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LAMP (Loop-Mediated Isothermal Amplification) primer group, kit and detection method for simultaneously detecting multiple capripoxvirus

A sheeppox virus and primer set technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of expensive instruments, difficult popularization and application, complicated operation, etc., and achieve simple operation and low cost Low and timely control of the epidemic

Active Publication Date: 2021-09-10
北京市动物疫病预防控制中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the most commonly used detection method in my country is fluorescent quantitative PCR. This detection method has good sensitivity and specificity, but the operation is complicated and the equipment is expensive, so it is difficult to popularize and apply it in the actual production at the grassroots level.

Method used

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  • LAMP (Loop-Mediated Isothermal Amplification) primer group, kit and detection method for simultaneously detecting multiple capripoxvirus
  • LAMP (Loop-Mediated Isothermal Amplification) primer group, kit and detection method for simultaneously detecting multiple capripoxvirus
  • LAMP (Loop-Mediated Isothermal Amplification) primer group, kit and detection method for simultaneously detecting multiple capripoxvirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] 1. Design the primer set shown in Table 1 and entrust Sangon Bioengineering (Shanghai) Co., Ltd. to synthesize it.

[0047] 2. Screening and verification of primers

[0048] (1) Virus strains and samples

[0049] Positive samples of goat pox virus (inactivated), positive samples of sheep pox virus (inactivated), positive samples of bovine nodular skin disease virus (inactivated) and 4 other pathogens that are similar in species to or cause similar symptoms to sheep pox virus : Peste des petits ruminants virus, sheep aphthous disease vaccine, vesicular stomatitis vaccine, and foot-and-mouth disease virus are all kept by the laboratory of Beijing Animal Disease Prevention and Control Center.

[0050] (2) Use the designed primer set A, primer set B, and primer set C to perform LAMP amplification on a fluorescent PCR instrument. The amplification reaction system (25 μL) is: 22 μL of fluorescent reaction solution, 1 μL of enzyme reaction solution, sample DNA 2 μL; the ampl...

Embodiment 2

[0055] Optimization of the annealing temperature of the LAMP method for the detection of goat pox virus, sheep pox virus and bovine nodular skin disease virus on an isothermal fluorescent PCR amplifier

[0056] 1. Select the primer combination as: primer set A in Table 1 of Example 1.

[0057] 2. The LAMP amplification reaction system is as in Example 1, that is, 22 μL of fluorescence reaction solution, 1 μL of enzyme reaction solution, and 2 μL of sample DNA.

[0058] 3. Carry out amplification on the loop-mediated isothermal amplification instrument, and set four temperatures of 58°C, 60°C, 63°C and 65°C.

[0059] 4. Analysis results: The results showed (as shown in Table 2) that the amplification effect was the best at 60°C, and 60°C was selected as the amplification temperature of the primer set A of the present invention.

[0060] Table 2 Amplification results at different reaction temperatures

[0061] 58℃ 60℃ 63℃ 65℃ TT value 06:55 06:26 09:17 2...

Embodiment 3

[0063] Detection of goat pox virus, sheep pox virus and bovine nodular skin disease virus using the prepared LAMP fluorescence kit

[0064] 1. Assembly of goat pox virus, sheep pox virus, bovine nodular skin disease virus LAMP fluorescence detection kit

[0065] Pack the following reagents in suitable outer packaging boxes, label them, and indicate the name, batch number, production date, expiration date, etc.

[0066] Specifications and quantity (48T / box).

[0067] Fluorescence reaction solution 1056 µL / tube; enzyme reaction solution 48 µL / tube; positive control 100 µL / tube; negative control 100 µL / tube.

[0068] The positive quality control standard used the recombinant plasmid puc57-ORF068 (genome sequence shown in SEQ ID NO.17) as the positive template, diluted with Tris-EDTA buffer (0.01 M pH8.0) and stored frozen. Use ddH 2 O is a negative control.

[0069] The preparation method of the above fluorescent reaction solution is: take 10×Thermopol buffer 120μL, 50mmol / L ...

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Abstract

The invention provides an LAMP (loop-mediated isothermal amplification) primer group, a kit and a detection method for simultaneously detecting multiple capripoxvirus, and relates to the technical field of biology. According to the LAMP primer group, the LAMP kit and the developed corresponding virus detection method, rapid identification of goatpox viruses, sheeppox viruses and lumpy skin disease viruses is directly achieved through fluorescence information, the LAMP primer group, the LAMP kit and the virus detection method have the advantages of being convenient, accurate, sensitive, specific, easy and convenient to operate and the like, the lowest detection limit is 6 copies, and the detection result is higher than that of real-time fluorescence quantitative PCR; and the primer group, the kit and the virus detection method are suitable for on-site rapid diagnosis of epidemic diseases and timely control of epidemic situations when the epidemic situations pre-outbreak.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a LAMP primer set, a kit and a detection method for simultaneously detecting several kinds of capipoxviruses. Background technique [0002] Goatpox virus (GTPV), sheep pox virus (SPPV) and bovine nodular skin disease virus (Lumpy skin disease Virus, LSDV) belong to Capripoxvirus (CPV), which can cause goat pox virus, respectively. , sheep and cattle skin, extensive nodules and edema on the surface of the organs, the milk production of sick animals is sharply reduced, and the quality of fur is greatly reduced, causing huge economic losses. [0003] Capripoxvirus (CPV) belongs to the genus capripoxvirus in the vertebrate poxvirus subfamily (chordopoxrinae) of the poxviridae family (poxviridae) in classification. Goatpox virus is a brick-shaped virus that replicates in the cytoplasm. Its genome is double-stranded DNA with a size of about 290nm✕270nm. Composed of the outer ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2531/119
Inventor 王林程汝佳韦海涛程敏姮张启龙栗云鹏刘海莹宋彦军周德刚赵浩军
Owner 北京市动物疫病预防控制中心
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