Loop-mediated isothermal amplification detection kit of influenza A3 viruses and detecting method

A ring-mediated isotherm and detection kit technology, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of high price of fluorescent quantitative PCR instruments and restrictions on popularization and use, and achieve flexible and simple judgment methods, Effect of reducing non-specific amplification product contamination and improving sensitivity

Inactive Publication Date: 2009-03-04
ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the high price of fluorescent quantitative PCR instruments, the promotion and use of this technology at the grassroots level is greatly restricted.

Method used

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  • Loop-mediated isothermal amplification detection kit of influenza A3 viruses and detecting method
  • Loop-mediated isothermal amplification detection kit of influenza A3 viruses and detecting method
  • Loop-mediated isothermal amplification detection kit of influenza A3 viruses and detecting method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment 1: LAMP method detects A type 3 influenza virus genome RNA

[0063] Influenza A3 / Shanghai / 1 / 98 strain was selected as influenza A3 virus, which was obtained from the National Influenza Center. Observation of cell infection by influenza virus and calculation of half cell infection dose (TCID) 50 ) method to calibrate the concentration, and obtain different concentrations of virus liquid (10 3 、10 2 、10 1 、10 0 、10 -1 、10 -2 TCID 50 ), carry out RNA extraction according to the following steps for each concentration of virus liquid:

[0064] 1. Genomic RNA extraction of type A influenza virus:

[0065] 1) Add 500 μL RLT and 6 μL β-mercaptoethanol to the centrifuge tube;

[0066] 2) Add 200 μL virus liquid to the above solution, shake and mix for 1 min;

[0067] 3) Add an equal volume of 70% (v / v) ethanol and mix;

[0068] 4) Add 700 μL of the mixed solution to the spin column in the collection tube, and centrifuge at 12000 rpm for 15 s;

[0069] 5) R...

Embodiment 2

[0080] Embodiment 2: Real-time fluorescent LAMP method detects influenza virus genome RNA of type A 3

[0081] The RNA obtained by extracting the virus liquid with different concentrations in Example 1 was subjected to the LAMP reaction according to the following method steps:

[0082] Composition of LAMP reaction system (primers were synthesized by Shanghai Yingjun Biotechnology Co., Ltd.):

[0083] F3, 5pmol; B3, 5pmol; BIP, 40pmol; FIP, 40pmol; LB, 20pmol; dNTP, 1.4mM; 4 , 6mM; AMV reverse transcriptase (Promega), 10U; Bst-DNApolymerase (NEB), 8U; 10×Bst-DNA polymerase Buffer, 2.5μL; 7.5μL of viral RNA obtained in Example 1; SYBR Green I 1μL, DEPC treatment Make up to 25 μL with water and mix well.

[0084] LAMP reaction conditions: MJ Opticon2 instrument detects fluorescence in real time, the reaction condition is 63°C for 45s, and the plate is read; a total of 40 cycles are performed.

[0085] figure 2 Middle: A, B, C, D, E indicate that the reaction contains 10 2 、...

Embodiment 3

[0087] Embodiment 3: detection of LAMP reaction of test sample (patient's gargle)

[0088] According to Example 1, the RNA method and reaction system for extracting the gargle samples of type A3 influenza patients were detected by the LAMP method. The reaction mixture was reacted at 63°C for 50min, and then 80°C for 2min to terminate the reaction. Each reaction took 5 μL for agarose gelation. Gel electrophoresis, observation of the gel shows typical ladder-like amplification bands, indicating that the LAMP method can be used to detect influenza A3 viruses contained in clinical case samples.

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Abstract

The invention provides a loop-mediated isothermal amplification test method of an influenza A3 virus. The method comprises the following steps: a specificity primer sequence is designed according to an influenza A3 virus hemagglutinin gene, a sample RNA to be tested is extracted as the template to have LAMP reaction; the LAMP reaction result is analyzed; if the LAMP amplification result is positive, the sample to be tested contains the influenza A3 virus. The LAMP influenza A3 virus test method is characterized by convenience, economy, fastness, sensitivity and specificity with flexible and convenient result judgment, is applicable in clinical or laboratory diagnosis, and has broad application prospect.

Description

(1) Technical field [0001] The invention relates to a detection kit and detection method for a loop-mediated isothermal amplification (loop-mediated isothermal amplification, LAMP) of type A3 influenza virus. (2) Background technology [0002] The clinical symptoms of influenza (flu) are easily confused with the common cold, and finally must be confirmed by laboratory methods. Among the laboratory diagnostic methods, the most direct and classic method is to collect the patient's gargle, inoculate chicken embryos or dog kidney passage cells (MDCK) for virus isolation and typing to determine. However, this method often takes a long time, and it is difficult to meet the needs of rapid diagnosis of outbreaks. [0003] The birth of Polymerase Chain Reaction (PCR) technology in 1985 has greatly promoted the development of nucleic acid detection technology and even the entire molecular biology technology, and has gradually become an important means in nucleic acid detection techno...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
Inventor 卢亦愚严菊英陈寅徐昌平姜晓慧李榛张严峻
Owner ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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