CN101962690B - Nucleotide sequences and kit for testing H1 and H3 subtypes of influenza viruses - Google Patents
Nucleotide sequences and kit for testing H1 and H3 subtypes of influenza viruses Download PDFInfo
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Abstract
The invention provides a group of nucleotide sequences and a kit for testing H1 and H3 subtypes of influenza viruses. The nucleotide sequences for testing H1 and H3 subtypes of influenza viruses are shown by the sequence table from SEQ ID NO:1 to SEQ ID NO:6, wherein the SEQ ID NO:1 and the SEQ ID NO:2 are respectively a sense primer and an antisense primer for testing the HA gene of H1 subtype of influenza viruses, the SEQ ID NO:3 is an LNA modified fluorescent probe for testing the HA gene of H1 subtype of influenza viruses, the SEQ ID NO:4 and the SEQ ID NO:5 are respectively a sense primer and an antisense primer for testing the HA gene of H3 subtype of influenza viruses, and the SEQ ID NO:6 is a fluorescent probe for testing the HA gene of H3 subtype of influenza viruses. The invention also provides a kit testing H1 and H3 subtypes of influenza viruses, which contains the primers and the probes. The kit has the advantages of quickness, high sensitivity, particularity, commonality and repeativeness, and is difficult to be polluted.
Description
Technical field
The present invention relates to detect the nucleotide sequence and the test kit of H1 and H3 subtype influenza virus; Especially finger detects nucleotide sequence and test kit based on the short probe substance and the double fluorescent RT-PCR of LNA and MGB modification fast; Be not only applicable to H1 and the viral accurate detection of H3 subtype influenza in the different animals tissue samples; Also applicable to different animals biopsy sample (being mainly the swab sample) H1 and the viral detection of H3 subtype influenza; Can be used for the examination of animal H1 and H3 subtype influenza virus, entry and exit quarantine etc. belong to inspection and quarantine field.
Background technology
Since 1934 isolate influenza A virus first, caused repeatedly the little popular of being very popular of global humans and animals and the interval that is very popular by the influenza virus of H1 or H3 hypotype.H1 and H3 subtype influenza virus infection host range are wide, and main infection host comprises the people, fowl, pig, horse and other multiple animals.And the some of them strain has the ability of between multiple animal, propagating, and for example causes that the cause of disease of many big influenza influenzas on the human history all can be propagated between people, pig.Betide the equine influenza strain (A/ horse/Jilin/1/89) of isolated H3N8 hypotype the equine influenza epidemic situation of China from 1989 with nineteen ninety, 6 gene fragments of this strain are fowl source fragment.Global pandemic H1N1virus in 2009 is a new type influenza variant, but studies Mammalss such as verified this strain natural infection pig, dog cat at present.
Adopt TaqMan fluorescence probe RT-PCR that influenza virus H1 and H3 hypotype are carried out the prefered method that fast typing detects has become the examination of present influenza virus rapid detection at present.But TaqMan method general requirement probe Tm value is apparently higher than primer Tm value, and therefore probe sequence is often longer when design primer probe.But for influenza virus; Between the virus sequence of different infection hosts and the HA sequence of novel variation strain and classical strains have very big-difference, this makes a lot of H1 that adopt long probes and H3 nucleic acid detection methods limited to aspect the H1 of detection different sources and the H3 subtype virus.
Adopt short probe can effectively solve H1 and the relatively poor problem of H3 subtype influenza virus classifying method versatility that the strain variation causes.
But adopt short probe must under the situation that does not change sequence, improve the Tm value of probe.In the recent period, (locked nucleic acid LNA) has caused people's extensive concern to lock nucleic acid in the biological study field as a kind of nucleotide derivative of novelty; It is a kind of special double-ring nucleotide derivative; Contain in the structure one or more 2 '-O, 4 '-C-methylene radical-β-D-ribofuranose nucleic acid monomer, 2 of ribose '-O position and 4 '-the C position forms oxygen methylene bridge, sulphur methylene bridge or amine methylene bridge through different shrink effects; And connect into annular; This annular bridge has locked the N configuration of furanose C3 '-Nei type, has reduced the snappiness of ribose structure, has increased the stability of phosphate backbone local structure.DNA, RNA there are good recognition capability and powerful avidity.LNA and DNA, RNA complementary two strands have very strong thermostability (Δ Tm=3～8 ℃); Stability with anti-3 ' deoxynucleotide enzyme liberating; Compound method is simple relatively, and the few nucleic acid chains available amino end of the LNA that partially or completely modifies phosphoric acid method is synthetic on automatic dna synthesizer.
