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LAMP visual detection kit for orf virus

A kind of technology of oral ulcer virus and detection kit, applied in the biological field, can solve the problems of inability to distinguish virus infection or vaccination, inconvenient nucleic acid detection means, inability to realize on-site detection, etc., to avoid the formation of primer dimers, specific High performance and high sensitivity

Pending Publication Date: 2020-11-27
陕西诺威利华生物科技有限公司 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] 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 instruments is relatively strong, and on-site detection cannot be realized, the present invention aims to provide a Mouth virus LAMP visual detection kit, in which the LAMP detection primers are aimed at the conserved VIR sequence of the aphthous aphthous virus, which can specifically detect the aphthous aphthous virus; and the constant temperature reaction detection method is adopted, and the whole reaction can be realized only by heating, eliminating It eliminates the dependence of traditional nucleic acid detection technology on PCR instruments; the LAMP detection kit can realize rapid on-site detection of aphthus 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 good repeatability , The detection speed is fast, and it can be used as an effective primary detection method for aphthus virus

Method used

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  • LAMP visual detection kit for orf virus
  • LAMP visual detection kit for orf virus
  • LAMP visual detection kit for orf virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1: Sample, primer design and preparation

[0034] (1) Plasmid and sample source

[0035] 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.

[0036] 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.

[0037] 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

[0044] Example 2: The establishment of the LAMP detection kit for aphthous ulcer virus

[0045] A kind of oral ulcer virus LAMP visual detection kit, described kit is made up of HNB-LAMP Mix, Primer Mix, 5MBetain, Bst DNA Polymerase, deionized water, negative control and positive control, and described Primer Mix comprises SEQ in table 1 Primer set of ID No. 1 - SEQ ID No. 6.

[0046] The HNB-LAMP Mix, 5M Betain, and Bst DNA Polymerase are all 2×HNB LAMP Mix, 5M Betain, and Bst2.0 DNA Polymerase in the LAMP-HNB Color Change Amplification Kit purchased from Haiji Biotechnology Co., Ltd., article number: A3802.

[0047] The molar ratio of the outer primer, loop primer and inner primer is 1:5:10.

[0048] 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

[0049] Embodiment 3: the establishment of the LAMP detection method of sheep oral disease virus

[0050] 3.1 Establishment of ORFV-LAMP reaction system

[0051] 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. Observe the color change with the naked eye to judge the test result. The 25 μL reaction system is shown in Table 2.

[0052] Table 2 25μl reaction system

[0053] name Dosage HNB LAMP Mix 12.5 μl Primer 7.5 μl 5M Betain 2 μl Template DNA 2 μl Bst DNA Polymerase 1 μl Total 25 μl

[0054] 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.

[0055]...

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Abstract

The invention relates to a LAMP visual detection kit for an orf virus. A detection primer in the kit can specifically detect the orf virus aiming at a conservative VIR sequence of the orf virus; a detection means of constant-temperature reaction is adopted, so that the whole reaction can be realized only by heating, and the dependence of a traditional nucleic acid detection technology on a PCR instrument is eliminated; the LAMP detection kit can realize rapid on-site detection of the orf virus, and a detection result can be directly observed by visual inspection, so that the LAMP detection kitis suitable for on-site detection; and the method is high in detection sensitivity, high in specificity, good in repeatability and high in detection speed, and can be used as the effective basic detection means for the orf virus.

Description

Technical field: [0001] The invention belongs to the field of biotechnology, and in particular relates to a LAMP visual detection kit for aphthous ulcer virus. Background technique: [0002] 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 industry. However, due to the unique ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2600/166C12Q2531/119C12Q2545/113
Inventor 吴玉江索朗达四朗玉珍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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