CN101665839A - Enterovirus type-71 nucleic acid amplification liquid chip test kit - Google Patents
Enterovirus type-71 nucleic acid amplification liquid chip test kit Download PDFInfo
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- CN101665839A CN101665839A CN200810042338A CN200810042338A CN101665839A CN 101665839 A CN101665839 A CN 101665839A CN 200810042338 A CN200810042338 A CN 200810042338A CN 200810042338 A CN200810042338 A CN 200810042338A CN 101665839 A CN101665839 A CN 101665839A
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Abstract
The invention belongs to the field of biotechnology and particularly relates to an enterovirus type-71 test kit. The invention discloses an enterovirus type-71 nucleic acid amplification liquid chip test kit, which comprises: a pair of EV type-71 specific primers A and A' having biotin-marked 5'-terminals; a pair of EV type-71 specific primers B and B' having biotin-marked 5'-terminals; fluorescence-encoded microspheres C cross-linked with probes E matched with the sequence of amplified fragments of the primers A and A'; fluorescence-encoded microspheres D cross-linked with probes F matched with the sequence of amplified fragments of the primers B and B'; and QRT-PCR reagent and fluorescence-marked avidin.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of detection kit of enterovirns type 71.
Background technology
(enterovirus71 EV71) is Picornaviridae (Picornaviridae) enterovirus genus (Enterovirus) member to enterovirns type 71.EV71 infects and mainly causes patient's hand foot mouth disease (HFMD), and is this sick main pathogens.In addition, EV71 also can cause the multiple neural system relative diseases such as paralysis of aseptic meningitis, BBE and poliomyelitis sample, but and outbreak of epidemic, cause death, the serious harm human health.
In order when the EV71 relative disease breaks out, can to find out the cause of disease rapidly, thereby in time take correct, effective measure of control, need badly at EV71 accurately, fast, highly sensitive, high specific, high-throughout novel diagnostic means.Enterovirus infection have " one is sick because of how, one sick many because of " characteristics, only be not apt to do correct diagnosis according to clinical symptom.For example, the enterovirus that causes hand foot mouth disease just has kind more than 20 (type), 16,4,5,9,10 types of CA group, 2,5 types of B group, and enterovirns type 71 is the more common pathogenic agent of hand foot mouth disease, wherein common with enterovirns type 71 and coxsackie virus A 16-type (Coxsakie A16 is called for short CoxA16).The traditional detection method of enterovirus is viral separation, serum neutralization test and diagnosis of molecular biology technology.Virus lock out operation complexity, length consuming time, expense height, separation rate are low.Though the immunological method score is easier, quick from culture method, still need the time about 1 week whole detection time.After the patient infection virus, producing specific antibody in the serum needs for some time.In addition, have only 2～10 days after EV71 infects latent period of (average 3～5 days), and infectivity starts from morbidity a few days ago, so the immunological method susceptibility is lower, especially is not suitable for early diagnosis.Strain antigenic variation, antigen-antibody cross reaction, patient individual difference etc. have all influenced the accuracy and the specificity of this method largely.In recent years, polymerase chain reaction (PCR) technology is widely used in the detection of virus-specific gene, although it also has sensitivity, special, advantage fast, the result judges needs electrophoresis, and reaction product is easy to generate and pollutes and cause false positive.
This shows that all there is bigger deficiency in enterovirns type 71 detection means commonly used at present, to a large amount of clinical samples, especially early stage sample is accurate, the requirement of quick diagnosis in the time of can not satisfying outbreak of disease.
Summary of the invention
Technical problem to be solved by this invention is that existing enterovirns type 71 detection means all exists bigger insufficient problem
In order to address the above problem, the invention discloses a kind of enterovirus type-71 nucleic acid amplification liquid chip test kit, comprising:
A pair of EV71 genome specificity primer A and A ', wherein 5 ' of A and/or A ' end is marked with vitamin H;
A pair of EV71 genome specificity primer B and B ', wherein 5 ' of B and/or B ' end is marked with vitamin H;
The fluorescence-encoded micro-beads C of a kind of crosslinked and primer A and A ' amplified fragments sequence paired probe E;
The fluorescence-encoded micro-beads D of a kind of crosslinked and primer B and B ' amplified fragments sequence paired probe F;
QRT-PCR reagent;
The avidin of fluorescent-substance markers.
Among the present invention, definition " EV71 genome specificity primer " is meant that with disclosed EV71 nucleotide sequence among the GeneBank be template, those skilled in the art obtain at EV71 genome specificity primer sequence by known primer-design software, and corresponding primer can prepare by the nucleic acid synthesizer.The sequence of A of primer described in the present invention and A ', B and B ' is all inequality.
