CN101560574A - Primers and probes for detecting influenza virus by rRT-PCR and method for detecting influenza virus - Google Patents
Primers and probes for detecting influenza virus by rRT-PCR and method for detecting influenza virus Download PDFInfo
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- CN101560574A CN101560574A CNA2009100854754A CN200910085475A CN101560574A CN 101560574 A CN101560574 A CN 101560574A CN A2009100854754 A CNA2009100854754 A CN A2009100854754A CN 200910085475 A CN200910085475 A CN 200910085475A CN 101560574 A CN101560574 A CN 101560574A
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Abstract
The invention discloses primers and probes for detecting influenza virus by rRT-PCR and a method for detecting the influenza virus. The primers and the probes comprise influenza A virus specificity primer and probe, two groups of novel influenza A H1N1 virus H1 specificity primers and probes, novel influenza A virus specificity primer and probe and the like, namely four kinds of total 12 oligonucleotide primer and probe sequences; and the invention simultaneously discloses treatment of a sample to be detected, a rRT-PCR reaction system and reaction condition, and result analysis. The invention can quickly and effectively determine influenza A virus, novel influenza A H1N1 virus and novel influenza A virus. The primers and the probes and the method have simple operation and convenient application, and provide feasible technical support for influenza epidemic early warning mechanisms in the fields such as clinical diagnosis, inspection and quarantine and the like.
Description
Technical field
The present invention relates to a kind of rRT-PCR detection technique, relate in particular to the method that a kind of influenza virus rRT-PCR detects primer and probe and detects influenza virus.
Background technology
The influenza virus gene group is made of 8 strand RNA sections.According to different A type, Type B, the C types of being divided into of nucleoprotein (NP) with stromatin (M).A type influenza virus is according to different 16 HA hypotypes and 9 NA hypotypes of being divided into of surperficial hemagglutinin (HA) and neuraminidase (NA); Type B and C type influenza virus are regardless of hypotype.The A type often causes worldwide being very popular; Type B, C type then break out with the part and distribute the Flow Behavior feature.
In March, 2009, the Mexico and the U.S. have successively found the H 1 N 1 influenza A virus infection case, and on May 21st, 2009, the whole world had 42 countries the Influenza A H1N1 confirmed cases to occur, and confirmed cases surpass 10,000, and number of the infected is continuous ascendant trend.Present data presentation, this virus has possessed interpersonal communication's ability, and the crowd is to this viral immunocompromised, and therefore virus is strong the more common influenza virus of transmission capacity between interpersonal.
In general 1 to 7 day of latent period of Influenza A H1N1, early symptom is similar to common influenza, comprise heating, cough, laryngalgia, physical distress, have a headache, feel cold and fatigue etc., some also can occur diarrhoea or vomiting, myalgia or tired, eyes are rubescent etc.The part conditions of patients breaks with tremendous force, high heat, body temperature surpass 39 ℃ suddenly, even the serious pneumonia of secondary, adult respiratory distress syndrome, pulmonary apoplexy, shock and Reye syndrome, respiratory insufficiency and multiple organ injury, causes death at last.The gene nucleic acid sequence of influenza A H1N1 subtype virus and seasonal influenza virus H1N1 are approaching, and the diagnosis of cause of disease need be adopted comparatively complicated and loaded down with trivial details gene sequencing, is difficult to carry out in common lab.
In the prior art, the influenza virus detection method of employing is chicken embryo virus isolation cultivation method, and is highly sensitive.
There is following shortcoming at least in above-mentioned prior art:
During operational cost, need 3 time-of-weeks, and to demand of laboratory also than higher.
Summary of the invention
The purpose of this invention is to provide a kind of simple to operate, use the method that influenza virus rRT-PCR easily detects primer and probe and detects influenza virus.
The objective of the invention is to be achieved through the following technical solutions:
Influenza virus rRT-PCR of the present invention detects primer and probe, comprises following one or more primers and probe:
Influenza A virus Auele Specific Primer and probe, two groups of new H1N1virus H1 Auele Specific Primers and probe, new influenza A virus Auele Specific Primer and probe;
Described influenza A virus Auele Specific Primer and probe are at the relative conserved regions of influenza A virus M gene;
Described two groups of new H1N1virus H1 Auele Specific Primers and probe are at the relative conserved regions of new H1N1virus HA gene;
Described new influenza A virus Auele Specific Primer and probe are at the relative conserved regions of new influenza A virus NP gene.
