CN102154520B - Enterovirus CoxA 16/EV 71/universal nucleic acid detection kit - Google Patents
Enterovirus CoxA 16/EV 71/universal nucleic acid detection kit Download PDFInfo
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- CN102154520B CN102154520B CN201110079878.5A CN201110079878A CN102154520B CN 102154520 B CN102154520 B CN 102154520B CN 201110079878 A CN201110079878 A CN 201110079878A CN 102154520 B CN102154520 B CN 102154520B
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Abstract
The invention discloses an enterovirus CoxA 16/EV 71/universal nucleic acid detection kit, which comprises the following components: RT-PCR reaction liquid, enzyme mixed liquid, CoxA16 type/EV 71 type/universal type triple reaction liquid and RNase removing water. The enterovirus CoxA 16/EV 71/universal nucleic acid detection kit can simultaneously detect the enterovirus CoxA16, EV71 or other enteroviruses in the same reaction tube, and solves the problem that the existing in-vitro detection kit cannot simultaneously detect the enteroviruses related to the hand-foot-and-mouth disease in the same reaction tube. The detection kit has the advantages of strong specificity, high sensitivity, simple and convenient operation, strong repeatability, quick and objective detection result and the like.
Description
Technical field
The present invention relates to a kind of external diagnosis reagent case of hand foot mouth disease, relate in particular to enterovirus Cox A 16 type/EV71 type/universal nucleic acid detection kit, belong to the in-vitro diagnosis field of hand foot mouth disease.
Background technology
Hand foot mouth disease (Hand-foot-mouth disease; HFMD) have another name called dermexanthesis property blister stomatitis; Be that the common transmittable that caused by one group of enterovirus is sick, clinical manifestation is a kind of acute infectious disease of main clinical characteristics with the fash at positions such as heating and hand, foot, oral cavity.Most HFMD prognosis of patients are good, generally can belong to self-limited disease recovery from illness in 5～7 days.Virus can also be invaded respiratory system, cns etc. and cause symptoms such as encephalitis, wet lung, flaccid paralysis, myocarditis, and indivedual critical infants can cause death because of multiple reason.This disease all can be fallen ill the whole year, and good being sent out summer and autumn is common in the children below 5 years old.The main route of transmission of HFMD is a fecal oral route, also can pass through respiratory infectious.The HFMD infectivity is strong, is prone to cause popular or breaks out.The HFMD distributed pole is extensive, does not have strict provincialism.HFMD all can fall ill the whole year, and tangible seasonal characteristic is arranged, to see summer and autumn more.
Cause 4 types, 5 types, 7 types, 9 types, 10 types, 16 types of the Coxsackie virus that is mainly Picornaviridae (Picornaviridae) enterovirus genus (Coxsacki evirus) the A group of HFMD; 2 types of CB group, 5 types; Part Echo virus (ECHO-viruses) and enterovirns type 71.The most common is CA group 16 types (CoxA16) and enterovirns type 71 (EV71).The people is to the general susceptible of the enterovirus that causes HFMD, but the equal infection morbidity of each age group, but be main with inapparent infection, inapparent infection is about 100: 1 with the ratio of apparent infection.Because after body was infected by the virus, the neutralizing antibody of generation can retain the long period in vivo, homologous virus infected producing firm immunizing power, therefore, the patient of HFMD is mainly the preschool children, children below 5 years old especially, it is several more than 90% to account for morbidity.And great majority adults' portabilities virus but do not fall ill.The ratio that severes such as myocarditis, encephalitis took place with interior children in 1 year old is high than children more than 1 years old.
China's hand foot mouth disease is popular, Shanghai reported first hand foot mouth disease in 1981; The hand foot mouth disease that nineteen eighty-three Tianjin generation CoxA16 causes is broken out; Institute of viruses, nineteen ninety-five Wuhan isolates EV71 from the hand foot mouth disease philtrum; Taiwan 129106 routine HFMD in 1998, wherein 405 examples are serious central nervous system infection, 78 examples are dead; Since 1999, the report EV71 of localized epidemics in area such as Chinese Guangdong, Fujian, Shanghai, Chongqing infects; Linyi, Shandong in 2007 is popular to be main hand foot mouth disease with EV71; Ground such as 2008～2009 years Fuyangs, Henan, Shandong are popular on a large scale.Be mainly CoxA16 and EV71 and circulate altogether and cause that hand foot mouth disease sends out, EV71 is an advantage virus in most of provinces and cities.