In an embodiment, described fluorescence-encoded micro-beads C is 144 microballoons of luminex company.
In an embodiment, described fluorescence-encoded micro-beads D is 146 microballoons of luminex company.
In some embodiments, fluorescence is selected from fluorescein isothiocyanate, tetraethylrhodamine, TRITC, phycoerythrin, 3 valency lanthanide chelates such as europium (Eu in the avidin of described fluorescent-substance markers
3+), terbium (Tb
3+), cerium (Ce
3+One of) in waiting, wherein preferred phycoerythrin.
In some embodiments, described fluorophor is FAM, TET, VIC, or HEX.
In an embodiment, the nucleotides sequence of described EV71 Auele Specific Primer A and A ' is classified 5 '-GCGGGTAGTGTGTCGTAAC-3 ' (SEQ ID NO:4) and 5 '-GGTGGTCACAGACTTCAAGGTT-3 ' (SEQ ID NO:5) as; Described nucleotides sequence with primer A and A ' amplified fragments sequence paired probe E is classified 5 '-NH-TTTTTTGGATTGGCCATCCGGTGTGCA-3 ' (SEQ ID NO:6) as.
In an embodiment, the nucleotides sequence of described EV71 Auele Specific Primer B and B ' is classified 5 '-GATTGAGACACGCTGTGTTCT-3 ' (SEQ ID NO:7) and 5 '-GCCTTCAAGAGGGAGGTCTATC-3 ' (SEQ ID NO:8) as; Described nucleotides sequence with primer B and B ' amplified fragments sequence paired probe F is classified 5 '-NH-TTTTTTCAGCAGAGCGGGATTAGTTGGAC-3 ' (SEQ ID NO:9) as.
Nucleic acid amplification liquid chip EV71 detection technique step
(1) design of primer and probe: design two pairs of EV71 Auele Specific Primers and corresponding hybridization probe pcr amplification and detection are carried out in two zones.
(2) probe and fluorescence-encoded micro-beads is crosslinked.
(3) preparation of EV71RT-PCR reaction solution: each reacted constituent is mixed by finite concentration.
(4) viral nucleic acid detects: by pcr amplification, the hybridization of fluorescence-encoded micro-beads probe and product is carried out liquid gene chip at last and is detected.
(5) result judges, detects data computation Cutoff value according to the reference product, thus the judgement sample attribute.
Liquid biochip disclosed in this invention detects and not only has more excellent specificity and sensitivity, more has the characteristics that the conventional sense technology can not have: high-throughput, and the result is stable simultaneously, and good reproducibility is easy and simple to handle.Unique amplification site is selected, especially the dual area of liquid chip detects, can reduce significantly because of single nucleotide polymorphism (single nucleotide polymorphism, SNP) and the new subtype virus strain occur and the detection false negative that causes, guarantee the accuracy of detected result, finally met quick, sensitive, easy, accurate, the high-throughout requirement of current enterovirus Clinical Laboratory.
Embodiment
Below in conjunction with specific embodiment, further illustrate the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.Ratio and per-cent are based on weight, unless stated otherwise.
Further specify the present invention with embodiment below, but the present invention is not limited.
(1) quantitative fluorescent PCR EV71 detection reagent is to the detection (liquid chip detects and carries out synchronously, and is described separately at this) of clinical sample
(1) design of primer and probe: design a pair of primer and one 5 ' the Taq-Man probe of holding mark fluorescent group and 3 ' end mark BHQ, probe mark FAM group at EV71 genome specificity conserved sequence.Probe sequence: 5 ' TCGCACAGCACAGCTGAGACCAC-3 ' (SEQ ID NO:3).Upstream primer: 5 '-GATTGAGACACGCTGTGTTCT-3 ' (SEQ IDNO:1); Downstream primer: 5 '-GCCTTCAAGAGGGAGGTCTATC-3 ' (SEQ ID NO:2);
(2) preparation of EV71QRT-PCR reaction solution: each reacted constituent is mixed, and composition and concentration (20 μ L system) are: 2 * reaction Mix, 10 μ L, upstream primer final concentration 0.4 μ M, downstream primer final concentration 0.2 μ M, probe final concentration 0.4 μ M.
(3) preparation of reference product: the nucleic acid with enterovirns type 71 is template, behind RT-PCR method acquisition PCR product, is diluted to specific concentrations and makes strong positive contrast and critical positive control.Negative control is prepared from the stool sample that adopts " gold mark " to confirm as the EV71 negative findings.
(4) viral nucleic acid extracting: the viral nucleic acid extracting is provided (utilizing Promega viral nucleic acid magnetic bead extraction agent box to finish) on Beckman Biomek 3000 nucleic acid automatic extracting instruments by Center of Diseases Prevention ﹠ Control, Shenzhen City (CDC), test experience is finished at this mechanism microorganism detection center.
