CN106011307A - Rapid detection method for H7 subtype avian influenza virus nucleic acid - Google Patents

Abstract

This invention, belonging to the field of biotechnology, establishes a rapid nucleic acid detection method for the H7 subtype avian influenza virus. It encompasses three key technical aspects: the identification of primer sequences required for nucleic acid detection; the identification of the detection reaction type; and the establishment of the detection reaction system and reaction conditions. This nucleic acid detection method can be used for scientific research on the H7 subtype avian influenza virus and for clinical diagnostic testing in animals or humans.

Description

H7 Rapid detection method for subtype avian influenza virus nucleic acid

technical field

The invention belongs to the field of biotechnology; specifically, the invention establishes a rapid detection method for H7 subtype avian influenza virus, which is used for the rapid detection of H7 subtype avian influenza virus, in vitro diagnosis, veterinary medicine, food safety, biological safety, etc. The field has a greater use value.

Background technique

According to different pathogenicity, avian influenza can be divided into highly pathogenic avian influenza, low pathogenic avian influenza and apathogenic avian influenza. Among them, highly pathogenic avian influenza is a disease caused by certain strains of H5 and H7 subtypes.

Viral nucleic acid detection is one of the important methods for virus detection. At present, various nucleic acid detection methods for H7 subtype avian influenza virus at home and abroad, including agricultural industry standards or national standards, take a long time, such as agricultural industry standards (RT-PCR detection method for avian influenza virus, NY/T 772 -2013) at least 2 hours, and the national standard (fluorescence RT-PCR detection method for H7 subtype avian influenza virus, GB/T 19438.3-2004) requires at least 1.2 hours. At present, there is no detection method with higher efficiency than these methods.

Contents of the invention

The present invention aims at the shortcomings of the above-mentioned existing H7 subtype avian influenza virus detection technology, carried out in-depth research and testing, and established a H7 subtype avian influenza virus nucleic acid rapid detection method based on recombinase polymerase amplification technology, which only takes 20 minutes The detection can be completed. This method includes the following three aspects:

(1) The primer sequence required for nucleic acid detection was established, and the primer sequence was designed for the conserved region in the HA gene of the H7 subtype avian influenza virus. The upstream primer sequence contained 30 bases, and the sequence was ggtattcgctctgattgcgatcattccaac (that is, the SEQ ID in the sequence list NO.1), the downstream upstream primer sequence contains 30 bases, and the sequence is ttccactgtatgtgaatcccattgcttcct (ie, SEQ ID NO.2 in the sequence listing);

(2) Established a rapid detection method based on recombinase polymerase amplification technology;

(3) The detection reaction system and reaction conditions were established, and 9.2 μl of pure water, 29.5 μl of reaction buffer (containing four nucleotides such as dATP, dGTP, dTTP, and dCTP), and the first 2.4 μl of upstream primer synthesized in one step (concentration is 10 μmol/L), 2.4 μl of downstream primer synthesized in the first step (concentration is 10 μmol/L), enzyme mixture [reverse transcriptase, capable of binding single-stranded nucleic acid ( oligonucleotide primer), single-strand DNA binding protein (SSB) and strand-displacing DNA polymerase] 1.0 μl, RNase inhibitor 1.0 μl, influenza virus nucleic acid to be detected 2.0 μl (from clinical samples or other samples extracted with a nucleic acid extraction kit), magnesium acetate (280 mmol/L) 2.5 μl; then the reaction system was sealed and placed on a thermostat (or water bath) for reaction, the reaction condition was 40°C for 20 min; the reaction was over Finally, add color-containing nucleic acid electrophoresis buffer to the reaction product and perform agarose nucleic acid gel electrophoresis. If a nucleic acid electrophoresis band with a size of about 395 bp appears, it is judged as positive, otherwise it is judged as negative.

detailed description

The following examples illustrate the technical solution of the present invention, but the protection scope of the present invention is not limited to this example.

