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Bovine-para-influenza virus 3 nano-PCR (polymerase chain reaction) detection kit and preparation method thereof

A technology of bovine parainfluenza virus and detection kit, which is applied in biochemical equipment and methods, microbiological-based methods, microbial measurement/testing, etc., and can solve the problems of expensive reagents, high false positives in serological testing, and low sensitivity problems, to achieve the effect of reagent cost reduction, cost reduction, and high safety in use

Inactive Publication Date: 2017-06-30
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 reagents imported from abroad in the existing methods for detecting bovine parainfluenza virus type 3 virus, the invention provides a nano-PCR detection reagent for bovine parainfluenza virus type 3 virus Cartridge and its preparation method

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  • Bovine-para-influenza virus 3 nano-PCR (polymerase chain reaction) detection kit and preparation method thereof
  • Bovine-para-influenza virus 3 nano-PCR (polymerase chain reaction) detection kit and preparation method thereof
  • Bovine-para-influenza virus 3 nano-PCR (polymerase chain reaction) detection kit and preparation method thereof

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Embodiment 1

[0044] Embodiment 1 Assembly of bovine parainfluenza virus type 3 virus nano-PCR detection kit of the present invention

[0045] (1) Preparation of nano-PCR reaction solution

[0046] ① Design a pair of specific primers according to the conserved gene sequence of the BPIV3 virus NP group. In order to identify the primers P1 and P2 of bovine parainfluenza virus type 3 (BPIV3), the sequences of the primers are as follows:

[0047] P1: 5'-GCTCTTCTCTTTTTGTCCCATTCTT-3',

[0048] P2: 5'-AACCCCTTCCTCAATCCTGATATAC-3'.

[0049] 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.

[0050] (2) Construction of positive control recombinant plasmid

[0051] ①Preparation of bovine parainfluenza virus type 3 virus (BPIV3) positive control plasmid: use MDBK cells to proliferate bovine ...

Embodiment 2

[0054] Embodiment 2 The using method of bovine parainfluenza virus type 3 virus nano-PCR detection kit of the present invention

[0055] (1) Extract sample DNA from the sample to be tested. The sample to be tested includes cell culture, blood, tissue culture, nasal swab and fecal swab, etc.

[0056] (2) Determination of nano-PCR reaction conditions

[0057] After optimizing the reaction concentration and annealing temperature of the gold nanoparticle sol, it was determined that in the 25 μL reaction system, the primer P1 was 0.5 μL, the primer P2 was 0.5 μL, the MightyAmp enzyme was 0.5 μL, the 2×buffer was 12.5 μL, and the positive template 0.5 μL of gold nanoparticle sol, make up the reaction system to 25 μL with deionized water, and 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 55°C to 62°C, annealing time of 2s to 10 s, 68°C for 1 min, 30 cycles, total ext...

Embodiment 3

[0061] Embodiment 3 The specificity experiment of bovine parainfluenza virus type 3 virus nano-PCR detection kit of the present invention

[0062] IBRV, BPIV3, BRSV and BVDV positive control recombinant plasmids and negative control (distilled water) were used as templates respectively, and primers P1 and P2 for identifying bovine parainfluenza virus type 3 virus (BPIV3) were added to carry out the specificity of the nano-PCR detection kit experiment.

[0063] Experimental results: if figure 1 As shown, BPIV3, IBRV, BVDV and BRSV positive control recombinant plasmids were used as templates to appear corresponding 422bp, negative, negative and negative bands respectively, and no band was amplified in the negative control.

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Abstract

The invention discloses a bovine-para-influenza virus 3 nano-PCR (polymerase chain reaction) detection kit and a preparation method thereof, relates to the field of bovine para-influenza virus detection, and solves the problems that an existing bovine-para-influenza virus 3 detection method is low in sensitivity, serological detection is high in false positive and foreign importing reagents are expensive. The kit comprises a MightyAmp enzyme, 2xBuffer Mix solution, gold nanoparticle sol, aseptic double-distilled water, a pair of specific primers P1 (5'-GCTCTTCTCTTTTTGTCCCATTCTT-3') and P2 (5'-AACCCCTTCCTCAATCCTGATATAC-3') and a bovine-para-influenza virus 3 positive control plasmid. The sequence of the bovine-para-influenza virus 3 positive control plasmid is as shown in SEQ ID NO: 2. The kit is high in sensitivity, simple, convenient, rapid, efficient and low in cost.

Description

technical field [0001] The invention relates to the technical field of bovine parainfluenza virus detection, in particular to a bovine parainfluenza virus type 3 virus nano-PCR detection kit and a preparation method thereof. Background technique [0002] Bovine parainfluenza virus (Bovine parainfluenza type-3 virus, BPIV3) is one of the important pathogens that can cause bovine acute respiratory disease complex (BRDC). Member of the subfamily Paramyxoviridae and the genus Respirovirus. The virus was isolated from cattle for the first time in the United States in 1959, and now it exists widely in various provinces in my country, causing huge economic losses to the cattle industry. Calves aged 2 to 6 months in large-scale breeding plants have a high incidence of infection, low mortality, and are prone to secondary bacterial infections, with a mortality rate of more than 20%. The virus has also been isolated from symptomatic cattle. Bovine parainfluenza disease is a common d...

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 郭利王超任静强孙娜朱红伟易立程世鹏
Owner INST OF SPECIAL ANIMAL & PLANT SCI OF CAAS
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