Through the viral HA gene of the H1 subtype influenza of different sources is carried out on the basis of sequence alignment, find that there is the conserved regions of 12nt in H1 subtype influenza virus HA gene afterbody, is fit to detect through being used for fluorescence RT-PCR as short probe after the LNA modification.
But when the HA gene to H3 subtype influenza virus carries out sequence alignment; Discovery can utilize the short probe of 14nt degeneracy to set up the fluorescence RT-PCR method; Annex base owing to exist, therefore be employed in the Tm value that probe 3 ' modification MGB (Minor Groove Binder) improves probe.
The present invention provides short probe, degenerated primer and the test kit based on LNA and MGB viral with the H3 subtype influenza to H1 first with substance with dual short fluorescence probe RT-PCR technology based on LNA and MGB is applied to H1 and H3 subtype influenza virus detects.
Summary of the invention
First purpose of the present invention provides strong, the highly sensitive detection of a group-specific to H1 and H3 subtype influenza viral nucleotide sequences, comprises primer sequence and based on the short probe sequence of LNA and MGB.
Another object of the present invention provides the substance and the double check test kit of detection H1 quick, accurate, easy to use and H3 subtype influenza virus.
For realizing above-mentioned purpose, the present invention adopts following technical scheme:
Select H1 and H3 subtype influenza virus HA gene coding region particular sequence as target region, on the basis of multiple sequence comparison, carry out primer and probe design.Primer length is about 20 bases, and GC content is 50%-60%, no secondary structure and repeatability in the primer, and with the interior no complementary sequence of primer, the melting temperature (Tm) between primer (Tm value) differs less than 5 ℃ between primer.The length of probe adopts LNA or MGB to modify about 12～14 bases, and the mark fluorescent group.
1 group of nucleotide sequence that detects influenza virus H1 hypotype, as follows:
1)5’-CAG?ATT?YTG?GCG?ATC?TAY?TC-3′(SEQ?ID?NO：1)
2)5’-GAC?CCA?TTA?GAR?CAC?ATC?CAG-3’(SEQ?ID?NO：2)
Y represents pyrimidine, is T or C here; R represents purine, is A or G here;
3)5’-[HEX]-CCCC
[TAMRA]-3’(SEQ?ID?NO：3)
1 group of nucleotide sequence that detects influenza virus H3 hypotype, as follows
4)5’-TGG?ATT?TCM?TTY?GCC?ATA?TCA?T-3′(SEQ?ID?N?O：4)
5)5’-GCA?AAT?GTT?GCA?YCT?RAT?RTT?G-3’(SEQ?ID?NO：5)
6)5’-[FAM]-TGG?CAR?GCC?CAC?AT-[MGB]-3’(SEQ?ID?NO：6)
Y represents pyrimidine, is T or C here; R represents purine, is A or G here; M is A or C here;
Wherein sequence 1) and 2) be respectively the sense primer and the antisense primer that detect H1 subtype influenza virus HA gene; Sequence 3) the short probe of modifying for the LNA that detects H1 subtype influenza virus HA gene of fluorescence; Base with italic is represented is modified with LNA; Promptly the 5th, 7,9,11 base is modified with LNA, 5 ' end mark report fluorophor HEX (5-hexachloro-resorcinolphthalein) of probe, 3 ' end mark cancellation fluorophor TAMRA; Sequence 4) and 5) be respectively the sense primer and the antisense primer that detect H3 subtype influenza virus HA gene; Sequence 6) the short probe of modifying for the MGB that detects H3 subtype influenza virus HA gene of fluorescence; 5 ' end mark report fluorophor FAM (6-carboxy-resorcinolphthalein) of probe, 3 ' end mark MGB group.
We adopt TaqMan fluorescent PCR detection technique to set up H1 and H3 subtype influenza virus substance and double check method, the various conditions of reaction are optimized, comprising the screening of primer probe, Mg
2+The optimization of working concentration, the optimization of primer concentration and probe concentration obtains following test kit.
Detect H1 and H3 subtype influenza virus substance and double check test kit, composed of the following components:
1) lysate: available from INVITROGEN company, carry out packing, 2 bottle/boxes by the 15ml/ bottle.
2) RT-PCR reaction solution, table 1 are H1 substance reaction solution prescription; Table 2 is a H3 substance reaction solution prescription;
Table 3 is for detecting the double reaction liquid formula of H1 and H3.