In this embodiment, the nucleic acid rapid detection of H7 subtype avian influenza virus is carried out by using the recombinant enzyme polymerase amplification technology, including the following steps:

The first step (synthetic primers): According to the nucleic acid sequence specified in the present invention (ie, SEQ ID NO.1 and SEQ ID NO.2 in the sequence listing), artificially synthesize the upstream primers and downstream primers required for the amplification reaction of recombinase polymerase ;

The second step (configuration of the reaction system): Add 9.2 μl of pure water, 29.5 μl of reaction buffer (containing four nucleotides such as dATP, dGTP, dTTP, and dCTP) to the polymerase chain reaction tube, and the Synthesized upstream primer 2.4 μl (concentration is 10 μmol/L), downstream primer synthesized in the first step 2.4 μl (concentration is 10 μmol/L), enzyme mixture [reverse transcriptase, capable of binding single-stranded nucleic acid (oligonucleoside acid primer), single-strand DNA binding protein (SSB) and strand-displacing DNA polymerase] 1.0 μl, RNase inhibitor 1.0 μl, influenza virus nucleic acid to be detected 2.0 μl (nucleic acid from clinical samples or other samples) extraction kit), magnesium acetate (280 mmol/L) 2.5 μl;

The third step (reaction): After sealing the reaction system configured in the second step, place it on a thermostat (or water bath) for reaction, and the reaction condition is 40°C for 20 min;

The fourth step (result detection): Add colored nucleic acid electrophoresis buffer to the reaction product of the third step, and perform agarose nucleic acid gel electrophoresis. If a nucleic acid electrophoresis band with a size of about 395 bp appears, it is judged as positive , otherwise it is judged as negative.

Practical application results: 96 standard positive clinical samples of H7 subtype avian influenza virus were detected, and 300 standard negative samples were tested with the above-mentioned rapid nucleic acid detection technology of H7 subtype avian influenza virus. The results showed that the sensitivity of this technology was 100.0 %, the specificity was 100.0%.

<110> China Animal Health and Epidemiology Center

<120> Rapid detection method for H7 subtype avian influenza virus nucleic acid

<160> 2

<210> 1

<211> 30

<212> dna

<213> Artificial sequence

<220>

<223> Designed according to the conserved nucleic acid sequence in the genome of the H7 subtype avian influenza virus, as the upstream primer required for the nucleic acid detection of the H7 subtype avian influenza virus

<400> 1

ggtattcgctctgattgcgatcattccaac 30

<210> 2

<211> 30

<212> dna

<213> Artificial sequence

<220>

<223> Designed according to the conserved nucleic acid sequence in the genome of the H7 subtype avian influenza virus, as a downstream primer required for nucleic acid detection of the H7 subtype avian influenza virus

<400> 2

ttccactgtatgtgaatcccattgcttcct 30

Claims (5)

1. The rapid detection method for H7 subtype avian influenza virus nucleic acid adopts the amplification reaction of recombinase polymerase, and is characterized in that all H7 subtype avian influenza viruses can be detected. 2. The method for rapid detection of H7 subtype avian influenza virus nucleic acid has two features: one is that all H7 subtype avian influenza viruses can be detected; the other is the use of recombinase polymerase amplification reaction. 3. The rapid detection method of H7 subtype avian influenza virus nucleic acid has three characteristics: one is that all H7 subtype avian influenza viruses can be detected; pair of primers; the third is to use recombinase polymerase amplification reaction. 4. The rapid detection method of H7 subtype avian influenza virus nucleic acid can adopt the amplification reaction of recombinase polymerase, which has two characteristics: one is that all H7 subtype avian influenza viruses can be detected; the other is a primer used in the detection reaction The sequence is ggtattcgctctgattgcgatcattccaac, or a sequence with 10 or more base sequences identical to the ttccactgtatgtgaatcccattgcttcct sequence (in this paragraph, the primer sequences are all from 5 prime to 3 prime ends, and a, t, g, and c represent corresponding bases). 5. The rapid detection method of H7 subtype avian influenza virus nucleic acid has two features: one is to use a recombinase polymerase amplification reaction; the other is to use two primers for the detection reaction, the sequences of which are respectively ggtattcgctctgattgcgatcattccaac and ttccactgtatgtgaatcccattgcttcct, or with These two sequences have 10 or more nucleotide sequences identical (in this paragraph, the primer sequences are all 5-primer to 3-primer, and a, t, g, c represent the corresponding bases).