With the fluorescence labeling probe is the real-time fluorescence PCR technology on basis, in Chinese domestic at present clinical diagnosis, uses the most extensive.In the real-time fluorescence PCR reaction system of probe method; According to what of detection pathogenic agent; Can also be divided into substance or multiple real time fluorescence round pcr, often comprise one or more pairs of specific PCR primers and probe in the reaction system, grope through the condition in the R&D process; Probe and primer only combine with corresponding template specificity ground, and its binding site is between two primers.5 of probe ' end be marked with reporter group (Reporter, R), like FAM, VIC, ROX etc., 3 ' end be marked with the fluorescent quenching group (Quencher, Q), like BHQ1, BHQ2, TAMRA etc.When probe was complete, reporter group institute emitted fluorescence energy was absorbed by quenching group, and instrument detecting is less than signal.Along with the carrying out of PCR, the Taq enzyme runs in the chain extension process and template bonded probe, and its 3 ' → 5 ' exonuclease activity will cut off probe, and reporter group is away from quenching group, and its energy can not be absorbed, and promptly produces fluorescent signal (Fig. 1).So every through a PCR circulation, fluorescent signal is also the same with the purpose fragment, the process that has a sync index to increase.
Defectives such as the external diagnosis reagent case of existing hand foot mouth disease has specificity mostly and susceptibility is lower, detection complex steps have much room for improvement.
Summary of the invention
It is a kind ofly can in same reaction tubes, detect simultaneously enterovirus EV 71 type, CoxA16 respectively or other causes the triple fluorescent PCR detection kit of the enterovirus of hand foot mouth disease that main purpose of the present invention provides, and this test kit has advantages such as high specificity, sensitivity height.
In order to achieve the above object, the present invention has adopted such technical scheme:
A kind of enterovirus Cox A 16 type/EV71 type/universal nucleic acid detection kit comprises following each component: RT-PCR reaction solution, enzyme mixed solution, CoxA16 type/EV71 type/universal triple response liquid, remove RNA enzyme water.
Wherein, described enterovirus Cox A 16 type/EV71 type/universal reaction solution comprises following 3 groups of components:
(1) primer of a pair of detection enterovirus Cox A 16 type, its base sequence are respectively shown in SEQ IDNo.1 and the SEQ ID No.2; A pair of detection enterovirus Cox A 16 type probe, its base sequence are respectively shown in SEQ ID No.3 and the SEQ ID No.4, wherein, SEQ ID No.3 and SEQ IDNo.4 ' to hold to be marked with the fluorescence report group, 3 ' end is marked with the fluorescent quenching group;
(2) primer of a pair of detection enterovirus EV 71 type, its base sequence are respectively shown in SEQ IDNo.5 and the SEQ ID No.6; Article one, detect the probe of enterovirus EV 71 type, its base sequence is shown in the SEQ ID No.7; Wherein, this probe ' to hold to be marked with the fluorescence report group, 3 ' end is marked with the fluorescent quenching group;
(3) primer of a pair of detection enterovirus universal, its base sequence are respectively shown in SEQ ID No.8 and the SEQ ID No.9; Article one, detect the probe of enterovirus universal, its base sequence is shown in the SEQID No.10; Wherein above-mentioned probe ' end is marked with the fluorescence report group, 3 ' end is marked with the fluorescent quenching group;
The present invention gropes the proportioning ratio of above-mentioned primer and probe and optimizes; Test-results is found; Different proportioning ratio has significant difference for the specificity and the susceptibility of detected result between them; The present invention finds that through high-throughout shaker test above-mentioned primer and probe have optimum detection effect under following proportioning:
The primer of enterovirus enterovirus Cox A 16 type and the proportioning of probe are respectively: SEQ IDNo.1: SEQ ID No.2: SEQ ID No.3: SEQ ID No.4=400nM: 400nM: 100nM: 100nM;
Detecting the primer of enterovirus Cox A 16 type and the proportioning of probe is respectively: SEQ ID No.5: SEQID No.6: SEQ ID No.7=600nM: 600nM: 200nM;
The primer of enterovirus universal primer and the proportioning of probe are respectively: SEQ ID No.8: SEQ IDNo.9: SEQ ID No.10=500nM: 500nM: 400nM.
