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Probe, primer and reagent kit for realtime fluorescent quantitative RT-PCR detection of classical swine fever virus

A real-time fluorescence quantitative, swine fever virus technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc. Simple, fast detection effect

Inactive Publication Date: 2009-01-21
黄萍
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The PCR primers and probes used are designed for the genome sequence of a specific region of CSFV, which can effectively distinguish BVDV, BDV, and CSFV in the tested samples, but this technology has not been used in animal clinical samples and animal-derived foods. CSFV has been applied in China, but its sensitivity and specificity need to be further improved

Method used

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  • Probe, primer and reagent kit for realtime fluorescent quantitative RT-PCR detection of classical swine fever virus
  • Probe, primer and reagent kit for realtime fluorescent quantitative RT-PCR detection of classical swine fever virus
  • Probe, primer and reagent kit for realtime fluorescent quantitative RT-PCR detection of classical swine fever virus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Positive control: Viral RNA was extracted from the attenuated swine fever rabbit vaccine.

[0074] Negative control: Use non-infected CSFV porcine tissue, add DEPC water at a volume of 1:10, grind in a tissue homogenizer, centrifuge at 3500 rpm for 15 min, and take the supernatant to extract sample RNA.

[0075] reaction system:

[0076] 5×buffer 5.0μl

[0077] 25mmol / L MgCl 2 2.0μl

[0078] RNase Inhibitor 0.5μl

[0079] Reverse Transcriptase 0.2μl

[0080] 10mmol / L dNTPs 0.5μl

[0081] 10μmol / L Primer 1 0.5μl

[0082] 10μmol / L Primer 2 0.5μl

[0083] 10μmol / L Probe 0.5μl

[0084] Taq enzyme 0.5μl (3U / μl)

[0085] RNA 5.0μl

[0086] DEPC H 2 O 9.8 μl

[0087]

[0088] 25 μl total

[0089] Reaction conditions: the first stage, reverse transcription 45°C / 15min;

[0090] In the second stage, pre-denaturation at 95°C / 5min;

[0091] In the third stage, 94°C / 30s, 58°C / 45s, 40 cycles. Fluorescent signal is detected upon ...

Embodiment 2

[0093] Embodiment 2 specificity research

[0094]In view of the high homology of the genome sequences of BVDV, BDV, and CSFV of the genus Pestivirus in the family Flaviviridae, and the widespread occurrence of animal virus infections such as PRRSV, PRV, PPV, and PCV-2 in intensive pig farms in my country, and These epidemics have certain similarities in epidemiology, clinical symptoms, pathological manifestations, and histological changes of affected animals. Therefore, the present invention adopts CSFV, BVDV, BDV, PRRSV, PRV, PPV, PCV-2 and other virus culture Liquid for comparative study to determine the specificity of the method of the present invention.

[0095] The experimental results can be found in the attached figure 2 , the two positive "S" curves represent the results of real-time RT-PCR amplification using RNA extracted from 20 μl and 10 μl CSFV culture medium as templates, while the other six viral RNAs did not amplify "S" Curve, no Ct value, the experimental res...

Embodiment 3

[0097] The comparative study of different detection methods of embodiment 3

[0098] Using RT-PCR, real-time RT-PCR and AC-ELISA three different detection methods to detect the same sample, and compare the detection results to determine the sensitivity of the method. 268 clinical tissue samples, 324 clinical blood samples, 122 salted sausage casings, 96 meat cuts, and 24 cured meat samples were tested.

[0099] The molecular biology detection method is: homogenate the tested sample, dilute it with sterilized physiological saline according to the ratio of 1:4, 1:8, 1:16, 1:32, 1:64, and extract the total RNA of the sample , and then perform CSFV RT-PCR and real-time RT-PCR detection according to the method designed in this study, instead of taking samples to extract RNA, diluting the RNA and then conducting sensitivity research, which is different from the detection required in real work The samples are basically the same; for the AC-ELISA detection method, the diluted homogen...

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Abstract

The invention provides a probe used for carrying out realtime fluorescence quantitative RT-PCR detection of hog cholera virus, a primer sequence and a reagent kit containing the probe and the primer sequence. By means of the principles of the reagent kit and fluorescence quantitative PCR, the realtime fluorescence quantitative RT-PCR method for detecting hog cholera virus in animal food and animal-derived food is established; moreover, comparative study is carried out from aspects of sensitivity, specificity and so on. The detection method has the advantages of strong specificity, high sensitivity, excellent repeatability, simple operation, fast detection speed, no influence caused by subjective consciousness of operating staff, high flux of detected sample, low biosafety risk, and the like.

Description

Technical field: [0001] The invention relates to a probe and a primer sequence for real-time fluorescent quantitative RT-PCR detection of swine fever virus, and a kit comprising the probe and the primer. Background technique: [0002] Classical swine fever (CSF) is an acute, febrile, and highly contagious infectious disease caused by classical swine fever virus (CSFV), which is characterized by rapid onset, wide epidemic range, and high mortality. It not only causes great economic losses to the pig industry, but also seriously hinders the international trade of pigs and pig products. OIE lists it as a class A infectious disease, and FAO and governments of various countries also list it as the main animal that needs to be closely monitored and quarantined. one of infectious diseases. [0003] At present, the traditional domestic CSFV laboratory testing methods mainly include: (1) Fluorescent antibody test, which is not sensitive enough to effectively detect the presence of C...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 黄萍朱中武龙新平黄志强唐连飞鲁杏华黄辉胡宇东
Owner 黄萍
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