CN105112407B - A kind of kit and its application for being used to detect enterovirus - Google Patents
A kind of kit and its application for being used to detect enterovirus Download PDFInfo
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Abstract
The invention discloses a kind of kit and its application for being used to detect enterovirus.Contain the primer pair group for being used for detecting enterovirus in kit provided by the present invention, be made up of 3 primer pairs, its sequence is sequence 16 in sequence table.Kit provided by the invention supports high flux, common enterovirus infection can quickly and accurately be detected, for clinic, the testing result that 3 kinds of enterovirus indexs can be obtained in 1 hour is not only faster than the real time fluorescence quantifying PCR method more generally used at present, and also significant for the treatment of quick auxiliary direction and medication.Meanwhile the detection of multi objective can be used for regional epidemiological survey and epidemic situation monitoring, to study popularity of the enterovirus infection in China.
Description
Technical field
The invention belongs to nucleic acid amplification technologies field, is related to a kind of kit and its application for being used to detect enterovirus.
Background technology
Enterovirus includes poliovirus, Coxsackie virus (Coxsackievirus), causes enterocyte lesion people
Orphan virus (enterocytopathichumanorphanvirusECHO abbreviations echovirus) and newtype enteroviru totally 71
Individual serotype.Infectious disease caused by enterovirus genus virus, clinical manifestation the lighter only have Juan Dai ﹑ Fa Li ﹑ low-heat etc., and severe one can be complete
The vitals such as body infection , Nao ﹑ Ji Sui ﹑ Xin ﹑ livers are damaged, and prognosis is poor, and can leave sequelae or cause death.This class disease
It is distributed in all over the world, has in Perenniporia martius whole year, summer is common in temperate zone, Wen Nuan ﹑ Chao Shi ﹑ sanitary condition Cha ﹑
The crowded regional incidence of disease is high.
Hand-foot-and-mouth disease (Hand-foot-mouth disease, HFMD) is the clinical disease as caused by enterovirus infection
Group, has the characteristics of diverse clinical manifestations, and majority of cases clinical manifestation is lighter, with the fash at the positions such as heating and hand, foot, oral cavity
Or bleb is principal character.There is respiratory system, central nervous system damage in a small number of cases, cause encephalitis, myocarditis, edema with the lung involved
The symptoms such as swollen, flaccid paralysis, indivedual children with serious disease rapid onsets, cause death.Triggering the enterovirus of hand-foot-and-mouth disease has more than 20
Kind, including Coxsackie virus A group, enterovirns type 71 etc..
Rapid＆Early diagnosis enterovirus infection can guiding clinical treatment be in time reasonable selection antiviral antibacterial medicine
Thing offer foundation is simultaneously significant in terms of abuse of antibiotics is prevented.
The method for detecting common enterovirus at present is more, but all Shortcomings, such as Electronic Speculum detection method complex and expensive, disease
Poison is separately cultured time-consuming extremely length and false negative is more, and generally clinical meaning, ELISA have been lost after cultivation results are taken
Detection is special viral antibody but is once only capable of detecting a kind of virus.And the detection of nucleic acids based on nucleic acid amplification, due to speed
Degree fast (can complete to detect in usual 3 hours), high sensitivity, specificity are good, and goldstandard is increasingly becoming in field of virus detection.
The amplification technique (Nucleic Acid Sequence Based Amplification) of nucleotide sequence is relied on, i.e.,
NASBA amplification techniques, it is to be mediated by pair of primers, be specific in vitro and continuous homogeneous to single stranded RNA progress constant temperature expansion
The process of increasing.Under 41 DEG C of constant temperatures, in terms of reaction speed, template ribonucleic acid can be expanded about 10 by NASBA amplifications 1h9~1012
Times, and common PCR reactions need 2~3h.In terms of detection sensitivity, NASBA, which can be detected, is less than 10gene in solution
Copies/ μ l trace target, and PCR test limit is in 100gene copies/ μ l or so.Therefore, NASBA technologies are either
Amplification efficiency or detection sensitivity are all higher than RT-PCR technology.And because NASBA reaction only needs one 41 DEG C of perseverance
Warm condition, water-bath can meet that reaction requires, it is not necessary to the temperature control device that the Standard PCR such as complicated heating and cooling is relied on, because
The instrument cost of this NASBA technology is very cheap.Combined with corresponding detection technique, NASBA also has easy to operate, specific
Many advantages, such as strong.
