CN105200161A - Real-time fluorescence RT-PCR method for quantitatively detecting human-infected avian influenza virus (A/H7N9) - Google Patents
Real-time fluorescence RT-PCR method for quantitatively detecting human-infected avian influenza virus (A/H7N9) Download PDFInfo
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Abstract
The invention belongs to the field of virus detection, and particularly relates to a real-time fluorescence RT-PCR method for quantitatively detecting a human-infected avian influenza virus (A/H7N9). According to comparative analysis with an existing human-infected H7N9 virus in a Genbank database, it is shown that a synthesized and designed primer and probe are completely conservative to the epidemic H7N9 virus. According to the method, a higher detection sensitivity is achieved on the condition that five genes are copied in each reaction, a cross reaction on 17 common respiratory tract infection viruses does not occur, and the H7N9 can be specifically detected; contrast detection between the method and a national CDC detection method is conducted on 80 clinical specimens, double-check is conducted on 13 inconsistent specimens by adopting rRT-PCR recommended by WTO for detecting an influenza A virus, detection results are all positive, and it is shown that the detection results are accurate and reliable. The method can conduct quantitative analysis on the copy number of the H7N9 virus in the clinical specimens, the specificity, the sensitivity and the accuracy are achieved, the method is suitable for large-scale detection of the clinical specimens, and an important basis is provided for optimizing clinical treatment strategies.
Description
Technical field
The invention belongs to field of virus detection, relate to people and infect avian influenza virus (A/H7N9) detection method, be specifically related to a kind of method that detection by quantitative people infects the real-time fluorescence RT-PCR of avian influenza virus (A/H7N9).
Background technology
It is reported, in March, 2013 infects avian influenza virus (avianinfluenzavirus, AIV) H7N9 hypotype Chinese somewhere Late Cambrian people.Monitored more than 100 routine people by the end of October 24 and infected avian influenza virus (A/H7N9), wherein more than 40 examples are dead.This SARS Epidemic causes showing great attention to of WHO and whole world national governments.Report before this and mainly contain H5N1, H7N2, H7N3, H7N7, H9N2 and H10N7 by poultry direct contagion to the AIV hypotype of people; Research display, except Highly Pathogenic Avian Influenza Virus (HPAIV) (highlypathogenicavianinfluenza, HPAI) H5N1 hypotype can cause the infected up to beyond 60% mortality ratio, other several hypotypes mainly cause conjunctivitis or upper respiratory tract infection symptoms, only report that 1 routine people infected H7N7 death in Finland.The H7N9 virus of above-mentioned discovery is that the novel people of Late Cambrian in global range infects fowl source and course Influenza Virus.There are some researches show that this virus comes from the chicken and duck group of Yangtze River Delta Area, it can have effect spread in poultry but and not pathogenic, and bird has the people of close contact history easily to infect this virus.People infects H7N9 and generally shows as influenza-like symptom, and patient with severe symptoms's PD is rapid, shows as severe pneumonia, occurs adult respiratory distress syndrome, mortality ratio about about 30%.It is effective that early application neuraminidase inhibitor class anti-influenza virus medicament shows as H7N9 avian influenza, but treatment plan still needs to optimize.There is no the vaccine for H7N9 avian influenza virus at present both at home and abroad.
The method of real-time fluorescence RT-PCR is because of its quick (2 ~ 3 hours), and highly sensitive (1 ~ 10 copy DNA/ reaction), the advantages such as specificity is good have been widely used in clinical diagnosis and qualification.But, study according to people such as Hu and find that H7N9 virus medicament-resistant mutation can occur and causes viral persistence positive in Tamiflu therapeutic process, therefore need to pay close attention to the virus load variation tendency of patient in antiviral therapy process, to judge antiviral therapy effect in time, for clinical optimizing therapeutic regimen provides foundation.Although the people such as national influenza center and foreign study group VMCorman have established real-time fluorescence RT-PCR method and detected H7 and N9 gene respectively, all quantitative examination is not carried out to the virus load in clinical samples; In order to tackle SARS Epidemic, about the method for novel avian influenza A/H7N9 to detect in control department in the urgent need to setting up a kind of Rapid identification, be especially badly in need of setting up a kind of special, sensitive, reliable and be applicable to the H7N9 virus load monitoring method of clinical extensive detection.
Prior art related to the present invention has:
[1]GaoR,CaoB,HuY,etal..HumanInfectionwithaNovelAvian-OriginInfluenzaA(H7N9)Virus.NEnglJMed.2013.
