CN111270018A - Influenza virus fluorescent quantitative PCR detection method - Google Patents

Influenza virus fluorescent quantitative PCR detection method Download PDF

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CN111270018A
CN111270018A CN202010264876.2A CN202010264876A CN111270018A CN 111270018 A CN111270018 A CN 111270018A CN 202010264876 A CN202010264876 A CN 202010264876A CN 111270018 A CN111270018 A CN 111270018A
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influenza
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magnetic beads
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刘凤鸣
马运国
师凤华
谭木秀
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Guangxi Botanical Garden of Medicinal Plants
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    • C12Q1/6851—Quantitative amplification

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Abstract

The invention discloses a fluorescence quantitative PCR detection method for influenza virus, which comprises the steps of mixing immunomagnetic beads and DEAE magnetic beads to obtain virus nucleic acid extraction magnetic beads; extracting the virus nucleic acid in the sample to be detected by using the obtained virus nucleic acid extraction magnetic beads; and performing fluorescence quantitative PCR amplification on the extracted virus nucleic acid by adopting a high-resolution melting curve method combining a Taqman probe and a fluorescence quantitative PCR technology, thereby realizing the typing detection of the influenza virus in the sample to be detected. The invention can be used for clinical rapid differential diagnosis, prevention and control during epidemic outbreak, virus epidemiological monitoring and the like, and overcomes the defect that the detection sensitivity is influenced by the virus RNA of the sample due to the multiple steps of extracting the RNA from the tissue sample.

Description

Influenza virus fluorescent quantitative PCR detection method
Technical Field
The invention relates to the field of quantitative PCR detection of influenza viruses, in particular to a fluorescence quantitative PCR detection method for directly detecting influenza A and B viruses.
Background
Influenza, which is called influenza for short, is a common acute respiratory infectious disease with strong infectivity and high transmission speed caused by influenza virus; it is common in winter and spring and is mainly transmitted by airborne droplets, human-to-human contact or contact with contaminated products. Clinically, the medicine is characterized by general poisoning symptoms such as high fever, hypodynamia, headache, general aching pain and the like.
Influenza viruses belong to the family orthomyxoviridae, are enveloped negative-strand RNA viruses whose viral genome comprises 8 segments, is single-stranded, negative-strand RNA, and encodes at least more than 10 proteins. Influenza virus comprises three types of A (A), B (B) and C (C), wherein the type A is most easy to generate variation, can infect human and various animals, is the main pathogen of human influenza and often causes pandemics and medium-small flows. Influenza b viruses are less variable and can infect humans, causing outbreaks or small epidemics. The type C is stable and can infect human, and is often sporadic. Among them, influenza a viruses can be classified into HA subtypes and NA subtypes. And influenza viruses causing infection are mainly influenza viruses of H1(N1), H3(N2) subtype in type a and type B. The influenza virus has strong variability and wide host range, is easy to spread across species, brings great difficulty to prevention and treatment work, and often causes influenza epidemics, so the rapid diagnosis of the influenza virus has great significance to the prevention and early treatment of diseases.
Influenza diagnosis relies primarily on etiology, specific nucleic acid testing, and serum antibody assays. In the traditional method, separation culture (such as chick embryo culture) is adopted, and then classification confirmation is carried out through a hemagglutination experiment, a hemagglutination inhibition experiment and the like, so that the overall operation period is long and the specificity is poor. With the development of molecular biology, a variety of new diagnostic means are used for diagnosis of various influenza viruses, such as real-time fluorescent quantitative PCR, isothermal amplification, and gene chip technology. The real-time fluorescent quantitative PCR technology utilizes the characteristics of high-efficiency amplification of nucleic acid by PCR and high specificity of a probe technology to realize the leap of PCR from nature determination to quantification, overcomes the defects of easy pollution and high false positive rate of conventional PCR detection, and has the advantages of strong specificity, high sensitivity, automatic operation and the like.