The content of the invention
First purpose of the present invention is to provide a kind of primer pair group for being used to detect enterovirus.
The primer pair group provided by the present invention for being used to detect enterovirus, is made up of following 3 primer pairs：
The primer pair 1 being made up of two single strand dnas shown in sequence in sequence table 1 and sequence 2；By sequence in sequence table
The primer pair 2 of two single strand dnas composition shown in row 3 and sequence 4；As two shown in sequence in sequence table 5 and sequence 6
The primer pair 3 of single strand dna composition.
Wherein, the primer pair 1 (Entv_TY) is used to expand enterovirus；The primer pair 2 (EV71_VP1) is used to expand
Increase enterovirus EV 71 type；The primer pair 3 (CA16_VP1) is used to expand enterovirus CA16 types.
Second object of the present invention is to provide a kind of complete single stranded DNA for being used to detect enterovirus.
The complete single stranded DNA provided by the present invention for being used to detect enterovirus, by probe groups and the primer pair group group
Into；The probe groups are made up of following 3 ssDNA probes：SsDNA probe 1 in sequence table shown in sequence 7；Sequence table
SsDNA probe 2 shown in middle sequence 8；SsDNA probe 3 in sequence table shown in sequence 9.
Wherein, the ssDNA probe 1 (Entv_TY_MB) is used for the amplification for monitoring the primer pair 1 in real time；Institute
State the amplification that ssDNA probe 2 (EV71_VP1_MB) is used to monitor the primer pair 2 in real time；The ssDNA probe 3
(CA16_VP1_MB) it is used for the amplification for monitoring the primer pair 3 in real time.
5 ' the ends mark of every ssDNA probe in the probe groups has FAM, and 3 ' ends mark
There is fluorescent quenching group TAMRA.
In the complete single stranded DNA provided by the present invention, each primer pair and its corresponding ssDNA probe can be single
Solely it is packaged in a packaging.As the primer pair 1 and the ssDNA probe 1 are packaged in first packaging；The primer
2 and the ssDNA probe 2 are packaged in second packaging；The primer pair 3 and the ssDNA probe 3 are packaged in
In three packagings.
Third object of the present invention is to provide a kind of kit for being used to detect enterovirus.
The kit provided by the present invention for being used to detect enterovirus, contains the primer pair group or described complete single-stranded
DNA。
The kit can also contain internal reference primer pair and internal reference probe.The internal reference primer pair is for expanding
House-keeping gene GAPDH mRNA primer pair GAPD_IC in human genome, as two shown in sequence in sequence table 10 and sequence 11
Bar single strand dna forms.The internal reference probe (GAPD_IC_MB) is that the single stranded DNA in sequence table shown in sequence 12 is visited
Pin, for monitoring the amplification of the internal reference primer pair (GAPD_IC) in real time.
5 ' ends of the internal reference probe are marked with fluorescent reporter group FAM, and 3 ' ends are marked with fluorescent quenching group
TAMRA。
Constant-temperature amplification buffer solution and constant-temperature amplification enzyme solutions can also be contained in the kit.
The solvent of the constant-temperature amplification buffer solution is water, and solute and concentration are as follows：200mM pH 8.0 Tris-HCL,
50mM DTT, 10mM dNTP, 10mM rNTP, 80mM MgCl2, 450mM KCl, 15% volumn concentration DMSO, 1M sorb
Alcohol, 20mM tetramethyl ammonium chlorides.
The solvent of the constant-temperature amplification enzyme solutions is water, and solute and concentration are as follows：AMV reverse transcriptase 1U/ μ l, T7RNA gather
Synthase 5U/ μ l, ribonuclease H 0.5U/ μ l, the μ g/ μ l of pyrophosphatase 0.5U/ μ l, RNase inhibitor 5U/ μ l, BSA 0.5.