[2]HuY,LuS,SongZ,etal..AssociationbetweenadverseclinicaloutcomeinhumandiseasecausedbynovelinfluenzaAH7N9virusandsustainedviralsheddingandemergenceofantiviralresistance.Lancet.2013；381:2273-79.
[3]CormanVM,EickmannM,LandtO,etal..Specificdetectionbyreal-timereverse-transcriptionPCRassaysofanovelavianinfluenzaA(H7N9)strainassociatedwithhumanspilloverinfectionsinChina.EuroSurveill.2013；18:20461.。
Summary of the invention
The object of the invention is the defect and the deficiency that overcome prior art, provide a kind of Rapid identification to detect the method for novel avian influenza A/H7N9, be specifically related to a kind of method that detection by quantitative people infects the real-time fluorescence RT-PCR of avian influenza virus (A/H7N9).Present method special, sensitive, reliable detection by quantitative people can infect avian influenza virus (A/H7N9), is applicable to clinical extensive monitoring and detects H7N9 virus load.
The present invention infects hemagglutinin gene (HA) full length sequence of H7N9 virus according to existing people current in Genbank, establish real-time fluorescence RT-PCR method (the quantitativereal-timeRT – PCR that a kind of detection by quantitative people infects avian influenza virus (A/H7N9) carrying capacity, qRT-PCR), both may be used for the quick diagnosis that people infects H7N9 virus, quantitative analysis can have been carried out to the virus load of sample simultaneously.Reference frame is provided further for people infects the clinical diagnosis of avian influenza virus (A/H7N9) and treatment and epidemic prevention and control.
The inventive method infects specificity fluorescent quantification PCR primer and the probe that the HA full length sequence design and synthesis of H7N9 detects H7N9 viral nucleic acid based on the people included in Genbank, special, sensitive, reliable detection by quantitative people can infect avian influenza virus (A/H7N9) doubtful sample
Concrete, detection by quantitative people of the present invention infects the method for the real-time fluorescence RT-PCR of avian influenza virus (A/H7N9), and it is characterized in that, it comprises step:
1) design of primers
Infect the HA full length sequence of H7N9 according to the people included in Genbank, use PrimerExpress3.0 design and synthesis to detect specificity fluorescent quantification PCR primer and the probe of H7N9 viral nucleic acid; The BLAST instrument on NCBI website and BioEdit software (version7.0.9) is used to analyze the conservative property of new design primer and probe.
Primer and probe are synthesized by Shanghai Invitrogen company.
2) virus culture, nucleic acid extraction, qRT-PCR
Choose the throat swab sample from different patient, be seeded in dog renal epithelial cell (mdck cell) and cultivate two generations more than, H7N9 positive culture carries out titration virus concentration (pfu/ml) by plaque-forming assay;
Virus genome RNA is extracted with QIAampViralRNAMiniKit (250) (Qiagen52906);
QRT-PCR adopts test kit (TakaraRR064A) to carry out;
PCR reaction conditions: 42 DEG C of reverse transcriptions, 10min; 95 DEG C of denaturation 30s; 95 DEG C of 10s, 60 DEG C of 45s, run 45 circulations, carry out fluorescent collecting at 60 DEG C.Reaction is at real-time fluorescence quantitative PCR instrument (AppliedBiosystems, ViiA
tM7 systems) on carry out;
Primer and probe sequence as shown in table 1;
Table 1 primer and probe sequence
* the H7N9 virus strain of reference is A/Shanghai/4664T/2013 (GenbankNo.KC853228)
3) T-A clone
RT-PCR adopts test kit (TakaraRR064A) to carry out;
Wherein, upstream primer HA-1144 (5 '-GGAATGATAGATGGNTGGTAYGG-3 '),
Downstream primer HA-Reverse (5 '-ATATCGTCTCGTATTAGTAGAAACAAGGGTGTTTT-3 '),
PCR reaction conditions: 42 DEG C of reverse transcriptions, 20min; 95 DEG C of denaturation 30s; 95 DEG C of 15s, 50 DEG C of 30s, 72 DEG C of 40s run 40 circulations, and 72 DEG C extend 5 minutes;
Reaction is carried out on EppendorfMastercyclerepgradientPCR instrument,
Get a RT-PCR positive products and carry out 1.5% agarose gel electrophoresis, adopt rubber tapping purification kit (Invitrogen, K2100-12) PCR primer is reclaimed, by T-A Cloning Kit (Takara, D101A) PCR primer is cloned into pMD18-T carrier, obtain cloned sequence sequence by the order-checking of M13 primer, then use BLAST instrument to compare to cloned sequence, confirm the exactness inserting object fragment.
