Primer group and probe group capable of simultaneously detecting various influenza viruses and kit capable of detecting various influenza viruses
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A technology for influenza virus and influenza A virus, applied in the field of molecular biology, can solve problems such as lack of detection of various influenza viruses, and achieve the effects of eliminating nucleic acid extraction steps, improving efficiency and easy operation.
Inactive Publication Date: 2017-08-11
SOUTHERN MEDICAL UNIVERSITY
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The direct amplification method is a technical method that realizes virus lysis, nucleic acid release, RNA reverse transcription, and PCR amplification steps in one tube on the basis of
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Embodiment 1
[0034] For influenza virus IVA, IVB, H1 and H3, design specific primers respectively, and the primer sequences are as follows:
[0035] IVA-F: 5'-CTTCTAAGCGAGGTCGAACC-3'
[0036] IVA-R: 5'-GTCTTAGCCATTCGATGAGT-3'
[0037] IVB-F: 5'-CGACATCCAAAGCCAATTCG-3'
[0038] IVB-R: 5'-CGGTGCTCTTGACCAAATTGC-3'
[0039] H1-F: 5'-CCTCAGGGAGCTATAAACAGA-3'
[0040] H1-R: 5'-TGACCTACTTTGGACACTCTCC-3'
[0041] H3-F: 5'-CAACTGCAATTCTGAATGCATC-3'
[0042] H3-R: 5'-GATCCTGTTTACATTTTGGAATG-3'
[0043] For each set of primers, design its specific probe, the sequence is as follows:
[0048] The RT-PCR reaction system is: 28 μl of RT-PCR reaction solution A, 2 μl of RT-PCR reaction solution B, and 20 μl of sample eluent.
[0049] The RT-PCR rea...
Embodiment 2
[0083] Composition and preparation of embodiment 2 kit
[0084] A kit for simultaneously detecting multiple influenza viruses, comprising: direct RT-PCR reaction solution A, direct RT-PCR reaction solution B, sample diluent, positive quality control substance, and negative quality control substance.
[0085] The RT-PCR reaction solution A consists of: the four influenza virus-specific primers and probe sequences described in Example 1 (the primers are all 15 pmol, and the probes are all 10 pmol), 10 mM Tris-HCl (pH7.5), 2mM MgCl 2 , 0.2% (V / V) NP-40, 0.01% (V / V) Tween20, 30mM KCl, and the rest is DEPC water.
[0086] The RT-PCR reaction solution B consists of: reverse transcriptase (2.5U / reaction), Taq DNA polymerase (5U / reaction), dNTPs (0.6mm / reaction), RNase inhibitor (20U / reaction).
[0088] The composition of the positive quality control product is as follows: it consists of a plasmid ...
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Abstract
The invention provides a primer group and a probe group capable of simultaneously detecting various influenza viruses. Sequences of the primer group are shown as SEQ ID NO:1-8, and sequences of the probe group are shown as SEQ ID NO:9-12; and the influenza viruses include influenza A virus, influenza B virus, seasonal influenza virus H1 sub-type and H3 sub-type. On the basis, a kit, which is capable of simultaneously detecting the various influenza viruses, is provided. According to the prime group, the probe group and the kit, direct amplification and multiplex PCR are combined; direct RT-PCR can be conducted on suspected influenza cases or epidemiological investigation throat swab samples; and in comparison with a conventional method, a nucleic acid extraction step is avoided, so that it is conducive to implementation of automatic integrated detection and to improvement of efficiency. The prime group, the probe group and the kit have obvious advantages in aspect of being applied to rapid discrimination and detection of people infected by the influenza viruses, and the invention is simple and convenient to operate, short in detection time and relatively low in detection cost. The prime group, the probe group and the kit are applicable to rapid detection at a site of epidemic situation; and the prime group, the probe group and the kit have a broad and practical application value.
Description
technical field [0001] The invention relates to the technical field of molecular biology, more specifically, to a primer set, a probe set and a kit for simultaneously detecting multiple influenza viruses. Background technique [0002] Influenza is a viral infection that primarily affects the nose, throat, bronchi, and occasionally the lungs. The infection usually lasts about a week and is characterized by sudden onset of high fever, muscle aches, headache and severe malaise, dry cough, sore throat and rhinitis. The virus spreads easily from person to person through droplets and particles produced when an infected person coughs or sneezes. Influenza tends to spread rapidly during seasonal epidemics. Most infected people recover within one to two weeks without medical treatment. However, in young children, the elderly, and those with other serious medical conditions, infection can lead to serious complications, pneumonia, and death, depending on the underlying condition. I...
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