Bovine respiratory syncytial virus nano-PCR detection kit and preparation method thereof

A detection kit, nano-PCR technology, applied in the field of bovine respiratory syncytial virus detection, can solve the problems of high false positives, low sensitivity and expensive imported reagents in serological detection, and achieve cost reduction, reagent cost reduction, Samples for a wide range of effects

Inactive Publication Date: 2017-08-18
INST OF SPECIAL ANIMAL & PLANT SCI OF CAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the problems of low sensitivity, high false positives in serological detection and expensive imported reagents in the existing bovine respiratory syncytial virus detection method, the invention provides a bovine respiratory syncytial virus na no-PCR detection kit and its Preparation

Method used

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  • Bovine respiratory syncytial virus nano-PCR detection kit and preparation method thereof
  • Bovine respiratory syncytial virus nano-PCR detection kit and preparation method thereof
  • Bovine respiratory syncytial virus nano-PCR detection kit and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0045] Example 1 Assembly of the bovine respiratory syncytial virus nano-PCR detection kit of the present invention

[0046] (1) Prepare nano-PCR reaction solution

[0047] ①Design a pair of specific primers based on the conservative gene sequence of the BRSV virus NP genome. To identify the primers P1 and P2 of bovine respiratory syncytial virus (BRSV), the primer sequences are as follows:

[0048] P1: 5'-TATGCTATGTCCCGATTGG-3',

[0049] P2: 5'-ACTGATTTGGCTAGTACACCC-3'.

[0050] ②The preparation of nano-PCRP reaction solution: The reaction solution is composed of MightyAmp enzyme, gold nanoparticle sol (20nm, concentration 0.5μg / μL), 2xBuffer Mix buffer, sterile double distilled water and a pair of specific primers mentioned in ① above.

[0051] (2) Construction of positive control recombinant plasmid

[0052] ①Preparation of bovine respiratory syncytial virus (BRSV) positive control plasmid: use MDBK cells to proliferate bovine respiratory syncytial virus, culture for 40h, extract bovin...

Embodiment 2

[0055] Example 2 Method for using the bovine respiratory syncytial virus nano-PCR detection kit of the present invention

[0056] (1) Extract DNA from the sample to be tested. The sample to be tested includes cell culture, blood, tissue culture, nasal swab and fecal swab.

[0057] (2) Determination of nano-PCR reaction conditions

[0058] After optimization of the gold nanoparticle sol concentration and reaction annealing temperature conditions, it is determined that in the 25μL reaction system, primer P10.5μL, primer P20.5μL, MightyAmp enzyme 0.5μL, 2×buffer12.5μL, template 1μL, gold nanoparticles 0.7μL of sol, deionized water to make up the reaction system to 25μL, BRSV standard positive plasmid as the positive template. The reaction conditions were set as follows: 98℃2min; 98℃10s, annealing temperature range 55℃~62℃, annealing time 2s~10s, 68℃1min, 30 cycles, 68℃ total extension 10min, double distilled water was used as negative control.

[0059] (3) Using the extracted sample rev...

Embodiment 3

[0062] Example 3 Specific experiment of the bovine respiratory syncytial virus nano-PCR detection kit of the present invention

[0063] Using BRSV, IBRV, BVDV and BPI3 positive control recombinant plasmids as templates, add primers P1 and P2 for identification of bovine respiratory syncytial virus (BRSV), and perform specific experiments of the nano-PCR detection kit;

[0064] Experimental results: such as figure 1 As shown, the corresponding negative, 596bp, negative and negative bands appeared with the IBRV, BRSV, BVDV and BPI3 positive control recombinant plasmids as templates, and the negative control did not amplify any bands;

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Abstract

The invention discloses a bovine respiratory syncytial virus nano-PCR detection kit and a preparation method thereof, relates to the field of bovine respiratory syncytial virus detection, and solves the problems that an existing bovine respiratory syncytial virus detection method is low in sensitivity and that serological test has high probability of false positive and high cost. The kit comprises an Mighty Amp enzyme, a 2xBuffer Mix buffer liquid, gold nanoparticle sol, sterile double distilled water, a pair of specific primers and bovine respiratory syncytial virus positive reference plasmid; the pair of specific primers include P1(5'-TATGCTATGTCCCGATTGG-3') and P2(5'-ACTGATTTGGCTAGTACACCC-3'). The sequence of the bovine respiratory syncytial virus positive reference plasmid is as shown in SEQ ID NO.2. The kit is high in sensitivity, simple in operation and low in cost.

Description

Technical field [0001] The invention relates to the technical field of detection of bovine respiratory syncytial virus, in particular to a bovine respiratory syncytial virus nano-PCR detection kit and a preparation method thereof. Background technique [0002] Bovine respiratory syncytial virus (bovineerspiartory syneytialviurs, BRSV) is one of the important pathogens that can cause bovine acute respiratory disease complex (BRDC), which brings huge economic losses to the cattle industry. In large-scale breeding plants, calves at the age of 2-6 months have a high incidence of infection, low mortality, prone to secondary bacterial infections, and the mortality rate can reach more than 20%. The symptoms of re-infection in calves are not obvious after the infection has recovered. The virus can also be isolated from symptomatic cattle. This disease exists in cattle for a long time, and it can cause an outbreak under stress conditions such as sudden temperature drops or long-distance ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/686C12Q1/701C12Q2563/137C12Q2563/155
Inventor 郭利李建友李家伟杨艳玲王超王建科程世鹏
Owner INST OF SPECIAL ANIMAL & PLANT SCI OF CAAS
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