Dish-style chip can also be contained in the kit, such as 24 reaction chamber dish-style chips.
In the present invention, the 24 reaction chamber dish-style chip is " the brilliant core of Capitalbio Corporation Co., Ltd.'s production
The supporting dish-style chip of RTisochipTM-A constant-temperature amplification micro-fluidic chips nucleic acids instrument ", its model 1 × 24.
The 1# of the dish-style chip deposits dry following (1)-(5) to 5# reaction chambers respectively：
(1) primer pair 1 and the ssDNA probe 1；(2) primer pair 2 and the ssDNA probe 2；(3)
The primer pair 3 and the ssDNA probe 3；(4) the internal reference primer pair and the internal reference probe；(5) negative control
Product.
The negative controls concretely water without RNase (Rnase-free water).
Wherein, the method for primer and probe being embedded into dish-style chip is：By primer, the spy corresponding with the primer
Pin, agarose mixing, are configured to mixed solution, make the primer, the probe and the agarose in the mixed solution
Final concentration be respectively 0.2 μM, 50nM, 0.1% (weight/mass percentage composition)；Mixed solution described in 1 μ l is taken to click and enter corresponding dish-style
Chip reaction chamber, after being dried in clean super-clean bench, tabletting encapsulation, punching press is made after vacuumizing.
The non-diagnostic mesh of the primer pair group or complete single stranded DNA or kit in detecting or aiding in detection enterovirus
Application fall within protection scope of the present invention.
In the application, by constant-temperature amplification enzyme solutions described in constant-temperature amplification buffer solution described in 15 μ l and 10 μ l, with 25 μ l
It is injected into after the mixing of sample to be tested solution in the dish-style chip, isothermal reaction 1h at 41 DEG C.
In the application, sample to be tested can be used as using Nasopharyngeal swabs；Accordingly, the sample to be tested solution can be from nose
The RNA extracted in throat swab.
In the present invention, the enterovirus is concretely at least one of following：Enterovirus, enterovirus EV 71
Type and enterovirus CA16 types.
Kit provided by the invention supports high flux, can quickly and accurately detect enterovirus infection, for clinical
Speech, can be obtained in 1 hour 3 kinds of enterovirus indexs testing result be not only faster than it is current more generally use it is real-time glimmering
Fluorescent Quantitative PCR method, and it is also significant for the treatment of quick auxiliary direction and medication.Meanwhile the detection of multi objective
Regional epidemiological survey and epidemic situation monitoring are can be used for, to study popularity of the enterovirus infection in China.
Brief description of the drawings
Fig. 1 is dish-style chip and primer sample application cavity schematic diagram.
Fig. 2 is 3 kinds of enterovirus reference material dish-style chip testing result figures.Wherein, A is to contain enterovirus target gene
Recombinant plasmid pUC19-Entv 1# reaction chambers testing result；B is the restructuring matter containing enterovirus EV 71 target gene
Testing results of the grain pUC19-EV71 in 2# reaction chambers；C is the recombinant plasmid pUC19- containing enterovirus CA16 target genes
Testing results of the CA16 in 3# reaction chambers.In A-C, E1 represents that template is 101Copy/μ l, by that analogy, E5 represent that template is 105
Copy/μ l；NC represents negative control.
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.
The preparation and its use of embodiment 1, kit for detecting enterovirus
First, for the preparation for the kit for detecting enterovirus
Kit forms provided by the present invention for detecting enterovirus are as follows：
1st, constant-temperature amplification buffer solution
The solvent of constant-temperature amplification buffer solution is water, and solute and concentration are as follows：200mM Tris-HCL (pH 8.0), 50mM
DTT, 10mM dNTP, 10mM rNTP, 80mM MgCl2, 450mM KCl, 15% volumn concentration DMSO, 1M sorbierite,
20mM tetramethyl ammonium chlorides.
