4-dichloro-6-carboxyfluorescein (VIC) at its 5' end and a secondary groove binder non-fluorescent quencher (MGB-NFQ) at its 3' end. In one embodiment, the reagent includes SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9.

[0013] The present invention provides a combination of primer probes for detecting bovine parvovirus 3 (BPV-3) genomic DNA in extracted DNA of a test sample, in combination with a combination of endogenous positive control primer probes for detecting bovine parvovirus 3 (BPV-3) genomic DNA. In one embodiment, the primer probe combination is selected from the group consisting of a) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; b) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6; c) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9; d) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12; e) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15; f) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22; g) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25; and h) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28. In one embodiment, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, or SEQ ID NO: 15 has a fluorescent reporter dye and / or a non-fluorescent quencher. In one embodiment, one or more of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 22, SEQ ID NO: 25, or SEQ ID NO: 28 has the fluorescent reporter dye 6-carboxyfluorescein (FAM) at its 5' end and / or either a sub-groove binder non-fluorescent quencher (MGB-NFQ) or ZEN-IB and an Iowa Black fluorescent quencher (IBFQ) at its 3' end. In one embodiment, the primer-probe combination detects DNA encoding the structural capsid (VP) protein and / or non-structural (NS) protein of bovine parvovirus 3 in the test sample.In one embodiment, a combination of endogenous positive control primer probes having the nucleic acid sequences of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18 is used in combination with a combination of primer probes used to detect bovine parvovirus 3 (BPV-3) genomic DNA in a test sample, as described above. In a related embodiment, SEQ ID NO: 18 has a fluorescent reporter dye and / or a non-fluorescent quencher. In a related embodiment, SEQ ID NO: 18 has the fluorescent reporter dye 2'-chloro-7'phenyl-1,4-dichloro-6-carboxyfluorescein (VIC) at its 5' end and a secondary groove binder non-fluorescent quencher (MGB-NFQ) at its 3' end.

[0014] The present invention provides a primer-probe combination for detecting DNA encoding bovine parvovirus 3 genomic DNA in extracted DNA of a test sample, including SEQ ID NOs: 7, 8, and 9, in combination with a combination of endogenous positive control primer probes including SEQ ID NOs: 16, 17, and 18, wherein SEQ ID NOs: 9 has the fluorescent reporter dye 6-carboxyfluorescein at its 5' end and a secondary groove binder non-fluorescent quencher (MGB-NFQ) at its 3' end, and SEQ ID NOs: 18 has the fluorescent reporter dye 2'-chloro-7'phenyl-1,4-dichloro-6-carboxyfluorescein at its 5' end and a secondary groove binder non-fluorescent quencher (MGB-NFQ) at its 3' end.

[0015] The present invention provides a kit for detecting BPV-3 genomic DNA contamination in extracted DNA of a test sample, comprising a combination of primer probes for detecting DNA encoding bovine parvovirus 3, selected from the group consisting of a) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; b) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6; c) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9; d) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12; and e) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15; f) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22; g) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25; and h) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28; and an optional combination of endogenous positive control primer probes having the nucleic acid sequences of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18.In one embodiment, the kit includes a combination of primer probes for detecting DNA encoding BPV-3, selected from the group consisting of a) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; b) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6; c) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9; d) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12; and e) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15; f) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22; g) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25; and h) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28; and a combination of endogenous positive control primer probes having the nucleic acid sequences of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18.

[0016] The present invention relates to a method for determining the presence or absence of bovine parvovirus 3 genomic DNA in extracted DNA of a test sample, comprising: 1) a combination of extracted DNA of a test sample, a positive control, BPV-3_IPC positive control plasmid DNA, a nucleic acid amplification reagent, and an endogenous positive control primer probe having the nucleic acid sequences of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18 (where SEQ ID NO: 18 has a fluorescent reporter dye at its 5' end and a non-fluorescent quencher at its 3' end); and a) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 (where SEQ ID NO: 3 has a fluorescent reporter dye at its 5' end and a non-fluorescent quencher at its 3' end); b) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 (where SEQ ID NO: 6 has a fluorescent reporter dye at its 5' end and a non-fluorescent quencher at its 3' end); c) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 (where SEQ ID NO: 9 has a fluorescent reporter dye at its 5' end and (i) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 10, 11, and 12 (where SEQ ID NO: 12 has a fluorescent reporter dye at its 5' end and a non-fluorescent quencher at its 3' end), (d) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 10, 11, and 12 (where SEQ ID NO: 12 has a fluorescent reporter dye at its 5' end and a non-fluorescent quencher at its 3' end), (e) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 13, 14, and 15 (where SEQ ID NO: 15 has a fluorescent reporter dye at its 5' end and a non-fluorescent quencher at its 3' end), (g) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 23, 24, and 25 (where SEQ ID NO: 25 has a fluorescent reporter dye at its 5' end and a non-fluorescent quencher at its 3' end), and (h) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 26, 27, and 28 (where SEQ ID NO: 28 has a fluorescent reporter dye at its 5' end and a non-fluorescent quencher at its 3' end), selected from the group consisting of (i) a combination of primer probes having a non-fluorescent quencher at its 3' end and (d) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 10, 11, and 12 (where SEQ ID NO: 12 has a fluorescent reporter dye at its 5' end and a non-fluorescent quencher at its 3' end), and (h) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 26, 27, and 28 (where SEQ ID NO: 28 has a fluorescent reporter dye at its 5' end and a non-The present invention provides a method comprising the steps of: 3) a reaction mixture containing a primer-probe combination selective to the DNA sequence; 2) subjecting the reaction mixture to quantitative PCR to obtain a copy of the target sequence; and 3) measuring the increase in fluorescence signal, wherein the increase in fluorescence signal indicates the presence of bovine parvovirus 3 genomic DNA in the test sample. In one embodiment, the fluorescent reporter dye is 6-carboxyfluorescein (FAM), and the non-fluorescent quencher is a sub-groove binder non-fluorescent quencher (MGB-NFQ) or ZEN-IB and Iowa Black fluorescent quencher (IBFQ). In one embodiment, the present invention further comprises one or more negative extract controls, template-less controls, positive extract controls, positive controls and / or inhibitor controls. In one embodiment, the sensitivity or analytical limit of detection is 22 genome copies per reaction. In one embodiment, the detection limit of the sample is 25 genome copies per reaction. In one embodiment, a primer-probe combination selective to the DNA sequence of Bovine parvovirus 3 detects genomic DNA encoding the non-structural (NS) protein and / or structural capsid (VP) protein of Bovine parvovirus 3. In one embodiment, a primer-probe combination selective to the DNA sequence of Bovine parvovirus 3 amplifies a 144 bp fragment. In one embodiment, Bovine parvovirus 33) A primer-probe combination selective to the DNA sequence includes the combination of SEQ ID NOs: 7, 8, and 9, wherein SEQ ID NOs: 9 has 6-carboxyfluorescein (FAM) at its 5' end and a secondary groove binder non-fluorescent quencher (MGB-NFQ) at its 3' end. In one embodiment, the method further includes an endogenous positive control primer-probe combination. In a related embodiment, the primer-probe combination has the nucleic acid sequences of SEQ ID NOs: 16, 17, and 18. In a related embodiment, SEQ ID NOs: 18 has a fluorescent reporter dye and / or a non-fluorescent quencher. In a related embodiment, SEQ ID NOs: 18 has the fluorescent reporter dye 2'-chloro-7'phenyl-1,4-dichloro-6-carboxyfluorescein (VIC) at its 5' end and a secondary groove binder non-fluorescent quencher (MGB-NFQ) at its 3' end.

[0017] The present invention relates to a method for quantifying 1E3-1E8 genome copies of bovine parvovirus 3 genomic DNA in a PCR reaction, comprising: 1) a combination of extracted DNA from a test sample, a positive control, BPV-3_IPC positive control plasmid DNA, a nucleic acid amplification reagent, and an endogenous positive control primer probe having the nucleic acid sequences of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18 (where SEQ ID NO: 18 has a fluorescent reporter dye at its 5' end and a non-fluorescent quencher at its 3' end); a) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 (where SEQ ID NO: 3 has a fluorescent reporter dye at its 5' end and a non-fluorescent quencher at its 3' end); b) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 (where SEQ ID NO: 6 has a fluorescent reporter dye at its 5' end and a non-fluorescent quencher at its 3' end); c) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 (where SEQ ID NO: 9 has a fluorescent reporter dye at its 5' end and a non-fluorescent quencher at its 3' end); d) A combination of primer probes having the nucleic acid sequences of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12 (where SEQ ID NO: 12 has a fluorescent reporter dye at its 5' end and a non-fluorescent quencher at its 3' end); e) A combination of primer probes having the nucleic acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15 (where SEQ ID NO: 15 has a fluorescent reporter dye at its 5' end and a non-fluorescent quencher at its 3' end); f) A combination of primer probes having the nucleic acid sequences of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22 (where SEQ ID NO: 22 has a fluorescent reporter dye at its 5' end and a non-fluorescent quencher at its 3' end); g) A combination of primer probes having the nucleic acid sequences of SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25 (where SEQ ID NO: 25 has a fluorescent reporter dye at its 5' end and a non-fluorescent quencher at its 3' end);The present invention provides a method comprising: h) a reaction mixture comprising a primer-probe combination selective to the DNA sequence of Bovine parvovirus 3, selected from the group consisting of a primer-probe combination having the nucleic acid sequences of SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28 (where SEQ ID NO: 28 has a fluorescent reporter dye at its 5' end and a non-fluorescent quencher at its 3' end); 2) subjecting the reaction mixture to quantitative PCR to obtain a copy of the target sequence; and 3) measuring the increase in the fluorescence signal. In one embodiment, the detection limit (LOD 95%) of the method is 27 genome copies of Bovine parvovirus 3 genomic DNA per reaction, with 95% confidence intervals of 22 and 34 genome copies per reaction. In one embodiment, the linearity of the method has a correlation coefficient (R2) of 0.98 or higher and a PCR amplification efficiency of 90-110%. In one embodiment, the method has a parallel precision value of CV% of 25% or less. In one embodiment, the method has an intra-intra-laboratory reproducibility value of CV% of an amount of 30% or less. In one embodiment, the method has an accuracy value within ±30% of the acceptable threshold (ST) across the entire dynamic range of the assay. In one embodiment, the method has a limit of quantification of CV% of an amount of ≤25% in parallel precision and ≤30% in intra-laboratory reproducibility, and an acceptable accuracy threshold within ±30% of the expected standard threshold. In another embodiment, the method has robustness with CV% of an amount of ≤25% in parallel precision and ≤30% in intra-laboratory reproducibility, and an accuracy of the average amount of the tested combined matrix conditions within ±30% of the average amount of the optimized conditions. In another embodiment, the method includes one or more of a template-less control, a positive control, a negative extract control, a positive extract control, an inhibitor control, an endogenous positive control, and a standard. [Brief explanation of the drawing]

[0018] [Figure 1] Probit analysis used for determining ALOD in BPV-3 qPCR with a 95% probability (LOD95%). [Figure 2] POD graph and LOD 95% analysis results. [Modes for carrying out the invention]

[0019] As described herein, the qualitative real-time PCR method was developed to detect potential bovine parvovirus 3 (BPV-3) genomic DNA contamination in extracts of test samples. The performance of the assay was tested and confirmed using eligibility parameters, such as specificity, limit of detection (LOD), robustness, and parallel precision.

[0020] This assay utilizes a primer-probe combination optimized for detecting bovine parvovirus 3 (BPV-3) genomic DNA in extracted DNA from test samples. The BPV-3 PCR primers amplify conserved regions in the genes encoding the BPV-3 non-structural (NS) and / or structural capsid (VP) proteins in the BPV-3 genome. The "Method Validation of US Environmental Protection Agency (EPA) Microbiological Methods of Analysis," REVISION: December 21, 2016, was referenced and considered in the design and development of this assay.

[0021] The present invention provides a combination of oligonucleotide primer probes that can be used in detection methods such as quantitative polymerase chain reaction (qPCR) technology for detecting DNA encoding the bovine parvovirus 3 genome (BPV-3), and a combination of oligonucleotide primer probes for use in a method for determining the presence or absence of BPV-3 in extracted DNA of a test sample.

[0022] As used herein, an "oligonucleotide" is a short single-stranded synthetic DNA or RNA molecule less than 200 nucleotides in length, typically in the range of 13 to 25 nucleotides in length. Oligonucleotides bind to their complementary oligonucleotides to form double-stranded structures. As a result, oligonucleotides are commonly used in a variety of applications where the detection of the presence or absence of a specific DNA or RNA sequence is desired. In particular, oligonucleotides are useful as primers for use in polymerase chain reaction (PCR). Methods for synthesizing oligonucleotides are known in the art, and devices for generating oligonucleotides are commercially available, and there are also service providers that can produce custom oligonucleotides upon request. Oligonucleotides can also be obtained from the degradation of larger nucleic acid molecules or naturally occurring oligonucleotides such as microRNAs.

[0023] The present invention provides a composition comprising an oligonucleotide selected from the group consisting of: a) an oligonucleotide having the nucleic acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; b) an oligonucleotide having the nucleic acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6; c) an oligonucleotide having the nucleic acid sequences of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9; d) an oligonucleotide having the nucleic acid sequences of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12; e) an oligonucleotide having the nucleic acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15; f) an oligonucleotide having the nucleic acid sequences of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22; g) an oligonucleotide having the nucleic acid sequences of SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25; and h) an oligonucleotide having the nucleic acid sequences of SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28. In one embodiment, the composition comprises an oligonucleotide having the nucleic acid sequences of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9.

