CN102559925B - Primer and method for detecting human influenza A virus H3 subtype - Google Patents
Primer and method for detecting human influenza A virus H3 subtype Download PDFInfo
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- CN102559925B CN102559925B CN 201110415387 CN201110415387A CN102559925B CN 102559925 B CN102559925 B CN 102559925B CN 201110415387 CN201110415387 CN 201110415387 CN 201110415387 A CN201110415387 A CN 201110415387A CN 102559925 B CN102559925 B CN 102559925B
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Abstract
The invention relates to a primer and a method for detecting human influenza A virus H3 subtype. The primer for detecting the human influenza A virus H3 subtype contains a random nucleotide sequence shown as SEQ ID NO: 1 to SEQ ID NO: 6. According to the technical scheme, the popular novel influenza A virus H1N1 can be diagnosed, important mutation of the virus can be analyzed through bioinformatics, and a scientific proof is provided for formulating effective prevention and control measures by the state.
Description
The present invention is dividing an application of patent application 200910303111.9.
Technical field
The present invention relates to primer and the detection method of a kind of influenza virus A hominis of detection H1 and/or H3 hypotype.
Background technology
Influenza be a kind of by influenza virus cause, communicable disease by respiratory infectious.Being very popular of influenza once caused very serious consequence to the mankind in history, for example " spanish influenza " that caused by H1N1 subtype influenza virus of outburst in 1918.It is the catastrophic influenza viruses of tool of 20th century, has infected half population of the whole world in less than one-year age, causes tens of millions of people's death.Flu outbreak is normally by a kind of emerging or cause with the virus that forefathers had not infected, human body does not have immunizing power to this new virus, add that its spread scope is wide, speed is fast, and the development of vaccine and production are difficult to catch up with immediately, so cause many people to infect easily even death.In recent years, continuous intensification along with the integrated degree of global economy development, people-to-people contacts between the countries in the world and mobile frequent day by day and close, thereby influenza virus is further accelerated sending out with rate of spread of the whole world, in case certain country flu outbreak, epidemic situation will be sent out rapidly to all over the world at short notice.The novel Type A Influenza H1N1 epidemic situation that took place in 2009 just so.
In April, 2009, the first novel Type A Influenza H1N1 case has been made a definite diagnosis by Mexico.During April to May subsequently, novel Type A Influenza H1N1 epidemic situation is rapid spread worldwide, and only in the time of a wheat harvesting period, novel Type A Influenza H1N1 epidemic situation has all taken place in global a plurality of countries and regions.Mexico and the U.S. the most serious with epidemic situation are example, and by on May 14th, 2009, Mexico made a definite diagnosis in the whole nation novel Influenza A H1N1 case and rises to 2720 examples, and wherein death toll rises to 64 people; And in 50 states of the U.S., novel Influenza A H1N1 case is announced to find in existing 47 states, and patient's sum also rises to 4298 people, increases by 946 people than the day before yesterday.The first novel Influenza A H1N1 case has also been made a definite diagnosis May 11 in ground in China, by on May 19th, 2009,4 routine novel Influenza A H1N1 cases occurred, is Introduced cases influenza case.The World Health Organization announces that on April 29th, 2009 global flu outbreak warning level is 5 grades, and along with novel Influenza A H1N1 epidemic situation constantly spreading worldwide, the risk that breaks out global flu outbreak promotes day by day, the World Health Organization has clearly represented not get rid of at the beginning of 5 months warning level is risen to 6 grades, namely announces the possibility that flu outbreak arrives.
After this time novel Type A Influenza H1N1 epidemic situation took place, the scientists of countries in the world had been launched relevant scientific research immediately.Up-to-date investigation and result of study show that each Mexico patient who infects novel Type A Influenza H1N1 can cause increasing by 1.2 to 1.6 new cases, proves that the interpersonal communication of influenza virus is in continuous generation.Up to the present the infectious rate of this influenza virus is 1/3, may have the infection that 2,000,000,000 people are subjected to this influenza virus in the global range, and global big influenza is first meeting clue now.But owing to still can not determine the origin of toxicity, propagability and the virus of novel Type A Influenza H1N1 virus at present, and in should virus latent period and infective stage, neither be very clear and definite, so scientists also can't be judged the degree that it worldwide spreads.