4) typical curve of qRT-PCR quantitative analysis is set up
Adopt T-A cloned plasmids production standard product, set up H7N9 viral nucleic acid quantitation curves, first micro-ultraviolet spectrophotometer (Thermo, NanoDropND-2000C) carries out nucleic acid quantification to cloned plasmids, then carry out 10 times of template dilutions, make 10
9~ 10
3the standard substance of copies/ml are that template carries out qRT-PCR with standard substance, obtain typical curve.
5) sensitivity test of method
6) the specificity test of method
7) test with the coincidence rate of reference method
H7N9 viral nucleic acid rRT-PCR (real-timeRT-PCR) detection method (reference method) adopting Chinese CDC to provide carries out Parallel testing.Verified the accuracy of present method by the coincidence rate analyzing two kinds of method detected results, the rRT-PCR method of the detection influenza A virus that incongruent sample adopts WHO to provide is verified.
Above-mentioned detected result display:
(1) primer and probe sequence conservative Analysis
Choose the HA nucleotide sequence that people in Genbank database infects H7N9 virus strain, totally 16 strains, use ClustalW program in Bioedit software that upstream and downstream primer and probe are carried out alignment analysis with the HA nucleotide sequence chosen respectively.Result shows, the HA sequence of upstream primer H7-1507F (as shown in Figure 1A), downstream primer H7-1593R (as shown in Figure 1B) and probe H7-1540P (as shown in Figure 1 C) and existing H7N9 is mated completely, result shows, the sequence that the primer synthesized in present method and probe sequence are directed to the H7N9 virus of described burst is guarded completely;
(2) sensitivity of method
Adopt cloned plasmids standard substance (10
9~ 10
3copies/ml), carry out qRT-PCR, obtain amplification curve and typical curve (as Suo Shi Fig. 2 A, 2B), the slope of typical curve is-3.71, its linearly dependent coefficient R
2be 0.998; Determine the minimum virus being limited to 0.5pfu/ml of the detection of present method, by typical curve, determine that the most low energy of qRT-PCR method set up detects the viral nucleic acid of each reaction 5 gene copies;
(3) specificity of method
Use BLAST (primerBLAST) instrument to carry out compare of analysis in Genbank database, do not find that in upstream and downstream primer or probe and database, other species have the template matches possibility of pcr amplification; Use this PCR method to detect the respiratory tract infection virus strain that 17 strains are common, detected result is feminine gender (as shown in 2C).
(4) methods comparison and clinical sample detected result
The H7N9 virus real-timeRT-PCR method adopting national CDC to provide is verified, result shows, the positive coincidence rate of qRT-PCR method of the present invention is 96.6% (28/29), negative match-rate is 76.5% (39/51), and overall coincidence rate is 83.8% (67/80) (as shown in table 2); Misfitting result to 13 parts adopts the method for WHO to detect, and the method positive coincidence rate that result shows present method and WHO reaches 92.3% (12/13) (as shown in table 3).
The detected result statistics of table 2qRT-PCR and reference method (rRT-PCR of national CDC)
Table 3 does not meet the rRT-PCR detected result statistics that sample adopts the detection InfluenzaA of WHO
| Sample situation | Number of samples | qRT-PCR(+) | Reference method (+) | WHO method (+) |
| False positive sample | 12 parts | 12 parts | 0 part | 12 parts |
| Undetected sample | 1 part | 0 part | 1 part | 1 part |
Method of the present invention, carries out sequence alignment analysis by infecting H7N9 virus with existing people in Genbank database, and the primer of display present method compounding design and probe are guarded completely for described popular H7N9 virus; By sensitivity and the display of specificity experiments result, present method has higher detection sensitivity (each reaction 5 gene copies), and the method (comprises sH1N1 to 17 kinds of common respiratory tract infection viruses, sH3N2, the influenza A hypotype of the infection people that 2009pandemicH1N1 is common at present) there is no cross reaction, H7N9 can be detected specifically; The detection method that we law and state CDC provides carries out comparison and detection at 80 parts of clinical samples, and adopt WHO to recommend to detect the rRT-PCR of influenza A virus to misfit sample to 13 parts and check, detected result is the positive, shows that the detected result of present method is accurately and reliably.