Two of the most commonly used fluorescent probes for real-time fluorescent quantitative PCR are SYBR Green I and Taqman probes. SYBRGreen I is a fluorescent dye type probe, which is inserted into a DNA double strand in the real-time fluorescent PCR reaction process to emit a fluorescent signal, so that the real-time monitoring of the whole reaction process is realized, and the intensity of the fluorescent signal is positively correlated with the increment of a DNA template. The Taqman probe is characterized in that in the reaction process of real-time fluorescence PCR, a specific oligonucleotide probe with two ends respectively labeled with a fluorescence reporter group and a fluorescence quenching group is added besides an original pair of specific primers, the probe is hydrolyzed into mononucleotide by utilizing the 5 '-3' exonuclease activity of TaqDNA polymerase during PCR amplification, a free fluorescence reporter group leads to the increase of a fluorescence signal, and the amplification product is quantitatively analyzed by the real-time dynamic detection of the intensity of the fluorescence signal.
In the detection of influenza virus, firstly, an RNA kit is required to be adopted to extract RNA from a tissue standard sample (such as a throat swab) to be detected, and the RNA extraction steps are various, and the RNA is unstable and easy to degrade, so that the accurate analysis is not facilitated.
The high-resolution probe melting curve method is a novel analysis technology, is improved on the basis of a high-resolution melting curve technology (HRM), saturated dyes in the HRM are replaced by probes, the detection result is more direct and specific, the maximum advantage is that typing detection can be carried out, and the detection flux is greatly improved by combining multicolor fluorescence detection, while one fluorescence channel of the traditional fluorescence PCR method can only detect one genotype, if the probe melting curve method is adopted, one fluorescence channel can detect 3 genotypes or even more, and the flux is amplified by 3 times.
Therefore, there is a need to develop a real-time fluorescent quantitative PCR detection method for directly detecting influenza a and b viruses.
Disclosure of Invention
It is an object of the present invention to address at least the above-mentioned deficiencies and to provide at least the advantages which will be described hereinafter.
In order to realize the purposes and other advantages of the invention, the invention provides a fluorescence quantitative PCR detection method for influenza viruses based on a high-resolution melting curve method, which is characterized in that RNA in a sample to be detected is extracted by a method of mixing immunomagnetic beads and DEAE magnetic beads to obtain virus nucleic acid extraction magnetic beads, then fluorescence quantitative PCR amplification is carried out by a high-resolution melting curve method combining a Taqman probe and a fluorescence quantitative PCR technology, and typing detection for the influenza viruses is realized at one time.
In the technical scheme, a method of combining immunomagnetic beads and nucleic acid extraction magnetic beads is adopted to extract RNA of a tissue sample (throat swab) at one time, the immunomagnetic beads can effectively enrich a small amount of influenza virus in the sample, and the defect that the detection sensitivity is affected due to sample virus RNA caused by multiple steps of extracting RNA from the tissue sample is overcome.
The immune magnetic beads are coated with anti-influenza A virus antibodies and anti-influenza B virus antibodies.
The primers and probes adopted by the fluorescent quantitative PCR amplification are respectively designed according to a matrix protein (M) gene and a Hemagglutinin (HA) gene of influenza virus, comprise influenza A virus H1N1 and H3N2 and influenza B virus Victoria line (2008) and Yamagata line, are respectively coupled by different fluorescent groups and are detected in different channels.
