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General real-time fluorescent RT (Reverse Transcription)-PCR (Polymerase Chain Reaction) method and kit for detecting porcine reproductive and respiratory syndrome virus

A technology for respiratory syndrome and pig reproduction, applied in the direction of fluorescence/phosphorescence, biochemical equipment and methods, and microbial measurement/inspection, can solve the problems of insufficient sensitivity, specificity or timeliness, and achieve good reliability, The effect of strong specificity and high sensitivity

Active Publication Date: 2012-09-26
CHINA ANIMAL DISEASE CONTROL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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

Method used

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  • General real-time fluorescent RT (Reverse Transcription)-PCR (Polymerase Chain Reaction) method and kit for detecting porcine reproductive and respiratory syndrome virus
  • General real-time fluorescent RT (Reverse Transcription)-PCR (Polymerase Chain Reaction) method and kit for detecting porcine reproductive and respiratory syndrome virus
  • General real-time fluorescent RT (Reverse Transcription)-PCR (Polymerase Chain Reaction) method and kit for detecting porcine reproductive and respiratory syndrome virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Construction of standard RNA

[0027] (1) Experimental reagents

[0028] Restriction enzyme Pst I and its buffer, DL2000Marker were purchased from TaKaRa Company;

[0029] Ligation kit, Taq DNA polymerase, AMV reverse transcriptase, RNase inhibitor, dNTPs were purchased from Promega;

[0030] DNA gel recovery kit, purchased from OMEGA company;

[0031] High-efficiency Escherichia coli competent cells DH5α were purchased from Beijing Quanshijin Biotechnology Co., Ltd.;

[0032] Primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.;

[0033] Other biochemical reagents are imported subpackages or domestic analytical pure.

[0034] (2) Experimental equipment

[0035] TGRANDIENT PCR instrument: purchased from HYBAID Company; DYFIII2 type electrophoresis instrument: purchased from Bio-Rad Company;

[0036] UVP gel imaging analysis system: purchased from Gene Company; Steril Biological safety cabinet: purchased from BAKER Company; co...

Embodiment 2

[0050] Extraction of sample RNA

[0051] According to conventional methods, using QIAGEN The Mini Kit kit (purchased from QIAGEN Company) was used to extract the RNA of porcine reproductive and respiratory syndrome virus (JXA1 strain).

Embodiment 3

[0053] Screening test of specific primer and probe combinations for porcine reproductive and respiratory syndrome virus general-purpose real-time fluorescent RT-PCR method

[0054] (1) Experimental reagents

[0055] One Step PrimeScript TM RT-PCR Kit (Perfect Real Time) kit was purchased from TaKaRa Company;

[0056] Porcine reproductive and respiratory syndrome virus universal TaqMan MGB probe and primers were synthesized by Shanghai Jikang Biotechnology Co., Ltd.

[0057] (2) Experimental equipment

[0058] Applied Biosystems 7500 Real-Time PCR System

[0059] (3) Experimental process

[0060] ① Design of primers and probes for porcine reproductive and respiratory syndrome virus (JXA1 strain)

[0061] The specific primers for porcine reproductive and respiratory syndrome virus refer to: oligonucleotide chains with a length of about 18 to 20 bases, specific nucleotides in the highly conserved region of the porcine reproductive and respiratory syndrome Nsp7 gene Fragmen...

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Abstract

The invention discloses a nucleotide primer, a probe sequence and a method for carrying out real-time fluorescent RT (Reverse Transcription)-PCR (Polymerase Chain Reaction) detection on porcine reproductive and respiratory syndrome virus as well as an application of the method in clinically detecting the porcine reproductive and respiratory syndrome virus. The method has the characteristics of rapidity and convenience, strong specificity, high sensitivity and good reliability, and is suitable for rapidly detecting the porcine reproductive and respiratory syndrome virus.

Description

technical field [0001] The invention relates to the field of biotechnology, and relates to a general-purpose real-time fluorescent RT-PCR detection method for porcine reproductive and respiratory syndrome. 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). Characteristic highly contagious infectious disease. The disease broke out in the United States for the first time in 1987, and then appeared in Canada, Europe and Asia, and has become popular worldwide, causing huge economic losses. PRRS first appeared in my country in 1995, and then spread rapidly. In 2006, a pig "high fever disease" with high body temperature, high morbidity and high mortality broke out in my country. Porcine reproductive and respiratory syndrome virus (highly pathogenic PRRS, HP-PRRS). So far, the...

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 田克恭夏应菊陈南华韩雪訾占超倪建强遇秀玲翟新验
Owner CHINA ANIMAL DISEASE CONTROL CENT
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