Bovine viral diarrhea virus nano-PCR detection kit and preparation method thereof

The technology of a bovine viral diarrhea and detection kit is applied in the detection field of bovine viral diarrhea virus, which can solve the problems of time-consuming and laborious, cross-infection, easy misdiagnosis, etc., and achieves the avoidance of cross-infection, high use safety, and high specificity Sex and Sensitivity 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

[0004] In order to solve the problems of cross-infection, easy misdiagnosis, and time-consuming and labor-intensive existing detection methods in BVD infection, the present invention provides a bovine viral diarrhea virus nano-PCR detection kit and its preparation method

Method used

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  • Bovine viral diarrhea virus nano-PCR detection kit and preparation method thereof
  • Bovine viral diarrhea virus nano-PCR detection kit and preparation method thereof
  • Bovine viral diarrhea virus nano-PCR detection kit and preparation method thereof

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Experimental program
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Effect test

Embodiment 1

[0045] Embodiment 1 Assembly of bovine viral diarrhea virus nano-PCR detection kit of the present invention

[0046] (1) Preparation of PCR reaction solution

[0047] ①A pair of specific primers were designed according to the conserved gene sequence of the BVDV virus 5'-UTR group. In order to identify the primers P1 and P2 of bovine viral diarrhea virus (BVDV), the sequences of the primers are as follows:

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

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

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

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

[0052] ①Preparation of bovine viral diarrhea virus (BVDV) positive control plasmid: use MDBK cells to proliferate bovine viral diarrhea virus, and after cult...

Embodiment 2

[0055] Embodiment 2 The use method of bovine viral diarrhea virus nano-PCR detection kit of the present invention

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

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

[0058] After optimizing the conditions of reaction concentration and annealing temperature, it was determined that in a 25 μL reaction system, 10.5 μL of primer P, 20.5 μL of primer P, 0.5 μL of MightyAmp enzyme, 12.5 μL of 2×buffer, 1 μL of template, 0.7 μL of gold nanoparticle sol, Make up the reaction system to 25 μL with deionized water, and use the BRSV standard positive plasmid as the positive template. The reaction conditions were set as follows: 98°C for 2 min; 98°C for 10 s, annealing temperature range of 52°C to 62°C, annealing time of 2s to 10 s, 68°C for 1 min, 30 cycles, 68°C total extension for 10 min, and double distilled water ...

Embodiment 3

[0062] Embodiment 3 The specificity experiment of bovine viral diarrhea virus nano-PCR detection kit of the present invention

[0063] Using IBRV, BVDV, BRSV and BPI3 positive control recombinant plasmids and negative control (distilled water) as templates respectively, add primers P1 and P2 for identifying bovine viral diarrhea virus (BVDV), and carry out the specificity experiment of the nano-PCR detection kit;

[0064] Experimental results: if figure 1 As shown, the corresponding negative, 136bp, negative and negative bands appeared when IBRV, BVDV, BRSV and BPI3 positive control recombinant plasmids were used as templates, and no band was amplified in the negative control.

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Abstract

The invention discloses a bovine viral diarrhea virus nano-PCR detection kit and a preparation method thereof, relates to the field of bovine viral diarrhea virus detection, and solves the problems that BVD infection frequently has cross infection and is easily misdiagnosed, and that existing detection methods waste much time and labor. 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 viral diarrhea virus positive reference plasmid; the pair of specific primers include P1(5'-GGTAGCAACAGTGGTGAGTTC-3') and P2(5'-CTCAGGTTAAGATGTGCTGTG-3'). The bovine viral diarrhea virus positive reference plasmid is pMD18-T recombinant plasmid formed by a nucleotide fragment containing 130 basic groups, and the sequence of the 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 bovine viral diarrhea virus detection, in particular to a bovine viral diarrhea virus nano-PCR detection kit and a preparation method thereof. Background technique [0002] Bovine viral diarrhea virus (BVDV) is an infectious disease caused by BVDV virus and is one of the important pathogens of bovine acute respiratory disease complex (BRDC). Cattle of all ages are susceptible to infection , The susceptibility of young cattle is the highest, and the source of infection is mainly sick animals. my country's cattle industry is in a period of rapid development, and the systemic diseases of cattle are increasing day by day, which is seriously harmful to the cattle industry. Bovine viral diarrhea (BVD) is one of the primary infectious diseases in cattle farms. It seriously affects the growth and development of cattle and can cause reproductive disorders in cows. BVD infection can also delay the first estrus tim...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/686C12Q1/701C12Q2545/113C12Q2563/137C12Q2563/157
Inventor 郭利程世鹏杨艳玲张淑琴
Owner INST OF SPECIAL ANIMAL & PLANT SCI OF CAAS
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