Table 1H1 substance reaction solution prescription
| Component | |
| 5 * RT-damping fluid | 0.5 * RT- |
| 10 * PCR damping fluid | 0.5 * PCR damping fluid |
| 25mM?MgCl 2 | 1.5mmol/L?MgCl 2 |
| 2.5mM?dNTP | 0.2mmol/L?dNTP |
| The H1 sense primer | 0.3μmol/L |
| The H1 antisense primer | 0.3μmol/L |
| The short probe (H1 fluorescent probe) of H1LNA | 0.3μmol/L |
Table 2H3 substance reaction solution prescription
| Component | |
| 5 * RT- |
1 * RT-damping fluid |
| 25mM?MgCl 2 | 4.0mmol/L?MgCl 2 |
| 2.5mM?dNTP | 0.05mmol/L?dNTP |
| The H3 sense primer | 0.4μmol/L |
| The H3 antisense primer | 0.4μmol/L |
| The short probe (H3 fluorescent probe) of H3MGB | 0.2μmol/L |
Table 3 detects the double reaction liquid formula of H1 and H3 subtype influenza virus
| Component | |
| 5 * RT-damping fluid | 1.0 * RT-damping fluid |
| 25mM?MgCl 2 | 4.0mmol/L?MgCl 2 |
| 2.0mM?dNTP | 0.05mmol/L?dNTP |
| The H1 sense primer | 0.4μmol/L |
| The H1 antisense primer | 0.4μmol/L |
| The short probe of H1LNA | 0.3μmol/L |
| The H3 sense primer | 0.4μmol/L |
| The H3 antisense primer | 0.4μmol/L |
| The short probe of H3MGB | 0.3μmol/L |
10 * PCR damping fluid, 25mM MgCl2 purchase the company in Promega; 5 * RT-damping fluid; 2.5mM dNTP is available from the precious biotinylated biomolecule in Dalian Engineering Co., Ltd; Primer and probe all entrust Dalian precious biotinylated biomolecule Engineering Co., Ltd synthetic; Consisting of of 10 * PCR damping fluid wherein: 500mmol/L KCl, 100mmol/L Tris-HCl (pH9.0,25 ℃), 1.0%Triton X-100.5 * RT-damping fluid consists of 375mmol/L KCl, 15mmol/L MgCl
2, 50mmol/L DTT, 250mmol/L Tris-HCl (pH8.3,25 ℃).
3) RT-PCR enzyme granulate, available from AMERCIA company, 12/box.
4) Taq archaeal dna polymerase 5U/ μ L is available from Promega company.
5) DEPC water; With twice distillation of tap water; Through Millipore MILLI-Q PF PLUS pure water appearance purifying, collect the water of resistivity >=18.0M Ω .cm, the Millipore-Q purified water add DEPC to final concentration be 0.1%; 37 ℃ of stir process 12hr, 15lbf/in2 (1.034 * 105Pa) high pressure steam sterilizations 15 minutes.
6) negative control: the negative tissue sample of influenza virus, process 20% suspension with 0.01mol/L pH7.2PBS BS, 70 ℃ act on 1 hour.
7) positive control: be the non-infectious RNA fragment of in-vitro transcription.Reclaim H1 subtype influenza virus of A/Swine/2003 (H1N1) HA and H3 subtype influenza virus of A/Swine/2003/GD (H3N2) HA gene RT-PCR amplified production; Obtain highly purified H1 hypotype and H3 subtype influenza virus HA gene coding region sequence gene respectively; Length is 1701bp; (available from Promega company) is connected with the pGEM-T carrier, transforms the JM109 competent cell, alkaline lysis method of extracting plasmid DNA; Cut through PCR and enzyme and to identify that the back obtains positive recombinant plasmid, distinguishes called after pGEM-H1 and pGEM-H3.Plasmid with purifying is a template, after linearization for enzyme restriction, carries out in-vitro transcription with the Ribo MAXTM Large Scale RNA Production System-T7 test kit of Promega company; The in-vitro transcription product is removed dna profiling wherein after after measuring after the TRIZOL extraction, promptly obtain preparing H1 and the required RNA positive reference substance mother liquor of H3 subtype influenza virus-positive reference substance with DNase.
H1 subtype influenza virus of A/Swine/2003 (H1N1) 1701bp HA purpose fragment gene sequence has the nucleotide sequence shown in the sequence table SEQ ID NO:7.
