CN101886138A - Three-color fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) combined detection method of enterovirus 71, Coxsackie virus A16 and other subtypes of enterovirus as well as kit thereof - Google Patents
Three-color fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) combined detection method of enterovirus 71, Coxsackie virus A16 and other subtypes of enterovirus as well as kit thereof Download PDFInfo
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Abstract
The invention provides a three-color fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) combined detection method of enterovirus 71, Coxsackie virus A16 and other subtypes of enterovirus as well as a kit thereof. The method can rapidly and accurately detect the enterovirus 71, the Coxsackie virus A16 and the other subtypes of nucleic acids of enterovirus in a sample. The method comprises the following steps of: (1) acquiring and conveying a sample of an infected patient or a suspected patient; (2) preprocessing the sample and extracting RNA; (3) detecting the sample by adopting a one-step PCR-three-color fluorescent probe in-vitro amplification method; and (4) analyzing the corresponding sample according to the fluorescence intensity of each amplification reaction after the amplification reaction is finished, thereby judging the existence of the enterovirus 71, the Coxsackie virus A16 and the other subtypes of nucleic acids of the enterovirus in the acquired sample and being capable of carrying out accurate quantitation (a figure 3) on the enterovirus 71, the Coxsackie virus A16 and the other subtypes of nucleic acids of the enterovirus. The invention realizes the aim of carrying out rapid and accurate combined detection of the enterovirus 71, the Coxsackie virus A16 and the other subtypes of nucleic acids of the enterovirus.
Description
One, technical field
This examination invention belongs to the viral nucleic acid detection range, the detection method that relates to other each subtype virus RNA of enterovirns type 71, coxsackie virus A 16-type and enterovirus particularly relates to and uses three fluorescence probe quantitative round pcr (PCR-three fluorescence probe method) to detect the method for enterovirns type 71, coxsackie virus A 16-type and other each hypotype of enterovirus in the sample (ight soil, throat swab sample, bleb liquid, cerebrospinal fluid or viral separation and Culture thing sample etc.) fast and accurately.The invention further relates to the test kit that is used for this virus clinical detection, be applicable to the laboratory rapid detection that human enteric virus infects.
Two, background technology
Hand foot mouth disease (Hand, foot and mouth disease HFMD) is the acute infectious disease that is caused by enterovirus, and virus is seen with enterovirns type 71 (EV71), coxsackie virus A 16-type (CAV16) more, pilosity is born in the preschool children, and is the highest with 3 years old following age group sickness rate especially.Cardinal symptom shows as maculopapule, the bleb at positions such as hand, foot, oral cavity, and neurological symptom etc. can appear in the minority severe cases, how to cause that by the EV71 infection cause of death is mainly severe BBE and neurogenic pulmonary edema.Patient and inapparent infection person are contagium, mainly by digestive tube, respiratory tract with the approach propagation such as contact closely.
(Enterovirus is the less RNA viruses of particle EV) to human intestine's virus, is 20 bodies, diameter 24～30nm does not conform to mixtinite, and core has singlestranded RNA, ether-resistant and other fatsolvent, acidproof, various microbiotic, antiviral drug, stain remover there are resistant function.Most viruses produce cytopathy in cell cultures.From nineteen fifty-nine reported first by human intestine's virus (Enterovirus, EV) cause hand foot mouth disease (Hand, foot and mouth disease, HFMD) report since, HFMD repeatedly breaks out in the whole world.It is popular that the U.S. in 1974 finds that enterovirns type 71 (EV71) also often causes HFMD, and COxsackie A group (CAV16, CAV9, CAV5 etc.) causes HFMD simultaneously also report.The Malaysian hand foot mouth disease that has taken place mainly to be caused by EV71 was popular in 1997, and 29 routine patient deaths are only just arranged 4～June.EV71 infection in 1998 causes a large amount of hand foot mouth diseases and herpangina and has a large amount of children due to illness to cause death in China Taiwan Province.