The inventive method, relative to the domestic and international method for detecting H7N9, can be carried out quantitative analysis to H7N9 viral copy number in clinical sample, and having good specificity, sensitivity and accuracy, being applicable to the extensive detection of clinical sample.In the present invention, adopt present method to accept to carry out in Tamiflu treatment patient treating the H7N9 virus load monitoring of front and back in District of Shanghai 14 example, detected result has pointed out the generation of Tamiflu resistance exactly, for the optimization of clinical management strategy provides important evidence.
Accompanying drawing explanation
Fig. 1 shows the conservative property that Bioedit analyzes primer and probe sequence, wherein,
(A) upstream primer H7-1507F and 16 strains 2013 are from Fujian China, Hangzhou, Nanjing, Nanchang, the comparison result of the H7 sequence of the H7N9 that Wuxi and Taiwan find;
(B) downstream primer H7-1593R and 16 strains 2013 are from Fujian China, Hangzhou, Nanjing, Nanchang, the comparison result of the H7 sequence of the H7N9 that Wuxi and Taiwan find;
(C) probe H7-1540P and 16 strains 2013 are from Fujian China, Hangzhou, Nanjing, Nanchang, the comparison result of the H7 sequence of the H7N9 that Wuxi and Taiwan find.
Fig. 2 shows sensitivity and the specificity experiments result of Real-timeRT-PCR method, wherein,
(A) cloned plasmids standard substance (10 are used
9~ 10
3copies/ml) fluorescence curve of pcr amplification acquisition is carried out;
(B) cloned plasmids standard substance (10 are used
9~ 10
3copies/ml) typical curve (the wherein log10 value of the copy number of DNA in X representative sample, the Ct value of Y representative amplification) of pcr amplification acquisition is carried out;
(C) (positive quality control product is 10 to use 17 kinds of respiratory tract infection common disease strains to carry out the fluorescence curve of pcr amplification
7copies/ml clones standard substance).
Embodiment
Embodiment 1
1 materials and methods
The collection of 2.1 clinical samples
During 4 to June in 2013, have collected 18 routine H7N9 infected patients totally 684 parts of clinical samples from Shanghai Public Health Clinical Center, wherein throat swab 253 parts, sputum 30 parts, urine 228 parts, 173 parts, ight soil.
2.2 design of primers
Infect the HA full length sequence of H7N9 according to the people included in Genbank, use PrimerExpress3.0 design and synthesis to detect specificity fluorescent quantification PCR primer and the probe of H7N9 viral nucleic acid.The BLAST instrument on NCBI website and BioEdit software (version7.0.9) is used to analyze the conservative property of new design primer and probe.Primer and probe are synthesized by Shanghai Invitrogen company.
2.3 virus culture, nucleic acid extraction, qRT-PCR
Choose 10 parts of throat swab samples from different patient, be seeded in dog renal epithelial cell (mdck cell) and cultivate two generations more than.Mdck cell is cultivated and is adopted the MEM substratum containing the calf serum of 10% and the mycillin of 5% and amphotericin B solution to cultivate.The MEM substratum containing the calf serum of 2% and the mycillin of 5% and amphotericin B solution is adopted to cultivate after virus inoculation.H7N9 positive culture carries out titration virus concentration (pfu/ml) by plaque-forming assay.
Virus genome RNA is extracted with QIAampViralRNAMiniKit (250) (Qiagen52906).140 μ l samples are added 560 μ l containing in the AVL of polyA (10 μ g/ml), place 10min for 15 ~ 25 DEG C; Add 560 μ l dehydrated alcohols, put upside down the rear brief centrifugation of mixing; Adsorb RNA with pillar, add AW1 solution 500 μ l, centrifugal 1 minute of 8000rpm; Add AW2 solution 500 μ l again, centrifugal 1 minute of 8000rpm; Centrifugal 2 minutes of 13000rpm; Finally use 60 μ lAVE eluted rnas.