H3 subtype influenza virus of A/Swine/2003/GD (H3N2) 1701bp HA purpose fragment gene sequence has the nucleotide sequence shown in the sequence table SEQ ID NO:8.：
Synthetic primer and probe are HPLC analyze, as the ratio that obtains single absorption peak collection of illustrative plates and measure OD260nm/OD280nm with ultraviolet spectrophotometer promptly is regarded as qualified primer between 1.6-2.0.From 1: 10
4The RNA that extracts in the H1 subtype influenza virus of A/Swine/2003 (H1N1) of dilution and H3 subtype influenza virus of A/Swine/2003/GD (H3N2) chicken embryo culture thing is that template increases; The result shows that primer provided by the invention pair and probe are used; The Ct value of amplification is relatively low, and increasing degree is higher.
The primer concentration that screening is good is that spacing increases progressively from 0.1 μ mol/L to 0.8 μ mol/L with 0.1 μ mol/L, and concentration and probe concentration increases progressively with 0.025 μ mol/L from 0.025 μ mol/L to 0.2 μ mol/L.Proportioning to primer and probe different concns compares, and from repeatedly finding the revision test: for the substance reaction, fluorescence amplification is higher relatively when adopting listed primer concentration of table 1 and table 2 and concentration and probe concentration respectively; For double reaction, fluorescence amplification is higher relatively when adopting listed primer of table 3 and concentration and probe concentration respectively.
We are to Roche Light Cycler2.0; Roche480 and ABI 7900HT fluorescent PCR detector; At 42 ℃/30min, behind the rt of 94 ℃/3min, in this experiment denaturation temperature and time, annealing and elongating temperature and time having been carried out optimization experiment.During amplification, adopt cycle index to be respectively 40,45 and 50, compare the Ct value of amplification, and then confirm that the cycle index of amplification is 40.Final definite expanding effect that adopts is better, and short scheme consuming time is the PCR reaction parameter: H1 hypotype substance reaction conditions is: 94 ℃/15s, and 52 ℃/5s, 60 ℃/35s, 40 circulations; H3 hypotype substance reaction conditions and double reaction condition are: 94 ℃/15s, and 50 ℃/5s, 60 ℃/35s, 40 circulations; When extending, each round-robin annealing collects fluorescence.
Research shows, Mg
2+Concentration has certain influence to the fluorescent PCR amplification, thus use the good primer probe of screening, with Mg
2+Concentration be that spacing increases progressively with 0.5mmol/L from 1.5mmol/L to 6.0mmol/L, to different Mg
2+Amplification under the concentration conditions is compared.Mg after the optimization
2+Concentration is for seeing table 1, table 2 and table 3.
The Taq enzyme that we select Promega company to produce, the definition of a unit: 74 ℃ act on 30 minutes, can the dNTPs of 10nM be mixed needed enzyme amount in the acid soluble material.The active requirement: tool dna polymerase activity and 5 ' → 3 ' exonuclease activity, do not have 3 ' → 5 ' exonuclease activity and endonuclease activity; The tool thermostability, 94 ℃ still kept 50% activity after 1 hour.From repeatedly selecting 1.25U Taq enzyme as the Taq enzyme amount of using the revision test result.
The substance of detection H1 according to the invention and H3 subtype influenza virus and the method for use of double check test kit:
1) sample preparation: for tissue sample, in mortar, fully grind, add 5mL PBS mixing again, then tissue suspension is changed in the aseptic Eppendorf pipe, number subsequent use with aseptic scissors and tweezers clip sample 1.0g to be checked.For liquid sample, directly draw to aseptic Eppendorf pipe with asepsis injector, number subsequent use.The sample of gathering or handling is preserved under 2 ℃～8 ℃ conditions and should be no more than 24h; If need prolonged preservation, must place-70 ℃ of refrigerators, but should avoid multigelation (freeze thawing is 2 times at most).After the sample sealing of gathering, adopt thermo jug or insulated tank sealing on the rocks, be transported to the laboratory as early as possible.
2) extraction of RNA: carry out in the sample process district.
Get n 1.5ml sterilization centrifuge tube (n=sample number+1 pipe negative control+1 pipe positive control), perform mark.(lysate has very strong corrodibility at first to add 600 μ L lysates; Be sure not to be stained with skin or clothing; Otherwise immediately with a large amount of flushing with clean water and dry), add each 200 μ L (a sample is used a suction nozzle instead) of sample to be tested, negative control, positive control then respectively; Add 200 μ L chloroforms again, vibration mixing 5sec (should not be too strong, in order to avoid produce emulsion layer, also available hand is put upside down mixing) on the vortex mixer.