[0024] In one embodiment, the composition optionally includes a second composition comprising an oligonucleotide having the sequences of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, in combination with one or more of the above primer-probe combinations. In one embodiment, the composition includes an oligonucleotide having a nucleic acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18. In one embodiment, the composition includes a first composition comprising an oligonucleotide having the nucleic acid sequences of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, and a second composition comprising an oligonucleotide having the sequences of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18.

[0025] In one embodiment, one or more of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 25, and SEQ ID NO: 28 may include at least one fluorescent reporter dye at the 5' end and / or may include at least one non-fluorescent quencher at the 3' end. In one embodiment, one or more of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 25, and SEQ ID NO: 28 have at least one fluorescent reporter dye at the 5' end and at least one non-fluorescent quencher at the 3' end. In one embodiment, one or more of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 25, and SEQ ID NO: 28 have a fluorescent reporter dye at the 5' end and a non-fluorescent quencher at the 3' end.

[0026] In one embodiment, one or more of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 22, 25, and 28 have a fluorescent reporter dye at their 5' end, selected from 6-carboxyfluorescein (FAM) or 2'-chloro-7'phenyl-1,4-dichloro-6-carboxyfluorescein (VIC). In one embodiment, SEQ ID NOs: 3, 6, 9, 12, 15, 22, 25, and 28 have the fluorescent reporter dye 6-carboxyfluorescein (FAM) at their 5' end. In one embodiment, SEQ ID NOs: 18 has the fluorescent reporter dye 2'-chloro-7'phenyl-1,4-dichloro-6-carboxyfluorescein (VIC) at its 5' end.

[0027] In one embodiment, one or more of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 22, 25, and 28 have a non-fluorescent quencher at their 3' end. In one embodiment, the 3'-terminus quencher is selected from a sub-groove binder non-fluorescent quencher (MGB-NFQ) and ZEN-IB and Iowa Black fluorescent quenchers (IBFQ). In one embodiment, one or more of SEQ ID NOs: 6, 9, 12, 15, and 18 have a sub-groove binder non-fluorescent quencher (MGB-NFQ) at their 3' end. In one embodiment, one or more of SEQ ID NOs: 3, 22, 25, and 28 have ZEN-IB and Iowa Black fluorescent quenchers (IBFQ) at their 3' end.

[0028] In one embodiment, SEQ ID NO: 3 has the fluorescent reporter dye 6-carboxyfluorescein (FAM) at its 5' end and the ZEN-IB and Iowa Black fluorescent quencher (IBFQ) at its 3' end. In one embodiment, SEQ ID NO: 6 has the fluorescent reporter dye 6-carboxyfluorescein (FAM) at its 5' end and the sub-groove binder non-fluorescent quencher (MGB-NFQ) at its 3' end. In one embodiment, SEQ ID NO: 9 has the fluorescent reporter dye 6-carboxyfluorescein (FAM) at its 5' end and the sub-groove binder non-fluorescent quencher (MGB-NFQ) at its 3' end. In one embodiment, SEQ ID NO: 12 has the fluorescent reporter dye 6-carboxyfluorescein (FAM) at its 5' end and the sub-groove binder non-fluorescent quencher (MGB-NFQ) at its 3' end. In one embodiment, SEQ ID NO: 15 has the fluorescent reporter dye 6-carboxyfluorescein (FAM) at its 5' end and a secondary groove binder non-fluorescent quencher (MGB-NFQ) at its 3' end. In one embodiment, SEQ ID NO: 18 has the fluorescent reporter dye 2'-chloro-7'phenyl-1,4-dichloro-6-carboxyfluorescein (VIC) at its 5' end and a secondary groove binder non-fluorescent quencher (MGB-NFQ) at its 3' end. In one embodiment, SEQ ID NO: 22 has the fluorescent reporter dye 6-carboxyfluorescein (FAM) at its 5' end and ZEN-IB and Iowa Black fluorescent quenchers (IBFQ) at its 3' end. In one embodiment, SEQ ID NO: 25 has the fluorescent reporter dye 6-carboxyfluorescein (FAM) at its 5' end and ZEN-IB and Iowa Black fluorescent quenchers (IBFQ) at its 3' end. In one embodiment, Sequence ID No. 28 has the fluorescent reporter dye 6-carboxyfluorescein (FAM) at its 5' end and ZEN-IB and Iowa Black fluorescent quencher (IBFQ) at its 3' end.

[0029] This invention provides a reagent for use in detecting bovine parvovirus 3 (BPV-3) genomic DNA. This reagent can be used to determine the presence or absence of bovine parvovirus 3 genomic DNA in extracted DNA from a test sample.

[0030] As used herein, “test sample” means any sample of extracted DNA for which the presence or absence of BPV-3 is to be determined. A test sample may be known or suspected to contain BPV-3. A test sample may be from raw materials, for example, raw materials used in the manufacture of biopharmaceuticals. Raw materials include those known or suspected to be of animal origin, or those containing components of animal origin, particularly bovine origin, or those known or suspected to have come into contact with other materials of animal origin, particularly bovine origin, such as fetal bovine serum and fetal calf serum. A test sample may also be from cell culture media, particularly cell culture media containing fetal bovine serum or fetal calf serum. The media may be obtained before or during cell culture, or after taking the cell culture media from the cell culture operation. Periodic samples may be taken during cell culture, which can be done multiple times a day, daily, at important points in the culture, particularly at the start of the culture and at the time of sampling. Test samples may also come from cell lines used in the manufacture of biopharmaceuticals, including bovine-derived cells and cell lines. Test samples may also originate from samples collected during downstream processing, such as eluents from downstream purification steps, and samples can be obtained from active pharmaceutical ingredients and formulations.

[0031] The present invention provides a reagent for detecting bovine parvovirus 3 (BPV-3) genomic DNA in extracted DNA of a test sample selected from the group consisting of a) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; b) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6; c) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9; d) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12; and e) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15. In one embodiment, the primer probe combination includes an oligonucleotide having the nucleic acid sequences of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9.

[0032] In one embodiment, the reagent optionally includes a combination of endogenous positive control primer probes. In one embodiment, the reagent further includes a combination of endogenous positive control primer probes. In one embodiment, the combination of endogenous positive control primer probes includes the nucleic acids of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18. In one embodiment, the reagent includes a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, and a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18.

[0033] In one embodiment, one or more of SEQ ID NOs: 3, 6, 9, 12, 15, or 18 may contain at least one fluorescent reporter dye at its 5' end and / or at least one non-fluorescent quencher at its 3' end. In related embodiments, one or more of SEQ ID NOs: 3, 6, 9, 12, 15, or 18 have at least one fluorescent reporter dye at their 5' end and at least one non-fluorescent quencher at their 3' end. In related embodiments, one or more of SEQ ID NOs: 3, 6, 9, 12, 15, and 18 have a fluorescent reporter dye at their 5' end and a non-fluorescent quencher at their 3' end.

[0034] In one embodiment, one or more of SEQ ID NOs: 3, 6, 9, 12, 15, and 18 have a fluorescent reporter dye at their 5' end, selected from 6-carboxyfluorescein (FAM) or 2'-chloro-7'phenyl-1,4-dichloro-6-carboxyfluorescein (VIC). In one embodiment, SEQ ID NOs: 3, 6, 9, 12, and 15 have the fluorescent reporter dye 6-carboxyfluorescein (FAM) at their 5' end. In one embodiment, SEQ ID NOs: 18 has the fluorescent reporter dye 2'-chloro-7'phenyl-1,4-dichloro-6-carboxyfluorescein (VIC) at its 5' end.

[0035] In one embodiment, one or more of SEQ ID NOs: 3, 6, 9, 12, 15, or 18 have a non-fluorescent quencher at their 3' end. In one embodiment, the 3'-terminated quencher is selected from a sub-groove binder non-fluorescent quencher (MGB-NFQ) and ZEN-IB and Iowa Black fluorescent quenchers (IBFQ). In one embodiment, one or more of SEQ ID NOs: 6, 9, 12, 15, and 18 have a sub-groove binder non-fluorescent quencher (MGB-NFQ) at their 3' end. In one embodiment, SEQ ID NOs: 3 has ZEN-IB and Iowa Black fluorescent quenchers (IBFQ) at their 3' end.

[0036] In one embodiment, SEQ ID NO: 3 has the fluorescent reporter dye 6-carboxyfluorescein (FAM) at its 5' end and the ZEN-IB and Iowa Black fluorescent quencher (IBFQ) at its 3' end. In one embodiment, SEQ ID NO: 6 has the fluorescent reporter dye 6-carboxyfluorescein (FAM) at its 5' end and the sub-groove binder non-fluorescent quencher (MGB-NFQ) at its 3' end. In one embodiment, SEQ ID NO: 9 has the fluorescent reporter dye 6-carboxyfluorescein (FAM) at its 5' end and the sub-groove binder non-fluorescent quencher (MGB-NFQ) at its 3' end. In one embodiment, SEQ ID NO: 12 has the fluorescent reporter dye 6-carboxyfluorescein (FAM) at its 5' end and the sub-groove binder non-fluorescent quencher (MGB-NFQ) at its 3' end. In one embodiment, SEQ ID NO: 15 has a fluorescent reporter dye 6-carboxyfluorescein (FAM) at its 5' end and a secondary groove binder non-fluorescent quencher (MGB-NFQ) at its 3' end. In another embodiment, SEQ ID NO: 18 has a fluorescent reporter dye 2'-chloro-7'phenyl-1,4-dichloro-6-carboxyfluorescein (VIC) at its 5' end and a secondary groove binder non-fluorescent quencher (MGB-NFQ) at its 3' end.

[0037] The present invention provides a primer-probe combination that targets the structural capsid (VP) protein of BPV-3. In one embodiment, the primer-probe combination includes a forward primer that targets oligo positions 3535-3554 of a BPV-3 isolate having GenBank accession number AF406967, a reverse primer that targets oligo positions 3667-3643 of a BPV-3 isolate having GenBank accession number AF406967, and a probe that targets oligo positions 3571-3588 of a BPV-3 isolate having GenBank accession number AF406967. In one embodiment, the primer-probe combination has the nucleic acid sequence of SEQ ID NO: 1, the reverse primer has the nucleic acid sequence of SEQ ID NO: 2, and the probe has the nucleic acid sequence of SEQ ID NO: 3. In one embodiment, the primer-probe combination includes a forward primer targeting oligo positions 1734-1716 of the BPV-3 isolate having GenBank accession number AF406967, a reverse primer targeting oligo positions 1618-1645 of the BPV-3 isolate having GenBank accession number AF406967, and a probe targeting oligo positions 1665-1678 of the BPV-3 isolate having GenBank accession number AF406967. In one embodiment, the primer-probe combination has the nucleic acid sequence of SEQ ID NO: 13, the reverse primer has the nucleic acid sequence of SEQ ID NO: 14, and the probe has the nucleic acid sequence of SEQ ID NO: 15. In one embodiment, the primer-probe combination includes a forward primer targeting oligo positions 2862-2878 of the BPV-3 isolate having GenBank accession number AF406967, a reverse primer targeting oligo positions 2963-2938 of the BPV-3 isolate having GenBank accession number AF406967, and a probe targeting oligo positions 2902-2930 of the BPV-3 isolate having GenBank accession number AF406967.In one embodiment, in the primer-probe combination, the forward primer has the nucleic acid sequence of SEQ ID NO: 20, the reverse primer has the nucleic acid sequence of SEQ ID NO: 21, and the probe has the nucleic acid sequence of SEQ ID NO: 22. In one embodiment, the primer-probe combination includes a forward primer targeting oligo positions 3051-3068 of the BPV-3 isolate having GenBank accession number AF406967, a reverse primer targeting oligo positions 3134-3114 of the BPV-3 isolate having GenBank accession number AF406967, and a probe targeting oligo positions 3079-3102 of the BPV-3 isolate having GenBank accession number AF406967. In one embodiment, in the primer-probe combination, the forward primer has the nucleic acid sequence of SEQ ID NO: 23, the reverse primer has the nucleic acid sequence of SEQ ID NO: 24, and the probe has the nucleic acid sequence of SEQ ID NO: 25. In one embodiment, the primer-probe combination includes a forward primer targeting oligo positions 3061-3079 of the BPV-3 isolate having GenBank accession number AF406967, a reverse primer targeting oligo positions 3140-3122 of the BPV-3 isolate having GenBank accession number AF406967, and a probe targeting oligo positions 3081-3103 of the BPV-3 isolate having GenBank accession number AF406967. In one embodiment, the primer-probe combination has the nucleic acid sequence of SEQ ID NO: 26, the reverse primer has the nucleic acid sequence of SEQ ID NO: 27, and the probe has the nucleic acid sequence of SEQ ID NO: 28.

[0038] The present invention provides a primer-probe combination that targets the non-structural (NS) protein and structural capsid (VP) protein of BPV-3. In one embodiment, the primer-probe combination includes a forward primer that targets oligo positions 2190-2209 of a BPV-3 isolate having GenBank accession number AF406967, a reverse primer that targets oligo positions 2256-2236 of a BPV-3 isolate having GenBank accession number AF406967, and a probe that targets oligo positions 2213-2226 of a BPV-3 isolate having GenBank accession number AF406967. In one embodiment, the forward primer has the nucleic acid sequence of SEQ ID NO: 4, the reverse primer has the nucleic acid sequence of SEQ ID NO: 5, and the probe has the nucleic acid sequence of SEQ ID NO: 6.