QRT-PCR adopts test kit (TakaraRR064A) to carry out, 2 × BufferMixIII12.5 μ l is comprised in 25 μ l reaction solutions, TaKaRaExTaqHS (5U/ μ l) 0.5 μ l, PrimeScriptRTEnzymeMix II 0.5 μ l, upstream primer H7F8pmol, downstream primer H7R8pmol, probe H7Pb4pmol, RNA template 2.5 μ l, DEPC water supplies 25 μ l.PCR reaction conditions: 42 DEG C of reverse transcriptions, 10min; 95 DEG C of denaturation 30s; 95 DEG C of 10s, 60 DEG C of 45s, run 45 circulations, carry out fluorescent collecting at 60 DEG C.Reaction is at real-time fluorescence quantitative PCR instrument (AppliedBiosystems, ViiA
tM7 systems) on carry out, primer and probe sequence as shown in table 1.
2.4T-A clone
RT-PCR adopts test kit (TakaraRR064A) to carry out, 2 × BufferMixIII25 μ l is comprised in 50 μ l reaction solutions, TaKaRaExTaqHS (5U/ μ l) 1 μ l, PrimeScriptRTEnzymeMix II 1 μ l, upstream primer HA-1144 (5 '-GGAATGATAGATGGNTGGTAYGG-3 ') 10pmol, downstream primer
HA-Reverse (5 '-ATATCGTCTCGTATTAGTAGAAACAAGGGTGTTTT-3 ') 10pmol, RNA template 5 μ l, DEPC water supplies 50 μ l.PCR reaction conditions: 42 DEG C of reverse transcriptions, 20min; 95 DEG C of denaturation 30s; 95 DEG C of 15s, 50 DEG C of 30s, 72 DEG C of 40s run 40 circulations, and 72 DEG C extend 5 minutes.Reaction is carried out on EppendorfMastercyclerepgradientPCR instrument.
Get a RT-PCR positive products 50 μ l and carry out 1.5% agarose gel electrophoresis.Rubber tapping purification kit (Invitrogen, K2100-12) is adopted to reclaim the PCR primer of 664bp size.By T-A Cloning Kit (Takara, D101A), PCR primer is cloned into pMD18-T carrier.Obtain cloned sequence sequence by the order-checking of M13 primer, then use BLAST instrument to compare to cloned sequence, confirm the exactness inserting object fragment.
2.5 typical curves setting up qRT-PCR quantitative analysis
Adopt T-A cloned plasmids production standard product, set up H7N9 viral nucleic acid quantitation curves.First micro-ultraviolet spectrophotometer (Thermo, NanoDropND-2000C) carries out nucleic acid quantification to cloned plasmids, then carries out 10 times of template dilutions, makes 10
9~ 10
3the standard substance of copies/ml are that template carries out qRT-PCR with standard substance, obtain typical curve.The sensitivity test of 2.6 methods
Choose the H7N9 culture of strain titration, carry out 10 times with virus-culturing fluid and be diluted to 10-
3pfu/ml virus titer.Extract the virus genome RNA of all dilution cultures.Every increment originally adopts newly-established qRT-PCR to carry out repetition and detects for 20 times.Arrange statistic mixed-state result from low to high by virus titer, first is occurred the virus titer being not less than 90% positive rate (>=18 positive amplification) is as possible lowest detection titre A.And then by this dilution culture, doubly dilute 3 times successively with virus-culturing fluid 2, obtaining virus titer is respectively the dilution of A/2, A/4, A/8.Adopt newly-established qRT-PCR to carry out repetition after extracting three dilution RNA to detect for 20 times, determine the lowest detection virus titer of this qRT-PCR method, virus-culturing fluid by dilute twice arranges statistic mixed-state result from low to high according to titre, and first virus titer occurring being not less than 90% positive rate is the lowest detection virus titer (pfu/ml) of this PCR.The lowest detection viral copy number (copies/ml) obtaining qRT-PCR method is calculated again by typical curve.
The specificity test of 2.7 methods
Have chosen the common virus that 17 strain human respiratories infect, comprise the strain of seasonal H1N1 influenza virus 2, the strain of seasonal H3N2 influenza virus 2,2009PandemicH1N1 influenza virus 2 strain, each 1 strain of parainfluenza virus 1 ~ 3 type, Type B influenza virus 2 strain, each 1 strain of respiratory syncytial virus A/B type, adenovirus 1 strain, bocavirus 1 strain, metapneumovirus 1 strain, coronavirus (229E) 1 strain, to verify the specificity of this PCR method.QIAampViralRNAMiniKit (250) (Qiagen52906) is adopted to extract the geneome RNA of these virus strain.With these viral RNAs for template chooses cloned plasmids (10 simultaneously
7copies/ml) for positive reference substance carries out qRT-PCR, amplification curve is observed.