12, the centrifugal 15min of 000g (as have ready conditions, carry out centrifugal in 4 ℃).Get with the last step in the 1.5ml sterilization centrifuge tube of equal amts, add 400 μ L Virahols (20 ℃ of precoolings), perform mark.Draw the supernatant phase transition of centrifugal back to corresponding pipe, put upside down mixing.
12, the centrifugal 15min of 000g.Remove supernatant gently, be inverted on the thieving paper, be stained with dry liquids (different samples should be stained with dried in the different places of thieving paper); Add 600 μ L, 75% ethanol (20 ℃ of precoolings), put upside down washing.
12, the centrifugal 10min of 000g.Remove supernatant gently, be inverted on the thieving paper, be stained with dry liquids as far as possible.4, the centrifugal 10sec of 000g is thrown to the pipe bottom with the residual liquid on the tube wall, it is blotted (a sample is used a suction nozzle instead, and the attention suction nozzle is not run into has the deposition one side) with micro sample adding appliance as far as possible, drying at room temperature 3min (should not be too dry, in order to avoid RNA is insoluble).
Add 11 μ L DEPC water, mixing dissolves the RNA on the tube wall gently, and 2, the centrifugal 5sec of 000g preserves subsequent use (note: the RNA of this moment is subject to the RNA enzyme liberating most, please in 2 hours, carries out pcr amplification) on ice.
3) RT-PCR amplification: from test kit, take out RT-PCR reaction solution, Taq enzyme, after at room temperature melting, 2, the centrifugal 5sec of 000g.If required PCR pipe number is n (a n=sample number+1 pipe negative control+1 pipe positive control), each test reaction system needs 15 μ L RT-PCR reaction solutions and 0.25 μ L Taq enzyme.Calculate the usage quantity of good each reagent, add in the proper volume test tube, to wherein adding n/4 RT-PCR enzyme granulate, thorough mixing is even, and each packing 15 μ L in each PCR pipe are transferred to the sample process district.Each the 10 μ L of RNA solution that in the PCR of each setting pipe, add preparation respectively, the tight pipe lid of lid is in the centrifugal 5sec of 800g.PCR pipe sequenced put into the fluorescent PCR detector, the record sample is put order.
Reaction parameter is provided with: H1 hypotype substance reaction conditions is: 94 ℃/15s, and 52 ℃/5s, 60 ℃/35s, 40 circulations; H3 hypotype substance reaction conditions and double reaction condition are: 94 ℃/15s, and 50 ℃/5s, 60 ℃/35s, 40 circulations; When extending, each round-robin annealing collects fluorescence.
4) result's judgement: the interpretation of result condition enactment, read detected result.The threshold setting principle is with the vertex of threshold line just above normal negative control article amplification curve.Different instruments can be adjusted according to instrument noise situation.Quality control standard, negative control do not have the Ct value and do not have amplification curve.The Ct value of positive control answers≤30.0, and specific amplification curve occurs.Like negative control and the discontented condition that is enough to of positive condition, it is invalid that experiment this time is regarded as.The result describes and judges, feminine gender, and no Ct value and do not have amplification curve shows in the sample that no H1 or H3 subtype influenza are viral; The positive, Ct value≤30.0, and specific amplification curve appears, there are H1 or H3 subtype influenza virus in the expression sample.
Advantage of the present invention is: the present invention selects H1 or H3 subtype influenza virus HA gene coding region as target region; The short probe of design primer and LNA and MGB is set up and has been optimized H1 and H3 subtype influenza virus substance and double fluorescent RT-PCR detection method, has obtained excellent technique effect:
1) quick: this method is monitored the PCR product in real time, and RT-PCR finishes to obtain the result.
2) sensitivity: owing to adopted specific fluorescent probe and highly sensitive fluorescent PCR appearance, make this method highly sensitive 100～1000 times than traditional P CR method; In-vitro transcription RNA to the known copy number detects.The result shows that the sensitivity of substance method all can reach 10 copy/reactions, and the sensitivity of double reaction can reach 100 copy/reactions, a little less than the substance detection method.
3) special: owing to not only adopted specific primer, and adopted specific probe, make the specificity of this method be higher than traditional RT-PCR method; The fluorescent RT-PCR method for detecting of setting up is detecting collected other subtype influenza virus (H4 hypotype, H5 hypotype, H7 hypotype and H9 hypotype) and NDVs, and the result is negative, does not find cross reaction.