The above-mentioned influenza virus rRT-PCR of application of the present invention detects the method for primer and probe in detecting influenza virus, comprises step:
At first, utilize nucleic acid extracting reagent from sample to be tested, to extract Influenza Virus RNA;
Then, utilize single stage method rRT-PCR test kit to add described primer and probe carries out nucleic acid amplification;
Afterwards, judge the rRT-PCR detected result, judge whether sample influenza virus to be checked is positive according to the Ct value.
As seen from the above technical solution provided by the invention, influenza virus rRT-PCR of the present invention detects primer and probe and detects the method for influenza virus, owing to comprise at influenza A virus M gene, new H1N1virus HA gene, the relative conserved regions primer of new influenza A virus NP gene and probe, can detect influenza virus by the rRT-PCR method, simple to operate, application convenience.
Embodiment
Influenza virus rRT-PCR of the present invention (real-time RT-PCR) detects primer and probe, and its preferable embodiment is to comprise following one or more primers and probe:
Influenza A virus Auele Specific Primer and probe, two groups of new H1N1virus H1 Auele Specific Primers and probe, new influenza A virus Auele Specific Primer and probe;
Described influenza A virus Auele Specific Primer and probe are at the relative conserved regions of influenza A virus M gene;
Described two groups of new H1N1virus H1 Auele Specific Primers and probe are at the relative conserved regions of new H1N1virus HA gene;
Described new influenza A virus Auele Specific Primer and probe are at the relative conserved regions of new influenza A virus NP gene.
Described primer and probe can comprise that length is the oligonucleotide fragment of 19～28bp;
Wherein, consistent at the probe of M gene and HA gene with the base sequence of goal gene minus strand, consistent at the probe of NP gene with the base sequence of goal gene normal chain.
Described influenza A virus Auele Specific Primer and probe comprise following two primer sequences and a probe sequence:
Primer sequence: 5 '-GACCRATCCTGTCACCTCTGAC-3 '; 5 '-GGGCATTYTGGACAAAKCGTCTACG-3 ';
Probe sequence: 5 ' FAM-TGCAGTCCTCGCTCACTGGGCACG-TAMARA3 ';
First group of described new H1N1virus H1 Auele Specific Primer and probe comprise following two primer sequences and a probe sequence:
Primer sequence: 5 '-ACATTCGAAGCAACTGGAAA-3 '; 5 '-GTRTTRCAATCGTGGACTGG-3 ';
Probe sequence: 5 ' FAM-TCCATTGCGAAKGCATATCTCGG-BHQ1-3 ';
Second group of described new H1N1virus H1 Auele Specific Primer and probe comprise following two primer sequences and a probe sequence:
Primer sequence: 5 '-GGGTAAARCTGGAATCRACAA-3 '; 5 '-CCAGGGAGACTAMCAGTACCA-3 ';
Probe sequence: 5 ' FAM-TTGGCGATCTAYTCAACTGYCGCC--BHQ1-3 ';
Described new influenza A virus Auele Specific Primer and probe comprise following two primer sequences and a probe sequence:
Primer sequence: 5 '-TCMGACATGCGAACRGAAGTT-3 '; 5 '-GGGYTCGTTGCCTTTTCGT-3 ';
Probe sequence: 5 ' HEX-CCAGAAGATTTGTCCTTCCA--BHQ1-3 '.
Flag F AM, the TAMARA at above-mentioned probe sequence two ends, BHQ1, HEX are fluorescein-labelled.
The present invention uses the method that above-mentioned influenza virus rRT-PCR detects primer and probe in detecting influenza virus, and its preferable embodiment is to comprise step:
At first, utilize nucleic acid extracting reagent from sample to be tested, to extract Influenza Virus RNA;
Then, utilize single stage method rRT-PCR test kit to add described primer and probe carries out nucleic acid amplification;
Described influenza virus can comprise following one or more viruses:
Influenza A virus, new H1N1virus, new influenza A virus.