HFMD often causes jointly by multiple enterovirus, and EV71 and the CAV16 main cause of disease of hand foot mouth disease outbreak of epidemic often, and the two is closely related on genetics, often for polyinfection with alternately infect.China's hand foot mouth disease is popular, and its cause of disease also is mainly EV71 type and CAV16.Generally, the HFMD that causes of EV71 and CAV16 is difficult to difference on clinical symptom.This sick Acute onset, heating, bleb appears being dispersed in oral mucosa, and maculopapule, bleb appear in hand, foot and buttocks, around the bleb inflammatory blush can be arranged, and liquid is less in the blister.Can be with symptoms such as cough, runny nose, poor appetites.Some cases only shows as fash or herpangina.The prognosis bona.Meningitis, encephalitis, encephalomyelitis, pulmonary edema, cycle penalty etc. can appear in the minority acute case, and the state of an illness is dangerous, can cause death to die or leave sequela.Clinically diagnosed cases have one of following person and can make a definite diagnosis: 1. enterovirus (EV71, CAV16 etc.) specific nucleic acid detects positive; 2. isolate enterovirus, and be accredited as EV71, CAV16 or other can cause the enterovirus of hand foot mouth disease; 3. acute phase and convalescent phase serum EV71, CAV16 or other can cause that the enterovirus neutralizing antibody of hand foot mouth disease has the rising more than 4 times.
Three, summary of the invention
Laboratory examination for this kind enterovirus infection mainly comprises following aspect at present: 1. the virus infection routine inspection comprises cell and biochemical analysis.2. etiological examination, enterovirus (EV71, CAV16 etc.) specific nucleic acid is positive or be separated to enterovirus.3. Serological testing, ELISA method detection acute phase and convalescent phase serum EV71, CAV16 or other enterovirus neutralizing antibody have the rising more than 4 times.Serological testing must be to be based upon virus infection takes place and to produce on the basis of antibody, and in view of the influenza disease the height variability and the intercrossing of learning with other serum virus, its specificity is difficult to guarantee, occurs false positive and false negative easily, also can't carry out rapid detection.From sample, separate swine influenza virus, carry out egg inoculation and cell cultures and may compare sensitivity, but this method needs several days time.Enterovirus (EV71, CAV16 etc.) specific nucleic acid detects and need carry out the RT-PCR test, and needs order-checking to confirm.Though tool remolding sensitivity aforesaid method slightly improves, but this method only is based on a pair of nucleic acid primer and increases, the result is easy to generate non-specific amplification, even can be because false positive appears in the reason of operation, and its result's of regular-PCR TRAP judgement need be carried out gel electrophoresis analysis, complex operation to product.
Three fluorescence quantitative PCR technique principle is based on monochromatic fluorescent quantitative PCR technique, be meant three goal gene that in same fluorescent quantitative PCR test, increase simultaneously, probe is that multiple fluorescent mark is such as FAM, VIC, JOE, NED, HEX etc., every kind of fluorescent signal difference that fluorescently-labeled probe is produced in the pcr amplification process.The test kit that utilizes this principle invention is in the detection of same sample, can detect other each hypotype of enterovirns type 71, coxsackie virus A 16-type and enterovirus simultaneously, realized detecting multiple enterovirus simultaneously in the sample, and virus carried out the purpose of somatotype, it is very convenient to use.Because this method introduced specificity amplification primer and fluorescent probe, make that the sensitivity and the specificity that detect are strengthened significantly, thereby avoided the problem of the not high and easy omission of other detection method specificitys.
