RT-LAMP detection kit and detection method of swine influenza virus

A technology of RT-LAMP and swine flu virus, applied in the field of veterinary biological products

Inactive Publication Date: 2010-01-20
CHINA INST OF VETERINARY DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no reports on the application of RT-LAMP kit and RT-LAMP method in the detection of swine influenza virus at home and abroad.

Method used

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  • RT-LAMP detection kit and detection method of swine influenza virus
  • RT-LAMP detection kit and detection method of swine influenza virus
  • RT-LAMP detection kit and detection method of swine influenza virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] SIV RT-LAMP primer design and screening

[0090] According to GenBank, SIV includes 54 subtypes of HA and NA sequences, and DNAMAN5.9 and DNAstar5.0 are used for sequence analysis. In the comparison of 54 SIV strains, it was found that there were few mutations between 550nt and 780nt in the PA sequence, and the length was suitable for the requirements of RT-LAMP experiments. Primers were designed with reference to the H1N1 SIV sequence. The designed primers were compared with the sequences of 54 strains of SIV again, and the sequences that were different in each subtype of SIV near the 5' end and 3' of the primers were degenerately processed ( figure 1 ), set up the reaction system of SIV RT-LAMP according to the instructions of the RNA LAMP kit and test and screen each set of primers. Through the analysis of the results of the test screening, it is determined that the following set of primers is the working primer of this method, and the sequence is as follows:

[00...

Embodiment 2

[0100] Preparation of components in the kit:

[0101] According to the formula (50 reaction volumes) of the following components, the various components are formulated and distributed into small glass or plastic containers and sealed with corresponding stoppers:

[0102] 1. LBB II-RNA, this reagent group consists of four parts: RA, RB, RC, and RD:

[0103] RA: Dissolve 0.6ml of 1% 2-mercaptoethanol, 0.6ml of pH 8.01% Tris-HCL, and 1ml of 10mmol / ml EDTA in 5.0ml of sterilized double-distilled water.

[0104] RB: 35ml of 6mmol / ml guanidine isothiocyanate.

[0105] RC: absolute ethanol 15ml.

[0106] RD: 100u / μl DNAse A 20μl dissolved in 10ml DEPC H 2 O,

[0107] 2. RT-LAMP Reagent

[0108] 1) PM: Take 100pmol / μl F3-PA 2.5μl, 100pmol / μl B3-PA 2.5μl, 100pmol / μl FIP-PA 20.0μl, 100pmol / μl BIP-PA 20.0μl, 100pmol / μl LF-PA 10.0μl, 100pmol / μl LB-PA10.0μl, add sterilized deionized water to make the total system 75.0μl. Take 1.5 μl PM for each reaction.

[0109] 2) RM: Take 50 μl ...

Embodiment 3

[0113] Use of SIV RT-LAMP Kit

[0114] 1. Use LBBII-RNA to extract sample RNA

[0115] Take 100 μl of cell culture (or swab, tissue grinding sample), after repeated freezing and thawing 3 times, follow the steps below:

[0116] Add an equal volume of RA to the sample, vortex for 15s, centrifuge at 12,000g for 1min, take the supernatant and put it in a small tube; add 3 times the RB solution to the supernatant, vortex or vigorously shake for 90s, and let it stand for 3min; add 1 / 3 volumes of RC solution, vortexed for 1 min, and centrifuged at 12000 g for 1 min. Discard the supernatant; after the sample is dry, add 11 μl RD to dissolve the precipitate, which is the sample RNA.

[0117] 2. SIV RT-LAMP reaction

[0118] ① Prepare RT-LAMP reaction solution: add 12.5 μl of RM, 1.0 μl of EM, and 1.5 μl of PM into the reaction tube;

[0119] ② Take 5.0 μl sample RNA and add it to the tube of the reaction solution prepared in item ①;

[0120] ③ After mixing, place the reaction tu...

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PUM

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Abstract

The invention relates to an RT-LAMP detection kit and a detection method of swine influenza virus (SIV). According to different subtypes of SIV gene sequences published by a GenBank, 6 SIV RT-LAMP degenerate primers are designed aiming at a relative conserved domain of a PA sequence, and an RT-LAMP method for detecting all the subtype SIV is established by taking H1, H3, H5 and H9 subtype strains of the swine influenza as templates. An LA-320 LAMP Tubidimeter is employed to analyze the reaction process and judge the results; the reaction at a temperature of 63 DEG C for 45min is displayed in the LA-320 LAMP Tubidimeter; RNAs of four subtypes of SIV samples used in all the tests are all subjected to high efficient specific amplification. Sensitivity tests prove that the method can still effectively amplify the RNAs of SIV samples after carrying out 10<-4> dilution on the RNAs, thus showing high sensitivity of the method; through specific tests and RT-LAMP detection of SIV pathogeny of 19 clinical samples, SIV nucleic acid in 5 samples is detected to be positive, which is identical with the results of RT-PCR and isolation of chicken eggs; typing and identification of gene chips verify that the accordance rate of the comprehensive result is 100%. The method is specific, sensitive and quick and is suitable for SIV detection under various test conditions.

Description

technical field [0001] The invention relates to a swine influenza virus RT-LAMP detection kit and a detection method thereof, belonging to the disease diagnosis technology in the field of veterinary biological products. Background technique [0002] Swine influenza virus (SIV) belongs to Orthomyxoviridae, which can cause an acute and highly contagious respiratory infectious disease in pigs—swine influenza. Pigs play the role of intermediate hosts and multiple hosts in the process of interspecies transmission of avian-pig-human influenza virus crossing species barriers and infecting new hosts. Since porcine epithelial cells have sialic acid α-2,3-galactoside (SAα-2,3-Gal) and sialic acid α-2,6-galactoside (SAα-2,6-Gal), human influenza virus can interact with The former binds, while avian influenza virus binds to the latter. Therefore, porcine epithelial cells can be infected by human influenza virus and avian influenza virus, and become live carriers of gene rearrangement ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/78C12R1/93
Inventor 刘业兵张磊宁宜宝陈蕾
Owner CHINA INST OF VETERINARY DRUG CONTROL
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