Real-time fluorescence reverse transcription-polymerase chain reaction (RT-PCR) detection method and kit for highly pathogenic porcine reproductive and respiratory syndrome live vaccine

A JXA1-R, RT-PCR technology, applied in the field of real-time fluorescent RT-PCR detection of highly pathogenic porcine reproductive and respiratory syndrome live vaccines, can solve the problem of insufficient sensitivity, specificity or timeliness, and distinguish vaccines strains, vaccine strains cannot be detected, etc., to achieve the effect of good reliability, low detection cost and high sensitivity

Active Publication Date: 2012-11-21
CHINA ANIMAL DISEASE CONTROL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Traditional PRRSV detection methods mainly include virus isolation and identification, immunoperoxidase monolayer assay (IPMA), indirect immunofluorescence assay (IFA), indirect enzyme-linked immunosorbent assay (indirect ELISA) and reverse transcription-polymerase Chain reaction test (RT-PCR), etc., they have played an important role in virus diagnosis and import and export inspection and quarantine, but each has shortcomings in sensitivity, specificity or timeliness, and it is difficult to isolate the vaccine strain (JXA1-R ) from wild strains to achieve the purpose of detecting vaccine strains (JXA1-R)
At present, although real-time fluorescent quantitative RT-PCR and other detection methods for highly pathogenic PRRSV variant strains have been established, they still cannot specifically detect the vaccine strain (JXA1-R)

Method used

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  • Real-time fluorescence reverse transcription-polymerase chain reaction (RT-PCR) detection method and kit for highly pathogenic porcine reproductive and respiratory syndrome live vaccine
  • Real-time fluorescence reverse transcription-polymerase chain reaction (RT-PCR) detection method and kit for highly pathogenic porcine reproductive and respiratory syndrome live vaccine
  • Real-time fluorescence reverse transcription-polymerase chain reaction (RT-PCR) detection method and kit for highly pathogenic porcine reproductive and respiratory syndrome live vaccine

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Construction of standard RNA

[0028] (1) Experimental reagents

[0029] Restriction enzyme Pst I and its buffer, DL2000 Marker were purchased from TaKaRa Company;

[0030] Ligation kit, Taq DNA polymerase, AMV reverse transcriptase, RNase inhibitor, dNTPs were purchased from Promega;

[0031] DNA gel recovery kit, purchased from OMEGA company;

[0032] High-efficiency Escherichia coli competent cells DH5α were purchased from Beijing Quanshijin Biotechnology Co., Ltd.;

[0033] Primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.;

[0034] Other biochemical reagents are imported subpackages or domestic analytical pure.

[0035] (2) Experimental equipment

[0036] TGRANDIENT PCR instrument: purchased from HYBAID Company; DYFIII2 type electrophoresis instrument: purchased from Bio-Rad Company;

[0037] UVP gel imaging analysis system: purchased from Gene Company; Steril Biological safety cabinet: purchased from BAKER Company; c...

Embodiment 2

[0051] Extraction of sample RNA

[0052] According to conventional methods, using QIAGEN Mini Kit kit (purchased from QIAGEN), or according to the GTC method (guanidine isothiocyanate method) ( The Total RNA Isolation System kit was purchased from Promega) to extract the RNA of the highly pathogenic porcine reproductive and respiratory syndrome live vaccine (JXA1-R strain).

Embodiment 3

[0054] Screening test of specific primer and probe combinations by real-time fluorescent RT-PCR method for highly pathogenic porcine reproductive and respiratory syndrome live vaccine (JXA1-R strain)

[0055] (1) Experimental reagents

[0056] One Step PrimeScript TM RT-PCR Kit (Perfect Real Time) kit was purchased from TaKaRa Company;

[0057] Highly pathogenic porcine reproductive and respiratory syndrome live vaccine (JXA1-R strain) TaqMan MGB probe and primers were synthesized by Shanghai Jikang Biotechnology Co., Ltd.

[0058] (2) Experimental equipment

[0059] Applied Biosystems 7500 Real-Time PCR System

[0060] (3) Experimental process

[0061] ①Design of primers and probes for highly pathogenic porcine reproductive and respiratory syndrome live vaccine (JXA1-R strain)

[0062] The specific primers for the highly pathogenic porcine reproductive and respiratory syndrome live vaccine (JXA1-R strain) refer to: an oligonucleotide chain with a length of about 20 base...

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Abstract

The invention discloses nucleotide primer and probe sequences and a method for real-time fluorescence reverse transcription-polymerase chain reaction (RT-PCR) detection of a highly pathogenic porcine reproductive and respiratory syndrome vaccine, live (JXA1-R strain), and application of the method in detecting the highly pathogenic porcine reproductive and respiratory syndrome vaccine, live (JXA1-R strain). The method overcomes the defects of the traditional detection technology, has the characteristics of quickness, simplicity, convenience, strong specificity, high sensitivity and high reliability, and is suitable for quickly detecting the highly pathogenic porcine reproductive and respiratory syndrome vaccine, live (JXA1-R strain) and monitoring quality in vaccine production.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a real-time fluorescent RT-PCR detection method for highly pathogenic porcine reproductive and respiratory syndrome live vaccine (JXA1-R strain). Background technique [0002] Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome, PRRS) is caused by porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus, PRRSV). Characterized by highly contagious infectious diseases. The disease broke out in the United States for the first time in 1987, and then appeared in Canada, Europe and Asia one after another. At present, it has become popular all over the world, causing huge economic losses every year. PRRS first appeared in my country in 1995, and then spread rapidly. In 2006, there was an outbreak of highly pathogenic PRRSV (highly pathogenic PRRSV, HP-PRRSV) in China, which was characterized by high b...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11G01N21/64
Inventor 田克恭王立林遇秀玲倪建强夏应菊曲萍翟新验陈西钊
Owner CHINA ANIMAL DISEASE CONTROL CENT
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