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LAMP detection primer and LAMP detection reagent kit for orf viruses

A canker sore virus and detection kit technology, applied in DNA/RNA fragments, microorganisms, recombinant DNA technology, etc., can solve the problems of inability to distinguish virus infection or vaccination, inconvenient nucleic acid detection methods, and inability to realize on-site detection, etc. Achieve the effect of avoiding the formation of primer dimers, high specificity and high sensitivity

Pending Publication Date: 2019-08-06
陕西诺威利华生物科技有限公司 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] In order to solve the technical problems that the serological detection method of aphthous ulcer virus cannot distinguish between virus infection or vaccination, and the conventional nucleic acid detection method is inconvenient, the dependence on the instrument is relatively strong, and the on-site detection cannot be realized, the present invention aims to provide a LAMP detection primers for oral ulcer virus and its kit, the LAMP detection primers are aimed at the conserved VIR sequence of oral oral ulcer virus, and can specifically detect oral oral ulcer virus; and the detection method of constant temperature reaction is adopted, and the whole reaction can be realized only by heating , got rid of the dependence of traditional nucleic acid detection technology on PCR instruments; the LAMP detection kit can realize rapid on-site detection of aphthous ulcer virus, and the detection results can be directly visually inspected, which is suitable for on-site detection; the method has high detection sensitivity, strong specificity, and repeatability Good performance and fast detection speed, it can be used as an effective primary detection method for aphthus virus

Method used

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  • LAMP detection primer and LAMP detection reagent kit for orf viruses
  • LAMP detection primer and LAMP detection reagent kit for orf viruses
  • LAMP detection primer and LAMP detection reagent kit for orf viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1: Sample, primer design and preparation

[0042] (1) Plasmid and sample source

[0043] According to the VIR gene sequence (GenBank: JN565697.1) provided on Genebank, a partial fragment (shown in SEQ ID No.7) was selected, and the plasmid pUC57-VIR was synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. , dissolved and diluted to 1.0×10 5 copies / μL.

[0044] Oral mouth disease virus (ORFV) nucleic acid, goat pox virus nucleic acid, Peste des Petits Ruminants virus nucleic acid, bluetongue virus nucleic acid, foot-and-mouth disease virus nucleic acid, sheep Escherichia coli nucleic acid, and sheep Salmonella nucleic acid were all provided by the Animal Husbandry and Veterinary Research Institute of the Tibet Autonomous Region Academy of Agriculture and Animal Science.

[0045] Lymph node nucleic acid extracts of healthy sheep were provided by Beijing Combaolihua Biotechnology Co., Ltd., and the healthy sheep were ORFV-negative healthy sheep through se...

Embodiment 2

[0052] Example 2: The establishment of the LAMP detection kit for aphthous ulcer virus

[0053] A kind of LAMP detection kit of aphthous ulcer virus, described kit comprises the primer set described in Table 1, Bst DNA polymerase, LAMP reaction solution, betaine, positive control and negative control.

[0054] The molar ratio of the outer primer, loop primer and inner primer is 1:5:10.

[0055] The LAMP reaction liquid contains 10mM dNTP, 10×ThermoPol reaction buffer, 150mM MgSO 4 aqueous solution.

[0056] The positive control is a plasmid DNA containing the VIR fragment of the target gene, and the sequence of the VIR fragment of the target gene is shown in SEQ ID No.7; the negative control is deionized water.

Embodiment 3

[0057] Embodiment 3: the establishment of the LAMP detection method of sheep oral disease virus

[0058] 3.1 Establishment of ORFV-LAMP reaction system

[0059] 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 the detection of LAMP of oropharynx virus, 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.

[0060] Table 2 25 μL reaction system

[0061]

[0062] Set the concentration to 10 3 The copied positive plasmid was used as the detection object, and the LAMP reaction temperature was determined to be 64° C., and the optimal reaction time was 35 minutes.

[0063] The results showed that the positive plasmid pUC57-VIR LAMP reaction tubes were all white and turbid after reacting at 64°C for 35 minut...

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Abstract

The invention relates to an LAMP detection primer and LAMP detection reagent kit for orf viruses. The LAMP detection primer is a conservative VIR sequence for orf viruses, and can specially detect theorf viruses. An isothermal reaction detection means is adopted, only heating is needed, the entire reaction can be realized, and the relying of a traditional nucleic acid detection technique on a PCRinstrument can be shaken off. The LAMP detection reagent kit can realize quick site detection on the orf viruses, the detection results can be directly detected by eyes, and the LAMP detection reagent kit is suitable for site detection. The method is high in detection sensitivity, high in specificity, good in repeatability and high in detection speed, and can be used as an effective orf virus basal detection means.

Description

[0001] Technical field: [0002] The invention belongs to the field of biotechnology, and in particular relates to a LAMP detection primer and a detection kit for aphthous ulcer virus. [0003] Background technique: [0004] Aphthous aphthous disease is an acute, contact, epithelial infectious disease caused by aphthous aphthus virus. Affected sheep are mainly manifested in severe ulceration and scabs on the lips, which lead to difficulty in feeding and insufficient nutrient intake, which greatly reduces the production performance of adult goats and the growth and development of lambs. Oral aphthous disease is one of the most prevalent viral diseases in the world. It mainly infects small ruminants such as goats and sheep. The survey found that the incidence of aphthous oral disease in lambs is as high as 100%, and the mortality rate caused by secondary infection is also 100%. Up to 15%, so far, high morbidity and mortality have brought huge economic losses to the sheep industr...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6844C12Q2531/119
Inventor 吴玉江四朗玉珍索朗达巴贵次仁德吉德吉张靖飞
Owner 陕西诺威利华生物科技有限公司
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