[0039] The present invention provides a primer-probe combination that targets the non-structural (NS) protein of BPV-3. In one embodiment, the primer-probe combination includes a forward primer that targets oligo positions 1331-1352 of a BPV-3 isolate having GenBank accession number AF406967, a reverse primer that targets oligo positions 1474-1453 of a BPV-3 isolate having GenBank accession number AF406967, and a probe that targets oligo positions 1377-1391 of a BPV-3 isolate having GenBank accession number AF406967. In one embodiment, the forward primer has the nucleic acid sequence of SEQ ID NO: 7, the reverse primer has the nucleic acid sequence of SEQ ID NO: 8, and the probe has the nucleic acid sequence of SEQ ID NO: 9. In one embodiment, the primer-probe combination includes a forward primer targeting oligo positions 1453-1474 of the BPV-3 isolate having GenBank accession number AF406967, a reverse primer targeting oligo positions 1562-1539 of the BPV-3 isolate having GenBank accession number AF406967, and a probe targeting oligo positions 1507-1522 of the BPV-3 isolate having GenBank accession number AF406967. In one embodiment, the forward primer has the nucleic acid sequence of SEQ ID NO: 10, the reverse primer has the nucleic acid sequence of SEQ ID NO: 11, and the probe has the nucleic acid sequence of SEQ ID NO: 12.

[0040] In one embodiment, a reagent for detecting BPV-3 genomic DNA in a sample is used in combination with a combination of endogenous positive control primers. The present invention provides a combination of primer probes for detecting DNA encoding bovine parvovirus 3 genomic DNA in extracted DNA of a test sample, in combination with a combination of endogenous positive control primer probes including SEQ ID NOs: 16, SEQ ID NOs: 17, and SEQ ID NOs: 18. The present invention provides a primer-probe combination for detecting DNA encoding bovine parvovirus 3 genomic DNA in extracted DNA of a test sample, including SEQ ID NOs: 7, 8, and 9, in combination with a combination of endogenous positive control primer-probes including SEQ ID NOs: 16, 17, and 18, wherein SEQ ID NOs: 9 has a fluorescent reporter dye 6-carboxyfluorescein at its 5' end and a secondary groove binder non-fluorescent quencher (MGB-NFQ) at its 3' end, and SEQ ID NOs: 18 has a fluorescent reporter dye 2'-chloro-7'phenyl-1,4-dichloro-6-carboxyfluorescein at its 5' end and a secondary groove binder non-fluorescent quencher (MGB-NFQ) at its 3' end. In one embodiment, SEQ ID NOs: 18 has a fluorescent reporter dye and / or a non-fluorescent quencher. In one embodiment, SEQ ID NO: 18 has a fluorescent reporter dye, 2'-chloro-7'phenyl-1,4-dichloro-6-carboxyfluorescein (VIC), at its 5' end and a secondary groove binder non-fluorescent quencher (MGB-NFQ) at its 3' end.

[0041] The present invention provides a primer-probe combination for detecting bovine parvovirus 3 (BPV-3) genomic DNA in extracted DNA of a test sample, in combination with an endogenous positive control primer-probe combination for detecting bovine parvovirus 3 (BPV-3) genomic DNA. In one embodiment, the primer-probe combination for detecting bovine parvovirus 3 genomic DNA is selected from the group consisting of a) a combination of primer-probes having the nucleic acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; b) a combination of primer-probes having the nucleic acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6; c) a combination of primer-probes having the nucleic acid sequences of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9; d) a combination of primer-probes having the nucleic acid sequences of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12; e) a combination of primer-probes having the nucleic acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15; f) a combination of primer-probes having the nucleic acid sequences of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22; g) a combination of primer-probes having the nucleic acid sequences of SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25; and h) a combination of primer-probes having the nucleic acid sequences of SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28. In one embodiment, in the primer-probe combination, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 22, SEQ ID NO: 25, and SEQ ID NO: 28 have a fluorescent reporter dye and / or a non-fluorescent quencher. In one embodiment, in a primer-probe combination, one or more of SEQ ID NOs: 3, 6, 9, 12, 15, 22, 25, and 28 have the fluorescent reporter dye 6-carboxyfluorescein (FAM) at the 5' end and / or have either a sub-groove binder non-fluorescent quencher (MGB-NFQ) or ZEN-IB and an Iowa Black fluorescent quencher (IBFQ) at the 3' end.In one embodiment, the primer-probe combination detects DNA encoding the structural capsid (VP) protein and / or non-structural (NS) protein of bovine parvovirus 3 in the test sample.

[0042] In one embodiment, the combination of endogenous positive control primer-probes for detecting bovine parvovirus 3 (BPV-3) genomic DNA in extracted DNA of a test sample, in combination with a primer-probe combination for detecting bovine parvovirus 3 (BPV-3) genomic DNA, has the nucleic acid sequences of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18. In one embodiment, SEQ ID NO: 18 has a fluorescent reporter dye and / or a non-fluorescent quencher. In one embodiment, SEQ ID NO: 18 has the fluorescent reporter dye 2'-chloro-7'phenyl-1,4-dichloro-6-carboxyfluorescein (VIC) at the 5' end and a secondary groove binder non-fluorescent quencher (MGB-NFQ) at the 3' end.

[0043] A primer-probe combination is also provided for detecting bovine parvovirus 3 genomic DNA in extracted DNA of a test sample, including SEQ ID NOs: 7, 8, and 9, in combination with a combination of endogenous positive control primer-probes including SEQ ID NOs: 16, 17, and 18, wherein SEQ ID NOs: 9 has the fluorescent reporter dye 6-carboxyfluorescein at its 5' end and a secondary groove binder non-fluorescent quencher (MGB-NFQ) at its 3' end, and SEQ ID NOs: 18 has the fluorescent reporter dye 2'-chloro-7'phenyl-1,4-dichloro-6-carboxyfluorescein at its 5' end and a secondary groove binder non-fluorescent quencher (MGB-NFQ) at its 3' end.

[0044] The present invention provides a method for determining the presence or absence of bovine parvovirus 3 (BPV-3) genomic DNA in extracted DNA from a test sample. The method comprises: extracted DNA from a test sample; a nucleic acid amplification reagent; a reaction mixture comprising a primer-probe combination selective for bovine parvovirus 3 (BPV-3) genomic DNA and optionally a combination of an endogenous positive control primer-probe; subjecting the reaction mixture to real-time PCR (qPCR) to obtain a copy of a target sequence; and measuring the increase in fluorescence signal, where an increase in fluorescence signal indicates the presence of bovine parvovirus 3 (BPV-3) genomic DNA in the test sample. The reagents described herein may also be used to determine the level of positivity and viral load, for example, with respect to the amount of viral genome per ml of test sample, for the quantification of BPV-3 genome copy number in the test sample.

[0045] The reaction mixture contains the components necessary to perform quantitative PCR techniques. Standard master mixes and component mixes are commercially available, including the 2×TAQMAN® Universal PCR Master Mix (Applied Biosystems). Components typically include dNTPs (dATP, dCTP, dGTP, dTTP, or dUTP), magnesium, TAQ DNA polymerase, buffer, and loading dye (if required by the PCR thermal cycler used). Other options include Quantabio, PerfeCTa qPCR Supermix, and Low ROX (Quantabio, Beverly, MA). Various commercially available thermal cyclers are also available. Those skilled in the art will be able to determine which meets their needs.

[0046] Real-time PCR, or qPCR, is a technique that requires relatively small amounts of DNA, cDNA, or RNA, enabling quantification and facilitating real-time monitoring of the PCR progress as the reaction progresses. This PCR technique utilizes a combination of oligonucleotide primers and a double-labeled oligonucleotide probe. The probe acts as a reporter, accumulating with each cycle of the PCR reaction when amplified.

[0047] Specific detection of amplified products can be performed using one or more oligonucleotide probes labeled with a reporter fluorescent dye and a quencher dye. Such probes are known to those skilled in the art and are commercially available, such as molecular indicators, dual-labeled probes, FRET (fluorescence resonance energy transfer) probes, and Scorpion® probes. Oligonucleotide probes can be labeled with a reporter fluorescent dye and one or more quencher dyes. Examples of fluorescent reporter dyes include 6-carboxyfluorescein (FAM or 6-FAM), 2'-chloro-7'phenyl-1,4-dichloro-6-carboxyfluorescein (VIC), TET(trademark), HEX(trademark), JOE, Cy(registered trademark)3, CY(registered trademark)5, CY(registered trademark)5.5, TAMRA, ROX(trademark), LC Red 610, Texas Red(registered trademark), LC640, SUN(trademark), MAX(trademark), ATTO(trademark)550, ATTO647(trademark), Cal Fluor Gold 540 and Orange 560, TxRd(sulforodamine 101-X), Quasar 570 and 670. Examples of fluorescent quenchers include the sub-groove binder non-fluorescent quencher (MGB-NFQ), ZEN-IB, Black Hole Quencher® (BHQ 1, 2, and 3), TAMRA, Iowa Black® FQ, and RQ.

[0048] For example, a dual-labeled probe can be labeled with one or more fluorescent reporter dyes that fluoresce in the presence of a complementary target at its 5' end, and one or more non-fluorescent quenchers at its 3' end. The dual-labeled probe is designed to hybridize to a template between two primers and is used in combination with a DNA polymerase enzyme that has intrinsic 5'→3' endonuclease activity. When the probe is intact, the fluorescence of the reporter dye is quenched by the proximity of the quencher dye. During the extension phase of each PCR cycle, the 5' endonuclease activity of the DNA polymerase enzyme cleaves the annealed probe, releasing the reporter dye from the probe and increasing fluorescence. This increase in fluorescence is directly proportional to the amount of amplified target DNA present during the reaction. Fluorescence is continuously monitored throughout the PCR reaction. During the initial cycles of the PCR reaction, the amount of fluorescence is below the instrument's detection threshold. Continue monitoring the fluorescence signal and record the first PCR cycle in which fluorescence is detected. The greater the amount of target DNA present in the sample at the start of the reaction, the faster the fluorescence is detected, and this is inversely correlated with the amount of target DNA in the sample.

[0049] In one embodiment, a primer-probe combination selective for the DNA sequence of bovine parvovirus 3 amplifies a 144 bp fragment. In another embodiment, a primer-probe combination having the nucleic acid sequences of SEQ ID NOs. 7, 8, and 9 amplifies a 144 bp fragment.

[0050] The reaction mixture also includes primer-probe combinations selective to the DNA sequence of bovine parvovirus 3. In one embodiment, the primer-probe combinations are: a) a combination of primer-probes having the nucleic acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 (where SEQ ID NO: 3 has a fluorescent reporter dye at its 5' end and a non-fluorescent quencher at its 3' end); b) a combination of primer-probes having the nucleic acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 (where SEQ ID NO: 6 has a fluorescent reporter dye at its 5' end and a non-fluorescent quencher at its 3' end); c) a combination of primer-probes having the nucleic acid sequences of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 (where SEQ ID NO: 9 has a fluorescent reporter dye at its 5' end and a non-fluorescent quencher at its 3' end); d) a combination of primer-probes having the nucleic acid sequences of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12 (where SEQ ID NO: 12 has a fluorescent reporter dye at its 5' end and a non-fluorescent quencher at its 3' end). );e) combinations of primer probes having the nucleic acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15 (where SEQ ID NO: 15 has a fluorescent reporter dye at its 5' end and a non-fluorescent quencher at its 3' end);f) combinations of primer probes having the nucleic acid sequences of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22 (where SEQ ID NO: 22 has a fluorescent reporter dye at its 5' end and a non-fluorescent quencher at its 3' end);g) combinations of primer probes having SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25 (where SEQ ID NO: 25 has a fluorescent reporter dye at its 5' end and a non-fluorescent quencher at its 3' end);and h) combinations of primer probes having the nucleic acid sequences of SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28 (where SEQ ID NO: 28 has a fluorescent reporter dye at its 5' end and a non-fluorescent quencher at its 3' end).

[0051] In one embodiment, the reaction mixture optionally includes a combination of endogenous positive control primer probes. In one embodiment, the combination of endogenous positive control primer probes has the nucleic acid sequences of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, wherein SEQ ID NO: 18 has a fluorescent reporter dye at its 5' end and a non-fluorescent quencher at its 3' end.

[0052] In one embodiment, the method further comprises a combination of endogenous positive control primer-probes. In one embodiment, the primer-probe combination has the nucleic acid sequences of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18. In one embodiment, SEQ ID NO: 18 has a fluorescent reporter dye and / or a non-fluorescent quencher. In one embodiment, SEQ ID NO: 18 has the fluorescent reporter dye 2'-chloro-7'phenyl-1,4-dichloro-6-carboxyfluorescein (VIC) at its 5' end and a secondary groove binder non-fluorescent quencher (MGB-NFQ) at its 3' end.