2nd, constant-temperature amplification enzyme solutions
The solvent of constant-temperature amplification enzyme solutions is water, and solute and concentration are as follows：AMV reverse transcriptase 1U/ μ l, t7 rna polymerase
5U/ μ l, ribonuclease H 0.5U/ μ l, pyrophosphatase 0.5U/ μ l, RNase inhibitor 5U/ μ l, BSA0.5 μ g/ μ l.
3rd, 24 reaction chamber dish-style chips of primer pair and ssDNA probe are mounted with
The 24 reaction chamber dish-style chip is " the brilliant core RTisochipTM-A perseverances of Capitalbio Corporation Co., Ltd.'s production
The supporting dish-style chip of temperature amplification micro-fluidic chip nucleic acids instrument ", its model 1 × 24, schematic diagram is as shown in Figure 1.
The 1# of the 24 reaction chamber dish-style chip deposits dry following (1)-(5) to 5# reaction chambers respectively：
(1) primer pair 1 and ssDNA probe 1；(2) primer pair 2 and ssDNA probe 2；(3) primer pair 3 and single stranded DNA
Probe 3；(4) internal reference primer pair and internal reference probe；(5) negative controls.The negative controls are specially without RNase
Water (Rnase-free water).
Wherein, the primer pair 1 (Entv_TY) is used to expand enterovirus, as shown in sequence in sequence table 1 and sequence 2
Two single strand dnas composition；The primer pair 2 (EV71_VP1) is used to expand enterovirus EV 71 type, by sequence table
Two single strand dnas composition shown in sequence 3 and sequence 4；The primer pair 3 (CA16_VP1) is used to expand enterovirus
CA16 types, it is made up of two single strand dnas shown in sequence in sequence table 5 and sequence 6.Internal reference primer pair (the GAPD_
IC) it is used to expand house-keeping gene GAPDH mRNA in human genome, as two lists shown in sequence in sequence table 10 and sequence 11
Ssdna molecule forms.
The ssDNA probe 1 (Entv_TY_MB) is used for the amplification for monitoring the primer pair 1 in real time, its nucleosides
Acid sequence is sequence 7 in sequence table；The ssDNA probe 2 (EV71_VP1_MB) is used to monitor the primer pair 2 in real time
Amplification, its nucleotides sequence are classified as sequence 8 in sequence table；The ssDNA probe 3 (CA16_VP1_MB) is used to supervise in real time
The amplification of the primer pair 3 is surveyed, its nucleotides sequence is classified as sequence 9 in sequence table.Internal reference probe (the GAPD_IC_
MB) it is used for the amplification for monitoring the internal reference primer pair (GAPD_IC) in real time, its nucleotides sequence is classified as sequence in sequence table
12.5 ' the ends mark of every ssDNA probe and the internal reference probe (GAPD_IC_MB) has base
Group FAM, 3 ' ends mark have TAMRA.
Wherein, the method for primer and probe being embedded into dish-style chip is：By primer, the spy corresponding with the primer
Pin, agarose mixing, are configured to mixed solution, make the primer, the probe and the agarose in the mixed solution
Final concentration be respectively 0.2 μM, 50nM, 0.1% (weight/mass percentage composition)；Mixed solution described in 1 μ l is taken to click and enter corresponding dish-style
Chip reaction chamber, after being dried in clean super-clean bench, tabletting encapsulation, punching press is made after vacuumizing.
2nd, for the application method for the kit for detecting enterovirus
1st, reaction system is prepared
15 μ l constant-temperature amplifications buffer solutions (see step 1 1), 10 μ l constant-temperature amplifications enzyme solutions (see step 1 2), 25 μ l are taken to treat
This liquid of test sample is mixed into 50 μ l reaction solutions, after vortex concussion uniformly injection be mounted with primer pair and the 24 of ssDNA probe anti-
Chamber dish-style chip (see step 1 3) is answered, seals Quick spin 30s after membrana oralis.