2.8 test with the coincidence rate of reference method
Choose the 684 parts of H7N9 case samples collected for 2013, carry out nucleic acid extraction and adopt this rebuilding method to detect; Therefrom randomly draw 80 parts of sample of nucleic acid, Chinese CDC is adopted to provide H7N9 viral nucleic acid rRT-PCR (real-timeRT-PCR) detection method (reference method) in this laboratory to carry out Parallel testing, verified the accuracy of this rebuilding method by the coincidence rate analyzing two kinds of method detected results, the rRT-PCR method of the detection influenza A virus that incongruent sample adopts WHO to provide carries out verifying (http://www.who.int/csr/resources/publications/swineflu/realtime ptpcr/en/);
Upper detected result display:
(1) primer and probe sequence conservative Analysis
Choose the HA nucleotide sequence that people in Genbank database infects H7N9 virus strain, totally 16 strain (GenbankNO.KC994453, KC853766, KF001513, KF001516, KF278745, KF226113, KF261988, KC896774, KF021597, KC853228, KF018045, KF018053, KF034911, KF034919, KC885956, KF055470).Use ClustalW program in Bioedit software that upstream and downstream primer and probe are carried out alignment analysis with the HA nucleotide sequence chosen respectively, result shows, the HA sequence of upstream primer H7-1507F (see Figure1A), downstream primer H7-1593R (see Figure1B) and probe H7-1540P (see Figure1C) and existing H7N9 is mated completely, according to above-mentioned analysis, the sequence that the primer of the present invention's synthesis and probe sequence are directed to the H7N9 virus of described burst is guarded completely;
(2) sensitivity of method
Adopt cloned plasmids standard substance (10
9~ 10
3copies/ml), carry out qRT-PCR, obtain amplification curve and typical curve (shown in Fig. 2 A and 2B), the slope of typical curve is-3.71, its linearly dependent coefficient R
2be 0.998;
Choosing a strain virus titre is 2*10
5the H7N9 virus culture of pfu/ml, carry out 10 times dilution 8 times after, the nucleic acid extracting all dilutions carries out qRT-PCR, result shows, and arranges from low to high, occur more than 18 times positive findingses at 2pfu/ml by virus titer, again the culture of 2pfu/ml is carried out 2 times dilution 3 times afterwards extract nucleic acid detect, result shows, and arranges from low to high, occur more than 18 times positive findingses at 0.5pfu/ml by virus titer; Determine the minimum virus being limited to 0.5pfu/ml of the detection of present method, determine that the most low energy of qRT-PCR method set up detects the viral nucleic acid of each reaction 5 gene copies by typical curve;
(3) specificity of method
The specificity of the PCR method that the present invention sets up, primaryly to be assessed by two aspects: first, after design completes primer and probe, use BLAST (primerBLAST) instrument to carry out compare of analysis in Genbank database, do not find that in upstream and downstream primer or probe and database, other species have the template matches possibility of pcr amplification; Secondly, use this PCR method to detect the respiratory tract infection virus strain that 17 strains are common, detected result is feminine gender (as shown in Figure 2 C).
(4) methods comparison and clinical sample detected result
To 684 parts of clinical samples nucleic acid extraction, the qRT-PCR method set up is adopted to carry out amplification experiment, wherein 109 parts of H7N9 positives, 575 parts of H7N9 feminine genders; 40 parts of nucleic acid have respectively been randomly drawed respectively from positive sample and ' negative ' specimens, the H7N9 virus real-timeRT-PCR method adopting national CDC to provide is verified, result shows, the positive coincidence rate of the qRT-PCR method that the present invention sets up is 96.6% (28/29), negative match-rate is 76.5% (39/51), and overall coincidence rate is 83.8% (67/80) (as shown in table 2); Misfitting result to 13 parts adopts the method for WHO to detect, and the method positive coincidence rate that result shows present method and WHO reaches 92.3% (12/13) (as shown in table 3).
Claims (8)
1. detection by quantitative people infects a method for the real-time fluorescence RT-PCR of avian influenza virus (A/H7N9), and it is characterized in that, it comprises step:
1) design of primers;
2) virus culture, nucleic acid extraction, qRT-PCR;
3) T-A clone;
4) typical curve of qRT-PCR quantitative analysis is set up;
5) sensitivity test of method;
6) the specificity test of method;
7) test with the coincidence rate of reference method.