[0053] In one embodiment, one or more of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 22, 25, and 28 have a fluorescent reporter dye at their 5' end, selected from 6-carboxyfluorescein (FAM) or 2'-chloro-7'phenyl-1,4-dichloro-6-carboxyfluorescein (VIC). In one embodiment, SEQ ID NOs: 3, 6, 9, 12, 15, 22, 25, and 28 have the fluorescent reporter dye 6-carboxyfluorescein (FAM) at their 5' end. In one embodiment, SEQ ID NOs: 18 has the fluorescent reporter dye 2'-chloro-7'phenyl-1,4-dichloro-6-carboxyfluorescein (VIC) at its 5' end.

[0054] In one embodiment, one or more of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 22, 25, and 28 have a non-fluorescent quencher at their 3' end. In one embodiment, the 3'-terminated quencher is selected from a sub-groove binder non-fluorescent quencher (MGB-NFQ) and ZEN-IB and Iowa Black fluorescent quenchers (IBFQ). In one embodiment, one or more of SEQ ID NOs: 6, 9, 12, 15, and 18 have a sub-groove binder non-fluorescent quencher (MGB-NFQ) at their 3' end. In one embodiment, one or more of SEQ ID NOs: 3, 22, 25, and 28 have ZEN-IB and Iowa Black fluorescent quenchers (IBFQ) at their 3' end.

[0055] In one embodiment, SEQ ID NO: 3 has the fluorescent reporter dye 6-carboxyfluorescein (FAM) at its 5' end and the ZEN-IB and Iowa Black fluorescent quencher (IBFQ) at its 3' end. In one embodiment, SEQ ID NO: 6 has the fluorescent reporter dye 6-carboxyfluorescein (FAM) at its 5' end and the sub-groove binder non-fluorescent quencher (MGB-NFQ) at its 3' end. In one embodiment, SEQ ID NO: 9 has the fluorescent reporter dye 6-carboxyfluorescein (FAM) at its 5' end and the sub-groove binder non-fluorescent quencher (MGB-NFQ) at its 3' end. In one embodiment, SEQ ID NO: 12 has the fluorescent reporter dye 6-carboxyfluorescein (FAM) at its 5' end and the sub-groove binder non-fluorescent quencher (MGB-NFQ) at its 3' end. In one embodiment, SEQ ID NO: 15 has the fluorescent reporter dye 6-carboxyfluorescein (FAM) at its 5' end and a secondary groove binder non-fluorescent quencher (MGB-NFQ) at its 3' end. In one embodiment, SEQ ID NO: 18 has the fluorescent reporter dye 2'-chloro-7'phenyl-1,4-dichloro-6-carboxyfluorescein (VIC) at its 5' end and a secondary groove binder non-fluorescent quencher (MGB-NFQ) at its 3' end. In one embodiment, SEQ ID NO: 22 has the fluorescent reporter dye 6-carboxyfluorescein (FAM) at its 5' end and ZEN-IB and Iowa Black fluorescent quenchers (IBFQ) at its 3' end. In one embodiment, SEQ ID NO: 25 has the fluorescent reporter dye 6-carboxyfluorescein (FAM) at its 5' end and ZEN-IB and Iowa Black fluorescent quenchers (IBFQ) at its 3' end. In one embodiment, Sequence ID No. 28 has the fluorescent reporter dye 6-carboxyfluorescein (FAM) at its 5' end and ZEN-IB and Iowa Black fluorescent quencher (IBFQ) at its 3' end.

[0056] In one embodiment, the primer-probe combination targets the non-structural (NS) protein of BPV-3. In another embodiment, the primer-probe combination targets both the non-structural (NS) protein and the structural capsid (VP) protein of BPV-3. In yet another embodiment, the primer-probe combination targets the structural capsid (VP) protein of BPV-3.

[0057] In one embodiment, the primer-probe combination targets the structural capsid (VP) protein of BPV-3. In one embodiment, the primer-probe combination includes a forward primer targeting oligo positions 3535-3554 of a BPV-3 isolate having GenBank accession number AF406967, a reverse primer targeting oligo positions 3667-3643 of a BPV-3 isolate having GenBank accession number AF406967, and a probe targeting oligo positions 3571-3588 of a BPV-3 isolate having GenBank accession number AF406967. In one embodiment, in the primer-probe combination, the forward primer has the nucleic acid sequence of SEQ ID NO: 1, the reverse primer has the nucleic acid sequence of SEQ ID NO: 2, and the probe has the nucleic acid sequence of SEQ ID NO: 3. In one embodiment, the primer-probe combination includes a forward primer targeting oligo positions 1734-1716 of the BPV-3 isolate having GenBank accession number AF406967, a reverse primer targeting oligo positions 1618-1645 of the BPV-3 isolate having GenBank accession number AF406967, and a probe targeting oligo positions 1665-1678 of the BPV-3 isolate having GenBank accession number AF406967. In one embodiment, the primer-probe combination has the nucleic acid sequence of SEQ ID NO: 13, the reverse primer has the nucleic acid sequence of SEQ ID NO: 14, and the probe has the nucleic acid sequence of SEQ ID NO: 15. In one embodiment, the primer-probe combination includes a forward primer targeting oligo positions 2862-2878 of the BPV-3 isolate having GenBank accession number AF406967, a reverse primer targeting oligo positions 2963-2938 of the BPV-3 isolate having GenBank accession number AF406967, and a probe targeting oligo positions 2902-2930 of the BPV-3 isolate having GenBank accession number AF406967.In one embodiment, in the primer-probe combination, the forward primer has the nucleic acid sequence of SEQ ID NO: 20, the reverse primer has the nucleic acid sequence of SEQ ID NO: 21, and the probe has the nucleic acid sequence of SEQ ID NO: 22. In one embodiment, the primer-probe combination includes a forward primer targeting oligo positions 3051-3068 of the BPV-3 isolate having GenBank accession number AF406967, a reverse primer targeting oligo positions 3134-3114 of the BPV-3 isolate having GenBank accession number AF406967, and a probe targeting oligo positions 3079-3102 of the BPV-3 isolate having GenBank accession number AF406967. In one embodiment, in the primer-probe combination, the forward primer has the nucleic acid sequence of SEQ ID NO: 24, the reverse primer has the nucleic acid sequence of SEQ ID NO: 25, and the probe has the nucleic acid sequence of SEQ ID NO: 26. In one embodiment, the primer-probe combination includes a forward primer targeting oligo positions 3061-3079 of the BPV-3 isolate having GenBank accession number AF406967, a reverse primer targeting oligo positions 3140-3122 of the BPV-3 isolate having GenBank accession number AF406967, and a probe targeting oligo positions 3081-3103 of the BPV-3 isolate having GenBank accession number AF406967. In one embodiment, the primer-probe combination has the nucleic acid sequence of SEQ ID NO: 26, the reverse primer has the nucleic acid sequence of SEQ ID NO: 27, and the probe has the nucleic acid sequence of SEQ ID NO: 28.

[0058] In one embodiment, the primer-probe combination targets the structural capsid (VP) protein and non-structural (NS) protein of BPV-3. In one embodiment, the primer-probe combination includes a forward primer targeting oligo positions 2190-2209 of a BPV-3 isolate having GenBank accession number AF406967, a reverse primer targeting oligo positions 2256-2236 of a BPV-3 isolate having GenBank accession number AF406967, and a probe targeting oligo positions 2213-2226 of a BPV-3 isolate having GenBank accession number AF406967. In one embodiment, the forward primer has the nucleic acid sequence of SEQ ID NO: 4, the reverse primer has the nucleic acid sequence of SEQ ID NO: 5, and the probe has the nucleic acid sequence of SEQ ID NO: 6.

[0059] In one embodiment, the primer-probe combination targets the non-structural (NS) protein of BPV-3. In one embodiment, the primer-probe combination includes a forward primer targeting oligo positions 1331-1352 of a BPV-3 isolate having GenBank accession number AF406967, a reverse primer targeting oligo positions 1474-1453 of a BPV-3 isolate having GenBank accession number AF406967, and a probe targeting oligo positions 1377-1391 of a BPV-3 isolate having GenBank accession number AF406967. In one embodiment, the forward primer has the nucleic acid sequence of SEQ ID NO: 7, the reverse primer has the nucleic acid sequence of SEQ ID NO: 8, and the probe has the nucleic acid sequence of SEQ ID NO: 9. In one embodiment, the primer-probe combination includes a forward primer targeting oligo positions 1453-1474 of the BPV-3 isolate having GenBank accession number AF406967, a reverse primer targeting oligo positions 1562-1539 of the BPV-3 isolate having GenBank accession number AF406967, and a probe targeting oligo positions 1507-1522 of the BPV-3 isolate having GenBank accession number AF406967. In one embodiment, the forward primer has the nucleic acid sequence of SEQ ID NO: 10, the reverse primer has the nucleic acid sequence of SEQ ID NO: 11, and the probe has the nucleic acid sequence of SEQ ID NO: 12.

[0060] In one embodiment, the primer-probe combination includes SEQ ID NOs: 7, 8, and 9 in combination with an endogenous positive control primer-probe combination including SEQ ID NOs: 16, 17, and 18, wherein SEQ ID NOs: 9 has the fluorescent reporter dye 6-carboxyfluorescein at its 5' end and a secondary groove binder non-fluorescent quencher (MGB-NFQ) at its 3' end, and SEQ ID NOs: 18 has the fluorescent reporter dye 2'-chloro-7'phenyl-1,4-dichloro-6-carboxyfluorescein at its 5' end and a secondary groove binder non-fluorescent quencher (MGB-NFQ) at its 3' end.

[0061] The reaction mixture used in the methods described herein may further include one or more controls. These controls include a template-free control containing all PCR reagents except the DNA template; a negative extraction control for monitoring cross-contamination during nucleic acid extraction and verifying that the assay does not produce false-positive results with nonspecific background DNA; a positive extraction control for evaluating DNA extraction performance during test sample preparation and verifying that the assay does not produce false-negative results; a positive control for verifying that the qPCR component acts correctly and provides a PCR amplification signal specific to the target sequence; an inhibitory control for determining sample interference / inhibition levels; a standard for determining PCR efficiency, linear range, and quantifying the absolute copy number of the target sequence in the extracted DNA of the test sample / control; and / or an endogenous positive control for monitoring cross-contamination of the positive control plasmid in the test sample used during sample preparation and assay execution.

[0062] The preferred levels of accuracy, accuracy, and linearity of an assay are indicated preferentially within the dynamic range of the analytical assays, particularly quantitative assays, described herein. The accuracy of an assay is based on parallel accuracy (intra-assay accuracy) and intra-laboratory reproducibility (laboratory accuracy). Parallel accuracy is the coefficient of variation (CV) of results obtained over a short period of time, by the same analyst, in the same laboratory, using the same equipment, on the same sample, and in the same manner. To determine the parallel accuracy of an assay, the mean and standard deviation (StdDev) (genomic copies of the target sequence per reaction, GC / rxn) and the CV% of the volume are calculated from each set of PCR reactions at each concentration. Intra-laboratory reproducibility takes into account inherent variability such as different analysts, different detection systems, and different time periods. To determine the intra-laboratory reproducibility of an assay, the mean and StdDev and the CV% of the volume are calculated from all sets of PCR reactions at each concentration in independent experiments over a set period.

[0063] Accuracy (also called trueness) is determined by comparing values ​​obtained from a series of samples (e.g., positive control plasmids at a given concentration) to an actual value or reference value called a standard (ST). To determine the accuracy of an assay, the average quantity (genomic copies of the target sequence per reaction, GC / rxn) is calculated from each set of PCR reactions at each concentration.

[0064] The limit of quantification (LOQ) of a quantitative qPCR assay is the minimum amount of the target sequence in a sample that can be quantitatively determined with adequate precision (parallel precision and intra-intra-laboratory precision) and accuracy. To determine the LOQ, several minimum, maximum, and intermediate ranges are tested. Positive control plasmids at minimum, intermediate, and upper limits are prepared and spiked into exogenous extracted DNA, and quantified by performing an appropriate number of replicates, preferably by different analysts on different days, in the presence of a calibration curve.

[0065] The limit of detection (LOD) or assay sensitivity is the minimum amount of the target sequence that can be detected by the assay but is not necessarily quantified as an accurate value.

[0066] The robustness of the assay can be determined by the Youden and Steiner method (Youden, Steiner, Statistical Manual of Association of Official Analytical Chemists, Association of Official Analytical Chemists ed., Arlington 1975, pps. 33-36, 70-71; 82083). For example, changes to specific critical reagent concentrations can be evaluated. Specific important PCR factors can be selected and slightly altered. Acceptance criteria require that the response obtained to robust conditions with respect to the applied minute changes meets the acceptance criteria for the established assay.

[0067] The present invention provides an assay for quantifying bovine parvovirus 3 genomic DNA in extracted DNA from a test sample. In one embodiment, an assay is provided for quantifying 1E3-1E8 genomic copies of bovine parvovirus 3 genomic DNA in a PCR reaction.