2nd, isothermal amplification reactions and detection
Dish-style chip is placed in rich Isochip-RT constant-temperature amplifications instrument difficult to understand, sets 41 DEG C to react 1 hour, and complete simultaneously
Real-time fluorescence scans.
3rd, result judges
After reaction terminates, the sample to be tested liquid is determined according to the amplification curve of sample in each reaction chamber as follows
In whether contain corresponding enterovirus genome：If the testing sample produces S type amplification curves, the sample to be tested liquid
In contain corresponding enterovirus genome；Conversely, the genome of corresponding enterovirus is not contained in the sample to be tested liquid then.
Embodiment 2, the analysis of the sensitivity and specificity of kit for detecting enterovirus
First, the preparation of reference material RNA nucleic acid
1st, structure includes the plasmid of enterovirus target gene
(1) plasmid containing enterovirus target gene
By 5~1705 sections of enterovirus Entv target gene sequences (enterovirus Entv target gene sequences
Genbank Sequence ID:Gb | KC755230.1 |, Update Date：2013-12-17) be inserted into pUC19 carriers (my god
With biochemical corp's product) multiple cloning sites EcoR V between, obtain recombinant plasmid pUC19-Entv.
(2) plasmid containing enterovirus EV 71 target gene
By 4~1695 sections of enterovirus EV 71 target gene sequence (enterovirus EV 71 target gene sequence
Genbank Sequence ID:Gb | HM245928.1 |, Update Date：2011-9-27) be inserted into pUC19 carriers (my god
With biochemical corp's product) multiple cloning sites EcoR V between, obtain recombinant plasmid pUC19-EV71.
(3) plasmid containing enterovirus CA16 target genes
By 1392~2564 sections of enterovirus CA16 target gene sequences (enterovirus CA16 target gene sequences
Genbank Sequence ID:Gb | AY790926.1 |, Update Date：2005-8-9) be inserted into pUC19 carriers (day with
Biochemical corp's product) multiple cloning sites EcoR V between, obtain recombinant plasmid pUC19-CA16.
2nd, the preparation of reference material RNA nucleic acid
For trying recombinant plasmid：3 kinds of recombinant plasmids for containing corresponding enterovirus target gene that step 1 is built.
(1) digestion：First 37 DEG C of digestion 2h of EcoRI restriction endonucleases are used by each for examination recombinant plasmid.
(2) transcribe：It polymerize by 5 μ l 5 × Transcription Optimized Buffer (Promega), 2U T7RNA
Enzyme, 10mM DTT (Promega), 10U recombinant RNAs enzyme inhibitor (Promega), 2mM rNTP, the μ l of digestion products 5 prepare overall
Product is 50 μ l reaction system, 37 DEG C of transcription 4h after shaking uniformly.
(3) digest：System after the completion of transcription is added to 1 μ l DNA digestive ferments (rDnaseI, 5U/ μ l), concussion centrifuges,