4) versatility is good: the H1 hypotype or the H3 subtype influenza virus that can detect the different infection hosts source of collecting.
5) stable: the replica test result shows having good stability of institute's establishment method; CRNA to the known copy number of different concns carries out replica test, and the result sees table 4.
Table 4 replica test result
6) be difficult for polluting: totally-enclosed reaction need not the PCR aftertreatment, operational safety.
Below in conjunction with Figure of description and embodiment the present invention is described further.
Description of drawings
Fig. 1 is A/Swine/2003 (H1N1) and A/equine/HB/1/07 (H3N8) HA gene RT-PCR amplification; Swimming lane 1 negative contrast; Swimming lane 2 is A/Swine/2003 (H1N1) HA gene amplification result, and swimming lane 3 is A/equine/HB/1/07 (H3N8) HA gene amplification result.
Fig. 2 is that H1 and H3 subtype influenza virus double check method limit of detection are measured the result.The figure middle and upper part is divided into the H1 limit of detection and measures the result, and lower section H3 limit of detection is measured the result.
Fig. 3 is the specificity test-results of H1 and H3 subtype influenza virus double fluorescent RT-PCR detection method.
Embodiment
The viral nucleic acid of using among research of table 5 the inventive method and the embodiment
The preparation of embodiment 1, test kit and use
1, the preparation of test kit is formed, and sees table 6.
The preparation of table 6 test kit is formed
Concrete material is formed as is noted earlier.
2, the method for use of test kit
2.1RNA extract:
Get n 1.5ml sterilization centrifuge tube (n=sample number+1 pipe negative control+1 pipe positive control), perform mark.(lysate has very strong corrodibility at first to add 600 μ L lysates; Be sure not to be stained with skin or clothing; Otherwise immediately with a large amount of flushing with clean water and dry), add each 200 μ L (a sample is used a suction nozzle instead) of sample to be tested, negative control, positive control then respectively; Add 200 μ L chloroforms again, vibration mixing 5sec (should not be too strong, in order to avoid produce emulsion layer, also available hand is put upside down mixing) on the vortex mixer.
12, the centrifugal 15min of 000g (as have ready conditions, carry out centrifugal in 4 ℃).Get with the last step in the 1.5ml sterilization centrifuge tube of equal amts, add 400 μ L Virahols (20 ℃ of precoolings), perform mark.Draw the supernatant phase transition of centrifugal back to corresponding pipe, put upside down mixing.
12, the centrifugal 15min of 000g.Remove supernatant gently, be inverted on the thieving paper, be stained with dry liquids (different samples should be stained with dried in the different places of thieving paper); Add 600 μ L, 75% ethanol (20 ℃ of precoolings), put upside down washing.
12, the centrifugal 10min of 000g.Remove supernatant gently, be inverted on the thieving paper, be stained with dry liquids as far as possible.4, the centrifugal 10sec of 000g is thrown to the pipe bottom with the residual liquid on the tube wall, it is blotted (a sample is used a suction nozzle instead, and the attention suction nozzle is not run into has the deposition one side) with micro sample adding appliance as far as possible, drying at room temperature 3min (should not be too dry, in order to avoid RNA is insoluble).
Add 11 μ L DEPC water, mixing dissolves the RNA on the tube wall gently, and 2, the centrifugal 5sec of 000g preserves subsequent use (note: the RNA of this moment is subject to the RNA enzyme liberating most, please in 2 hours, carries out pcr amplification) on ice.
2.2 amplifing reagent is prepared and preparation
From test kit, take out substance and dual RT-PCR reaction solution, Taq enzyme, after at room temperature melting, 2, the centrifugal 5sec of 000g.If required PCR pipe number is n (a n=sample number+1 pipe negative control+1 pipe positive control), each test reaction system needs 15 μ L RT-PCR reaction solutions and 0.25 μ L Taq enzyme.Calculate the usage quantity of good each reagent, add in the proper volume test tube, to wherein adding n/4 RT-PCR enzyme granulate, thorough mixing is even, and each packing 15 μ L in each PCR pipe are transferred to the sample process district.Each the 10 μ L of RNA solution that in the PCR of each setting pipe, add preparation respectively, the tight pipe lid of lid is in the centrifugal 5sec of 400g.PCR pipe sequenced put into the fluorescent PCR detector, the record sample is put order.