Although present novel H1N1virus is still in diffusion, and how to develop still unpredictablely, as long as the whole world works in concert, fully carry out monitoring and the prevention work of epidemic situation, just can reduce influenza virus to greatest extent to the mankind's harm.Making a definite diagnosis novel H 1 N 1 influenza A virus infection case in early days, in time carry out isolation and the treatment of case, is the important measures that stop the influenza epidemic situation to spread.Yet, along with influenza virus is constantly propagated between interpersonal, the possibility that it morphs and virulence changes from weak to strong all exists, particularly the variability with height of viral hemagglutinin (HA) gene and neuraminidase (NA) gene and probabilistic characteristics of variation make the diagnosis of influenza virus and the outburst that classifying method lags behind its epidemic situation always.Therefore, developing quick, special, sensitive detection method and diagnostic reagent in the short period of time, is the top priority of the novel H1N1virus of antagonism.
Because the widespread usage of monoclonal antibody technique, many based on monoclonal antibody, succeed in developing at ELISA and the colloidal gold diagnosis test kit of influenza A virus, for example influenza A virus colloidal gold diagnosis reagent carries out rapid screening to the passenger from the epidemic-stricken area aircraft, a kind of valuable method of can yet be regarded as
Along with the development of modern biotechnology, Protocols in Molecular Biology has been widely used in the quick diagnosis of influenza A virus.This time epidemic situation breaks out the influenza virus method for quick that the back World Health Organization announces successively, all is based on the fluorescence quantitative RT-RCR detection technique of novel H1N1virus nucleic acid.The reagent of this technical project is easy to preparation, and have fast, sensitive, be easy to the advantage promoted in basic unit.But also there is certain defective in these methods, sequence-specific as employed primer and probe is low, be difficult to the many places variation that the genome to current novel H1N1virus is dispersed in and carry out specific diagnosis, be easy to produce cross reaction with in the past seasonal H1N1virus and maybe can not amplify at primer or probe location and the novel H1N1virus strain of variation occurs, thereby cause false positive or false negative result.Therefore, WHO not with it as the diagnostic method of making a definite diagnosis.Based on these practical problemss, we are according in the past influenza virus and the novel H 1 N 1 influenza viruses genome sequence of up-to-date announcement, relative conservative region design primer in various influenza virus gene group, by sequencing and the bioinformatic analysis to amplified fragments, can detect influenza virus and the hypotype thereof that H1, H3, H5, H9 etc. have outbreak potential exactly.This method for detecting specificity based on the influenza nucleic acids sequencing not only can be used for quick diagnosis and the somatotype of novel H1N1 virus, and go in the monitoring that can also extend to other influenza virus with outbreak potential from now on.
Summary of the invention
Goal of the invention of the present invention is to propose the primer of a kind of influenza virus A hominis of detection H1 and/or H3 hypotype.
Another goal of the invention of the present invention is to propose the method for a kind of influenza virus A hominis of detection H1 and/or H3 hypotype.
In order to finish goal of the invention of the present invention, the technical solution used in the present invention is:
The present invention relates to the primer of a kind of influenza virus A hominis of detection H1 and/or H3 hypotype, comprise by the arbitrary nucleotide sequence shown in SEQ ID NO:1 to the SEQ ID NO:6.