The sequences of the primers and probes are as follows:
the upstream primer is influenza A H1-F: 5'-GAGCTAAGAGAGCAATTGA-3'
The downstream primer is influenza A H1-R: 5'-GTAGATGGATGGTGAATG-3'
Probe influenza a H1P: 5 '-Fam-TTGCTGAGCTTTGGGTATGA-3' -BHQ-1
The upstream primer is influenza A N1-F: 5'-TCCACGCCCTAATGATAA-3'
The downstream primer is influenza A N1-R: 5'-TTCTCCCTATCCAAACAC-3'
Probe influenza a N1P: 5 '-Fam-ATCCTTTTACTCCATTTGCTCC-3' BHQ-1
The upstream primer is influenza A H3-F: 5'-AGCAAAGCCTACAGCAA-3'
The downstream primer is influenza A H3-R: 5'-GACCTAAGGGAGGCATAA-3'
Probe influenza a H3P: 5'-Texas Red-CCGGCACATCATAAGGGTAACA3' -BHQ-2
The upstream primer is influenza A N2-F: 5'-GTCCAACCCTAAGTCCAA-3'
The downstream primer is influenza A N2-R: 5'-GCCACAAAACACAACAATAC-3'
Probe influenza a N2P: 5'-Texas Red-CTTCCCCTTATCAACTCCACA-3' -BHQ-2
The upstream primer is influenza B V-F: 5'-AGACCAGAGGGAAACTATGCCC-3'
Downstream primer influenza B V-R: 5'-TCCGGATGTAACAGGTCTGACTT-3'
Probe influenza b V P: 5 '-Cy 5-CAGACCAAAATGCACGGGGAAHATACC-3' -BHQ-3
The upstream primer is influenza B Y-F: 5'-AGACCAGAGGGAAACTATGCCC-3'
Downstream primer influenza B Y-R: 5'-TCCGGATGTAACAGGTCTGACTT-3'
Probe influenza b Y P: 5 '-Cy 5-CAGRCCAATGTGTGTGGGGAYCACACC-3' -BHQ 3.
Weighing silicon dioxide magnetic beads containing carboxyl groups, then sequentially adding EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide), and stirring at room temperature in a dark place for reaction to form activated ester magnetic beads; dissolving an anti-influenza A virus antibody or an anti-influenza B virus antibody in PBS (phosphate buffered saline), slowly dropwise adding the activated ester magnetic beads into the anti-influenza virus antibody solution, stirring at 4 ℃ for reacting overnight to obtain immune magnetic beads of the influenza A virus antibody or the influenza B virus antibody, and storing for later use.
Weighing silicon dioxide magnetic beads containing carboxyl groups, then sequentially adding EDC and NHS, stirring and reacting at room temperature in a dark place to form activated ester magnetic beads, dissolving DEAE in PBS, slowly dropwise adding the activated ester magnetic beads into a DEAE solution, stirring and reacting at 4 ℃ overnight to obtain DEAE magnetic beads, and storing for later use;
before use, the immunomagnetic beads of the influenza A virus antibodies, the immunomagnetic beads of the influenza B virus antibodies and the DEAE magnetic beads are mixed according to the volume ratio of 1:1:2 to obtain the virus nucleic acid extraction magnetic beads.
The extraction process of the virus nucleic acid comprises the following steps of 1) sampling a throat swab of a suspected influenza patient, uniformly oscillating the throat swab in Hanks virus preservation solution, and freezing and preserving the throat swab for later use; 2) during detection, the virus nucleic acid extraction magnetic bead solution is added, mixing is carried out, magnetic beads are adsorbed by a magnet, the supernatant is removed, a sample lysate (containing 50mM Tris-HCl, 1% Triton X-100 and 2M guanidine hydrochloride) is added, the magnetic beads are adsorbed by the magnet, the supernatant is removed, a washing buffer (containing 50mM Tris-HCl, proteinase K and 50% ethanol) is added, the magnetic beads are adsorbed by the magnet, the supernatant is removed, an elution buffer (containing 10mM Tris-HCl and pH8.0) is added, the magnetic beads are adsorbed by the magnet, and the supernatant is taken out and is an influenza virus-containing RNA sample.
The influenza virus fluorescence quantitative PCR detection method based on the high-resolution melting curve method is applied to the development of influenza virus fluorescence quantitative PCR detection kits.