Reaction parameter is provided with: H1 hypotype substance reaction conditions is: 94 ℃/15s, and 52 ℃/5s, 60 ℃/35s, 40 circulations; H3 hypotype substance reaction conditions and double reaction condition are: 94 ℃/15s, and 50 ℃/5s, 60 ℃/35s, 40 circulations; When extending, each round-robin annealing collects fluorescence.
2.3 the result judges
The interpretation of result condition enactment reads detected result.The threshold setting principle is with the vertex of threshold line just above normal negative control article amplification curve.Different instruments can be adjusted according to instrument noise situation.Quality control standard, negative control do not have the Ct value and do not have amplification curve.The Ct value of positive control answers≤30.0, and specific amplification curve occurs.Like negative control and the discontented condition that is enough to of positive condition, it is invalid that experiment this time is regarded as.The result describes and judges, feminine gender, and no Ct value and do not have amplification curve shows in the sample that no H1 or H3 subtype influenza are viral; The positive, Ct value≤30.0, and specific amplification curve appears, there are H1 or H3 subtype influenza virus in the expression sample.
The sensitivity test of embodiment 2, test kit
1, material:
Classical swine influenza virus A/Swine/2003 (H1N1) nucleic acid and equine influenza virus A/equine/HB/1/07 (H3N8) nucleic acid that the viral nucleic acid that is applied in the method research process is preserved for this laboratory
2, method
1) with classical swine influenza virus A/Swine/2003 (H1N1) and the synthetic cDNA of equine influenza virus A/equine/HB/1/07 (H3N8) RNA reverse transcription; The application specific primer amplification contains the dna fragmentation of the about 1.7kb of complete HA ORF, and amplified fragments is reclaimed rear clone in the pGEM-T carrier.The plasmid of purifying with restriction enzyme PvuII digestion process, to the dna fragmentation purifying and recovering, is carried out in-vitro transcription as template according to the Ribo MAXTM Large Scale RNAProduction System-T7 test kit of Promega company with it afterwards.(1u/ μ g sample DNA after Promega) dna profiling is wherein removed in digestion, extracts RNA with TRIZOL, to remove foreign protein and various ion to transcription product again through the Dnase of no RNase.RNA is precipitated in the aqua sterilisa of the no RNA enzyme that is dissolved in 1.0mL, packing, 20 μ L/ pipe, frozen under-80 ℃, promptly obtain the cRNA fragment for preparing.
2) the in-vitro transcription RNA that gets preparation does 200 times of dilutions respectively with the aqua sterilisa of no RNA enzyme, measures its A260 absorption value and calculates copy number.To the cRNA standard substance of above-mentioned preparation with 10 times of gradient dilutions after, carry out substance and double fluorescent RT-PCR measures by top condition, and with deionized water as negative standard.In detection, confirm the minimum cRNA concentration that fluorescence quantitative RT-RCR can detect, and draw the limit of detection of this method thus different extent of dilution standard substance.
3, result
1) sees shown in Figure 1ly,, obtain the dna fragmentation that A/Swine/2003 (H1N1) and the about 1.7kb of A/equine/HB/1/07 (H3N8) contain complete HA ORF through RT-PCR amplification.After in-vitro transcription with the pure article of cRNA that obtain.
2) result shows, the sensitivity of substance method all can reach 10 copy/reactions, and the sensitivity of double reaction can reach 100 copy/reactions, and a little less than the substance detection method, the sensitivity result of double check method sees Fig. 2.
The specificity test of embodiment 3, test kit
1, material
Listed like table 5
2, method
Use substance and the double fluorescent RT-PCR method set up that multiple influenza virus (comprising H1, fowl source H4, H5, H7 and H9 subtype virus) and NDV are detected, with the specificity of verification method.
3, result
As shown in Figure 3, the result shows method and the above-mentioned viral no cross reaction of being set up, and specificity is good.
The laboratory report that embodiment 4, test kit detect clinical sample
1, material
586 parts of the known samples that China provides directly under Entry-Exit Inspection and Quarantine Bureau.
2, method
China is detected for 586 parts directly under the known sample that Entry-Exit Inspection and Quarantine Bureau provides.The susceptibility of verification method and specificity in actual sample.
3, result
Detected result is seen table 7.
The detected result of table 7 clinical sample
| Sample | Quantity | The real-time fluorescence RT-PCR detected result |
| The H1 positive | 16 | 16/16 |
| The H3 positive | 12 | 12/12 |
| The H5 positive | 43 | 0/43 |
| The H4 positive | 2 | 0/2 |
| The H9 positive | 13 | 0/13 |
| The influenza negative sample | 500 | 0/500 |
586 parts of known samples are detected.The result shows that the method for foundation can detect wherein all H1 and H3 positive, and false positive results do not occur, and the result sees table 8.The method that shows foundation is reliable and practical.