(5) viral nucleic acid detects: get viral RNA nucleic acid 9.3 μ L, and QRT-PCR reaction solution 10.2 μ L, reaction enzymes 0.5 μ L is provided with critical positive control, strong positive contrast, negative control simultaneously.Reaction conditions: 50 ℃, 15min; 95 ℃, 2min; 95 ℃, 15sec, 60 ℃, 30sec, 50 circulations.Adopt Stratagene Mx3005P quantitative real time PCR Instrument, fluorescent signal is set at the FAM fluorescein when collecting, and fluorescent signal is collected and is located at 60 ℃.
(6) result judges: threshold value (threshold) can generate automatically, also can adjust according to instrument noise situation, and setting principle is with the vertex of threshold line just above normal negative control product amplification curve (random noise line), and Ct value=50.0 are as the criterion.
Detect 5＜Ct≤50 in the sample, sample is reported as the positive; The Ct value is " No Ct " in the detection sample, and sample is reported as feminine gender; For more weak amplification, single curve is wanted separate analysis, adjusts threshold line and just exceeds baseline, if amplification curve exceeds threshold line, then this sample is positive.
(2) EV71 nucleic acid amplification liquid chip detection reagent is to the detection of clinical sample
(1) design of primer and probe: design two pairs of EV71 Auele Specific Primers, 5 ' end is marked with vitamin H (biotin); Also have a pair of corresponding hybridization probe in addition, end has amino.Upstream primer 1:5 '-Biotin-GCGGGTAGTGTGTCGTAAC-3 '; Downstream primer 1:5 '-GGTGGTCACAGACTTCAAGGTT-3 '; Probe 1:5 '-NH-TTTTTTGGATTGGCCATCCGGTGTGCA-3 '; Upstream primer 2:5 '-GATTGAGACACGCTGTGTTCT-3 '; Downstream primer 2:5 '-Biotin-GCCTTCAAGAGGGAGGTCTATC-3 '; Probe 2:5 '-NH-TTTTTTCAGCAGAGCGGGATTAGTTGGAC-3 '.
(2) probe and fluorescence-encoded micro-beads is crosslinked: with two kinds of specific probes crosslinked with 144 and No. 146 fluorescence-encoded micro-beads respectively (cross-linking process slightly).
(3) preparation of EV71RT-PCR reaction solution: each reacted constituent is mixed, and composition and concentration (20 μ L system) are: 2 * reaction Mix, 10 μ L, the primer final concentration is 0.4 μ M.
(4) viral nucleic acid detects:
Pcr amplification: get viral RNA nucleic acid 9.18 μ L, RT-PCR reaction solution 10.32 μ L, reaction enzymes 0.5 μ L is provided with critical positive control, strong positive contrast, negative control (the same fluorescent quantitation of preparation method) simultaneously.Reaction conditions: 50 ℃, 15min; 95 ℃, 2min; 95 ℃, 15sec, 55 ℃, 20sec, 72 ℃, 30sec, 50 circulations.
Hybridization: get microballoon diluent 15 μ L and two kinds of crosslinked microballoons that probe arranged respectively 2 μ L mix, add above-mentioned amplified production 5 μ L then, blank is set simultaneously, and (blank is 1 * TE).Hybridization conditions: 95 ℃, 5min, 55 ℃, 20min.
Detect: (PE, Invitrogen) 36 μ L are hatched 30min for 55 ℃ to add 2 μ g/mL phycoerythrin.Go up at liquid chip 200 analysers (QIAGEN) then and detect.Sample panel is set to 55 ℃; Last sample volume is 50 μ L; Be 75sec sample time (timeout); Gate is provided with: lower limit 7500, the upper limit 15000; Unit is MFI; Minimum detection microballoon number is 100; Data are output as " data collection only ".Whole process is carried out according to Luminex 200 analyzer system instruction manuals.
(5) result judges: it is negative that middle fluorescence intensity (Medium) value is lower than the sample of Cutoff value, and it is positive that middle fluorescence intensity (Medium) value is higher than the sample of Cutoff value, Cutoff value=1.25 * NTC mean value+15.
(3) result and analysis
Clinical test results is as shown in table 1, the clinical positive sample of 30 routine EV71, and 28 examples are the fluorescence quantitative PCR detection positive, 2 routine quantitative fluorescent PCR negative sample wherein, liquid chip detects all positive.
This shows that though quantitative fluorescent PCR also can be to viral nucleic acid sensitivity, special detection, (single nucleotide polymorphism SNP) and the appearance of new subtype strain, and false negative usually occurs because single nucleotide polymorphism.Liquid biochip adopts multizone to detect, and can reduce false negative significantly.