Table 1 enterovirus Cox A 16 type, EV71 type, universal primer probe
Table 2 CoxA16 type/EV71 type/universal reaction solution component
| Reagent name | Add volume (μ l)/50 reaction |
| Enterovirus Cox A 16 type FP | 5 |
| Enterovirus Cox A 16 type RP | 5 |
| Enterovirus Cox A 16 type P | 1.25 |
| Enterovirus Cox A 16 type P | 1.25 |
| Enterovirus EV 71 type FP | 7.5 |
| Enterovirus EV 71 type RP | 7.5 |
| Enterovirus EV 71 type P | 2.5 |
| Enterovirus universal FP | 6.25 |
| Enterovirus |
5 |
| Enterovirus |
5 |
Described RT-PCR reaction solution comprises RNA enzyme water, 10 * PCR damping fluid, 25mM Mg
2+, 10mM dNTPs.(table 2)
The composition of table 2 RT-PCR reaction solution
| Reagent name | Add volume (μ l)/50 reaction |
| Remove RNA enzyme water | 290 |
| 10 * PCR damping fluid | 50 |
| 25mM? |
25 |
| 10mM? |
10 |
| Amount to | 375 |
Described enzyme mixed solution comprises RNA enzyme inhibitors, Taq enzyme, reversed transcriptive enzyme, 10 * damping fluid, removes RNA enzyme water.Reversed transcriptive enzyme and archaeal dna polymerase play important effect in pcr amplification, comprise the efficient and the specificity of rt, amplification, and in order to reach better effect, the present invention preferably adopts M-MLV reversed transcriptive enzyme and warm start enzyme (table 3).
The composition of table 3 enzyme mixed solution
In order to reach better detection effect, also can contain enterovirus Cox A 16/EV71 type/universal positive reference article (virus-culturing fluid through available from Jiangsu Prov. Disease Preventing and Controlling Center prepares) in the test kit of the present invention.
Another object of the present invention provides test kit of the present invention in the method for use that detects enterovirus Cox A 16 type, EV71 type or other enterovirus, comprising:
(1) RNA of extraction sample;
(2) prepare following component: RT-PCR reaction solution 12.5ul, enzyme mixed solution 1ul, CoxA16 type/EV71 type/universal reaction solution 4ul removes RNA enzyme water 2.5ul, RNA sample 5ul;
(3) RT-PCR amplification: carry out the RT-PCR amplification according to following program
50℃ 30min
95℃ 5min
(4) result judges: fluorescence curve is " S " type curve and CT≤36.0 in the FAM passage, and it is positive to be judged as the enterovirus Cox A 16 type; Do not have amplification of typical case's " S " type or CT>=36.0, it is negative to be judged as the enterovirus Cox A 16 type.Fluorescence curve is " S " type curve and CT≤36.0 in the VIC passage, and it is positive to be judged as the enterovirus EV 71 type; Do not have amplification of typical case's " S " type or CT>=36.0, it is negative to be judged as enterovirus EV7 type.Fluorescence curve is " S " type curve and CT≤35.5 in the FAM passage, and it is positive to be judged as enterovirus universal; Do not have amplification of typical case's " S " type or CT>=35.5, it is negative to be judged as enterovirus universal.
Before the detection, earlier according to the RNA process for extracting of routine to sample extraction RNA, sample comprises: ight soil, anus swab, throat swab and bleb are drawn the thing suspension.Sample to be measured is added in the PCR reaction tubes of ready enterovirus Cox A 16 type/EV71 type/universal reagent augmentation detection in the fluorescent quantitative PCR appearance.Amplification judges whether to infect enterovirus according to fluorescence curve, and is that a kind of enterovirus (Fig. 2) after finishing.
Enterovirus Cox A 16 type of the present invention/EV71 type/universal nucleic acid detection kit can detect enterovirus Cox A 16 type, EV71 type or other enterovirus simultaneously, has solved currently available products and can't take turns the problem that detects enterovirus in the reaction simultaneously one.That the present invention also has is easy and simple to handle, repeatable strong, advantage such as detected result is quick and objective, has great application prospect in enterovirus in-vitro diagnosis field.
Description of drawings
The fluorescent signal generation mechanism of Fig. 1 TaqMan probe.
Fig. 2 test kit of the present invention detects the graphic representation of two groups of samples.
Fig. 3 test kit of the present invention detects the sensitivity test result of enterovirus Cox A 16 type.
Fig. 4 test kit of the present invention detects the sensitivity test result of enterovirus EV 71 type.
Fig. 5 test kit of the present invention detects the specificity test-results of enterovirus Cox A 16 type.
Fig. 6 test kit of the present invention detects the specificity test-results of enterovirus EV 71 type.
Fig. 7 test kit of the present invention detects the amplification of poliovirus, Coxsackie virus, Echo virus, new enterovirus.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage of the present invention and characteristics will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment with form or replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall in protection scope of the present invention the details of technical scheme of the present invention.
The detection test of Test Example 1 test kit clinical sample of the present invention
1 sample process (RNA extraction)
1.1 get 200ul brothers mouth clinical sample, add 400ul Binding Buffer supplemented with Poly (A), fully change high purifying strainer tube over to behind the mixing, the centrifugal 15s of 8000rmp discards the waste liquid in the collection tube.