Claims (4)
1. one group is detected H3 subtype influenza viral nucleotide primer and probe; Its nucleotide primer and probe such as sequence table SEQ ID NO:4 to SEQ ID NO:6; Wherein sequence SEQ ID NO:4 and SEQ ID NO:5 are respectively sense primer and the antisense primer that detects H3 subtype influenza virus HA gene, and sequence SEQ ID NO:6 is for detecting the fluorescent probe of H3 subtype influenza virus HA gene.
2. the nucleotide primer and the probe of detection H3 subtype influenza virus according to claim 1 is characterized in that: 5 ' the end mark report fluorophor FAM of said probe sequence SEQ ID NO:6,3 ' end mark MGB group.
3. one kind is detected H1 and the viral test kit of H3 subtype influenza, composed of the following components:
1) lysate
2) H1 hypotype substance fluorescence RT-PCR reaction solution comprises: PCR damping fluid, RT damping fluid, MgCL
2, dNTP, H1 sense primer, H1 antisense primer and H1 fluorescent probe
3) H3 hypotype substance fluorescence RT-PCR reaction solution comprises: RT damping fluid, MgCL
2, dNTP, H3 sense primer, H3 antisense primer and H3 fluorescent probe
4) H1 and H3 double fluorescent RT-PCR reaction solution comprise: RT damping fluid, MgCL
2, dNTP, H1 sense primer, H1 antisense primer, H1 fluorescent probe, H3 sense primer, H3 antisense primer, H3 fluorescent probe
5) RT-PCR enzyme granulate
6) Taq archaeal dna polymerase
7) DEPC water
8) negative control: the negative tissue sample of influenza virus, process 20% suspension with 0.01mol/L pH7.2PBS BS, 70 ℃ act on 1 hour;
9) positive control: be the non-infectious RNA fragment of in-vitro transcription, its corresponding DNA sequences is the nucleotide sequence shown in sequence table SEQ ID No.7 and the SEQ ID No.8;
Wherein, The H1 sense primer is the nucleotide sequence shown in the sequence table SEQ ID No.1, and the H1 antisense primer is the nucleotide sequence shown in the sequence table SEQ ID No.2, and the H1 fluorescent probe is the nucleotide sequence shown in the sequence table SEQ ID No.3; Its 5 ' end mark report fluorophor HEX; 3 ' end mark cancellation fluorophor TAMRA, the 5th, 7,9,11 base is modified with LNA, and the H3 sense primer is the nucleotide sequence shown in the sequence table SEQ ID No.4; The H3 antisense primer is the nucleotide sequence shown in the sequence table SEQ ID No.5; The H3 fluorescent probe is the nucleotide sequence shown in the sequence table SEQ ID No.6, its 5 ' end mark report fluorophor FAM, 3 ' end mark MGB group.
4. the test kit of detection H1 according to claim 3 and H3 subtype influenza virus is characterized in that said H1 hypotype substance fluorescence RT-PCR reaction solution comprises: 0.5 * PCR damping fluid, 0.5 * RT damping fluid, 1.5mmol/L MgCL
2, 0.2mmol/L dNTP, 0.3 μ mol/L H1 sense primer, 0.3 μ mol/L H1 antisense primer and 0.3 μ mol/L H1 fluorescent probe;
Said H3 hypotype substance fluorescence RT-PCR reaction solution comprises: 1 * RT damping fluid, 4mmol/L MgCL
2, 0.05mmol/L dNTP, 0.4 μ mol/L H3 sense primer, 0.4 μ mol/L H3 antisense primer and 0.2 μ mol/L H3 fluorescent probe;
Said H1 and H3 double fluorescent RT-PCR reaction solution comprise: 1 * RT damping fluid, 4mmol/LMgCL
2, 0.05mmol/L dNTP, 0.4 μ mol/L H1 sense primer, 0.4 μ mol/L H1 antisense primer, 0.3 μ mol/L H1 fluorescent probe, 0.4 μ mol/L H3 sense primer, 0.4 μ mol/L H3 antisense primer, 0.3 μ mol/L H3 fluorescent probe.
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| 韩雪清 等.禽流感病毒H1、H3、H5、N2亚型多重RT-PCR检测方法的初步研究.《中国人兽共患病学报》.2007,第23卷(第10期),993－996. * |
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