Primer among the present invention and probe sequence (5 ' → 3 ') and the target fragment that detects thereof are as follows:
Specific embodiment:
Processing, detection and the analysis of the design of Oligonucleolide primers, sample to be checked have been comprised.Detect primer and application method for further specifying influenza virus rRT-PCR, describe with reference to the following example:
Embodiment one: the design of influenza A virus H1N1rRT-PCR Oligonucleolide primers is with synthetic
Download pig source influenza virus H1N1 and hypotype M gene, NP gene and HA gene complete sequences such as influenza A virus H1N1, H3N2, H5N1 in the GenBank database (http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html) and in the hiv sequence database of China national influenza center, the consistence of all influenza A virus M gene orders of software compare of analysis is selected relative conserved regions design primer and probe; With the comparison of putting together of pig source H1N1, new H1N1 and all NP genes of seasonal influenza A virus H1N1 and HA gene order, select relative conserved regions design primer and probe.Allow same variant sites to allow 2 and 2 following degeneracy bases in primer and the probe design.The alternative primer that is extracted is satisfied following the requirement to be screened: 1. probe length L is between 19 ~ 28bp; 2. the Tm value is between 42 ~ 59 ℃; 3. GC% is between 25 ~ 75%; 4. polyN≤4bp; 5. Hairpin≤4bp; 6. fraction of coverage＞90%; 7. carry out the BLAST screening, specificity mark＞L * 0.4.Best Tm value is set in 47 ℃, and best probe length is 20-25bp.
Embodiment two: the present invention detects the applicating example of unknown virus
1. the extraction of viral RNA:
Get viral sample solution 200 μ L, add 500 μ L lysates, (Qiagen company, catalog#74104) specification sheets extracts viral RNA 50 μ L to press RNeasy Mini Kit.
2.rRT-PCR reaction
1) system configurations: use QIAGEN QuantiTectTM Probe rRT-PCR Kit (catalog#20443) reaction solution
2) rRT-PCR: the reaction tubes that will add above-mentioned reaction system is put in the PCR instrument and carries out rRT-PCR, and response procedures is as follows
60 ℃ act on 5 minutes;
50 ℃ act on 30 minutes;
95 ℃ act on 15 minutes;
94 ℃ of sex change 15 seconds;
Annealed 30 seconds for 50 ℃;
72 ℃ were extended 30 seconds;
Got back to for the 5th step, 45 circulations.
3. the result judges:
The situation that the negative and positive results of comparison the is set up result that judges, and negative with reference to there not being Ct value, and positive reference has the Ct value.The following Ct of generalized case less than 38 can be judged as the positive.
The invention provides specific oligonucleotide primer and probe sequence that 4 covers are used to screen influenza A virus, new H1N1virus, new influenza A virus; And detection architecture and the application in the influenza virus rapid detection thereof of rRT-PCR are provided.
Detect and estimate:
Specificity is estimated: choose 20 strains of domestic nearly 3 years epidemic season influenza virus H1N1 hypotypes altogether and detect, qFluA all can detect, and qSW-H1-1, qSW-H1-2 and qSWNP all can not detect; Choose 10 strain swine influenza virus H1N1 hypotypes, 2 strain new influenza A virus H1N1 hypotypes detect, the four pairs of primers and probe all can detect; Choose influenza A virus H3N2 and detect, except that the qFluA test positive, its excess-three is all negative to detecting; Choose the Type B influenza virus and detect, four pairs of primers detect all negative.
Susceptibility is estimated: using from U.S.'s HAU is H1N1 strain (A/California/09/2009) the 200 μ L extraction RNA 50 μ L of 32HA/50 μ L, and 10 times of gradient dilutions show that susceptibilitys reach 10
-10
-7Extent of dilution.
The present invention can be applied to the rRT-PCR technology in the examination, detection architecture of new H1N1virus (porcine influenza).For the laboratory diagnosis of H1N1virus newly provides effective detection technique, for the formulation of discovery morning, prevention early and the early treatment and the public health strategy of H1N1virus newly provides technique means and detects foundation.
The above; only for the preferable embodiment of the present invention, but protection scope of the present invention is not limited thereto, and anyly is familiar with those skilled in the art in the technical scope that the present invention discloses; the variation that can expect easily or replacement all should be encompassed within protection scope of the present invention.
