African swine fever virus ASFV-LAMP detection primer set and reagent kit

An African swine fever virus, detection kit technology, applied in DNA/RNA fragments, microorganisms, recombinant DNA technology, etc., can solve the problems of strong instrument dependence, low efficiency, inability to achieve on-site detection, etc. Simple, avoids the effect of primer-dimer formation

Pending Publication Date: 2019-08-06
陕西诺威利华生物科技有限公司
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  • Abstract
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Problems solved by technology

[0009] In order to solve the technical problems of low efficiency of conventional detection methods for African swine fever detection, strong dependence on instruments, and inability to realize on-site detection, the present invention aims to provide a primer set, kit and detection method for detection of African swine fever virus LAMP , the LAMP detection method can realize the rapid detection of African swine f

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  • African swine fever virus ASFV-LAMP detection primer set and reagent kit
  • African swine fever virus ASFV-LAMP detection primer set and reagent kit
  • African swine fever virus ASFV-LAMP detection primer set and reagent kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1: Sample, primer design and preparation

[0053] 1.1 Plasmid, sample source and experimental site

[0054] According to the African swine fever virus P72 protein gene (shown in SEQ ID No.7) provided on Genebank, the plasmid pUC57-P72 was synthesized by Sangon Bioengineering (Shanghai) Co., Ltd., dissolved and diluted to 1.0×10 7 copies / μL.

[0055] Porcine Japanese encephalitis virus, classical swine fever virus, porcine parvovirus, porcine pseudorabies virus, porcine circovirus, and porcine transmissible gastroenteritis virus were provided by Shaanxi Nuowei Lihua Biotechnology Co., Ltd.

[0056] SPF pig lymph nodes and SPF pig serum were collected from Shaanxi Nuowei Lihua Biotechnology Co., Ltd.

[0057] 1.2 Identification of positive plasmids

[0058] The SalI-XbaI site of the positive plasmid pUC57-P72 was digested to verify the correctness of the plasmid. The SalI-XbaI site of the positive plasmid pUC57-P72 was digested and identified. The results s...

Embodiment 2

[0063] Embodiment 2: the establishment of African swine fever virus LAMP detection kit

[0064] A detection kit for African swine fever virus LAMP, said kit comprising the above-mentioned primer set.

[0065] Preferably, the kit includes the primer set described in Example 1 above, Bst DNA polymerase, LAMP reaction solution, betaine, positive control and negative control.

[0066] Preferably, the molar ratio of outer primer, loop primer and inner primer is 1:(4-6):(8-12).

[0067] Preferably, the LAMP reaction solution contains 10mM dNTP, 10×ThermoPol reaction buffer, 150mM MgSO 4 aqueous solution.

[0068] Preferably, the positive control is plasmid DNA containing the target gene fragment, and the negative control is sterile water, preferably double-distilled water, triple-distilled water or DEPC water.

[0069] Preferably, the positive control is plasmid DNA containing p72 gene.

[0070] Preferably, the sequence of the p72 gene is shown in SEQ ID No.7.

[0071] 【Precaut...

Embodiment 3

[0075] Embodiment 3: the establishment of African swine fever LAMP detection method

[0076] 3.1 Establishment of LAMP reaction system

[0077] According to the kit in Example 2, the above primers were used to determine the content and proportion of each component in the 25 μl reaction system for African swine fever virus LAMP detection, which was placed in a constant temperature container for amplification. The test result can be judged by combining the white turbidity with naked eyes and gel electrophoresis. The 25 μL reaction system is shown in Table 2.

[0078] Table 2 25μL reaction system

[0079]

[0080] 3.2 LAMP reaction

[0081] 3.2.1 Determination of LAMP reaction time

[0082] According to the LAMP reaction system determined above, assuming that the positive plasmid pUC57-P72 was specifically amplified at 65°C for 20 min, 30 min, 40 min, 50 min, and 60 min, record the results and determine the optimal reaction time.

[0083] respectively set the concentratio...

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Abstract

The invention relates to an African swine fever virus ASFV-LAMP detection primer set, reagent kit and detection method. In order to cope with the epidemic situation of African swine fever in Chin at 2018, an African swine fever virus P72 gene is used as an LAMP detection primer set. The primer set specially detects the African swine fever virus gene P72, ASFV can be effectively detected, a new technique means is provided for prevention and control of the African swine fever, and toxin strain detection and entry and exit quick screening are facilitated. The LAMP detection method can realize quick detection of the African swine fever viruses, detection results can be directly tested visually, and the entire reaction can be realized only needing heating. The relying of a traditional nucleic acid detection technique on a PCR instrument can be shaken off. The method is high in detection sensitivity, high in specificity, good in repeatability and high in detection speed, and can be used as an effectively on-field detection means for African swine fever.

Description

[0001] Technical field: [0002] The invention belongs to the field of biotechnology, and in particular relates to a detection primer set, kit and detection method for African swine fever virus LAMP. [0003] Background technique: [0004] African swine fever (ASF) is an acute, febrile, highly contagious infectious disease caused by African swine fever virus (ASFV), with a short onset time and high fatality rate. ASF is the most serious disease to the pig industry. It is listed as a statutory reportable disease by the World Organization for Animal Health (OIE) and a class I infectious disease in my country. ASFV belongs to the African swine fever virus genus of the African swine fever virus family of the double-stranded DNA virus order. There is currently only one ASFV virus species in this genus. The genome of ASFV is a single-molecule linear DNA with a length of about 170-190 kb. ASFV is relatively large, with a diameter of up to 200nm. The surface has an icosahedral struc...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6844C12Q2531/119
Inventor 陈瑞张磊张志刚董剑辉张满义潘玉洪金郭晶莹
Owner 陕西诺威利华生物科技有限公司
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