Special primer for one-tube multiplex fluorescence PCR detection of viruses BVDV, BRV and BCV and application of special primer

A multiple fluorescence and virus technology, applied in the direction of DNA / RNA fragments, microorganisms, recombinant DNA technology, etc., can solve the problems of rapid transmission, great harm, and difficulty in pathogen diagnosis, and achieve specific technical means, good repeatability, and high sensitivity Effect

Active Publication Date: 2021-01-05
HEBEI AGRICULTURAL UNIV. +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the process of clinical diagnosis, these three causes have relatively similar clinical symptoms and often mixed infection, which has caused great difficulty in the diagnosis of the pathogen, and spreads quickly, is harmful, and has no specific drug treatment, causing huge economic losses to the dairy industry.
[0003] Currently, diagnostic methods for BVDV, BRV, and BCV mainly include pathogen cell isolation, electron microscopy, single PCR, multiplex PCR, ELISA, gene chips, and loop-mediated isothermal amplification. However, the above detection methods have obvious limitations.

Method used

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  • Special primer for one-tube multiplex fluorescence PCR detection of viruses BVDV, BRV and BCV and application of special primer
  • Special primer for one-tube multiplex fluorescence PCR detection of viruses BVDV, BRV and BCV and application of special primer
  • Special primer for one-tube multiplex fluorescence PCR detection of viruses BVDV, BRV and BCV and application of special primer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] According to the reference strain sequences of BVDV (MK170077), BRV (MNo47454) and BCV (MK903505) published in GenBank, three pairs of specific primers were designed using DNA Star and Primer5.0 software to amplify BVDV E2 gene and BRVVP6 gene and BCV N gene. The primers were synthesized by Changchun Kumei Bioengineering Co., Ltd., and the primer sequences are shown in Table 1.

[0035] Table 1 PCR primer sequences

[0036]

Embodiment 2

[0038] The preparation of BVDV, BRV, BCV recombinant plasmid standard products, the steps are as follows:

[0039] (1) Clinical sample processing: 150 fresh diarrhea feces samples were collected from 10 cattle farms in Xinle and Xinji, Shijiazhuang, and 150 fresh feces samples were added to sterilized centrifuge tubes containing 5 mL of pH7.2 PBS solution, and oscillated repeatedly for 1 min , centrifuged at 5000r / min for 10min, and took the supernatant; according to the instructions of the viral genome RNA extraction kit (magnetic bead method), the virus nucleic acid was extracted by using a fully automatic nucleic acid extraction instrument, and the extracted nucleic acid product was reversed by the reverse transcription kit. Transcribed, obtained cDNA, and stored at -20°C for future use.

[0040] (2) The cDNAs of BVDV, BRV, and BCV were respectively amplified by PCR using the specific primers designed in Example 1, and sterile deionized water was used as a negative control....

Embodiment 3

[0047] Dilute the prepared three plasmid standards by 10-fold gradient, and take the concentration interval as 10 7 -10 0 After copy / μL, the fluorescent quantitative PCR amplification was carried out respectively, and the BVDV recombinant plasmid standard was prepared at a concentration of 10 7 -10 2 copy / μL, BRV, BCV recombinant plasmid standard at a concentration of 10 7 -10 1 The fluorescent signal can be detected between copies / μL, and analyzed by the system automatic analysis software, the standard curve of fluorescent quantitative PCR can be drawn with the logarithm of the copy number as the ordinate and the Ct value as the abscissa, and the results are as follows Figure 2a-2c shown, where Figure 2a is the standard curve of the BVDV recombinant plasmid, Figure 2b is the standard curve of the BRV recombinant plasmid, Figure 2c It is the standard curve of BCV recombinant plasmid. Depend on Figure 2a-2c It can be seen that BVDV in 10 7 -10 2 Within the range ...

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Abstract

The invention discloses a special primer for one-tube multiplex fluorescence PCR detection of viruses BVDV, BRV and BCV and application of the special primer, and belongs to the technical field of molecular biological detection. The optimal multiplex fluorescence PCR reaction conditions are designed for the special primer; the lowest detection limits of the primer and the reaction conditions on plasmid standard substances of BVDV, BRV and BCV are 1.19*10<2> copies/mu L, 3.89*10<1> copies/mu L and 3.74*10<1> copies/mu L respectively, the lowest sensitivity is 100 times higher than that of conventional PCR, and the sensitivity is high. The method only specifically amplifies BVDV, BRV and BCV, has no cross reaction with Escherichia coli, salmonella and infectious bovine rhinotracheitis virus,and has strong specificity. The intra-group variable coefficient is less than 1%, the inter-group variable coefficient is less than 1%, and the repeatability is good.

Description

technical field [0001] The invention belongs to the technical field of molecular biology detection, in particular to special primers for multiple fluorescent PCR detection of viruses BVDV, BRV and BCV and the application thereof. Background technique [0002] In recent years, with the expansion of the scale of dairy farming, the incidence of calf diarrhea has been increasing year by year. Calf diarrhea seriously affects the early growth and development of calves and the stability of later production performance. The mortality rate is as high as 90%, which seriously affects cattle. The expansion of the herd and the healthy development of the dairy farming industry. The factors causing diarrhea in calves are complex, among which the diarrhea caused by infectious viruses is the most serious, and most of them are mixed infections, bovine viral diarrhea virus (BVDV), bovine rotavirus (BRV) and bovine coronavirus (BCV) are the most common pathogen. In the process of clinical dia...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/701C12Q1/686C12Q2600/16C12Q2537/143C12Q2547/101C12Q2563/107Y02A50/30
Inventor 常丽云刘志勇秦建华李颖赵月兰
Owner HEBEI AGRICULTURAL UNIV.
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