＜110〉China Sickness Prevention Control Center Virus Disease Prevention Control Institute
＜120〉method of primers for detecting influenza virus by RT-PCR and detection influenza virus
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<211>22
<212>DNA
＜213〉artificial sequence
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＜223〉influenza A virus Auele Specific Primer
<400>1
gaccratcct?gtcacctctg?ac
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<211>25
<212>DNA
＜213〉artificial sequence
<220>
＜223〉influenza A virus Auele Specific Primer
<400>2
gggcattytg?gacaaakcgt?ctacg
<210>3
<211>24
<212>DNA
＜213〉artificial sequence
<220>
＜223〉influenza A virus specific probe
<400>3
tgcagtcctc?gctcactggg?cacg
<210>4
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉new H1N1virus H1 Auele Specific Primer
<400>4
acattcgaag?caactggaaa
<210>5
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉new H1N1virus H1 Auele Specific Primer
<400>5
gtrttrcaat?cgtggactgg
<210>6
<211>23
<212>DNA
＜213〉artificial sequence
<220>
＜223〉new H1N1virus H1 specific probe
<400>6
tccattgcga?akgcatatct?cgg
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<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉new H1N1virus H1 Auele Specific Primer
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gggtaaarct?ggaatcraca?a
<210>8
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉new H1N1virus H1 Auele Specific Primer
<400>8
ccagggagac?tamcagtacc?a
<210>9
<211>24
<212>DNA
＜213〉artificial sequence
<220>
＜223〉new H1N1virus H1 specific probe
<400>9
ttggcgatct?aytcaactgy?cgcc
<210>10
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉new influenza A virus Auele Specific Primer
<400>10
tcmgacatgc?gaacrgaagt?t
<210>11
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉new influenza A virus Auele Specific Primer
<400>11
gggytcgttg?ccttttcgt
<210>12
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉new influenza A virus specific probe
<400>12
ccagaagatt?tgtccttcca
09-05-22
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Claims (6)
1, a kind of influenza virus rRT-PCR detects primer and probe, it is characterized in that, comprises following one or more primers and probe:
Influenza A virus Auele Specific Primer and probe, two groups of new H1N1virus H1 Auele Specific Primers and probe, new influenza A virus Auele Specific Primer and probe;
Described influenza A virus Auele Specific Primer and probe are at the relative conserved regions of influenza A virus M gene;
Described two groups of new H1N1virus H1 Auele Specific Primers and probe are at the relative conserved regions of new H1N1virus HA gene;
Described new influenza A virus Auele Specific Primer and probe are at the relative conserved regions of new influenza A virus NP gene.
2, primers for detecting influenza virus by RT-PCR according to claim 1 is characterized in that, described primer and probe comprise that length is the oligonucleotide fragment of 19～28bp;
Wherein, consistent at the probe of M gene and HA gene with the base sequence of goal gene minus strand, consistent at the probe of NP gene with the base sequence of goal gene normal chain.
3, influenza virus rRT-PCR according to claim 2 detects primer and probe, it is characterized in that:
Described influenza A virus Auele Specific Primer and probe comprise following two primer sequences and a probe sequence:
Primer sequence: 5 '-GACCRATCCTGTCACCTCTGAC-3 '; 5 '-GGGCATTYTGGACAAAKCGTCTACG-3 ';
Probe sequence: 5 ' FAM-TGCAGTCCTCGCTCACTGGGCACG-TAMARA3 ';
First group of described new H1N1virus H1 Auele Specific Primer and probe comprise following two primer sequences and a probe sequence:
Primer sequence: 5 '-ACATTCGAAGCAACTGGAAA-3 '; 5 '-GTRTTRCAATCGTGGACTGG-3 ';
Probe sequence: 5 ' FAM-TCCATTGCGAAKGCATATCTCGG-BHQ1-3 ';
Second group of described new H1N1virus H1 Auele Specific Primer and probe comprise following two primer sequences and a probe sequence:
Primer sequence: 5 '-GGGTAAARCTGGAATCRACAA-3 '; 5 '-CCAGGGAGACTAMCAGTACCA-3 ';
Probe sequence: 5 ' FAM-TTGGCGATCTAYTCAACTGYCGCC--BHQ1-3 ';
Described new influenza A virus Auele Specific Primer and probe comprise following two primer sequences and a probe sequence:
Primer sequence: 5 '-TCMGACATGCGAACRGAAGTT-3 '; 5 '-GGGYTCGTTGCCTTTTCGT-3 ';
Probe sequence: 5 ' HEX-CCAGAAGATTTGTCCTTCCA--BHQ1-3 '.