The invention at least comprises the following beneficial effects:
1. the invention adopts the method of combining the immunomagnetic beads and nucleic acid extraction magnetic beads, extracts the RNA of the tissue sample (pharyngeal swab) at one time, effectively enriches a small amount of influenza virus in the sample by the immunomagnetic beads, and overcomes the defect that the detection sensitivity is influenced by the sample virus RNA caused by the multiple steps of extracting the RNA from the tissue sample.
2. The invention adopts the primer and the probe respectively designed according to the matrix protein (M) gene and the Hemagglutinin (HA) gene of the influenza virus, and adopts the Taqman probe method fluorescent quantitative PCR technology combined with the high resolution melting curve method, so that the typing detection of the influenza virus can be realized at one time.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Detailed Description
The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.
Example 1
Extraction of tissue sample RNA:
1. preparing immunomagnetic beads:
1) preparation of influenza A virus immunomagnetic beads: weighing 8mg of silica magnetic beads containing carboxyl groups, then sequentially adding 12mg of EDC and 7mg of NHS, and stirring at room temperature in the dark for reaction for 16h to form activated ester magnetic beads. Dissolving 50ug of anti-influenza A virus antibody in 6mL of PBS, slowly dripping the activated ester magnetic beads into the anti-influenza A virus antibody solution, and stirring at 4 ℃ for reaction overnight to obtain the immune magnetic beads of the anti-influenza A virus antibody.
2) Preparation of influenza B virus immunomagnetic beads: weighing 8mg of silica magnetic beads containing carboxyl groups, then sequentially adding 12mg of EDC and 7mg of NHS, and stirring at room temperature in the dark for reaction for 16h to form activated ester magnetic beads. Dissolving 50mg of anti-influenza B virus antibody in 6mL of PBS, slowly and dropwise adding the activated ester magnetic beads into the anti-influenza B virus antibody solution, and stirring at 4 ℃ for reacting overnight to obtain the immunomagnetic beads of the influenza B virus antibody.
3) Preparation of DEAE magnetic beads: weighing 8mg of silica magnetic beads containing carboxyl groups, then sequentially adding 12mg of EDC and 7mg of NHS, and stirring at room temperature in the dark for reaction for 16h to form activated ester magnetic beads. 50mg of DEAE is dissolved in 6ml of PBS (phosphate buffer solution), the activated ester magnetic beads are slowly dripped into the DEAE solution, and the mixture is stirred and reacts overnight at 4 ℃ to obtain the DEAE magnetic beads.
2. Preparing magnetic beads for extracting virus nucleic acid from tissue samples:
mixing DEAE magnetic beads, influenza A virus immunomagnetic beads and influenza B virus immunomagnetic beads according to the proportion of 2:1:1 to obtain the tissue sample virus nucleic acid extraction magnetic beads.
3. The nucleic acid extraction process comprises the following steps:
1) suspected influenza patients with fever (body temperature is more than or equal to 38 ℃), cough, sore throat, whole body muscle ache or hypodynamia and other influenza symptoms are sampled, throat swab specimens are strictly executed according to the technical scheme (trial) of laboratory detection of influenza A H1N1 virus, the specimens are placed in 3ml Hanks virus preservation solution to be uniformly oscillated, and the specimens are refrigerated and transported to a laboratory to be preserved at-70 ℃ for detection.
2) Adding 30ul of the prepared tissue sample virus nucleic acid extraction magnetic beads into the virus preservation solution, mixing for 15min, adsorbing the magnetic beads by a magnet, removing the supernatant, adding 100ul of sample lysate (containing 50mM Tris-HCl, 1% TritonX-100 and 2M guanidine hydrochloride), adsorbing the magnetic beads by a magnet, removing the supernatant, adding 100ul of cleaning buffer (containing 50mM Tris-HCl, proteinase K and 50% ethanol), adsorbing the magnetic beads by a magnet, removing the supernatant, adding 100ul of elution buffer (containing 10mM Tris-HCl and pH8.0), adsorbing the magnetic beads by a magnet, and taking out the supernatant, wherein the sample is an RNA sample containing influenza virus.