[0068] The present invention relates to 1) a combination of extracted DNA from a test sample, a positive control, BPV-3_IPC positive control plasmid DNA, a nucleic acid amplification reagent, and an endogenous positive control primer probe having the nucleic acid sequences of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18 (where SEQ ID NO: 18 has a fluorescent reporter dye at its 5' end and a non-fluorescent quencher at its 3' end), and a) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 (where SEQ ID NO: 3 has a fluorescent reporter dye at its 5' end and a non-fluorescent quencher at its 3' end); b) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 (where SEQ ID NO: 6 has a fluorescent reporter dye at its 5' end and a non-fluorescent quencher at its 3' end); c) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 (where SEQ ID NO: 9 has a fluorescent reporter dye at its 5' end). (i) a combination of a primer probe having the nucleic acid sequences of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12 (where SEQ ID NO: 12 has a fluorescent reporter dye at its 5' end and a non-fluorescent quencher at its 3' end); (ii) a combination of a primer probe having the nucleic acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15 (where SEQ ID NO: 15 has a fluorescent reporter dye at its 5' end and a non-fluorescent quencher at its 3' end); (iii) a combination of a primer probe having the nucleic acid sequences of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22 (where SEQ ID NO: 22 has a fluorescent reporter dye at its 5' end and a non-fluorescent quencher at its 3' end); (iv) a combination of a primer probe having the nucleic acid sequences of SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25 (where SEQ ID NO: 25 has a fluorescent reporter dye at its 5' end and a non-fluorescent quencher at its 3' end);The present invention provides a method for quantifying 1E3-1E8 genome copies of Bovine parvovirus 3 genomic DNA in a PCR reaction, comprising: h) a reaction mixture containing a primer-probe combination selective to the DNA sequence of Bovine parvovirus 3, selected from the group consisting of a combination of primer-probes having the nucleic acid sequences of SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28 (where SEQ ID NO: 28 has a fluorescent reporter dye at its 5' end and a non-fluorescent quencher at its 3' end); 2) subjecting the reaction mixture to quantitative PCR to obtain a copy of the target sequence; and 3) measuring the increase in the fluorescence signal.

[0069] In one embodiment, the dynamic range is 1E3 to 1E8 GC / rxn. In one embodiment, the lower limit of quantification is 1E3 GC / rxn. In one embodiment, the upper limit of quantification is 1E8 GC / rxn. In one embodiment, the limit of detection (LOD 95%) of the quantification assay is 27 genome copies per reaction, and the 95% confidence intervals are 22 and 34 genome copies per reaction. In one embodiment, the assay has linearity with a correlation coefficient (R2) ≥ 0.98 and a PCR amplification efficiency of 90-110%. In one embodiment, the assay has a parallel precision value of CV% of 25% or less. In one embodiment, the assay has an intra-laboratory reproducibility value of CV% of 30% or less. In one embodiment, the assay has an accuracy value within ±30% of the acceptable threshold (ST) across the entire dynamic range of the assay. In one embodiment, the assay has a limit of quantification which is a CV% of the amount with parallel precision of ≤25% and intra-intra

[0070] The present invention includes a combination of primer probes selected from the group consisting of a) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; b) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6; c) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9; d) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12; e) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15; f) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22; g) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25; and h) a combination of primer probes having the nucleic acid sequences of SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28; and an optional combination of endogenous positive control primer probes having the nucleic acid sequences of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, for detecting DNA encoding bovine parvovirus 3 in extracted DNA of a test sample. 3) Provide a kit for use in detecting (BPV-3) genomic DNA contamination.

[0071] The terminology used in this application is standard in the art; however, definitions of specific terms are provided herein to ensure clarity and unambiguity with respect to the meaning of the claims. Units, prefixes, and symbols may be expressed in their International System of Units (SI) acceptable forms. Numerical ranges described herein include the number defining the range, each integer within the defined range, and supporting it. Unless otherwise stated, methods and techniques described herein are generally carried out in accordance with conventional methods well known in the art, such methods and techniques are described in the various general and specific references cited and discussed throughout this specification. These include, but are not limited to, patents, patent applications, articles, books, and academic papers, and all or parts of such documents cited herein are expressly incorporated herein by reference.

[0072] The present invention is not limited in scope by the specific embodiments described herein, which are intended as single descriptions of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. In fact, various modifications of the invention, in addition to those shown and described herein, will be apparent to those skilled in the art from the above and the accompanying drawings. Such modifications are intended to be included within the scope of the accompanying claims. Any aspect or embodiment described herein can be combined with other aspects and / or embodiments of the invention.

[0073] The following examples, including experiments conducted and results obtained, are provided for illustrative purposes only and should not be construed as limiting the scope of the appended claims. [Examples]

[0074] Example 1: Development of a BPV-3 primer / probe set To identify the conserved regions of the BPV-3 genome, all publicly available full-length genome sequences of BPV-3 isolates were obtained from GenBank (Table 1).

[0075] [Table 1]

[0076] Multiple alignments were performed to select different conserved regions in the genes encoding the non-structural (NS) and structural capsid (VP) proteins of BPV-3, and used to design eight different primer / probe sets (BPV-3v1.0-BPV-3v8.0). The 5' end of the probes was labeled with the fluorescent reporter dye 6-carboxyfluorescein (FAM), and the 3' end was labeled with a sub-groove binder non-fluorescent quencher (MGB-NFQ) or a ZEN-IB and Iowa Black fluorescent quencher (IBFQ) (Applied Biosystems, Carlsbad, CA, or Integrated DNA Technologies, Inc., Coralville, IA). Table 2 shows the oligo positions and sequences of the probe and primer sets. The primer / probe sets were synthesized and tested in 6 replicates using a 5-log concentration range (1e7-1e3) of the positive control plasmid.

[0077] [Table 2]

[0078] [Table 3]

[0079] [Table 4]

[0080] LOD, dynamic range, PCR efficiency and R 2Based on the values, BPV-3 v3.0 was selected for use in assay development and validation. The primers amplified a 144 bp fragment of BPV 3. The probe has the fluorescent reporter dye 6-carboxyfluorescein (FAM) at the 5' end and a secondary groove binder non-fluorescent quencher (MGB-NFQ) at the 3' end (Applied Biosystems, Carlsbad, CA). The designed BPV-3 v3.0 primer / probe sequence perfectly matched all five sequences of the Parvovirus (Parvovirinae) subfamily by in silico analysis. Multiple alignments with other parvoviruses did not produce a match indicating that the primer-probe combination sequence is specific to BPV-3.

[0081] Endogenous positive control primer / probe set and BPV-3_IPC positive control plasmid DNA An endogenous positive control (IPC) was designed to monitor extraction efficiency and accidental cross-contamination between the spiked positive control plasmid and the extracted DNA from the test sample. The 5' end of the IPC probe (IPC v2.0) was labeled with the fluorescent reporter dye 2'-chloro-7'phenyl-1,4-dichloro-6-carboxy-fluorescein (VIC), and the 3' end was labeled with a sub-groove binder non-fluorescent quencher (MGB-NFQ) (Applied Biosystems, Carlsbad, CA). The IPC primer / probe set was amplified from a 67 bp fragment to facilitate differentiation from the BPV-3 primer / probe. Table 3 shows the oligofunctionality and sequences of the probe and primer. This probe / primer set was used for the development and validation of further BPV-3 assays.

[0082] [Table 5]

[0083] To construct a BPV-3_IPC positive control plasmid DNA, the target sequences of BPV-3 and IPC were synthesized and cloned into the pUC57 vector. The 5' end of the positive control sequence contained the BPV-3 target genome, and the 3' end contained the IPC target sequence. Therefore, the positive control plasmid contains both BPV-3 and IPC sequences and functions as a BPV-3 positive control and an endogenous positive control for monitoring extraction efficiency and accidental cross-contamination. The sequence of the positive control DNA is shown in Table 4.

[0084] [Table 6]

[0085] Example 2: BPV-3 Qualitative Assay Sample preparation For test samples containing cells, the samples were subjected to low-speed centrifugation (LSC) (320×g to 1000×g, 10 minutes at room temperature), followed by DNA extraction. The supernatant was collected for DNA extraction. Cell-free samples were processed directly.

[0086] To a 250 μL test sample, 10 μL of RNase Cocktail® enzyme mix (Thermo Fisher, Carlsbad, CA) and a 0.5 M EDTA, pH 8.0 mixture (9 μL:1 μL) were added. DNA was extracted from the test sample using an AutoMate Express® nucleic acid extraction system (Thermo Fisher). For the DNA extraction procedure, the PrepSEQ(PS) Express 1-2-3 protocol with the following parameters was selected for AutoMate: 1 hour of proteinase K (PK) digestion and 100 μL elution.

[0087] The extracted DNA was visually inspected, and if any magnetic beads remained in the eluted DNA, they were removed using a DynaMag®-2 magnetic benchtop workstation (Thermo Fisher). The DNA was either used immediately or stored at -30°C.

[0088] In parallel with DNA extraction from the test samples, BPV-3_IPC (PEC) plasmid controls and negative extraction controls (NECs) were also prepared and performed as described above. For the positive extraction control, 62,000 genome copies (GC) of BPV-3_IPC plasmid DNA were spiked into 250 μL of sterile 1× phosphate-buffered saline (PBS). The DNA extraction efficiency during sample preparation was evaluated using the positive extraction control. Assuming a 100% DNA extraction efficiency, the final eluted DNA should contain 500 GC / μL. The negative extraction control contained 250 μL of sterile 1× PBS. The negative extraction control was used to monitor for accidental cross-contamination that may occur during nucleic acid extraction.

[0089] Preparation of additional BPV-3 detection assay controls Template-free control (NTC): The template-free control contained all PCR reagents except the DNA template. This was used as a negative control to confirm that the qPCR raw materials were not contaminated with DNA.

[0090] Positive control (PC): The positive control contained 2000 GC / reaction of BPV-3_IPC recombinant plasmid DNA. The positive control was used to confirm the accuracy of the assay.

[0091] Inhibitory Control (IHN): The inhibitory control is used to monitor the level of interference or inhibition imposed by PCR amplification and the test sample matrix. The final extracted DNA from the test sample was spiked with the same amount of BPV-3_IPC plasmid (2000 GC / reaction) as the positive control (+S). CT (cycle threshold) is the number of cycles required for the fluorescence signal to exceed the threshold (i.e., exceed the background level). The mean cycle threshold (CT) value of the inhibitory control (+S) was compared to the mean CT value of the positive control as a measure of interference or inhibition. ΔCT is defined as less than 3.32 CT (equivalent to a 1 log DNA concentration). Inhibition or interference was calculated as ΔCT = |PC (mean CT) - IHN (mean CT)|.

[0092] Setup of BPV-3 qPCR and IPC qPCR detection assays Prepare two different master mixes: a BPV-3 qPCR master mix for the BPV-3 pPCR assay and an IPC qPCR master mix for the IPC qPCR assay.

[0093] The BPV-3 qPCR detection assay setup included a BPV-3 v3.0 primer / probe set for detecting BPV-3 DNA in DNA extracted from the test sample. The BPV-3 qPCR master mix included a BPV-3 v3.0 primer-probe set, water, and 2×TAQMAN® Universal PCR Master Mix (Applied Biosystems). The 2×TAQMAN® Universal PCR Master Mix contained essential components for the qPCR reaction, including optimized buffer for amplifying G / C-rich sequences, dNTPs with dUTP, AmpliTaq Gold® DNA polymerase, ROX dye (as a passive internal reference), and AmpErase® UNG (uracil DNA glycosylase enzyme for removing carryover-contaminated PCR products containing dU).

[0094] Table 5 shows the amounts of each reaction component for each sample tested in the BPV-3 qPCR reaction: DNA extracted from the test sample and five controls: negative extract control (NEC), no-template control (NTC), positive extract control (PEC), inhibitor control (IHN), and positive control (PC) at two different concentrations (above detection limit (ADL) and limit of detection (DL)).

[0095] [Table 7]

[0096] For the IPC qPCR reaction, an IPC prime / probe master mix was prepared containing IPC primers-probes, water, and 2×TAQMAN® Universal PCR Master Mix (Applied Biosystems). Table 6 shows the amounts of each reaction component for the test sample and four controls, namely two different concentrations (above detection limit (ADL) and limit of detection (DL)), a negative extract control, a template-less control, a positive extract control, and a positive control.

[0097] [Table 8]

[0098] Both the BPV_3 qPCR and IPC qPCR reactions were performed in 96-well reaction plates, with at least three sets (three PCR reaction wells) for each sample and control. The reaction components could be set up manually or using a liquid handler robot such as the QIAgility HEPA / UV Liquid Handler (QIAGEN, Inc., Germantown, MD). The qPCR reaction mixture was placed in a MicroAmp optical 96-well plate (Applied Biosystems) and covered with a MicroAmp optical adhesive film (Applied Biosystems). Tables 6 and 7 show the amounts of PCR reagent components, samples, and controls for each PCR reaction. The sealed plates were then loaded into a QuantStudio® 7 Flex real-time PCR system (Applied Biosystems).

[0099] qPCR reactions were performed using the QuantStudio® 7 Flex real-time PCR system (Applied Biosystems). The thermal cycling program was set to the following reaction times and temperatures: 40 cycles of 50°C for 2 minutes, 95°C for 10 minutes, and 95°C for 15 seconds, followed by 60°C for 1 minute (data acquisition). Table 7 shows the selected experimental properties.

[0100] [Table 9]

[0101] Before evaluating the test sample, the assay must meet system compatibility and assay acceptance criteria to be considered a valid assay. If the assay does not meet the acceptance criteria, the assay must be repeated. If the test sample is valid and positive, the assay must be repeated to confirm the positive result. Table 8 outlines the valid assay criteria and evaluation of test results for the BPV-3 qualitative assay. If the test sample shows a positive amplification signal, the IHN and ΔCT controls are irrelevant and should not be considered part of the system compatibility and assay acceptance criteria.

[0102] [Table 10]

[0103] Acceptable criteria for test sample results If the assay meets system compatibility and assay acceptance criteria, the acceptance criteria for test samples are shown in Table 9. The results of the qualitative assay are reported as "positive" or "negative".