Wherein, first preferred version of detection influenza virus A hominis primer of the present invention is: the primer of detection influenza virus A hominis H1 and/or H3 hypotype is selected from least one group in following four groups of primers:
(1) it is right to be used for the primer of amplification influenza A virus H1 target nucleic acid sequence: comprise by the nucleic acid primer of at least 15 continuous nucleotides of nucleotide sequence shown in the SEQ ID NO:1, by the nucleic acid primer of at least 15 continuous nucleotides of nucleotide sequence shown in the SEQ ID NO:2 with by the nucleic acid primer of at least 15 continuous nucleotides of nucleotide sequence shown in the SEQ ID NO:3;
(2) it is right to be used for the primer of amplification influenza A virus H3 target nucleic acid sequence: comprise by the nucleic acid primer of at least 15 continuous nucleotides of nucleotide sequence shown in the SEQ ID NO:4, by the nucleic acid primer of at least 15 continuous nucleotides of nucleotide sequence shown in the SEQ ID NO:5 with by the nucleic acid primer of at least 15 continuous nucleotides of nucleotide sequence shown in the SEQ ID NO:6;
Second preferred version of detection influenza virus A hominis primer of the present invention is: the primer of detection influenza virus A hominis H1 and/or H3 hypotype is selected from least one group in following four groups of primers:
(1) it is right to be used for the primer of amplification influenza A virus H1 target nucleic acid sequence: comprise by the nucleic acid primer of nucleotide sequence shown in the SEQ ID NO:1, by the nucleic acid primer of nucleotide sequence shown in the SEQ ID NO:2 with by the nucleic acid primer of nucleotide sequence shown in the SEQ ID NO:3;
(2) it is right to be used for the primer of amplification influenza A virus H3 target nucleic acid sequence: comprise by the nucleic acid primer of nucleotide sequence shown in the SEQ ID NO:4, by the nucleic acid primer of nucleotide sequence shown in the SEQ ID NO:5 with by the nucleic acid primer of nucleotide sequence shown in the SEQ ID NO:6;
The invention still further relates to the method for a kind of influenza virus A hominis of detection H1 and/or H3 hypotype, may further comprise the steps:
(1) extracts sample of nucleic acid;
(2) nucleic acid that extracts with step (1) is template, uses primer sequence of the present invention, the target nucleic acid sequence of pcr amplification pathogenic agent;
(3) amplified production is carried out sequencing, and analytical results.
The gene sequencing technology is recommended as the method for making a definite diagnosis of the novel Influenza A H1N1 of diagnosis owing to possess special, advantage accurately by WHO.Therefore, according in the past influenza virus and the novel H 1 N 1 influenza virus gene group sequence of the up-to-date announcement of WHO, relative conservative region design special primer in each subtype gene group of influenza A virus, use RT-PCR amplicon virus HA gene order, use the data analysing method after checking order then, identify the Virus Type of sequence correspondence.
Based on above-mentioned strategy, the relevant primer principle of design is: 1) primer is selected in certain conservative relatively position of virus subtypes such as H1, H3; 2) the primer amplification frag info can be in order to distinguishing H1, H3, and H5, the hypotype of four kinds of infected person of H9 particularly can be distinguished the novel influenza A virus in North America in 2009 and conventional H 1N1 influenza virus; 3) primer should meet conventional PCR design of primers principle.We select H1, and H3, H5 and H9 be as distinguishing object because these several hypotypes have been found that can infection population, or may propagate the potentiality that also have outburst that have in the human world now or in the future.
Up-to-date influenza virus sequence data and relevant descriptor have been downloaded (by in May, 2009 from the Influenza Virus Resource of NCBI; Totally 187327 genbank entries, the sample that relates to is 23094), made up local influenza integrated information system (http://lifecenter.sgst.cn/flu) simultaneously.Because the influenza sequence is a lot, and various hypotypes are inconsistent at the number of database the inside storage, therefore, go up feasible for guaranteeing calculating, we have selected 50 nearest HA sequences that sample information is detailed and sequence data is complete by local system respectively in chronological order to various hypotypes.After gathering, obtain 300 HA sequences altogether, wherein, the host is 197 of bird, is 69 of people, is 15 of pig, and the host is 1 of pika (Plateau pika), and the host is labeled as 18 of environment.Subsequently, utilize parallelization Clustalw software (Bioinformatics, 2003,19 (12), it is right 1585-1586) these 300 sequences to be carried out multiple ratio, selects candidate's primer at conservative region relatively.
