Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) visual detection kit for H1N2 avian influenza viruses
An avian influenza virus and kit technology, which can be used in DNA/RNA fragments, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problems of high cost, unsuitable for grass-roots promotion and use, and time-consuming.
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Embodiment 1
[0045] Embodiment 1, complete set of primers
[0046]The set of primers for loop-mediated isothermal amplification for detection or auxiliary detection of H1N2 subtype avian influenza virus in this embodiment is composed of the complete set of primers for detection or auxiliary detection of H1 subtype avian influenza virus and the set of primers for detection or auxiliary detection of N2 Primer sets for loop-mediated isothermal amplification of subtypes of avian influenza virus. Among them, the set of primers for loop-mediated isothermal amplification for detection or auxiliary detection of H1 subtype avian influenza virus consists of degenerate primer FIP-H1, degenerate primer BIP-H1, degenerate primer F3-H1, degenerate primer B3-H1 , degenerate primer LF-H1 and degenerate primer LB-H1, and their nucleotide sequences are respectively Sequence 1, Sequence 2, Sequence 3, Sequence 4, Sequence 5 and Sequence 6 in the sequence listing (Table 1). Among them, F3-H1 and B3-H1 are ou...
Embodiment 2
[0052] Example 2. Optimization of Reverse Transcription Loop-Mediated Isothermal Amplification Reagent for Detection or Assisted Detection of H1 Subtype and N2 Subtype Avian Influenza Virus
[0053] In this embodiment, strains A / Duck / Guangxi / 030D / 2009 (H1N1) and A / Duck / Guangxi / D / 2011 (H1N2) are used as samples to be tested for the following detection or auxiliary detection of H1 subtype and N2 subtype avian influenza The viral reverse transcription loop mediates the effect of isothermal amplification reagents:
[0054] Methods as below:
[0055] 1. RNA extraction of H1N2 subtype avian influenza virus
[0056] Total RNA was extracted from clinically isolated strain A / Duck / Guangxi / D / 2011 (H1N2) identified by hemagglutination test, hemagglutination inhibition test and sequence determination using TRIZOL method or RNA extraction kit.
[0057] 2. Optimization of RT-LAMP reaction conditions
[0058] Optimize the reagents that affect the efficiency of the amplification reaction wi...
Embodiment 3
[0065] Embodiment 3, utilize the RT-LAMP kit of detection or auxiliary detection H1 subtype, N2 subtype avian influenza virus to detect chicken source sample
[0066] A. In this example, the following RT-LAMP kits for detection or auxiliary detection of H1 subtype avian influenza virus were used for detection. The kit is a product obtained by assembling together RT-LAMP reaction reagent A, positive control A, negative control and fluorescent reagent for detection or auxiliary detection of H1 subtype avian influenza virus.
[0067] Among them, 19 μl of RT-LAMP reaction reagent for detection or auxiliary detection of H1 subtype avian influenza virus (hereinafter referred to as RT-LAMP reaction reagent A) is composed as follows: 2.5 μl 10-fold reaction buffer (20mM Tris-HCl, 10mM KCl, 10mM ( NH 4 ) 2 SO 4 , 2mM MgSO 4 , 0.1%Triton X-100), 6mmol / L MgSO 4 , 1mmol / L betaine, outer primers F3-H1 and B3-H1 each 0.2μmol / L, inner primers FIP-H1 and BIP-H1 each 1.6μmol / L, loop prime...
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