37 DEG C of incubation 20min.
(4) purify：Using the RNA purification kits products of Macherey-Nage companies, (its catalog number is
740948) transcription product RNA is purified as follows：A) RA1-C is prepared2H5OH mixed liquors：With RA1:C2H5OH=1:1 (volume
Than) ratio prepare., it is necessary to add 600 μ l RA1-C in every 100 μ l transcription product2H5OH mixed liquors, i.e. 300 μ l RA1+
300μl C2H5OH solution, need to calculate the volume for matching somebody with somebody mixed liquor according to the pipe number of transcription product herein.If product is insufficient
100 μ l, then the amount of product is mended to 100 μ l with water and (50 μ l Tiangeng Rnase-free H are added in every pipe product2O)。
B) the ready RA1-C by before2H5OH mixed liquors are assigned in 1.5ml centrifuge tubes, two pipes, often the μ l of pipe 600, and 100 μ l products are turned
Enter into corresponding centrifuge tube, totally 700 μ l, fully shaking centrifuge in pipe.C) prepare two adsorption columns, and carry out corresponding mark
Note, 700 μ l product mixtures are transferred in adsorption column (adsorption column maximum capacity is 700 μ l), 1.2 ten thousand rpm centrifugation 2min, are fallen
Fall down a layer solution.D) 700 μ l RA3 is added in adsorption column, 1min is placed, it is sufficiently dispersed in the bottom of adsorption column
Portion, 1.2 ten thousand rpm centrifugation 2min, outwells lower floor's solution.E) 350 μ l RA3 is added in adsorption column, places 2min, 1.2 ten thousand rpm
2min is centrifuged, outwells lower floor's solution.F) 300 μ l RA3 is added in adsorption column, 2min is placed, 1.2 ten thousand rpm centrifugation 2min, falls
Fall down a layer solution.G) after the lid 3min for opening adsorption column, 1.2 ten thousand rpm skies outwell lower floor's solution, repeat this step from 2min
Once.H) it is empty from end after, adsorption column is transferred in corresponding centrifuge tube, opens adsorption column lid and place 15min, make
Ethanol volatilization is complete, and 60 μ l Rnase-H are added in adsorption column2O (carries) elution in kit, place 2min, and 1.2 ten thousand
Rpm centrifuges 2min.I) centrifugation gained template is sucked back in adsorption column, places 2min, 1.2 ten thousand rpm centrifugation 2min, repeat this step
Twice.J) adsorption column is lost, the template in centrifuge tube is taken out into 2 μ l, determines its concentration with Nano drop, and record its concentration
And its 260/280,260/230 ratio.
2nd, for the sensitivity and specificity analysis for the kit for detecting enterovirus
1st, prepared by the reference material template of various concentrations
It is fixed that each RNA templates (the 3 kinds of recombinant plasmids built in corresponding step 1 1) of above-mentioned steps one after purification are calculated
Measure to 1010Copy/μ l, and gradient dilution, obtain 105Copy/μ l, 104Copy/μ l, 103Copy/μ l, 102Copy/μ l, 10 are copied
The template dilution of the various concentrations such as shellfish/μ l.
2nd, reaction system is prepared
15 μ l constant-temperature amplifications buffer solutions are taken (see the step 1) of embodiment 1,10 μ l constant-temperature amplifications enzyme solutions (see the step of embodiment 1
2), the template dilution of 25 μ l concentration is mixed into 50 μ l reaction solutions, after vortex concussion uniformly injection be mounted with primer pair and
24 reaction chamber dish-style chips of ssDNA probe (see the step 3) of embodiment 1, seal Quick spin 30s after membrana oralis.
3rd, isothermal amplification reactions and detection
Dish-style chip is placed in rich Isochip-RT constant-temperature amplifications instrument difficult to understand, sets 41 DEG C to react 1 hour, and complete simultaneously
Real-time fluorescence scans.And result is judged according to the method for the step 23 of embodiment 1.
3rd, result
As a result it is as shown in Figure 2：
(1) for recombinant plasmid pUC19-Entv RNA sample, the 1# reaction chambers pair of 24 reaction chamber dish-style chips
105Copy/μ l, 104Copy/μ l, 103Copy/μ l, 102The testing result of copy/μ l, 10 copies/μ l templates has obvious S
Type amplification curve, it is shown as positive；And 2# and 3# reaction chambers are shown as negative without amplification curve.
(2) for recombinant plasmid pUC19-EV71 RNA sample, the 2# reaction chambers pair of 24 reaction chamber dish-style chips
105Copy/μ l, 104Copy/μ l, 103Copy/μ l, 102The testing result of copy/μ l, 10 copies/μ l templates has obvious S
Type amplification curve, it is shown as positive；And 1# and 3# reaction chambers are shown as negative without amplification curve.
(3) for recombinant plasmid pUC19-CA16 RNA sample, the 3# reaction chambers pair of 24 reaction chamber dish-style chips
105Copy/μ l, 104Copy/μ l, 103Copy/μ l, 102The testing result of copy/μ l, 10 copies/μ l templates has obvious S
Type amplification curve, it is shown as positive；And 1# and 2# reaction chambers are shown as negative without amplification curve.