The two check reagent box clinical test results of table 1EV71 nucleic acid amplification fluorescent quantitative and liquid chip
Annotate: on behalf of quantitative fluorescent PCR, the Ct value obvious amplification is arranged, and " No Ct " expression does not have amplification."-" expression does not detect.
Scope of the present invention is not subjected to the restriction of described specific embodiments, and described embodiment is only desired also to comprise the method and the component of functional equivalent in the scope of the invention as the single example of illustrating all respects of the present invention.In fact, except content as herein described, those skilled in the art can easily grasp multiple improvement of the present invention with reference to above description and accompanying drawing.Described improvement also falls within the scope of appended claims.
Sequence table
＜110〉Shanghai Geneprotech Co., Ltd.
＜120〉enterovirus type-71 nucleic acid amplification liquid chip test kit
＜130〉sequence table
<160>6
<170>PatentIn?version?3.3
<210>1
<211>19
<212>DNA
<213>artificial?sequence
<220>
<223>artificial?sequence
<400>1
gcgggtagtg?tgtcgtaac 19
<210>2
<211>22
<212>DNA
<213>artificial?sequence
<220>
<223>artificial?sequence
<400>2
ggtggtcaca?gacttcaagg?tt 22
<210>3
<211>27
<212>DNA
<213>artificial?sequence
<220>
<223>artificial?sequence
<400>3
ttttttggat?tggccatccg?gtgtgca 27
<210>4
<211>21
<212>DNA
<213>artificial?sequence
<220>
<223>artificial?sequence
<400>4
gattgagaca?cgctgtgttc t 21
<210>5
<211>22
<212>DNA
<213>artificial?sequence
<220>
<223>artificial?sequence
<400>5
gccttcaaga?gggaggtcta?tc 22
<210>6
<211>29
<212>DNA
<213>artificial?sequence
<220>
<223>artificial?sequence
<400>6
ttttttcagc?agagcgggat?tagttggac 29
Claims (7)
1. enterovirus type-71 nucleic acid amplification liquid chip test kit comprises:
A pair of EV71 genome specificity primer A and A ', wherein 5 ' of A and/or A ' end is marked with vitamin H;
A pair of EV71 genome specificity primer B and B ', wherein 5 ' of B and/or B ' end is marked with vitamin H;
The fluorescence-encoded micro-beads C of a kind of crosslinked and primer A and A ' amplified fragments sequence paired probe E;
The fluorescence-encoded micro-beads D of a kind of crosslinked and primer B and B ' amplified fragments sequence paired probe F;
QRT-PCR reagent;
The avidin of fluorescent-substance markers.
2. enterovirus type-71 nucleic acid amplification liquid chip test kit as claimed in claim 1, the nucleotide sequence that it is characterized in that described EV71 Auele Specific Primer A and A ' is shown in SEQ ID NO:4 and SEQ ID NO:5; The nucleotide sequence of described and A and A ' amplified fragments sequence paired probe E is shown in SEQ ID NO:6.
3. enterovirus type-71 nucleic acid amplification liquid chip test kit as claimed in claim 1, the nucleotide sequence that it is characterized in that described EV71 Auele Specific Primer B and B ' is shown in SEQ ID NO:7 and SEQ ID NO:8; The nucleotide sequence of described and primer B and B ' amplified fragments sequence paired probe F is shown in SEQ ID NO:9.
4. enterovirus type-71 nucleic acid amplification liquid chip test kit as claimed in claim 1 is characterized in that 144 microballoons of described fluorescence-encoded micro-beads C luminex company.
5. enterovirus type-71 nucleic acid amplification liquid chip test kit as claimed in claim 1 is characterized in that 146 microballoons of described fluorescence-encoded micro-beads D luminex company.
6. enterovirus type-71 nucleic acid amplification liquid chip test kit as claimed in claim 1 is characterized in that in the avidin of described fluorescent-substance markers that fluorescence is selected from fluorescein isothiocyanate, tetraethylrhodamine, TRITC, phycoerythrin or the 3 valency lanthanide chelates.
7. enterovirus type-71 nucleic acid amplification liquid chip test kit as claimed in claim 6 is characterized in that described fluorescence is a phycoerythrin.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104278079A (en) * | 2013-07-08 | 2015-01-14 | 嘉兴朝云帆生物科技有限公司 | Test strip and method for detecting nucleic acid through nucleic acid chromatographic technique |
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Cited By (1)
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| CN104278079A (en) * | 2013-07-08 | 2015-01-14 | 嘉兴朝云帆生物科技有限公司 | Test strip and method for detecting nucleic acid through nucleic acid chromatographic technique |
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Open date: 20100310 |