Based on three fluorescence quantitative PCR technique principle, in order to overcome defective and the deficiency in the existing detection technique, the invention provides the method that a kind of use three fluorescence quantitative PCR technique (PCR-three fluorescence probe method) quick and precisely detects the RNA of enterovirns type 71, coxsackie virus A 16-type and other each subtype virus of enterovirus in the sample (patient suspected's ight soil, throat swab sample, bleb liquid, cerebrospinal fluid or viral separation and Culture thing sample etc.).This method comprises: (1) is gathered and is transported and infect or patient suspected's sample (patient suspected's ight soil, throat swab sample, bleb liquid, cerebrospinal fluid or viral separation and Culture thing sample etc.); (2) sample pre-treatment and extraction RNA; (3) one step PCR-three fluorescence probe amplification in vitro methods detect sample: synthetic specific primer and fluorescent probe, also carry out augmentation detection with three fluorescence PCR reaction solution (PCR MIX) to sample with three fluorescence quantitative PCR reaction technology; (4) amplified reaction is analyzed respective sample according to the fluorescence intensity of each amplified reaction after finishing, thereby judges the existence of road virus 71 types, coxsackie virus A 16-type and other each hypotype of enterovirus in the sample of being gathered and according to quality control product the virus in the sample is carried out accurate quantification.
Download the gene order of all enteroviruses from Genbank, compare repeatedly through bioinformatic analysis, select as the specificity target area of lower area as amplification enterovirns type 71 (EV71), coxsackie virus A 16-type (CAV19) and other each hypotype (EV-U) of enterovirus:
(1) enterovirns type 71 (EV71) upstream primer:
5’-GCGTTTTGACGCAGAGTTCA-3’(SEQ?ID?NO.1)
(2) enterovirns type 71 (EV71) downstream primer:
5’-GGAGCAATTGCGGGACAA-3’(SEQ?ID?NO.2)
(3) enterovirns type 71 (EV71) fluorescent probe:
FAM-5’CTTTGTTGCATGCACCCCTACCGG--3’TAMRA(SEQ?ID?NO.3)
(4) coxsackie virus A 16-type (CAV16) upstream primer:
5’-GGGACCGGGAATGAAAATTC-3’(SEQ?ID?NO.4)
(5) coxsackie virus A 16-type (CAV16) downstream primer:
5’-ATGCGTCAGAA/GCTGCACCA/G-3’(SEQ?ID?NO.5)
(6) coxsackie virus A 16-type (CAV16) fluorescent probe:
JOE-5’TCCTCCCTACGCCACTACACAGCCTG-3’TAMRA(SEQ?ID?NO.6)
(7) each hypotype of enterovirus (EV-U) upstream primer:
5’-AGC/TGGGTA/GGIGIGICGTAACG-3’(SEQ?ID?NO.7)
(8) each hypotype of enterovirus (EV-U) downstream primer:
5’-TCACCATAAGCAGCCAA/GTATAA/T-3’(SEQ?ID?NO.8)
(9) each hypotype of enterovirus (EV-U) fluorescent probe:
VIC-5’TAACTCTGCAGCGGAACCGACTACTT-3’TAMRA(SEQ?ID?NO.9)
According to a preferred embodiment of the invention, the fluorescent mark of wherein said detection enterovirns type 71, coxsackie virus A 16-type and other each subtype virus of enterovirus is respectively FAM, JOE and VIC, detects so be called three fluorescence.
According to a preferred embodiment of the invention, at first use the qualitative amplification positive sample of common RT-PCR, the PCR product electrophoresis result of positive shows, as seen consistent with purpose fragment feature band, and negative sample does not all have this feature band, and Fig. 1 proves that primer is correct.
According to a preferred embodiment of the invention, wherein said test kit is a kind of test kit that quick real-time quantitative detects enterovirus EV 71 type, coxsackie virus A 16-type and each hypotype of enterovirus that is used for, and this test kit comprises that (1) viral nucleic acid extracts reagent; (2) reversed transcriptive enzyme system and Taq enzyme system; (3) three fluorescence PCR reaction system (PCRMIX); (4) positive and negative quality control product etc.
According to a preferred embodiment of the invention, wherein said detection enterovirns type 71, other each hypotype three fluorescence quantitative PCR reaction system (PCR MIX) of coxsackie virus A 16-type and enterovirus is by enterovirns type 71 (EV-71) specificity upstream and downstream primer,, a specificity fluorescent probe (Fam fluorescent mark), the specificity upstream and downstream primer of coxsackie virus A 16-type (CAV-16),, a specificity fluorescent probe (JOE fluorescent mark), each hypotype of enterovirus (EV-U) specificity upstream and downstream primer,, a specificity fluorescent probe (VIC fluorescent mark), (FQ-Buffer includes magnesium ion to the PCR reaction buffer, Tris-HCl etc.), four kinds of nucleotide monomers (dNTPs), the reaction system that compositions such as pcr amplification toughener and deionized water constitute.
