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A technology of RT-PCR and detection kit, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., which can solve the problems of cumbersome experimental operation, cost increase, and high detection cost, and achieve high detection efficiency and accuracy , the effect of short time period
Inactive Publication Date: 2011-08-10
WUHAN UNIV
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But the main disadvantage of this technique is the high cost of detection due to the use of fluorescently labeled probes
In addition, when fluorescent RT-PCR technology is used t
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Embodiment 1
[0067] 1. Example 1---Influenza virus one-step RT-PCR detection kit
[0068] Detect 12 throat swab samples from patients with suspected influenza virus infection, and take 200 μl of each sample solution for virus nucleic acid extraction.
[0069] 1. The composition of the kit:
[0070] This kit includes one-step 5xRT-PCR Buffer, positive control, negative control, three pairs of specific primers and Enzyme Mix;
[0071] 2. Design specific primers based on the M gene of influenza virus type A:
[0072] AF: 5'-ATTCTAACCGAGGTCGAAACGT-3' 22bp,
[0073] AR: 5'-GACAAAGCGTCTACGCTGCAGTC-3'23bp;
[0074] The specific primers designed according to the NS gene of influenza virus type B are:
[0075] BF: 5'-AGGGACATGAACAACAAAGATT-3' 22bp,
[0076] BR: 5'-GATGTCAGCTATTATGGAGCTC-3' 22bp;
[0077] The specific primers designed according to the HA gene of influenza virus type A H1N1 are:
[0078] H1N1F: 5'-AAATAACATTMGAAGCAACTGGT-3'23bp,
[0079] H1N1R: 5'-GAGGCTGGTGTTTATRGCACCC-3' 22...
Embodiment 2
[0117] Using the above-mentioned method to detect another 20 sputum samples from patients with suspected influenza virus infection, 2 cases were positive for influenza virus type A, 4 cases were positive for influenza virus type B, and 5 cases were positive for influenza virus type A H1N1, and the detection rate was 100%.
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Abstract
The invention discloses a one-step RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection kit for an influenza virus, relating to a PCR kit in the technical field of biology. The kit comprises one-step 5xRT-PCR Buffer, positive control, negative control, three pairs of specific primers and Enzyme Mix; a specific primer AF: 5'-ATTCTAACCGAGGTCGAAACGT-3' and a specific primer AR: 5'-GACAAAGCGTCTACGCTGCAGTC-3' are designed according to a gene M of an influenza virus type A; a specific primer is designed according to a gene NS of an influenza virus type B; and a specific primer is designed according to a gene HA of an influenza virus A type H1N1. The detection kit has a short detection time period, high detection efficiency, high virus detection specificity, high accuracy, higher detection sensitivity than an immunological detection method and high experiment result repeatability, can be applied to qualitative analysis of viruses, and is easy to operate and popularize.
Description
technical field [0001] The invention relates to a PCR kit in the field of biotechnology, in particular to a one-step RT-PCR (reverse transcription-polymerase chain reaction, reverse transcriptase-polymerase chain reaction) detection kit for influenza virus. Background technique [0002] Influenza viruses belong to the Orthomyxoviridae family and are segmented single-stranded negative-sense RNA viruses. According to the different antigenicities of their nucleoproteins and matrix proteins, influenza viruses are mainly divided into types A, B, and C, or called A, B, and C. Among them, influenza A virus can infect humans, birds and some other mammals, such as pigs. Influenza types B and C mainly infect humans. According to epidemiological investigations, it is mainly influenza virus type A and type B that currently cause influenza in the population. The influenza virus that broke out in Mexico in April 2009 and caused widespread worldwide spread and hundreds of deaths was nam...
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