Example 2
Real-time fluorescent quantitative PCR:
1. design of primers and probes: a plurality of gene sequences covering influenza A virus and influenza B virus at home and abroad are downloaded from an NCBI gene library, homology comparison is carried out, Primer Express 3.0 software is used for designing highly specific primers and Taqman probes in a conserved region, and an upstream Primer, a downstream Primer and corresponding specific probe sequences are as follows:
the upstream primer is influenza A H1-F: 5'-GAGCTAAGAGAGCAATTGA-3'
The downstream primer is influenza A H1-R: 5'-GTAGATGGATGGTGAATG-3'
Probe influenza a H1P: 5 '-Fam-TTGCTGAGCTTTGGGTATGA-3' -BHQ-1
The upstream primer is influenza A N1-F: 5'-TCCACGCCCTAATGATAA-3'
The downstream primer is influenza A N1-R: 5'-TTCTCCCTATCCAAACAC-3'
Probe influenza a N1P: 5 '-Fam-ATCCTTTTACTCCATTTGCTCC-3' BHQ-1
The upstream primer is influenza A H3-F: 5'-AGCAAAGCCTACAGCAA-3'
The downstream primer is influenza A H3-R: 5'-GACCTAAGGGAGGCATAA-3'
Probe influenza a H3P: 5'-Texas Red-CCGGCACATCATAAGGGTAACA3' -BHQ-2
The upstream primer is influenza A N2-F: 5'-GTCCAACCCTAAGTCCAA-3'
The downstream primer is influenza A N2-R: 5'-GCCACAAAACACAACAATAC-3'
Probe influenza a N2P: 5'-Texas Red-CTTCCCCTTATCAACTCCACA-3' -BHQ-2
The upstream primer is influenza B V-F: 5'-AGACCAGAGGGAAACTATGCCC-3'
Downstream primer influenza B V-R: 5'-TCCGGATGTAACAGGTCTGACTT-3'
Probe influenza b V P: 5 '-Cy 5-CAGACCAAAATGCACGGGGAAHATACC-3' -BHQ-3
The upstream primer is influenza B Y-F: 5'-AGACCAGAGGGAAACTATGCCC-3'
Downstream primer influenza B Y-R: 5'-TCCGGATGTAACAGGTCTGACTT-3'
Probe influenza b Y P: 5 '-Cy 5-CAGRCCAATGTGTGTGGGGAYCACACC-3' -BHQ 3.
2. Reaction system and reaction conditions
Reaction system: contains 10ul of buffer solution (Fast qPCR Mix of Madean company, containing magnesium chloride, deoxynucleotide triphosphate mixture), 2ul of enzyme mixture (containing 2:1:1 heat-resistant TAQ DNA polymerase, RNase inhibitor and MMLV reverse transcriptase), 2ul of primer probe mixture and 5ul of RNase removing water.
Reaction conditions are as follows: reverse transcription is carried out for 30min at 42 ℃; pre-denaturation at 95 ℃ for 2 min; amplifying at 95 ℃ for 10s and 55 ℃ for 40s for 40 cycles; the dissolution curves were 95 ℃ for 15s, 45 ℃ for 2min, and 75 ℃ for 1 s.
1ul of influenza virus-containing RNA sample obtained in example 1 was added to the reaction system, and the sample was analyzed for the melting curve and the peak pattern to determine the genotype.
3. Establishment of a Standard Curve
The standard synthetic fragments (107copies/ml) with calibrated copy number are respectively diluted in ten-fold gradient, and then the standard with concentration gradient is used as a template to amplify under the optimal reaction condition. And after the reaction is finished, taking the logarithm of the initial template copy number as an x axis, and taking the CT value as a y axis to draw a standard curve.