According to a preferred embodiment of the invention, the preparation in the following manner of wherein said PCR reaction system (PCR MIX):
Every part of reaction solution (PCR MIX):
Each 0.2 μ l of each primer (50pmol/ μ l)
Each 0.1 μ l of each fluorescent probe (30pmol/ μ l)
DNTPs (each 2.5mM) 2 μ l
PCR reaction buffer (FQ-Buffer) 16.5 μ l
?????????????????????????????????????????????????????????
Total???????????????????????????????????????????20.0μl
Need to be placed on-20 ℃ of preservations after the above system preparation, wherein the PCR reaction buffer is formed: 50mMTris-HCl (pH8.0), 3.5mM MgCl2,50mM KCl, 25mmol/L (NH4) 2SO4 form.
According to a preferred embodiment of the invention, through clone's plasmid, its accurate copy number is 10 behind the specificity purpose fragment amplification that wherein said positive quality control product is EV71
7/ ml, negative quality control product handle and autoclaved PCR damping fluid through DEPC.
According to a preferred embodiment of the invention, the fluorescent quantitative PCR condition that is wherein adopted is: ABI PRISM 7700, ABI PRISM5700, ABI GeneAmp 7000 (need cut the reaction lid), ABIPRISM7300/7500 (using 8 pipes), MJ Opticon (using 8 pipes) etc. use the instrument of thin-walled tube, cycling condition is 42 ℃ → 20 minutes, 93 ℃ then → 2 minutes, then 93 ℃ 30 seconds → 55 ℃ 45 seconds, 40 circulations; LightCycler etc. use instrument capillaceous, and cycling condition is 42 ℃ → 20 minutes, 93 ℃ → 2 minutes, then 93 ℃ 5 seconds → 55 ℃ 45 seconds, totally 40 circulations.
According to a preferred embodiment of the invention, after reaction finishes, use the manual setting threshold value to make negative quality control product Ct value more than 40, the Ct value is positive less than 35 round-robin; The Ct value is gray area between 35-40 circulation, need carry out revision test.After the revision test, if the Ct value still between 35-40 circulation, is judged as the positive, there do not have fluorescent value to increase to be negative, and the amplification dynamic curve is seen Fig. 2.
According to a preferred embodiment of the invention, the measuring method of limit up and down of the detection by quantitative of test kit is as follows:
Make dilution positive criteria product (10 with TE buffer
7Copies/ml), respectively as linearity and sensitivity reference material, it is 1.0 * 10 that standard substance concentration is respectively
7/ ml, 1.0 * 10
6/ ml, 1.0 * 10
5/ ml, 1.0 * 10
4/ ml, 1.0 * 10
3/ ml, 1.0 * 10
2/ ml, 1.0 * 10
1/ ml.Each extent of dilution is made 3 parts of replications.Analyze | the r| value all can reach | and r| 〉=0.99, the result confirms that the linear limit up and down of this test kit calibrating is: 1.0 * 10
2Copies/ml～1.0 * 10
7Copies/ml.Electrophorogram such as Fig. 3.
According to a preferred embodiment of the invention, the detection by quantitative accuracy determination method of test kit is as follows: the similar test kit of three batches of these test kits of usefulness quantity-produced and contrast company detects on same pcr amplification instrument observes it | the variation of r| value and detection sensitivity.Experiment conclusion is: | r| 〉=0.97, quantitative precision CV＜30%.
Be limited to 1.0 * 10 under the detection by quantitative of this test kit
2Copies; Sensitivity is 1.0 * 10
2Copies.