[0104] [Table 11]

[0105] Repeated testing strategy If ΔCT does not meet the acceptable criteria using a pure DNA sample, the assay may be repeated using a 1:2 dilution of the extracted DNA. Dilution reduces sample-matrix interference. In this case, all sample and control DNA must be diluted 1:2 with molecular biology-grade water before repeating the assay.

[0106] If the test sample shows a positive amplification signal for the target IPC, this indicates cross-contamination of the positive control plasmid DNA by the test sample. In this case, the assay is invalid and should be repeated after decontamination using in-situ applicable procedures.

[0107] Example 3: A qualitative assay for BPV3 is performed. Specificity: Sample matrix effect (to test for false positives) To demonstrate that there were no false-positive results between the culture medium components and the BPV-3 v3.0 and IPC v2.0 primer / probe sets, three sample matrices (Dulbeccoo's Modified Eagle Medium (DMEM) (Thermo Fisher) and two fetal bovine serums (FBS) (SAFC St. Louis, MO and Hyclone Logan, UT)) were tested. The assay procedure and setup were as described in Example 2.

[0108] Table 10 shows the test samples and the results of qPCR testing using BPV-3 and IPC assays. The acceptance criteria for this procedure required all test samples to show negative results with both primer / probe sets. Valid experimental results, assuming all controls and system compatibility met the acceptance criteria, revealed that all test samples were negative and showed no amplification signal (CT=40.00).

[0109] [Table 12]

[0110] BPV-3 detection (to test for false negatives) Since neither BPV-3 virus stocks nor BPV-3 genomic DNA are commercially available, the specificity of the BPV-3 v3.0 primer / probe set was tested using a BPV-3_IPC positive control plasmid. The BPV-3_IPC plasmid was sequenced, and the authenticity of the BPV-3 NS gene was confirmed by BLAST analysis.

[0111] The assay described in Example 2 was used. As part of the specificity test, two fetal bovine serum samples and an untreated non-GMP bulk sample containing FBS, both confirmed to be positive for BPV-3 DNA (using Bioreliance (BREL) qPCR Bovine Parvo Panel, Bioreliance, Rockville, MD), were used as true positive samples. A positive control (PC) was confirmed by sequencing.

[0112] The acceptance criteria for this procedure required that all test samples produce a positive result in the BPV-3 assay, and that the assay does not produce false negative results in BPV-3 samples that have been previously confirmed positive.

[0113] Table 11 shows the results of BPV-3 detection in PC, all of which met the acceptance criteria. Notably, the IPC v2.0 primer / probe was negative in positive FBS and, as expected, in untreated non-GMP bulk samples containing FBS, and only produced a positive result when using the positive control plasmid (BPV-3_IPC). The results of the IPC qPCR reaction were as expected and met the acceptance criteria.

[0114] [Table 13]

[0115] Cross-reactivity To test the cross-reactivity of the BPV-3 v3.0 and IPC v2.0 primer / probe sets, extracted nucleic acids from various viruses and cell lines were evaluated using the assay described in Example 2. The commercially available parvoviruses closest to BPV-3 are bovine parvovirus 1 (BPV-1) (ATCC® VR-767®) and mouse minute virus (MMV, rodent parvovirus), which were included to test for cross-reactivity. Several other DNA viruses were also included to test for cross-reactivity: porcine circovirus 2 (PCV 2), herpes simplex virus 1 (HSV 1), and pseudorabies virus (Prv). DNA from the following four cell lines was also tested: African green monkey kidney (VERO), baby hamster kidney cells (BHK), Chinese hamster ovary (CHO), and MSV-transformed cat brain (PG-4). DNA was extracted as described in Example 2.

[0116] The authenticity of nucleic acids extracted from all viruses was confirmed using in-house specific qPCR or next-generation sequencing (NGS) methods.

[0117] The acceptance criteria for this procedure required that all tested species show a negative result (CT=40.00) using the BPV-3 v3.0 and IPC v2.0 primer / probe set. Table 12 shows that the results of the cross-reactivity test revealed that all tested species were negative for BPV-3 and met the acceptance criteria.

[0118] [Table 14]

[0119] Limit of Detection (LOD) The BPV-3 qPCR method is for obtaining qualitative results, and proof of linearity is not required. In practice, the LOD is determined as the cutoff point in the form of the minimum number of amplified target sequences, based on the positive rate detected in 95% of a series of samples (referred to as LOD 95%).

[0120] To determine the detection sensitivity or analytical limit (ALOD) of this method, BPV-3_IPC plasmid DNA was diluted to different concentrations (1, 5, 10, 20, 25, 50, 75, 100, 250, and 500 GC / PCR reactions). ALOD is the concentration of target DNA at which the amplified product is detected with a probability of at least 0.95 (LOD 95%). To test this, 12 PCR repeat measurements from each plasmid DNA concentration were evaluated in the absence of the sample matrix using the assay described in Example 2. The concentration level with the lowest genome copy number at which all 12 repeats were positive was considered an approximation of the LOD 95%.

[0121] The ALOD of the BPV-3 qPCR assay was calculated using probit analysis with a 95% association probability. The results showed that the ALOD of the BPV-3 qPCR assay was approximately 22 genome copies per reaction (Figure 1).

[0122] The sample detection limit (SLOD) is the minimum amount of the target sequence that can be detected with a given level of confidence in the presence of the sample matrix (LOD 95%). Experiments to determine the SLOD were performed in the same manner as the ALOD experiment, except that positive control plasmid DNA at different concentration levels (25, 50, 75, and 100 GC / reaction) was spiked into DNA extracted from different sample matrices instead of molecular biology grade water (MBGW).

[0123] In this study, different sample matrices were used (see Table 13). Using the QIAgility liquid handler (QIAGEN, Inc.), each sample was spiked with 25, 50, 75, and 100 genome copies (BPV-3_IPC positive control plasmid) per reaction and tested using the BPV-3 qPCR detection assay described in Example 2. All samples were evaluated with 12 replicates. Tables 14 and 15 show the SLOD results. The results revealed that the SLOD for the BPV-3 qPCR assay was 25 genome copies per PCR reaction. Similar results were obtained for the IPC qPCR assay (Table 16).

[0124] [Table 15]

[0125] [Table 16]

[0126] [Table 17]

[0127] [Table 18]

[0128] Robustness To evaluate the robustness of the BPV-3 qPCR detection assay, untreated non-GMP bulk samples containing FBS and FBS samples were tested (Table 17). Acceptance criteria were that the proposed system compatibility and assay acceptance criteria, as well as the sample acceptance criteria, were met, as described in Example 2. The samples had been previously confirmed to be negative or positive for BPV-3 DNA by alternative methods such as NGS, Sanger DNA sequencing, or Bioreliance (BREL) Bovine Parvo Panel qPCR assay.

[0129] The samples were subjected to DNA extraction as described in Example 2. All controls were prepared and subjected to qPCR in parallel with the test samples, and then subjected to the BPV-3 qPCR detection assay as described in Example 2. The results of the BPV-3 qPCR assay were interpreted as described in Example 2 and reported as "positive" or "negative".

[0130] [Table 19]

[0131] The results for the negative test samples in Table 17 are shown in Tables 18-20. The reliability of this assay was demonstrated by negative results in NTC and NEC in three BPV-3 and IPC assays with three PCR replicates; detection of the target amplification signal in PC in three BPV-3 and IPC assays with three PCR replicates; detection of amplification signals by BPV-3 and IPC assays against PEC in three PCR replicates showing accurate extraction and recovery; detection of BPV-3 in spiked test samples (+S) in three PCR replicates; and assay control results including ΔCT results that did not show sample matrix interference. The test samples were negative in three IPC assays with three PCR replicates, indicating no cross-contamination between the positive control and the sample, and the test samples were also negative in three BPV-3 assays with three PCR replicates, indicating the absence of BPV-3 DNA. In summary, the BPV-3 assay results indicate that the assay and sample acceptance criteria were met and the test samples were valid and "negative" for BPV-3.

[0132] [Table 20]

[0133] [Table 21]

[0134] [Table 22]

[0135] Tables 21-24 show the results of the BPV-3 assay for positive test samples (two untreated non-GMP bulk samples containing FBS and two FBS samples (FBS#2 and FBS#3)). The reliability of the assay was demonstrated, and the assay control results included predicted negative results for NTC and NEC in three BPV-3 and IPC assays with three PCR replicates, as well as detection of targeted amplification signals in PC in three BPV-3 assays with three PCR replicates.

[0136] When the test sample showed a positive amplification signal and detection of BPV-3, the evaluation of spiked test samples (IHC) and ΔCT became irrelevant and did not need to be considered as part of the assay's acceptance criteria. Detection of amplification signals by the BPV-3 assay and IPC assay for three PECs of three replicates of the PCR reaction indicated accurate extraction and recovery, and negative signals of the test sample by the three IPC assays of three replicates of the PCR reaction indicated no cross-contamination between the positive control and the sample. The test sample showed a positive amplification signal in the three BPV-3 assays of three replicates of the PCR reaction, indicating the presence of BPV-3 DNA. In summary, the results showed that the assay and sample acceptance criteria were met and the samples were acceptable, indicating that the test samples were valid and positive for BPV-3.

[0137] [Table 23]

[0138] [Table 24]

[0139] [Table 25]

[0140] [Table 26]

[0141] Parallel precision To test parallelism accuracy, DNA extracted from BHK and Vero cells (30 ng / PCR reaction) was prepared as either unspiked or spiked (spiked at the SLOD level) versions of 25 genome copies of a BPV-3_IPC-positive control plasmid (spiked at the SLOD level). The BPV-3 detection assay (as described in Example 2) was performed with 12 PCR reaction repeats on different days.

[0142] The acceptance criteria for this procedure required that samples without spikes in at least 11 of the 12 replicates of the BPV-3 and IPC qPCR assays should not show an amplification signal, and samples with spikes in at least 11 of the 12 replicates of the BPV-3 and IPC qPCR assays should show a positive amplification signal. The results of the parallel precision assay showed that 12 samples without spikes (Table 25) and 12 samples with spikes (Table 26) met the acceptance criteria, demonstrating the parallel precision of the BPV-3 detection assay.

[0143] [Table 27]

[0144] [Table 28]

[0145] In summary, all test samples met the qualification specifications required for qualitative assays, including specificity, detection limit, robustness, and parallel precision. A qualified BPV-3 detection assay can reliably detect 25 BPV-3 genome copies per PCR reaction. All test samples met the proposed system compatibility and assay acceptance criteria, as well as the test sample acceptance criteria. Therefore, a qualified BPV-3 real-time PCR assay enabled the detection of BPV-3 DNA in the test samples.

[0146] Example 4: Real-time quantitative polymerase chain reaction assay for bovine parvovirus 3 (BPV-3) Sample preparation For test samples containing cells, the samples were subjected to slow centrifugation (LSC) (320 × g, 10 minutes at room temperature), followed by DNA extraction. The supernatant was collected for DNA extraction. Cell-free samples were processed directly.

[0147] To a 250 μL test sample, 10 μL of RNase Cocktail® enzyme mix (Thermo Fisher, Carlsbad, CA) and a 0.5 M EDTA, pH 8.0 mixture (9 μL:1 μL) were added. 200 μL of the test sample was extracted using an AutoMate Express® nucleic acid extraction system (Thermo Fisher). For the DNA extraction procedure, the PrepSEQ(PS) Express 1-2-3 protocol with the following parameters was selected for AutoMate: 1 hour of proteinase K (PK) digestion and 100 μL elution. The extracted DNA was visually inspected, and if magnetic beads remained in the eluted DNA, they were removed using a DynaMag®-2 magnetic benchtop workstation (Thermo Fisher). The DNA was either used immediately or stored at -20°C.

[0148] Along with the test samples, BPV-3_IPC(PEC) plasmid controls and negative extraction controls (NECs) were also prepared and performed as described above. For the positive extraction control, 62,000 genomic copies (GC) of BPV-3_IPC plasmid DNA were spiked into 250 μL of sterile 1× phosphate-buffered saline (PBS). This was used to evaluate the DNA extraction performance during test sample preparation and to confirm that the assay did not produce false negative results. Assuming a 100% DNA extraction efficiency, the final eluted DNA should contain 500 GC / μL. For the negative extraction control, approximately 300 ng of human genomic DNA in 1× PBS was used. This NEC was used to monitor cross-contamination during nucleic acid extraction and to verify that the assay did not produce false positive results due to nonspecific background DNA.

[0149] Preparation of other assay controls Template-free control (NTC): This contained all PCR reagents except for the DNA template. It was used as a negative control to confirm that the qPCR raw materials were free from DNA contamination.

[0150] Positive controls (PCs): Three PC concentrations of BPV-3_IPC positive control plasmid DNA were included: 2E4, 2E5, and 2E7 GC / reactions. These were used to confirm that the qPCR components functioned correctly and produced PCR amplification signals specific to the target sequence.

[0151] Positive extraction control (PEC): Add approximately 400 ng of human genomic DNA (approximately 3 μL) to 250 μL (in the case of 1×PBS), spike 62,500 copies of BPV-3_IPC positive control plasmid DNA (6.25 μL), and then extract.

[0152] Inhibition control (IHN): Contains DNA extracted from a test sample spiked with BPV-3_IPC plasmid DNA in a 2E4 GC / reaction. This is used to determine the sample interference / inhibition level.