Influenza virus quantitative standard curve: CT is axlogx + B
4. Analysis of results
Comparison: negative control has no CT value and no amplification curve; the CT value of the positive control is less than or equal to 28.0, and a specific amplification curve appears; such as negative control and positive control, the above conditions are not satisfied, and the test is regarded as invalid. And (3) negative control has no obvious melting peak, if the melting peak appears after analysis, the reagent is possibly polluted or polluted in the operation process, and the detection is carried out again after a pollution source is removed. After the melting curve analysis of the positive control, each channel of each tube has 2 melting peaks, if the positive control tube has no melting peak or only has 1 melting peak, please confirm whether the used reagent is still in the valid period, and determine whether the operation is strictly performed according to the instruction.
The results describe: negative, no characteristic amplification curve; positive, CT value less than or equal to 30.0, and specific amplification curve. The dissolution temperatures were respectively: H1N 1: 58-59 ℃; H3N 2: 60-63 ℃; BV/BY: 66-69 ℃.
The reaction system can be made into a fluorescence quantitative PCR detection kit for the influenza virus and used in cooperation with the detection method.
While embodiments of the invention have been disclosed above, it is not intended to be limited to the uses set forth in the specification and examples. It can be applied to all kinds of fields suitable for the present invention. Additional modifications will readily occur to those skilled in the art.

Claims (9)

1. A fluorescence quantitative PCR detection method for influenza viruses is characterized by comprising the following steps:
mixing the immunomagnetic beads and DEAE magnetic beads to obtain virus nucleic acid extraction magnetic beads;
extracting the virus nucleic acid in the sample to be detected by using the obtained virus nucleic acid extraction magnetic beads;
and performing fluorescence quantitative PCR amplification on the extracted virus nucleic acid by adopting a high-resolution melting curve method combining a Taqman probe and a fluorescence quantitative PCR technology, thereby realizing the typing detection of the influenza virus in the sample to be detected.
2. The method for fluorogenic quantitative PCR detection of influenza virus of claim 1, wherein said immunomagnetic beads are coated with anti-influenza A virus antibody and anti-influenza B virus antibody.
3. The method for fluorescence quantitative PCR detection of influenza virus of claim 1, wherein the primers and probes used for fluorescence quantitative PCR amplification are designed according to the matrix protein M gene and hemagglutinin HA gene of influenza virus, respectively, and comprise influenza A virus H1N1 and H3N2 and influenza B virus Victoria linkage 2008 and Yamagatalinkage, which are coupled with different fluorescent groups respectively and detected in different channels.
4. The method for fluorogenic quantitative PCR detection of influenza virus according to claim 3, wherein the sequences of said primers and probes are as follows:
the upstream primer is influenza A H1-F: 5'-GAGCTAAGAGAGCAATTGA-3'
The downstream primer is influenza A H1-R: 5'-GTAGATGGATGGTGAATG-3'
Probe influenza a H1P: 5 '-Fam-TTGCTGAGCTTTGGGTATGA-3' -BHQ-1
The upstream primer is influenza A N1-F: 5'-TCCACGCCCTAATGATAA-3'
The downstream primer is influenza A N1-R: 5'-TTCTCCCTATCCAAACAC-3'
Probe influenza a N1P: 5 '-Fam-ATCCTTTTACTCCATTTGCTCC-3' BHQ-1
The upstream primer is influenza A H3-F: 5'-AGCAAAGCCTACAGCAA-3'
The downstream primer is influenza A H3-R: 5'-GACCTAAGGGAGGCATAA-3'
Probe influenza a H3P: 5'-Texas Red-CCGGCACATCATAAGGGTAACA3' -BHQ-2
The upstream primer is influenza A N2-F: 5'-GTCCAACCCTAAGTCCAA-3'
The downstream primer is influenza A N2-R: 5'-GCCACAAAACACAACAATAC-3'
Probe influenza a N2P: 5'-Texas Red-CTTCCCCTTATCAACTCCACA-3' -BHQ-2
The upstream primer is influenza B V-F: 5'-AGACCAGAGGGAAACTATGCCC-3'
Downstream primer influenza B V-R: 5'-TCCGGATGTAACAGGTCTGACTT-3'
Probe influenza b V P: 5 '-Cy 5-CAGACCAAAATGCACGGGGAAHATACC-3' -BHQ-3
The upstream primer is influenza B Y-F: 5'-AGACCAGAGGGAAACTATGCCC-3'
Downstream primer influenza B Y-R: 5'-TCCGGATGTAACAGGTCTGACTT-3'
Probe influenza b Y P: 5 '-Cy 5-CAGRCCAATGTGTGTGGGGAYCACACC-3' -BHQ 3.