Accordingly, our the connection coordination of having chosen the HA sequence is put near 750nt and the 1100nt sequence respectively as forward and reverse primer.The connection situation of joining of the direct sequence of this positive anti-primer is seen Fig. 1.Fig. 1 center line zone is exactly the subregion of popular now novel H1N1virus HA gene.Because the authentication method of the influenza A virus of this paper design is based on the sequence that aligns between anti-primer and checks order to realize, so we draw clustering tree with the effect (see figure 2) of the sequence between the judgement primer to viral somatotype to the sequence among Fig. 1 according to sequence similarity.As can be seen from Figure 2, the sequence between the primer that we choose can be distinguished H1 in theory well, H2,300 sequences that H3, H5, H7 and H9(choose, all correct assign in the hypotype of this branch).In addition, as arrow indication position, novel H1N1virus in 2009 independently is divided into cluster and other H1 subtype virus has obviously distinguished.This explanation is at this both sides, zone over-designed primer, and the sequence of corresponding product is checked order can be H1, H2, and H3, H5, H7 and H9 distinguish, and particularly can also identify novel H1N1virus.
WHO is on April 28th, 2009, announced the fluorescence quantifying PCR method that detects influenza A virus H1N1, and this reagent is easy to preparation, and have fast, sensitive, be easy to the advantage promoted in basic unit.But, use fluorescence quantifying PCR method, the specificity of designed probe sequence is low, is difficult to distinguish the genome of different H1N1viruses, therefore be easy to produce cross reaction with in the past seasonal H1N1virus, and cause false-positive result.In addition, because the variation right and wrong of influenza virus usually see, the variation that primer or probe go out can cause amplifying at primer or probe location and the novel H1N1virus strain of variation occurs, and produces false negative result.
Sequencing technologies is recommended as the method for making a definite diagnosis of the novel Influenza A H1N1 of diagnosis owing to possess special, advantage accurately by WHO.Therefore, we are according in the past influenza virus and the novel H 1 N 1 influenza virus gene group sequence of the up-to-date announcement of WHO, relative conservative region design special primer in each subtype gene group of influenza A virus, use RT-PCR amplicon virus HA gene order, use the data analysing method after checking order then, identify the Virus Type of sequence correspondence.
Use technical scheme of the present invention, not only can make a definite diagnosis this popular novel H1N1virus, and can analyze its important variation by information biology, and then provide scientific basis for country formulates effective prevention and control measure.For example, because the high anomaly of influenza virus occurs in the variation of influenza virus sialic acid binding site (on the HA albumen), may influence the power of the invasiveness of virus.In addition, this epitope information that obtains based on order-checking, the development to effective vaccine and medicine has important value undoubtedly.
Description of drawings
Fig. 1 is the sequence comparison diagram in primer amplification zone;
Fig. 2 is that sequence between each serotype specific primer is according to the design sketch of similarity to viral somatotype;
Fig. 3 is H1 and the H3 serotype specific primer PCR product electrophorogram of different specimens;
Fig. 4 is the electrophorogram of primer susceptibility optimization experiment;
Fig. 5 is the somatotype situation after people and influenza virus A porcine H1 and the H3 changeable zone order-checking.
Embodiment
The embodiment that the present invention proposes only limits to technical scheme of the present invention is made further explanation, and technical scheme of the present invention is not made restriction.
