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Fluorescent quantitative PCR rapid diagnosis reagent box for specific detection of classical swine fever virus wild virus infection

A technology for swine fever virus and fluorescence quantification, which is applied in the direction of fluorescence/phosphorescence, biochemical equipment and methods, and microbial measurement/inspection. Vaccine immune differential diagnosis technology and other issues, to achieve the effect of fast detection, good specificity and high accuracy

Inactive Publication Date: 2008-12-24
INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Prior art about classical swine fever virus antigen, antibody detection and the document of fluorescent quantitative PCR technology, mainly contain following several items, as the document " the method for detecting classical swine fever virus specific antibody and enzyme-linked immunoassay kit thereof that the patent number application number is 200710176125X "Disclosed a method for detecting CSFV antigen preparation and antibody detection, but did not involve the detection of CSFV antigen; the patent number is 200610109404 "CSFV fluorescent RT-PCR diagnostic kit", the patent application number is 02109350.4 "A Fluorescent Quantitative PCR Kit and Its Application for Rapid Quantitative Detection of CSFV and CSF Rabbitized Attenuated Vaccine" discloses the detection methods of two CSFV antigens, but does not involve CSFV wild virus infection and vaccine immunity differential diagnosis techniques

Method used

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  • Fluorescent quantitative PCR rapid diagnosis reagent box for specific detection of classical swine fever virus wild virus infection
  • Fluorescent quantitative PCR rapid diagnosis reagent box for specific detection of classical swine fever virus wild virus infection
  • Fluorescent quantitative PCR rapid diagnosis reagent box for specific detection of classical swine fever virus wild virus infection

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Effect test

Embodiment 1

[0032] Composition and preparation of a fluorescent quantitative PCR rapid diagnostic kit for the specific detection of wild virus infection of classical swine fever virus

[0033] 1. Reagent composition:

[0034] Trizol RNA lysate is a product of Invitrogen; Taq enzyme (5U / μl), dNTPs (10mM), MgCl 2 (25mM), M-MLV enzyme (50U / μl) were purchased from Promega Company; PCR primer pair FCSFV and RCSFV, quantitative standard primers were synthesized by Shanghai Sangong Bioengineering Company; Taqman-MGB probe was purchased from Shanghai Jikang Bioengineering Co., Ltd. Synthesized by the company; Shimen standard strain of classical swine fever virus is preserved by our laboratory.

[0035] 2. Reagent preparation:

[0036] A) Reverse transcription reaction solution: 1×RT Buffer, dNTPs 0.5mM, M-MLV enzyme 50U, primer RCSFV 0.4μM; the sequence of primer RCSFV is 5′-TGCCCACAGTAGGACTAGCAAAC-3′23nt;

[0037] B) Fluorescence quantitative reaction solution: 1×PCR Buffer, MgCl 2 4mM, dNTP...

Embodiment 2

[0043] Application method of the fluorescence quantitative PCR rapid diagnostic kit for specific detection of classical swine fever virus wild virus infection

[0044] 1. Sample processing

[0045] A) The sample to be tested is a tissue sample: take 50-100 mg of the sample to be tested in a clean, sterilized and dried homogenizer, add PBS solution at a volume ratio of 1:5, fully homogenate, centrifuge at 4000 rpm for 10 minutes, and take 100 μl Transfer the supernatant (for each reaction) into a sterile 1.5ml centrifuge tube, numbered for later use;

[0046] B) The sample to be tested is a liquid sample (such as: whole blood, serum, nasal swab, etc.), directly take 100 μl and transfer it into a sterile 1.5ml centrifuge tube, numbered for future use.

[0047] 2. Extraction of RNA from the tested sample

[0048] Take n sterilized 1.5ml centrifuge tubes, where n is the sum of the number of tested samples and the positive control, critical positive quality control and negative q...

Embodiment 3

[0064] Application of Fluorescence Quantitative PCR Rapid Diagnosis Kit for Specific Detection of Wild Virus Infection of Classical Classical Classical Swine Fever Virus

[0065] 1. Sensitivity test

[0066] 10-fold serially diluted standard (1.0×10 1 ~1.0×10 7 copy / μl) as a template, detected on a fluorescent quantitative PCR instrument, to obtain a real-time PCR amplification curve and a standard curve, see the attached figure 1 And attached figure 2 . attached figure 1 It shows that when the standard plasmid concentration ≥ 10 copies / μl, the kinetic curve is on the rise, therefore, the detection sensitivity of the method is 10 copies / μl (the detection sensitivity is the cDNA template concentration of the tested sample, if converted to the detected The sample concentration should be 2.5 x 10 3 copy / m

[0067] 1). attached figure 2 It shows that the linear range of quantitative detection of this method is 1.0×10 1 ~1.0×10 7 copies / μl, correlation coefficient R=0....

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Abstract

The invention relates to a fluorescence quantitative PCR rapid diagnosis kit for specifically detecting the Hog cholera virus and wild virus infection and an application method thereof, belonging to the virus nucleic acid detection field. The fluorescence quantitative PCR rapid diagnosis kit is applied to the rapid quantitative detection of the CSFV wild virus clinically and in scientific research and can remove the false positive caused by the CSFV HCLV vaccine immunization. Compared with the prior art, the fluorescence quantitative PCR rapid diagnosis kit has the advantages that: the Hog cholera virus and wild virus infection and the vaccine immunization can be verified and diagnosed, the problem of the incapability of removing the false positive caused by the vaccine immunization is solved; the specificity is good, the high specificity hybridization double control of the primer high specificity amplification and the fluorescence probe is realized, the accuracy is high, the false positive is low; the sensitivity is high; the detection speed is high, only one hour is needed and two to three hours are needed when the extraction process of the nucleic acid and the preparation process of the cDNA are added; and the post treatment is not necessary, the processes such as hybridization, electrophoresis and photo are not necessary and no pollution is caused.

Description

technical field [0001] The present invention relates to a fluorescent quantitative PCR rapid diagnostic kit for specifically detecting CSFV wild virus infection and its application method, which belong to the field of viral nucleic acid detection, and are suitable for rapid quantitative detection of CSFV wild virus in clinical and scientific research. False positives caused by immunization with CSFV rabbitized attenuated virus (HCLV) vaccine were excluded. Background technique [0002] Classical Swine Fever (CSF) is an acute and contagious infectious disease of pigs caused by Classical Swine Fever Virus (CSFV), with a mortality rate of over 90%. Swine fever occurs and spreads all over the world, causing serious harm and huge economic losses to the pig industry. It is listed as one of the legal infectious diseases that must be declared by the International Organization for Animal Health (OIE). my country is also one of the countries with a high incidence of swine fever. In r...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68G01N21/64
Inventor 温国元邵华斌杨峻罗青平张蓉蓉艾地云王红琳罗玲李坤
Owner INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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