5. The method for fluorescence quantitative PCR detection of influenza virus according to claim 1, wherein the preparation of immunomagnetic beads comprises the following steps: weighing silicon dioxide magnetic beads containing carboxyl groups, then sequentially adding EDC and NHS, and stirring and reacting in a dark place to form activated ester magnetic beads;
dissolving an anti-influenza A virus antibody or an anti-influenza B virus antibody in PBS (phosphate buffer solution), slowly dripping the activated ester magnetic beads into an anti-influenza virus antibody solution, stirring at 4 ℃ for reacting overnight to obtain immune magnetic beads of the influenza A virus antibody or the influenza B virus antibody, and storing for later use.
6. The fluorescence quantitative PCR detection method of influenza virus according to claim 5, wherein the DEAE magnetic beads are prepared by steps of weighing silica magnetic beads containing carboxyl groups, then adding EDC and NHS in sequence, stirring at room temperature in the dark to react to form activated ester magnetic beads;
and (3) dissolving DEAE in PBS, slowly dropwise adding the activated ester magnetic beads into the DEAE solution, stirring at 4 ℃ for reacting overnight to obtain DEAE magnetic beads, and storing for later use.
7. The fluorescence quantitative PCR detection method for influenza virus according to claim 6, wherein the magnetic beads for extracting virus nucleic acid are obtained by mixing immunomagnetic beads for influenza A virus antibodies, immunomagnetic beads for influenza B virus antibodies and DEAE magnetic beads in a volume ratio of 1:1: 2.
8. The method for fluorogenic quantitative PCR detection of influenza virus according to claim 7, wherein said viral nucleic acid extraction process comprises the steps of:
1) sampling a suspected influenza patient with a throat swab, uniformly oscillating the suspected influenza patient in Hanks virus preservation solution, and freezing and preserving the suspected influenza patient for later use;
2) adding the virus nucleic acid extraction magnetic bead solution during detection, mixing, adsorbing magnetic beads by using a magnet, removing the supernatant, adding a sample lysate, adsorbing the magnetic beads by using a magnet, removing the supernatant, adding a cleaning buffer, adsorbing the magnetic beads by using a magnet, removing the supernatant, adding an elution buffer, adsorbing the magnetic beads by using a magnet, and taking out the supernatant to obtain an RNA sample containing influenza virus.
9. The use of the method for fluorescence quantitative PCR detection of influenza virus according to any one of claims 1 to 8 in the development of a fluorescence quantitative PCR detection kit for influenza virus.
CN202010264876.2A 2020-01-21 2020-04-07 Influenza virus fluorescent quantitative PCR detection method Pending CN111270018A (en)

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CN115058417A (en) * 2022-06-24 2022-09-16 中国科学院苏州生物医学工程技术研究所 LAMP primer group for detecting influenza B virus
CN115786326A (en) * 2022-11-04 2023-03-14 重庆医科大学 Viral nucleic acid extraction kit and method for extracting complete viral particle nucleic acid by using same

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Application publication date: 20200612