[0153] Standard (ST): 10-fold diluted BPV-3_IPC plasmid DNA (1E8, 1E7, 1E6, 1E5, 1E4, 1E3GC / reaction) used to determine PCR efficiency, linear range, and quantify the absolute copy number of the target sequence in the test sample / control.

[0154] Setup of BPV-3 qPCR and IPC qPCR detection assays The BPV-3 qPCR detection assay setup included a BPV-3 v3.0 prime / probe set for detecting BPV-3 DNA in the test sample and an IPC v2.0 primer / probe set for monitoring accidental cross-contamination between positive and negative samples. Two different master mixes, the BPV-3 qPCR master mix and the IPC qPCR master mix, were prepared.

[0155] The BPV-3 qPCR master mix contained BPV-3 v3.0 primer-probe, water, and 2× TAQMAN® Universal PCR Master Mix (Applied Biosystems). The TAQMAN® master mix contained essential components for the qPCR reaction, including optimized buffer for amplifying G / C-rich sequences, dNTPs with dUTP, AmpliTaq Gold® DNA polymerase, ROX dye (as a passive internal reference), and AmpErase® UNG (uracil DNA glycosylase enzyme for removing carryover-contaminated PCR products containing dU). Table 27 shows the amounts of each reaction component for the test samples and five controls (negative extract control (NEC), no-template control (NTC), positive extract control (PEC), inhibitor control (IHN), and positive control (PC)) for each sample tested.

[0156] [Table 29]

[0157] Each sample and control reaction was performed in a triple (3-well) configuration on a 96-well reaction plate. The qPCR reaction setup could be performed manually or using a liquid handler robot such as the QIAgility HEPA / UV Liquid Handler (Qiagen, Inc., Germantown, MD). The qPCR reaction mixture was placed in a MicroAmp optical 96-well plate (Applied Biosystems) and then covered with a MicroAmp optical adhesive film (Applied Biosystems). Table 27 shows the amounts of PCR reagent components, sample, and control for each PCR reaction. The sample arrangement in the 96-well plate could be configured per analyst, and the sealed plate was loaded into a QuantStudio® 7 Flex real-time PCR system (Applied Bioscience).

[0158] qPCR reactions were performed using the QuantStudio® 7 Flex real-time PCR system (Applied Bioscience). The thermal cycling program was set to the following reaction times and temperatures: 50°C for 2 minutes (AmpErase® UNG activation), 95°C for 10 minutes (AmpliTaq Gold® DNA polymerase activation / denaturation), and 95°C for 15 seconds, 60°C for 1 minute for 40 cycles (annealing / extension / data acquisition). Table 28 shows the selected experimental properties.

[0159] [Table 30]

[0160] The assay uses the QS7 real-time PCR system (Thermo Fisher Scientific, Waltham, MA) in combination with the BPV_3 and IPC primers and probes described herein.

[0161] For an assay to be considered valid, it must meet the following assay acceptance criteria (see Table 29). If an assay does not meet the acceptance criteria, the assay must be repeated.

[0162] [Table 31]

[0163] Statistical analysis of the data was performed using Excel software (Microsoft, Redmond, WA) and / or TIBCO SPOTFIRE® software (Palo Alto, CA). CT values ​​reported as "undetermined" indicated the absence of target DNA and were converted to 40.00 for further statistical analysis. Samples, standards, and controls were tested in three replicates. Outlier rules based on the Grubbs test or Dixon's Q test may be applied to CT values ​​from samples, controls, or standards. Outlier analysis tests may be applied to three consecutive data points to determine whether one of the results can be excluded from the analysis as an outlier. If the exclusion of an outlier is statistically significant (significance level: α=0.05) and acceptable, the data analysis is repeated using two valid consecutive results. Results based on two consecutive data points must satisfy all acceptance criteria for the control group and standard to be valid. Outlier determination (two-tailed test at significance level alpha 0.05) can be performed using online or offline resources known to those skilled in the art. Examples of data reporting and interpretation are shown in Table 30.

[0164] [Table 32]

[0165] Example 5 Eligibility of the quantitative assay Acceptable criteria for test sample results The eligibility of the quantitative analysis was confirmed. The following parameters—accuracy including specificity, limit of detection (LOD), linearity, dynamic range, parallel precision (intra-assay specificity), and intra-laboratory reproducibility (intra-laboratory precision), accuracy, limit of quantification (LOQ), and robustness—were deemed eligible, and assay validation was approved.

[0166] Specificity: Sample matrix effect (to test for false positives) To demonstrate that the assay does not produce false-positive results (an error in which qPCR incorrectly yields a positive result in the absence of the target sequence), the following sample matrices were tested.

[0167] Cell lines tested: PG4 (transformed feline brain Moloney sarcoma virus), Vero (African green monkey kidney), 324K (SV40 transformed human neonatal kidney), L929 (mouse fibroblast cell line), CHO (Chinese hamster ovary), HEK293 (human embryonic kidney), MDBK (Maddin-Derby bovine kidney), NRK (normal rat kidney), human leukocytes (Promega, San Luis Obispo, CA). Culture media tested: DMEM, McCoy 9A, HAM F12. Animal serum tested: fetal bovine serum and equine serum.

[0168] The acceptance criteria required all tested samples to show negative results in at least 5 of the 6 PCR replicates. No false positives were detected in any of the 6 replicates.

[0169] Specificity: BPV-3 detection (to test for false negatives) To demonstrate that the assay does not produce false negative results (an error in which qPCR incorrectly shows a negative result in the presence of the target sequence), the above cell lines, culture media, and animal serum were spiked with a BPV-3 positive control plasmid (50,000 genome copies / 200 μL sample), and subsequently, DNA was extracted and the BPV-3 qPCR assay was performed as described in Example 4. The acceptance criteria required a positive result in at least 5 out of 6 replicates. All tested samples showed detection of BPV-3 in 6 out of 6 replicates that detected BPV-3. No false negatives were detected.

[0170] Specificity: Cross-reactivity To determine the cross-reactivity of the assay, we evaluated the exclusivity and inclusivity of closely related virus species / isopropyl groups.

[0171] The exclusivity demonstrated that the assay does not detect non-target virus species / isopropyl alcohols closely related to the target sequence. BPV-1 (Bovine Parvovirus 1, VR-767®, ATCC®, Manassas, VA), BPV-2 (Bovine Parvovirus 2), and MMVp (Mouse Minute Virus prototype) viruses were extracted as described above. Since BPV-2 is not commercially available, the full-length non-structural (NS) gene sequence of BPV-2 (GenBank accession number: NC_006259) was synthesized, cloned into a plasmid, and confirmed by sequencing. The plasmid was used as a BPV-2 reference isolate with a concentration of 1E6GC per PCR reaction.

[0172] The virus species / isopropyl alcohol was used in the BPV-3 qPCR assay as described in Example 4. Acceptance criteria required that all tested samples show negative results in at least 5 of 6 replicates. The BPV-3 qPCR results met the acceptance criteria by showing no cross-reactivity with these closely related species and not detecting non-target species / isopropyl alcohol in 6 of the 6 replicates.

[0173] Inclusiveness demonstrates that the assay can specifically detect BPV-3 target sequence species / isopropyl alcohol. Previously confirmed BPV-3-positive fetal bovine serum samples (confirmed by full-length BPV-3 genome sequencing) were extracted and subsequently subjected to BPV-3 qPCR assay. Acceptance criteria for this procedure required that the tested samples show positive results in at least 5 of 6 replicates. The BPV-3 qPCR assay results met the acceptance criteria by specifically detecting BPV-3 DNA and detecting the target isolate in 6 out of 6 replicates.

[0174] Limit of Detection (LOD) The limit of detection (LOD) is the minimum amount of the target sequence that can be detected, but it is not always quantified as an exact value. Probit analysis using the LOD 95% detection limit approach was used to determine the assay LOD. LOD 95% is the number of copies of the target DNA sequence required to guarantee a 95% probability of detection (POD) in a qPCR assay.

[0175] BPV-3 positive control DNA (1000, 100, 50, 25, 20, 10, 5, and 1 genome copy / reaction) was spiked into exogenous extracted DNA (approximately 300 ng of human genomic DNA) and tested using the BPV-3 qPCR assay as described in Example 4. Each concentration was tested with 12 PCR repeats to obtain statistically reliable POD curves, from which LOD95% with a 95% confidence interval (CI) was derived. The experiment was repeated by three different analysts. CT values ​​less than 39.99 were considered positive signals, and undetermined CT values ​​(CT=40.00) were considered negative signals. 36 set points for each concentration (collected from all analysts) were obtained for probit analysis to create statistically reliable POD curves, from which LOD95% with a 95% confidence interval (CI) was derived (see Table 31 and Figure 2). Probit analysis revealed that the BPV-3 qPCR assay LOD 95% was 27 GC / rxn, with 95% confidence intervals of 22 and 34 GC / rxn.

[0176] [Table 33]

[0177] Dynamic range and linearity Adequate precision, accuracy, and linearity must be demonstrated within the dynamic range of the analytical procedure. To evaluate linearity (minimum and maximum dynamic range of the calibration curve), preliminary calibration curves were constructed and evaluated at concentrations ranging from eight orders of magnitude, including a predetermined number of positive control plasmids. In the BVP-3 assay, three replicates were used for each of the 1E8, 1E7, 1E6, 1E5, 1E4, 1E3, 1E2, and 1E1 genome copies / reactions.

[0178] At least six different sizes were selected for the working range, not only for the assay application and expected use, but also for the acceptable calibration curve performance parameters derived from preliminary test results. In this procedure, the linearity of the calibration curve within the six selected sizes (including the maximum and minimum setpoints) must meet the acceptable criteria of (i) a correlation coefficient (R²) of ≥0.98 and (ii) a PCR amplification efficiency of 90–110%.

[0179] The dynamic range at the seven setpoints (1E2–1E8 genome copies / reaction) met the assay's acceptable criteria, but the use of the seven setpoints from 1E2–1E8 as the minimum and maximum standard ranges was not expected to be robust. Therefore, the upper limit of quantification (LOQ) size (1E3) was selected, and the minimum and maximum dynamic ranges of the calibration curve were defined as six data setpoints between 1E3 and 1E8 genome copies / reactions (Table 32).

[0180] [Table 34]

[0181] Accuracy and precision The accuracy and precision (parallel precision and intra-intra

[0182] For parallel precision (intraassay precision), the mean and standard deviation of the quantity (genomic copies of the target sequence / reaction), as well as the CV% (coefficient of variation) of the quantity, were calculated in triplicate for each concentration. A CV% of ≤25% was the acceptable criterion for this procedure.

[0183] For intra-laboratory reproducibility (laboratory precision), the mean and standard deviation of the quantities, as well as the CV% (coefficient of variation), were calculated from all three PCR reactions at all concentrations in three independent experiments. The acceptable criterion was a CV% of ≤30%.

[0184] For accuracy, the average volume (genomic copies of the target sequence / reaction) was calculated from each triple PCR reaction at each concentration (1E8, 1E7, 1E6, 1E5, 1E4, and 1E3), taking into account inherent variability among different analysts performing the assay, different detection systems, and assays performed at different time intervals. The acceptable criterion was that the CV% of the volume was calculated for each of the above sample concentrations. The acceptable criterion was that the average quantitative value was within ±30% of the threshold value across the entire dynamic range of the assay. In the acceptable criterion for this procedure, linearity of each independent experiment was used as the correlation coefficient (R) 2 It was also required to demonstrate that the ratio was ≥ 0.98 and that the PCR amplification efficiency was between 90% and 110%.

[0185] The results of three independent studies showed that the assay met established acceptable standards for linearity (see Tables 33-36). Parallel precision was ≤25%, intra-intra 2 The ratio was ≥0.98, and the PCR amplification efficiency was within the acceptable range of 90-110%.

[0186] [Table 35]

[0187] [Table 36]

[0188] [Table 37]

[0189] [Table 38]

[0190] Limit of Quantitative Analysis The limit of quantification (LOQ) of a quantitative qPCR assay is the minimum amount of the target sequence in the sample that can be quantitatively determined with appropriate precision and accuracy. To determine the LOQ, the lowest setpoint (1E3 genome copies / reaction), intermediate setpoint (1E4 and 1E6 genome copies / reaction), and upper setpoint (1E8 genome copies / reaction) of the dynamic range were tested. Positive control samples with concentrations at the lowest, intermediate, and upper setpoints of the dynamic range were prepared, spiked into exogenous extracted DNA samples (approximately 300 ng of human genomic DNA per reaction, serving as background sample matrix DNA), and evaluated by performing 12 consecutive BVP-3 assays in the presence of a calibration curve. The assays were performed by three analysts on different days. Precision (parallel precision and intra-intra

[0191] The results of the three independent LOQ experiments were within established acceptable limits for parallelism, intra-laboratory repeatability and accuracy (Tables 37-39), and linearity (Table 40).

[0192] [Table 39]

[0193] [Table 40]

[0194] [Table 41]

[0195] [Table 42]

[0196] [Table 43]

[0197] [Table 44]

[0198] [Table 45]

[0199] Robustness To determine the robustness of the assay, changes in the concentration of key reagents up to 20% were evaluated according to the methods of Youden and Steiner (above). Several key qPCR factors were selected and slightly altered (Table 41). BPV-3 positive fetal bovine serum test samples were extracted and tested in 12 replicates using the optimal and altered conditions of the BPV-3 assay in parallel with the calibration curve.