According to a preferred embodiment of the invention, the detection by quantitative specific assay method of test kit is as follows: with this test kit 500 routine patients suspected are being carried out conventional sense, wherein detect positive 76 examples of EV71, the CAV1634 example, enterovirus universal 43 examples, after identifying, order-checking confirms that identical rate is 100%, false positive and false negative do not occur.
Test kit of the present invention confirms to have good good reproducibility and stable good performance through repeatedly different repeated experiments checkings.But the test kit validity period can reach 12 months under-20 ℃ of conditions.
At other each hypotype nucleotide sequence of enterovirns type 71, coxsackie virus A 16-type and enterovirus designed primer and three fluorescence probe amplification specific gene fragment, not only can detect enterovirns type 71, simultaneously can also detect coxsackie virus A 16-type and other each hypotype nucleic acid of enterovirus, thereby realized detecting simultaneously in the sample purpose of three kinds of viruses, to use very convenient.Because this method has been introduced specificity amplification primer and three fluorescence probe, make to detect to have better sensitivity and specificity, thereby avoided the not high problem of other detection method specificitys.The present invention has simultaneously introduced contrast and the quantitative criterion of positive quality control product as experiment, and the enteroviral rna that infects is carried out rapid detection and quantitative.The another one aspect, the present invention has used single stage method fluorescent quantitative PCR technology, has simplified process of the test and has improved detection efficiency.In general, the judgement from the processing sample to the result only needed about 2 hours.So compare with traditional method, tool of the present invention also has simple to operate, advantage is quickly and easily arranged.Therefore, test kit of the present invention provides new method for the rapid detection of clinical samples and large sample generaI investigation, and uses for patient suspected's examination reliable methodology foundation is provided.
Four, description of drawings
Fig. 1 show according to routine the qualitative amplification positive sample of common RT-PCR, the PCR product of positive shows through 2% agarose gel electrophoresis result can see consistent with purpose fragment feature band, and negative sample does not all have this feature band, proves that primer is correct.Wherein sample number 1-3 is Enterovirus 71 (EV71), and 4-6 is that coxsackie virus A 16 (CAV16), 7-10 are enterovirus universal (EV-U).Fig. 2, the amplification of the gradient dilution of positive quality control product, the amplification kinetic curve of display standard, S-type, expanding effect is better.Fig. 3 test kit detection limit test electrophoretogram, wherein 1～5 is the quality control standard product PCR reaction product of gradient dilution, 2% agar sepharose, sample concentration is respectively 1.0 * 10
6/ ml, 1.0 * 10
5/ ml, 1.0 * 10
4/ ml, 1.0 * 10
3/ ml, 1.0 * 10
2/ ml).
Five, embodiment
Embodiment 1: the collection of sample and transporting
Be suitable for the sample type and comprise patient suspected's ight soil, throat swab sample, bleb liquid, cerebrospinal fluid or viral separation and Culture thing sample etc.The throat swab sample: gather fall ill throat swab sample in 3 days of patient, with special-purpose sampling cotton swab, appropriateness firmly swabs pharynx rear wall and tonsilla position, both sides, should avoid touching tongue; Rapidly cotton swab is put into the sampling tube that 1～2ml preserves liquid (keeping liquid or physiological saline) is housed,, screw pipe lid and airtight censorship at the cotton swab bar that fractures near top end.Bleb liquid: the alcohol with 75% carries out disinfection to the skin around the bleb, with sterile needle bleb is needled to dip in cotton swab then and get bleb liquid, rapidly cotton swab is put into the sampling tube that 1～2ml preserves liquid (keeping liquid or physiological saline) is housed, at the cotton swab bar that fractures near top end, screw pipe lid and sealing.Samples of CSF: occur behind the neurological symptom in 3 days, collection capacity is 1.0～2.0ml.Pack into immediately after the collection in the frozen pipe of aseptic belt washer.Airtight censorship is surveyed.Ight soil: twist with the fingers swab with sterilization and wipe away and get ight soil or the diarrhoea thing is inserted sterile glass tube (containing the 0.4ml sterile saline), with aseptic cotton balls with the test tube jam-pack after, airtight censorship.Above-mentioned sample can be stored in-20 ℃ in a short time, and prolonged preservation can be put-70 ℃, but can not surpass 6 months, and sample transports and should adopt 0 ℃ of curling stone, and collect specimen (in the 12h) is immediately sent to the laboratory.
