Method for detection CPV 2a, 2b, and 2c and for discrimination wild type from vaccine type
Inventors
WONG, Jr Winston • Kao, Stephen Chang-Chi • Lai, Ying-Ta • Hung, Ming-Lung • Wang, Wei-Chen
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-10233509-B2
Publication Date
2019-03-19
Expiration Date
Abstract
The present invention disclose a method for detection CPV 2a, 2b, and 2c in a sample by detecting VP2 gene. The signals can be detected by both fluorescent detection system and lateral flow immunochromatographic assay. The second object of this present invention is to provide a method to discriminate the wild type from the vaccine type. The first approach to achieve this object is discriminating wild type from vaccine type by SNP 36, which could be used on both fluorescent detection system and lateral flow immunochromatographic assay. The second approach to achieve this object is discriminating wild type from vaccine type by SNP 899 and SNP 963.
Core Innovation
The invention relates to a method of discriminating a subject of wild type from vaccine type in a sample by analyzing a CPV-2 VP2 gene nucleic acid sequence. The method provides primers and amplifies the subject with a template-dependent polymerase, then anneals a pair of probes to form a hybridized product, where the probe pair is specific to SNP 899 and SNP 963 of the VP2 gene of CPV 2a, 2b, or 2c.
During detection, two distinct fluorescent signals generating from the hybridized product indicate the presence of the vaccine type. The fluorescent signals correspond to probe hybridization at the SNP targets so that a signal pattern discriminates between wild type and vaccine type. The disclosed discrimination framework is further tied to specific vaccine products such that the vaccine type is associated with a sample previously vaccinated with a named vaccine product.
The document also provides additional refinements for probe selection and fluorescent reporter and quencher configurations. It further describes detection using exonuclease hydrolysis cleavage quantification of a fluorescent reporter fragment released from a probe hybridized to a target nucleic acid.
Claims Coverage
The independent claim is directed to discriminating wild type versus vaccine type by using CPV-2 VP2 gene SNP-specific probes and detecting two distinct fluorescent signals. The inventive coverage includes a template-dependent polymerase amplification step, SNP 899/SNP 963 probe specificity, and interpretation of the two-signal fluorescence pattern as vaccine type versus wild type.
Wild type vs vaccine type discrimination by two SNP-specific probes
providing a pair of primers and amplifying with a template-dependent polymerase; annealing P1 and a P2 probes to form a hybridized product in which P1 is specific to SNP 899 of the VP2 gene of CPV 2a, 2b, or 2c and P2 is specific to SNP 963 of the VP2 gene of CPV 2a, 2b, or 2c; and detecting two distinct fluorescent signals generating from the hybridized product to indicate the presence of the vaccine type.
Primer selection from defined SEQ ID ranges
the first primer consists of at least contiguous 12 nucleotides selected from nucleic acid sequence SEQ ID NO:16 and the second primer consists of at least contiguous 12 nucleotides selected from complementary nucleic acid sequences SEQ ID NO:22 or 23, with P1 and P2 selected from specified groups of SEQ ID NO: 24 to 27 and SEQ ID NO:29 to 33, respectively.
Probe labeling with distinct fluorescent reporters and specified quenchers
annealing probes P1 and a P2 probes to form a hybridized product during amplification wherein the 5′ ends have distinct fluorescent reporters on P1 and P2 that are different from each other, and the 3′ ends include quenchers selected from TMARA, MGB, or BHQ.
Exonuclease hydrolysis cleavage-based quantification of fluorescent reporter
detecting two distinct fluorescent signals from a hybridized product by quantifying the cleaved fluorescent reporter fragment released from a probe hybridized to a target nucleic acid via exonuclease hydrolysis of a DNA-dependent polymerase.
Vaccine type defined by previously vaccinated sample with named vaccine products
defining vaccine type based on a sample previously vaccinated with one of the listed vaccine group products including Duramune MX5, Canivac 5, Vanguarad plus 5 L4 CV, Nobivac Puppy DP, Canine 6II-SL, Eurican5, and Virbagen DA2Parvo.
Across the independent claim and its dependents, the inventive subject matter centers on discriminating wild type versus vaccine type by using CPV-2 VP2 gene SNP 899 and SNP 963 specific probes together with template-dependent polymerase amplification and detection of two distinct fluorescent signals. Additional claim coverage specifies defined primer/probe SEQ ID selections, fluorescent reporter and quencher configurations, an exonuclease hydrolysis cleavage quantification detection mechanism, and a definition of vaccine type using named vaccine products.
Stated Advantages
Discriminates a subject of wild type from vaccine type in a sample.
Detects vaccine type by detecting two distinct fluorescent signals generating from the hybridized product.
Uses exonuclease hydrolysis of a DNA-dependent polymerase for cleavage-based detection by quantifying a cleaved fluorescent reporter fragment.
Defines vaccine type using samples previously vaccinated with named vaccine products.
Documented Applications
Discriminating wild type versus vaccine type in a sample for subjects associated with canine parvovirus type 2 (CPV-2) VP2 gene analysis.
Interested in licensing this patent?