1. material
1.1 strain sample, RNA and cDNA
The people source:
H1 subtype influenza virus type strain (disease prevention and control center, Shanghai, disease prevention and control center, Guangzhou)
H3 subtype influenza virus type strain (disease prevention and control center, Shanghai, disease prevention and control center, Guangzhou)
The pig source:
H1N1 type strain hypotype type strain cDNA (China Agriculture Academe Shanghai Veterinary Institute)
H1N1 type strain HA gene clone plasmid (China Agriculture Academe Shanghai Veterinary Institute)
H3N2 type strain HA gene clone plasmid (China Agriculture Academe Shanghai Veterinary Institute)
1.2 key instrument and reagent
Dry type constent temperature heater (GL-150, its woods Bel instrument Manufacturing Co., Ltd of Haimen), the transmission-type uv analyzer (day energy 2500, Tanon), high speed freezing centrifuge (Eppendorf, 5810R), the desk type high speed refrigerated centrifuge (Eppendorf, 5417R), cryogenic refrigerator (SanYo Medical freezer), electronic analytical balance (JT601N, smart day), digital display pH meter (PSH-37, the sky, Shanghai reaches Instr Ltd.), nucleic acid electrophoresis apparatus (HE-120, Tanon), clean bench (SDJ-DS, Microcystins in The Dianshan Lake cleaning apparatus factory), spectrophotometer (98090A, Agilent); Enzymes commonly used such as taq archaeal dna polymerase, M-MLVRM reversed transcriptive enzyme are all available from Takara and Invitrogen company; The Trizol test kit is available from Invitrogen company; Other biochemical reagents commonly used are all analytical pure, available from Research Center of Shanghai Human Genome warehouse.
1.3 primer sequence
Table 1
Annotate: primer SEQ ID NO:1 and SEQ ID NO:2 equal proportion are mixed in the experiment, and as H1 somatotype reaction upstream primer, primer SEQ ID NO:5 and SEQ ID NO:6 equal proportion are mixed, as H3 somatotype reaction downstream primer.
2. method
2.1 the cDNA of the total RNA of strain obtaining and gathering
Adopt the Trizol test kit of Invitrogen company, handle each influenza subtype strain culture supernatant of preserving from the disease prevention and control center, the total RNA of extracting and purifying, the entire operation step is carried out according to the specification sheets of producer.Identify the quality of the total RNA of above-mentioned purifying in 1% agarose electrophoresis after, carry out reverse transcription according to manufacturer's specification sheets with the ThermoScript II of Invitrogen company, obtain the cDNA of total RNA.
2.2 primer is synthetic: finish (synthetic method is with reference to modern molecular biology experimental technique (second edition) Lu Shengdong chief editor) by the super generation bio tech ltd in Shanghai
2.3 carry out the primer specificity analysis
Utilize synthetic good H1 and H3 subtype typing primer, the specificity of check primer.CDNA or plasmid with the above-mentioned HA of containing different subtype are template, choose H1 somatotype upstream primer (SEQ ID NO:1 and SEQ ID NO:2 mix primer) and downstream primer SEQ ID NO:3 respectively, and H3 serotype specific primer upstream primer SEQ ID NO:4 and downstream primer (SEQ ID NO:5 and SEQ ID NO:6 mix primer) are that template is carried out pcr amplification with above-mentioned all cDNA.
The PCR reaction system
| Composition | Volume |
| CDNA100 times of diluent | 2μl |
| 10×Taq?buffer | 2μl |
| dNTP（25mM） | 2μl |
| Upstream primer (10 μ M) | 0.6μl |
| Downstream primer (10 μ M) | 0.6μl |
| Taq polysaccharase (5U/ μ l) | 0.3μl |
| H 2O | 12.5μl |
The PCR reaction conditions of above system: 94 ℃ of pre-sex change 5min; Carry out 35 circulating reactions by following condition then, 94 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 30s; Last 72 ℃ are extended 5m; Agarose gel electrophoresis check PCR product.Electrophorogram such as Fig. 3.
Test-results is as shown in Figure 3: A figure is the PCR product of H1 serotype specific primer correspondence; B figure is the PCR product of H3 serotype specific primer correspondence; 1 swimming lane: 100bp DNA Ladder; The 2-8 swimming lane is the PCR product of different templates, 2 is deionized water, 3 is people H1 hypotype type strain cDNA, 4 is people H1 subtype virus strain cDNA, 5 is pig H1N1 hypotype type strain cDNA, 6 is pig H1N1 hypotype HA gene clone plasmid, and 7 is people H3 hypotype type strain cDNA, and 8 is pig H3N2 hypotype HA gene clone plasmid.The result shows, all can excellent specificity the increase cDNA of each self-corresponding H1 and H3 hypotype strain of H1 serotype specific primer and H3 serotype specific primer; And the H1 serotype specific primer is template with the cDNA of H3 hypotype strain, can not obtain effective amplified production, and the H3 primer can not be the amplification that template is carried out effective fragment with the cDNA of H1 hypotype strain.This explanation, H1 and H3 serotype specific primer do not intersect amplified reaction mutually, have the specificity of hypotype.
