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CDA primer group and kit for detecting African swine fever virus and application of CDA primer group and kit

A technology of African swine fever virus and kit, which is applied in the field of CDA primer set for detection of African swine fever virus, can solve the problems of difficult detection of ASFV carriers, epidemic spread, high false detection rate, etc., achieve shortened detection time, simple operation, The effect of fast and accurate detection

Active Publication Date: 2021-09-21
国科宁波生命与健康产业研究院 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the test strips made by immunoassay technology have the advantage of being simple and quick, but this method has a high false detection rate and low sensitivity, making it difficult to detect ASFV carriers in the incubation period, which may cause the spread of the epidemic

Method used

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  • CDA primer group and kit for detecting African swine fever virus and application of CDA primer group and kit
  • CDA primer group and kit for detecting African swine fever virus and application of CDA primer group and kit
  • CDA primer group and kit for detecting African swine fever virus and application of CDA primer group and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1 uses Eva Green to verify respectively the amplification reaction of four pairs of primer sets to the African swine fever virus gene DNA fragment

[0046] Similar to SYBR Green I, Eva Green is a dye with a green excitation wavelength that binds to the minor groove region of all dsDNA double helices, and its inhibition on nucleic acid amplification reactions such as PCR is much smaller than that of the latter. The intensity of the fluorescent signal of Eva Green correlates with the amount of double-stranded DNA. In the free state, Eva Green emits weak fluorescence, but when combined with double-stranded DNA, its fluorescence is greatly enhanced. Therefore, the amount of double-stranded DNA present in the nucleic acid amplification system can be detected based on the fluorescent signal.

[0047] The reaction solution combination is as follows (all the other add ddH 2 0 to 25 μL):

[0048] 20mM Tris-HCl pH8.8

[0049] 10mM KCl

[0050] 10mM (NH 4 ) 2 SO ...

Embodiment 2

[0069] Embodiment 2 uses Eva Green to verify that four pairs of primer sets are to the amplification reaction of African swine fever virus gene DNA fragment (negative and positive each setting 8 groups of repeated experiments)

[0070] Method is with embodiment 1.

[0071] The reaction solution combination is as follows (all the other add ddH 2 0 to 200 μL):

[0072] 160mM Tris-HCl pH8.8

[0073] 80mM KCl

[0074] 80mM (NH 4 ) 2 SO 4

[0075] 112mM MgSO 4

[0076] 0.8% Triton X-100

[0077] 8M betaine

[0078] 10mM dNTPs

[0079] 64U Bst DNA polymerase (NEW ENGLAND Biolabs)

[0080] 8X Eva Green (Biotum)

[0081] Primers:

[0082] 12800nM ASFV-MF-1 (shown in SEQ ID NO.1)

[0083] 12800nM ASFV-MR-1 (shown in SEQ ID NO.2);

[0084] 12800nM ASFV-MF-2 (shown in SEQ ID NO.3)

[0085] 12800nM ASFV-MR-2 (shown in SEQ ID NO.4);

[0086] 12800nM ASFV-MF-3 (shown in SEQ ID NO.5)

[0087] 12800nM ASFV-MR-3 (shown in SEQ ID NO.6);

[0088] 12800nM ASFV-MF-4 (shown in S...

Embodiment 3

[0093] Embodiment 3 application Eva Green verification primer group 3 is to the amplification reaction of African swine fever virus gene DNA fragment (repeat experiment)

[0094] Method is with embodiment 1.

[0095] The reaction solution combination is as follows (all the other add ddH 2 0 to 1000 μL):

[0096] 800mM Tris-HCl pH8.8

[0097] 400mM KCl

[0098] 400mM (NH 4 ) 2 SO 4

[0099] 560mM MgSO 4

[0100] 4.0% Triton X-100

[0101] 40M betaine

[0102] 50mM dNTPs

[0103] 320U Bst DNA polymerase (NEW ENGLAND Biolabs)

[0104] 40X Eva Green (Biotum)

[0105] Primers:

[0106] 64000nM ASFV-MF-3 (shown in SEQ ID NO.5)

[0107] 64000nM ASFV-MR-3 (shown in SEQ ID NO.6);

[0108] No target.

[0109] At the same time, the primer group 3 is provided with a target control group, and the reaction solution group is the same as in Example 2. Target: African swine fever virus genomic DNA dsDNA (SEQ ID NO.9).

[0110] Set the SLAN 96 real time PCR reaction temperatu...

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Abstract

The invention discloses a CDA primer group and a kit for detecting African swine fever virus (ASFV), and an application of the CDA primer group and the kit. The CDA primer group is a primer group for amplifying a conserved region fragment of the African swine fever virus, namely ASFV-MF-3 / ASFV-MR-3, wherein the nucleotide sequence of the ASFV-MF-3 is as shown in SEQ ID NO. 5, and the nucleotide sequence of the ASFV-MR-3 is as shown in SEQ ID NO. 6. The primer group provided by the invention is high in sensitivity and strong in specificity, and the kit prepared from the primer group can quickly and accurately detect whether a sample to be detected contains the African swine fever virus or not. In addition, the visual kit provided by the invention can provide great convenience for on-site detection in places such as farms, customs, ports and community vegetable markets with low professional degrees.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a CDA primer set, a kit and an application thereof for detecting African swine fever virus. Background technique [0002] African swine fever virus (ASFV) can cause a febrile, acute, highly contagious disease - African swine fever (ASF). The disease spreads quickly and has a high fatality rate. It is extremely harmful to the pig industry and is the most serious disease. The clinical manifestations are high fever, cyanosis of the skin, hemorrhage of various organs, and respiratory disturbance. It can be transmitted by insect vectors and body fluids, and remains active for a long time. In addition, virus-contaminated feed, water sources, utensils, and even farm staff and clothing, as well as polluted air near the farm are potential sources of infection. On August 3, 2018, the African swine fever epidemic occurred in my country for the first time, and the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2531/119C12Q2537/1376C12Q2563/107
Inventor 毛瑞赵月蔡挺
Owner 国科宁波生命与健康产业研究院
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