2., by method according to claim 1, it is characterized in that, described step 1) in,
Infect the HA full length sequence of H7N9 according to the people included in Genbank, use PrimerExpress3.0 design and synthesis to detect specificity fluorescent quantification PCR primer and the probe of H7N9 viral nucleic acid; The BLAST instrument on NCBI website and BioEdit software (version7.0.9) is used to analyze the conservative property of new design primer and probe.
3., by method according to claim 1, it is characterized in that, described step 2) in,
Choose the throat swab sample of different patient, be seeded in dog renal epithelial cell (mdck cell) and cultivate two generations more than, H7N9 positive culture carries out titration virus concentration (pfu/ml) by plaque-forming assay;
Virus genome RNA is extracted with QIAampViralRNAMiniKit (250) (Qiagen52906);
QRT-PCR adopts test kit (TakaraRR064A) to carry out;
PCR reaction conditions: 42 DEG C of reverse transcriptions, 10min; 95 DEG C of denaturation 30s; 95 DEG C of 10s, 60 DEG C of 45s, run 45 circulations, carry out fluorescent collecting at 60 DEG C, react at real-time fluorescence quantitative PCR instrument (AppliedBiosystems, ViiA
tM7 systems) on carry out;
Primer and probe sequence as shown in table 1;
Table 1
4., by method according to claim 1, it is characterized in that, described step 3) in,
RT-PCR adopts test kit (TakaraRR064A) to carry out;
Wherein, upstream primer HA-1144 (5 '-GGAATGATAGATGGNTGGTAYGG-3 '),
Downstream primer HA-Reverse (5 '-ATATCGTCTCGTATTAGTAGAAACAAGGGTGTTTT-3 '),
PCR reaction conditions: 42 DEG C of reverse transcriptions, 20min; 95 DEG C of denaturation 30s; 95 DEG C of 15s, 50 DEG C of 30s, 72 DEG C of 40s run 40 circulations, and 72 DEG C extend 5 minutes;
Reaction is carried out on EppendorfMastercyclerepgradientPCR instrument,
Get RT-PCR positive products and carry out 1.5% agarose gel electrophoresis, adopt rubber tapping purification kit (Invitrogen, K2100-12) PCR primer is reclaimed, by T-A Cloning Kit (Takara, D101A) PCR primer is cloned into pMD18-T carrier, obtain cloned sequence sequence by the order-checking of M13 primer, then use BLAST instrument to compare to cloned sequence, confirm the exactness inserting object fragment.
5., by method according to claim 1, it is characterized in that, described step 4) in,
Adopt T-A cloned plasmids production standard product, set up H7N9 viral nucleic acid quantitation curves, first micro-ultraviolet spectrophotometer (Thermo, NanoDropND-2000C) carries out nucleic acid quantification to cloned plasmids, then carry out 10 times of template dilutions, make 10
9~ 10
3the standard substance of copies/ml are that template carries out qRT-PCR with standard substance, obtain typical curve.
6., by method according to claim 5, it is characterized in that, described step 4) in, the slope of typical curve is-3.71, its linearly dependent coefficient R
2be 0.998.
7., by method according to claim 1, it is characterized in that, described step 5) in,
Detect the minimum virus being limited to 0.5pfu/ml, determine that the most low energy of qRT-PCR method set up detects the viral nucleic acid of each reaction 5 gene copies by typical curve.
8. by method according to claim 1, it is characterized in that, described step 7) in, the positive coincidence rate of described method is 96.6% (28/29), negative match-rate is 76.5% (39/51), and overall coincidence rate is 83.8% (67/80); 92.3% (12/13) is reached with the method positive coincidence rate of WHO.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107586776A (en) * | 2016-07-05 | 2018-01-16 | 上海市计量测试技术研究院 | Suitable for the plasmid control material of H7N9 avian influenza virus real-time fluorescence quantitative PCR detection |
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| CN106282414B (en) * | 2016-10-08 | 2020-01-07 | 深圳市检验检疫科学研究院 | Reagent for detecting H5N6 avian influenza virus, detection method and application |
| CN106350611A (en) * | 2016-10-28 | 2017-01-25 | 深圳市检验检疫科学研究院 | Reagent for detecting H5N8 avian influenza virus as well as detecting method and application thereof |
| CN107523649A (en) * | 2017-08-30 | 2017-12-29 | 上海伯杰医疗科技有限公司 | H7N9 subtype influenza virus genome sequencing methods |
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