Method for synchronously detecting porcine circovirus type 2 (PCV2), classical swine fever virus (CSFV) and porcine reproductive and respiratory syndrome virus (PRRSV)

A technology for porcine circovirus and respiratory syndrome, applied in the field of in vitro molecular diagnosis of biomedicine, can solve the problems of easy contamination, limited application of multiplex PCR, decreased sensitivity and specificity of multiplex PCR detection, etc., and achieves good stability, high sensitivity, specific effect

Inactive Publication Date: 2012-03-21
ZHENGZHOU COLLEGE OF ANIMAL HUSBANDRY ENG
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  • Application Information

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Problems solved by technology

However, in addition to the shortcomings of conventional PCR such as easy contamination, high false positives and false negatives, multiplex PCR amplification generally involves 2 or more pairs of primers, cross-reaction between primers often leads to multiplex PCR detection sensitivity and specificity decreased to varying degrees, limiting the application of multiplex PCR

Method used

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  • Method for synchronously detecting porcine circovirus type 2 (PCV2), classical swine fever virus (CSFV) and porcine reproductive and respiratory syndrome virus (PRRSV)
  • Method for synchronously detecting porcine circovirus type 2 (PCV2), classical swine fever virus (CSFV) and porcine reproductive and respiratory syndrome virus (PRRSV)
  • Method for synchronously detecting porcine circovirus type 2 (PCV2), classical swine fever virus (CSFV) and porcine reproductive and respiratory syndrome virus (PRRSV)

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1 loop-mediated indirect PCR detects the method for PCV, PRRSV and CSFV 3 kinds of porcine origin viruses

[0052] 1.1 Design of probes and primers

[0053] The PCV2, PRRSV and CSFV gene sequences published in the GenBank database were analyzed, and the conserved region of the gene was used as a template to design probes for different viral targets using Primer Premier 5.0 software to label the reporter gene. The reporter gene used in this experiment Because the soybean Lectin gene, primers used for PCR labeling and reverse PCR amplification primers were all synthesized by Shanghai Sangong, the 5' ends of primers P1-P6 were phosphorylated, and the underlined part was the probe sequence. The primer sequences are listed in Table 1.

[0054] Table 1 Sequences and characteristics of marker primers and reverse PCR amplification primers

[0055]

[0056]

[0057] 1.2 Preparation of viral nucleic acid

[0058] Preparation of viral DNA: The classic phenol: c...

Embodiment 2

[0066] Example 2 Evaluation of loop-mediated indirect PCR detection of PCV, PRRSV and CSFV 3 kinds of pig-derived virus methods

[0067] 2.1 Specificity assessment

[0068] Prepare PCV2DNA, PRVDNA, PPVDNA, Haemophilus parasuis DNA, enterotoxigenic Escherichia coli DNA, CSFV cDNA and PRRSV cDNA solutions, and use this solution as a template for single-virus loop-mediated indirect PCR detection and multi-virus loop-mediated indirect PCR Detection, evaluation ring-mediated indirect PCR detection specificity, the results are shown in the figure (attached Figure 4 , 5 ), regardless of single-virus detection or multi-virus detection, loop-mediated indirect PCR showed a high degree of specificity, a single amplification background, and no cross-reaction with other pathogens.

[0069] 2.2 Sensitivity assessment

[0070] After measuring the concentration of the prepared PCV, PRRSV and CSFV nucleic acid solutions with a spectrophotometer, adjust the PCV2 DNA concentration to 200pg / μ...

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Abstract

The invention discloses a method for synchronously detecting a porcine circovirus type 2 (PCV2), a classical swine fever virus (CSFV) and a porcine reproductive and respiratory syndrome virus (PRRSV). The method comprises the following steps of: designing three pairs of specific probes by the PRRSV nucleic acid, PCV2 nucleic acid and CSFV nucleic acid, preparing reporter genes of probe markers by using polymerase chain reaction (PCR), and implementing loop-mediated indirect PCR detection of three viruses by cyclization and reverse PCR amplification of the reporter genes. the pathogens of PRRSV, PCV2 and CSFV in sample tissues can be synchronously detected by designing three pairs of specific probes aiming at three viruses, marking the same reporter genes, designing a pair of reverse PCR amplification primers and establishing a loop-mediated indirect PCR detection system, so that certain reference is provided for scientifically preventing and controlling porcine diseases. Clinical tests show that the method has the advantages of single detection background, high sensitivity, strong specificity, good stability and the like, can be used for single-virus detection, can also be used for synchronous detection of multiple viruses, and provides a tool for viral clinical diagnosis and epidemiological study of PRRSV, PCV2 and CSFV.

Description

technical field [0001] The invention relates to a method for detecting porcine circovirus type 2 (PCV2), swine fever virus (CSFV) and porcine reproductive and respiratory syndrome virus (PRRSV) by loop-mediated indirect PCR, which belongs to biomedical in vitro molecular diagnosis technology. Background technique [0002] my country is a big pig raising country, and the scale and intensification of breeding are constantly improving. Pig diseases, headed by viral infectious diseases, are currently one of the main factors affecting the efficiency of pig raising, which has caused great harm to the pig raising industry. Pig high fever caused by pathogenic PRRSV is the main one. PRRSV plays an important role in many viral factors causing high fever symptoms in pigs, and is the main pathogen, followed by PCV2, and CSFV again. In recent years, PRRSV infection has been very common in pigs in my country, and the immunosuppression of infected pigs often leads to mixed infection of oth...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 边传周郑鸣王永芬王老七
Owner ZHENGZHOU COLLEGE OF ANIMAL HUSBANDRY ENG
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