4, influenza virus rRT-PCR according to claim 3 detects primer and probe, it is characterized in that flag F AM, the TAMARA at described probe sequence two ends, BHQ1, HEX are fluorescein-labelled.
5, a kind of application rights requires 1 to 4 each described influenza virus rRT-PCR to detect the method for primer and probe in detecting influenza virus, it is characterized in that, comprises step:
At first, utilize nucleic acid extracting reagent from sample to be tested, to extract Influenza Virus RNA;
Then, utilize single stage method rRT-PCR test kit to add described primer and probe carries out nucleic acid amplification;
Afterwards, judge the rRT-PCR detected result, judge whether sample influenza virus to be checked is positive according to the Ct value.
6, the method for detection influenza virus according to claim 5 is characterized in that described influenza virus comprises following one or more viruses:
Influenza A virus, new H1N1virus, new influenza A virus.
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| CN101948934A (en) * | 2010-09-13 | 2011-01-19 | 中国医学科学院医学实验动物研究所 | Kit for detecting seasonal influenza virus H1N1 through real-time PCR |
| CN102140539A (en) * | 2011-01-28 | 2011-08-03 | 中国科学院北京基因组研究所 | Kit for detecting influenza A H1N1 viruses and detection method |
| CN102296127A (en) * | 2011-08-26 | 2011-12-28 | 中国检验检疫科学研究院 | Fluorescence quantitative PCR (polymerase chain reaction) kit for detecting novel influenza A H1N1 virus and non-diagnostic detection method thereof |
| CN101845517B (en) * | 2009-12-08 | 2012-11-14 | 江苏省疾病预防控制中心 | Fast detection method of nucleic acid of A H1N1 influenza virus and kit thereof |
| CN105296673A (en) * | 2015-12-03 | 2016-02-03 | 江苏省疾病预防控制中心 | Influenza A virus molecular detection kit and preparation method thereof |
| CN111910016A (en) * | 2020-04-27 | 2020-11-10 | 江苏派森杰生物科技有限公司 | Universal primer, probe and kit for detecting influenza A virus nucleic acid |
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| CN103160617A (en) * | 2013-04-10 | 2013-06-19 | 中国疾病预防控制中心病毒病预防控制所 | H7N9 subtype influenza virus rRT-PCR (Real-Time Reverse Transcription-Polymerase Chain Reaction) detection primer and probe, and method for detecting influenza virus and influenza virus standard substance |
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| CN101845517B (en) * | 2009-12-08 | 2012-11-14 | 江苏省疾病预防控制中心 | Fast detection method of nucleic acid of A H1N1 influenza virus and kit thereof |
| CN101948934A (en) * | 2010-09-13 | 2011-01-19 | 中国医学科学院医学实验动物研究所 | Kit for detecting seasonal influenza virus H1N1 through real-time PCR |
| CN101948934B (en) * | 2010-09-13 | 2015-12-09 | 中国医学科学院医学实验动物研究所 | Based on the kit for detecting seasonal influenza virus H 1 N 1 through real-time PCR of dye method |
| CN102140539A (en) * | 2011-01-28 | 2011-08-03 | 中国科学院北京基因组研究所 | Kit for detecting influenza A H1N1 viruses and detection method |
| CN102140539B (en) * | 2011-01-28 | 2012-03-07 | 中国科学院北京基因组研究所 | Kit for detecting influenza A H1N1 viruses and detection method |
| CN102296127A (en) * | 2011-08-26 | 2011-12-28 | 中国检验检疫科学研究院 | Fluorescence quantitative PCR (polymerase chain reaction) kit for detecting novel influenza A H1N1 virus and non-diagnostic detection method thereof |
| CN105296673A (en) * | 2015-12-03 | 2016-02-03 | 江苏省疾病预防控制中心 | Influenza A virus molecular detection kit and preparation method thereof |
| CN111910016A (en) * | 2020-04-27 | 2020-11-10 | 江苏派森杰生物科技有限公司 | Universal primer, probe and kit for detecting influenza A virus nucleic acid |
| CN112126713A (en) * | 2020-07-16 | 2020-12-25 | 上海之江生物科技股份有限公司 | Coronavirus and influenza virus combined detection product and application thereof |
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