In addition, for the RNA sample of 3 kinds of all recombinant plasmids, the 4# reaction chambers of 24 reaction chamber dish-style chips are (interior
Control) and 5# reaction chambers (negative control) without amplification curve.4# reaction chambers (internal reference) why without amplification curve be because
Sample to be tested is the RNA nucleic acid of 3 kinds of recombinant plasmids, does not contain the RNA of house-keeping gene GAPDH in human genome.
It these results suggest that the kit has higher amplification sensitivity and specific amplification.
The detection of embodiment 3, actual clinical sample
First, clinical sample type
The present embodiment use clinical sample from Shenzhen San Yuan in collection Nasopharyngeal swabs sample (in line with this picker from
The principle of hope), adopt swab and deposit in 3mL physiological saline, totally 560.
2nd, in clinical sample viral nucleic acid extraction
Clinical sample viral nucleic acid extraction kit used is QIAamp Viral RNA Mini Kit (Qiagen),
Extracted as follows：
(1) take and the μ l of physiological saline 140 of clinical sample swab are deposited in step 1 in 1.5ml centrifuge tubes；
(2) Buffer AVL (the i.e. 5.6 μ l Carrier RNA mixing that 560 μ l contain Carrier RNA mixed liquors is added
The μ l Buffer of liquid+560 AVL are in centrifuge tube, slight whirlpool 15s；
(3) after the completion of brief centrifugation, (15-25 DEG C) placement 10-20min of room temperature, to ensure Buffer AVL in centrifuge tube
In have abundance time cracked；
(4) 560 μ l ethanol (96-100%, volumn concentration) are added in centrifuge tube, whirlpool mixes 15s, centrifugation；
(5) 630 μ l sample solutions are taken in adsorption column, 2min is placed, it is fully contacted with adsorption column, 8000rpm, from
Heart 1min, change clean collecting pipe；
(6) if sample solution is more than 630 μ l, repeatedly previous step；
(7) 500 μ l AW1 washing lotions are added in adsorption column, 2min is placed, it is fully contacted with adsorption column, 8000rpm
1min is centrifuged, changes clean collecting pipe；
(8) 500 μ l AW2 washing lotions are added in adsorption column, 2min is placed, it is fully contacted with adsorption column,
12000rpm centrifuges 3min, changes clean collecting pipe；
(9) 300 μ l AW2 washing lotions are added in adsorption column, 2min is placed, it is fully contacted with adsorption column,
12000rpm centrifuges 3min, changes clean collecting pipe；
(10) adsorption column lid is opened and places 2min, make that its inside and outside pressure is consistent, and 8000rpm skies are from 1min.It is empty from knot
Shu Yihou, repeat this step once.
(11) it is empty from end after, adsorption column is transferred in 1.5ml centrifuge tubes, open adsorption column lid, place 20-30min,
Make ethanol volatilization complete；
(12) 50 μ l Buffer AVE elution nucleic acid, 8000rpm centrifugation 1min, after centrifugation is completed, by under elution are added
Nucleic acid suck back adsorption column, lose adsorption column after centrifuging 1min.
3rd, the detection of actual clinical sample
1st, reaction system is prepared
15 μ l constant-temperature amplifications buffer solutions (see the step 11 of embodiment 1), 10 μ l constant-temperature amplifications enzyme solutions are taken (see the step of embodiment 1
Rapid 1 2), the clinical sample solution that 25 μ l step 2 are extracted to obtain is mixed into 50 μ l reaction solutions, is injected after vortex concussion uniformly
It is mounted with the 24 reaction chamber dish-style chips (see the step 13 of embodiment 1) of primer pair and ssDNA probe, seals rapid after membrana oralis
Centrifuge 30s.
2nd, isothermal amplification reactions and detection
Dish-style chip is placed in rich Isochip-RT constant-temperature amplifications instrument difficult to understand, sets 41 DEG C to react 1 hour, and complete simultaneously
Real-time fluorescence scans.And result is judged according to the method for the step 23 of embodiment 1.