Embodiment 2: the extraction of enteroviral rna
Sample preparation: get 500 μ l sample liquid and add 500 μ l virus concentrated solution and fully shake 4 ℃ of refrigerated centrifuges 13, the centrifugal 10min of 000rpm, remove supernatant, keep the sample liquids about 50 μ l, add fully concussion of 150ul Trizolreagents (RNA extracting solution), room temperature is placed 5min.Add the 100ul chloroform, firmly shake 15s, room temperature leaves standstill 5min, and 4 ℃ 13, the centrifugal 10min of 000rpm.Carefully with the upper water phase transition in clean centrifuge tube, add the equal-volume Virahol, abundant mixing, 13, the centrifugal 10min of 000rpm.Abandon supernatant, add the DEPC ethanol of 500 μ l 75%, abundant mixing, 13, the centrifugal 10min of 000rpm, the careful suction removed most of ethanol.With extraction tube uncovered in air at room temperature dry 5min treat that ethanol volatilization is clean, with 20 μ l DEPC H2O dissolution precipitations.Negative quality control product: get the negative quality control product of 50 μ l and add fully concussion of 150 μ l Trizol reagents (RNA extracting solution), room temperature is placed 10min.Aforesaid operations, EV71 positive quality control product are pressed in the back: directly get 3 μ l and carry out pcr amplification.
Embodiment 3: the instrument that this detection method and test kit are suitable for
Suitable instrument mainly comprises ABI GeneAmp PCR System7700, ABI GeneAmp PCRSystem7500, ABI PRISM 7300, LightCycler etc., instrument capillaceous such as LightCycler, the quantitative PCR instrument of the MJOpticon series or the authentication that qualifies.
Embodiment 4: the three fluorescence PCR method detects enteroviral rna
From test kit, take out PCRMIX, Taq enzyme system, reversed transcriptive enzyme system, behind room temperature thawing and the vibration mixing, 10, the centrifugal 10s of 000rpm.If needed PCR reaction tubes pipe number is N (a N=sample number+1 pipe negative control+1 pipe positive control), each test reaction system is formulated as follows table:
| Reagent | ??PCR?MIX | Taq enzyme system | Reversed transcriptive enzyme system |
| Consumption | ??20μl | ??1μl | ??1μl |
Calculate the usage quantity of good each reagent, add in the clean 0.2ml centrifuge tube of a proper volume, abundant mixing, 10, the centrifugal 10s of 000rpm adds 22 μ l respectively in N the PCR reaction tubes of setting, add to handle back sample (extracting RNA) or positive quality control product and negative quality control product 3 μ l in every pipe, 10, instantaneous centrifugal 10 seconds of 000rpm.Each reaction tubes is put into the reactive tank of quantitative PCR instrument, negative quality control product is set in proper order by correspondence, positive quality control product and key sample not, and sample title, mark fluorescent radical species (reporter group FAM, quencher group TAMRA) and cycling condition are set:
ABI PRISM 7700, ABI PRISM5700, ABI GeneAmp 7000 (need cut the reaction lid), ABI PRISM7300/7500 (using 8 pipes), MJ Opticon (using 8 pipes) etc. use the instrument of thin-walled tube, cycling condition: 42 ℃ → 20 minutes, 93 ℃ then → 2 minutes, then 93 ℃ 30 seconds → 55 ℃ 45 seconds, 40 circulations.LightCycler etc. use instrument capillaceous, cycling condition: 42 ℃ → 20 minutes, 93 ℃ → 2 minutes, back 93 ℃ 5 seconds → 55 ℃ 45 seconds, totally 40 circulations.