[0200] [Table 46]

[0201] The acceptance criteria for this procedure required that the response obtained from robust conditions for the applied microchanges met the acceptance criteria for established assays. These acceptance criteria included precision, with the CV% for parallel precision and intra-intra

[0202] The robustness test results showed that slight variations in key qPCR reagents were acceptable compared to optimal conditions, and the robustness results met the acceptable criteria for precision and accuracy (118,100±35,430) (Table 42).

[0203] [Table 47]

[0204] [Table 48]

[0205] verification Assay system compatibility and assay acceptance criteria were defined to verify assay performance according to the accredited protocol (see Table 44). To demonstrate that the assay performed as expected, test samples containing the target sequence (Sample A) and test samples without the target sequence (Sample B) were subjected to DNA extraction and then BPV-3 qPCR. The validation test was performed three times on different days by different analysts. The assay control, system compatibility, assay acceptance criteria, and reporting and interpretation of the test results are shown in Tables 43-44.

[0206] [Table 49]

[0207]

Table 50

[0208] Results of the verification test Tables 45, 47, and 49 show the results of assay system suitability and assay controls for three independent experiments. All three independent experiments met the established system suitability and assay control acceptance criteria defined herein. Tables 45, 47, and 49 show the results of the test samples (Sample A: a sample whose claim was previously confirmed and Sample B: a sample whose negativity was previously confirmed). The results of the verification test showed that in all three independent experiments, BPV-3 was not detected in the negative sample (Sample B), and in all three independent experiments, BPV-3 was detected and quantifiable in the positive sample (Sample A).

[0209]

Table 51

[0210]

Table 52

[0211] BPV-3 was detected in Sample A at a concentration of Log 10 7.01 ± 0.01 genome copies / mL (mean ± StdDev). In Sample B, BPV-3 was not detected.

[0212]

Table 53

[0213]

Table 54

[0214] BPV-3 was detected in Sample A at a concentration of Log 10It was detected at a concentration of 7.04 ± 0.01 genome copies / mL (mean ± StdDev). BPV-3 was not detected in sample B.

[0215] [Table 55]

[0216] [Table 56]

[0217] BPV-3, in sample A, Log 10 It is detected at a concentration of 7.04 ± 0.01 genome copies / mL (mean ± StdDev). BPV-3 is not detected in sample B.

[0218] Table 51 shows the established eligibility criteria for BPV-3 assays. A qualified quantitative BPV-3 assay allows for the quantification of 1E3–1E8 BPV-3 genome copies per PCR reaction.

[0219] [Table 57]

[0220] Test samples A and B met the qualification specifications required for quantitative assays, including specificity, detection limit, quantification limit, linearity, precision, accuracy, and robustness.

[0221] All test samples (Samples A and B) met the proposed system compatibility and assay acceptance criteria. The quantitative levels of the three independent tests showed similar results (Tables 45, 47, and 49), demonstrating the accurate, highly precise, and robust performance of the qualified assay. The quantitative BPV-3 assay and associated qualification data demonstrate that the BPV-3 quantitative real-time PCR assay enables the detection and quantification of BPV-3 DNA in test samples.

Claims

1. A composition comprising an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 7 as a forward primer, an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 8 as a reverse primer, and an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 9 as a probe.

2. The composition according to claim 1, further comprising a second composition containing an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 16 as a forward primer, an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 17 as a reverse primer, and an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 18 as a probe.

3. A reagent for detecting bovine parvovirus 3 (BPV-3) genomic DNA in extracted DNA of a test sample, comprising a primer-probe combination having an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 7 as a forward primer, an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 8 as a reverse primer, and an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 9 as a probe.

4. The reagent according to claim 3, wherein the oligonucleotide having the nucleic acid sequence of Sequence ID No. 9 comprises a fluorescent reporter dye and / or a non-fluorescent quencher.

5. The reagent according to claim 3, wherein the oligonucleotide having the nucleic acid sequence of Sequence ID No. 9 has a fluorescent reporter dye 6-carboxyfluorescein (FAM) at its 5' end, and / or a sub-groove binder non-fluorescent quencher (MGB-NFQ) or ZEN-IB and an Iowa Black fluorescent quencher (IBFQ) at its 3' end.

6. The reagent according to claim 3, wherein the primer-probe combination detects DNA encoding the structural capsid (VP) protein and / or non-structural (NS) protein of bovine parvovirus 3 in the test sample.

7. The reagent according to claim 3, combined with a combination of endogenous positive control primers.

8. A reagent for use as an endogenous positive control primer-probe combination in an assay for detecting bovine parvovirus 3 (BPV-3) genomic DNA in extracted DNA of a test sample, comprising a primer-probe combination wherein the primer-probe combination comprises an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 16 as a forward primer, an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 17 as a reverse primer, and an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 18 as a probe.

9. The reagent according to claim 8, wherein the oligonucleotide having the nucleic acid sequence of SEQ ID NO: 18 comprises a fluorescent reporter dye and / or a non-fluorescent quencher.

10. The reagent according to claim 9, wherein the oligonucleotide having the nucleic acid sequence of Sequence ID No. 18 has a fluorescent reporter dye 2'-chloro-7'phenyl-1,4-dichloro-6-carboxyfluorescein (VIC) at its 5' end and a secondary groove binder non-fluorescent quencher (MGB-NFQ) at its 3' end.

11. A primer-probe combination for detecting bovine parvovirus 3 (BPV-3) genomic DNA in extracted DNA of a test sample, combined with an endogenous positive control primer-probe combination for detecting bovine parvovirus 3 (BPV-3) genomic DNA, wherein the primer-probe combination comprises an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 7 as a forward primer, an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 8 as a reverse primer, and an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 9 as a probe.

12. The primer-probe combination according to claim 11, wherein the oligonucleotide having the nucleic acid sequence of SEQ ID NO: 9 comprises a fluorescent reporter dye and / or a non-fluorescent quencher.

13. The primer-probe combination according to claim 11, wherein the oligonucleotide having the nucleic acid sequence of Sequence ID No. 9 has the fluorescent reporter dye 6-carboxyfluorescein (FAM) at its 5' end, and / or the sub-groove binder non-fluorescent quencher (MGB-NFQ) or ZEN-IB and the Iowa Black fluorescent quencher (IBFQ) at its 3' end.

14. The primer-probe combination according to claim 11 for detecting DNA encoding the structural capsid (VP) protein and / or non-structural (NS) protein of bovine parvovirus 3 in a test sample.

15. An endogenous positive control primer-probe combination for detecting bovine parvovirus 3 (BPV-3) genomic DNA in a test sample, comprising an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 16 as a forward primer, an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 17 as a reverse primer, and an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 18 as a probe.

16. The combination of an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 18 and an endogenous positive control primer probe according to claim 15, comprising a fluorescent reporter dye and / or a non-fluorescent quencher.

17. The primer-probe combination according to claim 15, wherein the oligonucleotide having the nucleic acid sequence of Sequence ID No. 18 has a fluorescent reporter dye 2'-chloro-7'phenyl-1,4-dichloro-6-carboxyfluorescein (VIC) at its 5' end and a secondary groove binder non-fluorescent quencher (MGB-NFQ) at its 3' end.

18. A primer-probe combination for detecting bovine parvovirus trigenomic DNA in extracted DNA of a test sample, The primer-probe combination includes an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 7 as a forward primer, an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 8 as a reverse primer, and an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 9 as a probe. The primer-probe combination is combined with an endogenous positive control primer-probe combination that includes an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 16 as a forward primer, an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 17 as a reverse primer, and an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 18 as a probe. A primer-probe combination in which an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 9 has the fluorescent reporter dye 6-carboxyfluorescein at its 5' end and a secondary groove binder non-fluorescent quencher (MGB-NFQ) at its 3' end, and an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 18 has the fluorescent reporter dye 2'-chloro-7'phenyl-1,4-dichloro-6-carboxyfluorescein at its 5' end and a secondary groove binder non-fluorescent quencher (MGB-NFQ) at its 3' end.

19. A primer-probe combination for detecting DNA encoding BPV-3, comprising a primer-probe combination having an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 7 as a forward primer, an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 8 as a reverse primer, and an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 9 as a probe, for detecting bovine parvovirus 3 (BPV-3) genomic DNA contamination in extracted DNA of a test sample.

20. The kit according to claim 19, further comprising a combination of an endogenous positive control primer probe having an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 16 as a forward primer, an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 17 as a reverse primer, and an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 18 as a probe.

21. A method for determining the presence or absence of bovine parvovirus 3 genome DNA in extracted DNA of a test sample, 1) A step of preparing a reaction mixture comprising: 1) a test sample, a positive control, BPV-3_IPC positive control plasmid DNA, nucleic acid amplification reagent, an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 16 as a forward primer, an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 17 as a reverse primer, and an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 18 as a probe (where the oligonucleotide having the nucleic acid sequence of SEQ ID NO: 18 has a fluorescent reporter dye at its 5' end and a non-fluorescent quencher at its 3' end), and a primer-probe mixture selective for the DNA sequence of bovine parvovirus 3 comprising an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 7 as a forward primer, an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 8 as a reverse primer, and an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 9 as a probe (where the oligonucleotide having the nucleic acid sequence of SEQ ID NO: 9 has a fluorescent reporter dye at its 5' end and a non-fluorescent quencher at its 3' end), 2) The step of subjecting the reaction mixture to quantitative PCR to obtain a copy of the target sequence, 3) A step to measure the increase in the fluorescence signal and A method comprising the addition of a fluorescence signal, wherein an increase in the fluorescence signal indicates the presence of bovine parvovirus 3 genome DNA in the test sample.

22. The method according to claim 21, wherein the fluorescent reporter dye is 6-carboxyfluorescein (FAM), and the non-fluorescent quencher is a sub-groove binder non-fluorescent quencher (MGB-NFQ) or a ZEN-IB and Iowa Black fluorescent quencher (IBFQ).

23. The method according to claim 21, further comprising one or more negative extract controls, template-free controls, positive extract controls, positive controls and / or inhibitor controls.

24. The method according to claim 21, wherein the detection sensitivity or analytical limit is 22 genome copies per reaction.

25. The method according to claim 21, wherein the detection limit of the sample is 25 genome copies per reaction.

26. The method according to claim 21, wherein the primer-probe combination, which is selective for the DNA sequence of bovine parvovirus 3, detects DNA encoding the non-structural (NS) protein and / or structural capsid (VP) protein of bovine parvovirus 3.

27. The method according to claim 21, wherein the primer-probe combination, which is selective for the DNA sequence of bovine parvovirus 3, amplifies a 144 bp fragment.

28. The method according to claim 21, wherein the oligonucleotide having the nucleic acid sequence of Sequence ID No. 9 has 6-carboxyfluorescein (FAM) at its 5' end and a secondary groove binder non-fluorescent quencher (MGB-NFQ) at its 3' end.

29. The method according to claim 21, wherein the oligonucleotide having the nucleic acid sequence of Sequence ID No. 18 has a fluorescent reporter dye 2'-chloro-7'phenyl-1,4-dichloro-6-carboxyfluorescein (VIC) at its 5' end and a secondary groove binder non-fluorescent quencher (MGB-NFQ) at its 3' end.

30. A method for quantifying 1E3-1E8 genome copies of bovine parvovirus 3 genome DNA in a PCR reaction, 1) A step of preparing a reaction mixture comprising: 1) Extracted DNA from a test sample, a positive control, BPV-3_IPC positive control plasmid DNA, nucleic acid amplification reagent, an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 16 as a forward primer, an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 17 as a reverse primer, and an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 18 as a probe (where the oligonucleotide having the nucleic acid sequence of SEQ ID NO: 18 has a fluorescent reporter dye at its 5' end and a non-fluorescent quencher at its 3' end), and a primer-probe mixture selective for the DNA sequence of bovine parvovirus 3 comprising an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 7 as a forward primer, an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 8 as a reverse primer, and an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 9 as a probe (where the oligonucleotide having the nucleic acid sequence of SEQ ID NO: 9 has a fluorescent reporter dye at its 5' end and a non-fluorescent quencher at its 3' end), 2) The step of subjecting the reaction mixture to quantitative PCR to obtain a copy of the target sequence, 3) A step to measure the increase in the fluorescence signal and A method that includes this.

31. The method according to claim 30, wherein the detection limit (LOD 95%) of the method is 27 genome copies of three bovine parvovirus genomic DNA per reaction, and the 95% confidence intervals are 22 and 34 genome copies per reaction.

32. The linearity of the method is such that it has a correlation coefficient (R²) of 0.98 or higher and a PCR amplification efficiency of 90 to 110%.

33. The method according to claim 30, wherein the parallel precision value is CV% of 25% or less.

34. The method according to claim 30, wherein the indoor reproducibility value is CV% of 30% or less.

35. The method according to claim 30, wherein the accuracy value is within ±30% of the acceptable reference value (ST) over the entire dynamic range of the assay.

36. The method according to claim 30, having a limit of quantification which is a CV% of an amount with parallel accuracy of ≤25% and intra-intra

37. The method according to claim 30, having robustness with a CV percentage of an amount of ≤25% in parallel accuracy and ≤30% in intra-intra

38. The method according to claim 30, comprising one or more of a template-less control, a positive control, a negatively extracted control, a positively extracted control, an inhibitory control, an endogenous positive control, and a standard.

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