Experiment sets reliability demonstration of the conventional RT-PCR method for kit testing result of the present invention simultaneously.For examining
The specific RT-PCR primer sequence for surveying each enterovirus is as shown in table 1.
Table 1 is used for the specific RT-PCR primer sequence for detecting 3 kinds of enteroviruses
| Index name | RT-PCR sense primers (5 ' -3 ') | RT-PCR anti-sense primers (5 ' -3 ') |
| Enterovirus | CCCCTGAATGCGGCTAATCCYAAC | CCAATCCAATAGCTATATGGYAACAA |
| Enterovirus EV 71 type | AACACCCGTAYGTGCTTGATGCTGG | TAGGGATTACTGGCGTCGCTCCTTG |
| Enterovirus CA16 types | CACCCCCATGCARCGCTTGTGYTTT | TRGACTGCCACGGRCCATCTCTTCC |
4th, result
The testing result of kit of the present invention is shown, for each enterovirus, is detected using RT-PCR method positive
Sample example using kit of the present invention detection show positive findings.Indicated above, kit of the present invention is to corresponding enteron aisle disease
The testing result of poison is accurately and reliably.The statistical result of kit and RT-PCR method of the present invention detection enterovirus clinical sample referring to
Table 2.
The statistical result of 2 kit of the present invention of table and RT-PCR methods detection enterovirus clinical sample
Claims (2)
1. the kit for detecting enterovirus, contains complete single stranded DNA；The complete single stranded DNA, by probe groups and primer
To a group composition；
The primer pair group, it is made up of following 3 primer pairs：As two single stranded DNAs shown in sequence in sequence table 1 and sequence 2
The primer pair 1 of molecular composition；The primer pair 2 being made up of two single strand dnas shown in sequence in sequence table 3 and sequence 4；By
Sequence 5 and the primer pair 3 of two single strand dnas composition shown in sequence 6 in sequence table；
The probe groups are made up of following 3 ssDNA probes：SsDNA probe 1 in sequence table shown in sequence 7；Sequence table
SsDNA probe 2 shown in middle sequence 8；SsDNA probe 3 in sequence table shown in sequence 9；
5 ' the ends mark of every ssDNA probe in the probe groups has FAM, and 3 ' ends mark has
Optical quenching group TAMRA；
The kit also contains internal reference primer pair and internal reference probe；The internal reference primer pair is by sequence in sequence table 10
With two single strand dnas composition shown in sequence 11；The internal reference probe is the single stranded DNA shown in sequence 12 in sequence table
Probe；5 ' ends of the internal reference probe are marked with fluorescent reporter group FAM, and 3 ' ends are marked with fluorescent quenching group TAMRA；
Also contain dish-style chip in the kit；
The 1# of the dish-style chip deposits dry as follows respectively to 6# reaction chambers（1）-（5）：
（1）The primer pair 1 and the ssDNA probe 1；
（2）The primer pair 2 and the ssDNA probe 2；
（3）The primer pair 3 and the ssDNA probe 3；
（4）The internal reference primer pair and the internal reference probe；
（5）Negative controls；
The dish-style chip is 24 reaction chamber dish-style chips.
2. kit according to claim 1, it is characterised in that：In the kit also containing constant-temperature amplification buffer solution and
Constant-temperature amplification enzyme solutions；
The solvent of the constant-temperature amplification buffer solution is water, and solute and concentration are as follows：200mM pH 8.0 Tris-HCL, 50mM
DTT, 10mM dNTP, 10mM rNTP, 80mM MgCl2, 450mM KCl, 15% volumn concentration DMSO, 1M sorbierite,
20mM tetramethyl ammonium chlorides；
The solvent of the constant-temperature amplification enzyme solutions is water, and solute and concentration are as follows：AMV reverse transcriptase 1U/ μ l, T7 RNA polymerize
Enzyme 5U/ μ l, ribonuclease H 0.5U/ μ l, the μ g/ μ l of pyrophosphatase 0.5U/ μ l, RNase inhibitor 5U/ μ l, BSA 0.5.
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