Embodiment 5: interpretation of result and judgement
After reaction finishes, at first select a kind of fluorescence (the corresponding EV-U of corresponding CAV, 16VIC of AM corresponding EV71, JOE), use the manual setting threshold value to make negative quality control product Ct value more than 40, the Ct value is positive less than 35 round-robin; The Ct value is gray area between 35-40 circulation, need carry out revision test.After the revision test, if the Ct value still between 35-40 circulation, is judged as the positive, there do not have fluorescent value to increase to be negative.Need to satisfy: negative quality control product should be total negative; The Ct value of positive quality control product all should be less than 30, and are standard S type amplification curve.Above condition should satisfy simultaneously, otherwise that test this time is considered as is invalid, and Total Test should carry out again.
This test kit is put the process of reverse transcription and quantitative PCR detection together, need not two the step send detection, simplified testing process greatly and saved detection time.With this test kit can be in 2 hours the rapid detection sample whether infect other each hypotype of enterovirns type 71, Coxsackie virus CAV16 and enterovirus arranged, and be any type enterovirus.The every part of reagent one-time detection that is to say this test kit just has the ability that detects three kinds of viruses simultaneously.
Embodiment 6: the clinical application of the inventive method
Use method of the present invention and test kit, a large amount of samples are carried out quantitative PCR detection, and finally through the order-checking experimental verification.The result shows: the test kit of the inventive method, sensitivity are 10
2Copies, accuracy and specificity reach 100%, and repeatability and stability are better, and preservation condition of this test kit and validity period are :-20 ℃, 12 months.The invention of present method provides molecular biology experiment data science, individuation for the formulation of the prevention and control scheme that increases the detection of enterovirns type 71 (EV71), coxsackie virus A 16-type (CAV19) and other each hypotype (EV-U) of enterovirus and epidemic situation, for patient's diagnosis provides reliable Molecular Virology evidence.As occurring the existence of above-mentioned virus in the fruit disease sample, just prompting may be the infection of corresponding virus, then needs to take immediately corresponding measure, also avoids producing unnecessary consequence.
Six, sequence table
SEQUENCE?LISTING
＜110〉Beijing Suo Ao Bioisystech Co., Ltd
＜120〉other each hypotype three fluorescence RT-PCR associated detecting method and test kit thereof of enterovirns type 71, coxsackie virus A 16-type and enterovirus
<130>
<160>9
<170>PatentIn?version?3.3
<210>1
<211>20
<212>DNA
＜213〉artificial sequence
<400>1
gcgttttgac?gcagagttca????????????????????????????20
<210>2
<211>18
<212>DNA
＜213〉artificial sequence
<400>2
ggagcaattg?cgggacaa??????????????????????????????18
<210>3
<211>24
<212>DNA
＜213〉artificial sequence
<400>3
ctttgttgca?tgcaccccta?ccgg????????????????????24
<210>4
<211>20
<212>DNA
＜213〉artificial sequence
<400>4
gggaccggga?atgaaaattc?????????????????????????20
<210>5
<211>21
<212>DNA
＜213〉artificial sequence
<400>5
atgcgtcaga?agctgcacca?g???????????????????????21
<210>6
<211>26
<212>DNA
＜213〉artificial sequence
<400>6
tcctccctac?gccactacac?agcctg????????????????????????26
<210>7
<211>23
<212>DNA
＜213〉artificial sequence
<400>7
agctgggtag?gtgtgtcgta?acg???????????????????????????23