2.4PCR product order-checking:
Add 3 μ l Exo SAP-IT in the PCR product, carry out digestion reaction: 37 ℃ 20 minutes, 80 ℃ 15 minutes.After digesting after product and Sequencing Mix mixing, carry out sequencing reaction.As sequencing primer, the reaction of H3 somatotype uses H3F731 as sequencing primer to the reaction of H1 somatotype with H1R1062.
2.5 software analysis:
After two sections products order-checking for PCR, the two is compared and splice with the ssearch of Fasta bag the inside.Spliced sequence and consensus sequence mix, utilize the Clustalw(Bioinformatics of parallelization, 2003,19,1585-1586), these sequences are carried out multiple sequence connection join (multiple sequence alignment), according to the clustering tree of drawing (Fig. 2) judge the dependent of dead military hero that checks order in which kind of influenza virus.
By the sequence similarity cluster, can be referred to the virus strain of test usefulness accurately in the branch of anticipation.Fig. 5 is the somatotype situation after influenza A virus H1 and the H3 changeable zone order-checking.A, B subgraph are H1 and the H3 branches of classification tree among Fig. 2.3～8(3 in this experiment be people H1 hypotype type strain cDNA, 4 for people H1 subtype virus strain cDNA, 5 for pig H1N1 hypotype type strain cDNA, 6 for pig H1N1 hypotype HA gene clone plasmid, 7 for people H3 hypotype type strain cDNA, 8 for pig H3N2 hypotype HA gene clone plasmid) virus strain sequence and 300 benchmark HA sequences carry out cluster analysis as can be seen, 3～8 virus strain can be referred on the clustering tree position of anticipation exactly.Therefore, use this series primer and not only can make a definite diagnosis this popular novel H1N1virus, and might make a definite diagnosis other people the seasonal influenza A virus HA hypotype (as the H3 type) of susceptible.In addition, this method also should be found the new variant in influenza A virus HA hypotype, by the evolution rule of this type of " newly " of bioinformatic analysis variation, has potential using value for understanding and viral variation and the fashion trend of prediction; Long-term accumulation data and research may provide scientific basis for country formulates effective prevention and control measure.
1. material and instrument be with embodiment 1,
2. primer is synthetic: finished by the super generation bio tech ltd in Shanghai
The cDNA product of people source H1 and H3 hypotype standard strain is carried out doubling dilution, be respectively 10
-2, 10
-3, 10
-4, 10
-5, 10
-6And get 2ul respectively as template, choose H1 somatotype upstream primer (SEQ ID NO:1 and SEQ ID NO:2 mix primer) and downstream primer SEQ ID NO:3 respectively, and H3 serotype specific primer upstream primer SEQ ID NO:4 and downstream primer (SEQ ID NO:5 and SEQ ID NO:6 mix primer).
The PCR reaction system
The PCR reaction conditions of above system: 94 ℃ of pre-sex change 5min; Carry out 35 circulating reactions by following condition then, 94 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 30s; Last 72 ℃ are extended 5m; Agarose gel electrophoresis check PCR product.Electrophorogram such as Fig. 4.
Test-results: as shown in Figure 4,10
-5Extent of dilution, visible clear target stripe still, and can obtain sequencing result clearly, point out this group primer to have higher sensitivity.
Claims (1)
1. primer that detects influenza virus A hominis H3 hypotype, it is characterized in that described primer for amplification influenza A virus H3 target nucleic acid sequence is right: comprise by the nucleic acid primer of nucleotide sequence shown in the SEQ ID NO:4, by the nucleic acid primer of nucleotide sequence shown in the SEQ ID NO:5 with by the nucleic acid primer of nucleotide sequence shown in the SEQ ID NO:6.
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