<210>8
<211>24
<212>DNA
＜213〉artificial sequence
<400>8
tcaccataag?cagccaagta?taat??????????????????????????24
<210>9
<211>26
<212>DNA
＜213〉artificial sequence
<400>9
taactctgca?gcggaaccga?ctactt????????????????26
Claims (4)
1. enterovirns type 71, coxsackie virus A 16-type and an enterovirus other each hypotype three fluorescence RT-PCR associated detecting method and test kit thereof, download the gene order of all enteroviruses from Genbank, through bioinformatic analysis, comparison repeatedly, select as the specificity target area of lower area as amplification enterovirns type 71 (EV71), coxsackie virus A 16-type (CAV19) and other each hypotype (EV-U) of enterovirus:
(1) enterovirns type 71 (EV71) upstream primer:
5’-GCGTTTTGACGCAGAGTTCA-3’(SEQ?ID?NO.1)
(2) enterovirns type 71 (EV71) downstream primer:
5’-GGAGCAATTGCGGGACAA-3’(SEQ?ID?NO.2)
(3) enterovirns type 71 (EV71) fluorescent probe sequence:
5’-CTTTGTTGCATGCACCCCTACCGG--3’(SEQ?ID?NO.3)
(4) coxsackie virus A 16-type (CAV16) upstream primer:
5’-GGGACCGGGAATGAAAATTC-3’(SEQ?ID?NO.4)
(5) coxsackie virus A 16-type (CAV16) downstream primer:
5’-ATGCGTCAGAA/GCTGCACCA/G-3’(SEQ?ID?NO.5)
(6) coxsackie virus A 16-type (CAV16) fluorescent probe sequence:
5’-TCCTCCCTACGCCACTACACAGCCTG-3’(SEQ?ID?NO.6)
(7) each hypotype of enterovirus (EV-U) upstream primer:
5’-AGC/TGGGTA/GGTGTGTCGTAACG-3’(SEQ?ID?NO.7)
(8) each hypotype of enterovirus (EV-U) downstream primer:
5’-TCACCATAAGCAGCCAA/GTATAA/T-3’(SEQ?ID?NO.8)
(9) each hypotype of enterovirus (EV-U) fluorescent probe sequence:
5’-TAACTCTGCAGCGGAACCGACTACTT-3’(SEQ?ID?NO.9)。
2. a kind of look fluorescence RT-PCR associated detecting method according to claim 1 and test kit thereof, coxsackie virus A 16-type (CAV16) downstream primer (SEQ ID NO.5), each hypotype of enterovirus (EV-U) upstream primer (SEQ ID NO.7), each hypotype of enterovirus (EV-U) downstream primer (SEQ ID NO.8), wherein, wherein A/G represents that base is A or G herein, C/T represents that base is C or T herein, and A/T represents that base is A or T herein.
3. according to claim 1, probe 5 ' the fluorescent mark of wherein said detection enterovirns type 71 (SEQ ID NO.3), coxsackie virus A 16-type (SEQ ID NO.6) and other each subtype virus of enterovirus (SEQ ID NO.9) is respectively FAM, JOE and VIC, detect so be called three fluorescence, probe 3 ' is labeled as TAMRA.
4. comprise that according to described this test kit of claim 1 (1) viral nucleic acid extracts reagent; (2) reversed transcriptive enzyme system and Taq enzyme system; (3) three fluorescence PCR reaction system (PCR MIX); (4) positive and negative quality control product etc., the preparation in the following manner of wherein said PCR reaction system (PCR MIX):
Every part of reaction solution (PCR MIX):
Each 0.2 μ l of each primer (50pmol/ μ l)
Each 0.1 μ l of each fluorescent probe (30pmol/ μ l)
DNTPs (each 2.5mM) 2 μ l
PCR reaction buffer (FQ-Buffer) 16.5 μ l
?????????????????????????????????????????????????????????
Total?????????????????????????????????????20.0μl
The fluorescent quantitative PCR condition that is wherein adopted is: ABI PRISM 7700, ABI PRISM5700, ABI GeneAmp 7000 (need cut the reaction lid), ABI PRISM7300/7500 (using 8 pipes), MJ Opticon (using 8 pipes) etc. use the instrument of thin-walled tube, cycling condition is 42 ℃ → 20 minutes, 93 ℃ then → 2 minutes, then 93 ℃ 30 seconds → 55 ℃ 45 seconds, 40 circulations; LightCycler etc. use instrument capillaceous, and cycling condition is 42 ℃ → 20 minutes, 93 ℃ → 2 minutes, then 93 ℃ 5 seconds → 55 ℃ 45 seconds, totally 40 circulations.
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