1.2 add 500ul Inhibitor Removal Buffer to strainer tube, the centrifugal 1min of 8000rmp discards the waste liquid in the collection tube.
1.3 add 450ul Washing Buffer to strainer tube, the centrifugal 1min of 8000rmp discards the waste liquid in the collection tube.
1.4 repeating step 3, high speed centrifugation 10s then, this step must be removed waste liquid clean.
1.5 add 50ul Elution Buffer to strainer tube, room temperature leaves standstill 2min, the centrifugal 1min of 8000rmp, and centrifugal gained solution is purified RNA.
2 reagent are prepared
The 3PCR amplification program
50℃ 30min
95℃ 5min
4 interpretations of result: 5 clinical sample concrete outcomes are following:
Test Example 2 test kit sensitivity test results of the present invention
To the extraction nucleic acid of enterovirus Cox A 16 type canonical reference strain and enterovirus EV 71 type canonical reference strain (virus-culturing fluid through available from Jiangsu Prov. Disease Preventing and Controlling Center prepares), the CoxA16 and the total RNA of EV71 of extracting gained carried out 10 times of doubling dilutions.Experimental result shows that this test kit can detect to 0.001PFU, and the CT value reduces change (Fig. 3, Fig. 4) in gradient with concentration.Test-results shows that test kit of the present invention has the responsive type of height for the diagnosis of enterovirus Cox A 16 type/EV71 type.
Test Example 3 test kit specificity test-results of the present invention
In order to detect the specificity of enterovirus Cox A 16 type of the present invention/EV71 type/universal nucleic acid detection kit; Detect enterovirus strain, adenovirus, rotavirus, cytomegalovirus, influenza virus etc. with enterovirus Cox A 16 type/EV71 type/universal nucleic acid detection kit, the result shows that the FAM passage is only to enterovirus Cox A 16 type increase (Fig. 5);
The VIC passage is only to enterovirus EV 71 type increase (Fig. 6); The ROX passage increases to poliovirus, Coxsackie virus, Echo virus, new enterovirus.Show that detection kit of the present invention can specific amplification enterovirus virus, and not with other viral nucleic acid generation cross reaction (Fig. 7).
Claims (9)
1. enterovirus Cox A 16 type/EV71 type/universal nucleic acid detection kit is characterized in that, comprises following each component: RT-PCR reaction solution, enzyme mixed solution, CoxA16 type/EV71 type/universal triple response liquid and remove RNA enzyme water;
Wherein, described enterovirus Cox A 16 type/EV71 type/universal reaction solution comprises following 3 groups of components:
Component (1): the primer of a pair of detection enterovirus Cox A 16 type, its base sequence are respectively shown in SEQID No.1 and the SEQ ID No.2; A pair of detection enterovirus Cox A 16 type probe, its base sequence are respectively shown in SEQ ID No.3 and the SEQ ID No.4, and wherein, 5 of SEQ ID No.3 and SEQ ID No.4 ' end is marked with the fluorescence report group, and 3 ' end is marked with the fluorescent quenching group;
Component (2): the primer of a pair of detection enterovirus EV 71 type, its base sequence are respectively shown in SEQ IDNo.5 and the SEQ ID No.6; Article one, detect the probe of enterovirus EV 71 type, its base sequence is shown in the SEQ ID No.7; Wherein, 5 of SEQ ID No.7 ' end is marked with the fluorescence report group, and 3 ' end is marked with the fluorescent quenching group;
Component (3): the primer of a pair of detection enterovirus universal, its base sequence are respectively shown in SEQ IDNo.8 and the SEQ ID No.9; Article one, detect the probe of enterovirus universal, its base sequence is shown in the SEQ ID No.10; Wherein 5 of SEQ ID No.10 ' end is marked with the fluorescence report group, and 3 ' end is marked with the fluorescent quenching group.
2. according to the described detection kit of claim 1, it is characterized in that: the proportioning in the component (1) between primer and the probe is: SEQ ID No.1:SEQ ID No.2:SEQ ID No.3:SEQ ID No.4=400nM:400nM:100nM:100nM.
3. according to the described detection kit of claim 1, it is characterized in that: the proportioning in the component (2) between primer and the probe is: SEQ ID No.5:SEQ ID No.6:SEQ ID No.7=600nM: 600nM: 200nM.
4. according to the described detection kit of claim 1, it is characterized in that: the proportioning in the component (3) between primer and the probe is: SEQ ID No.8:SEQ ID No.9:SEQ ID No.10=500nM: 500nM: 400nM.
5. according to the described detection kit of claim 1, it is characterized in that described RT-PCR reaction solution comprises: remove RNA enzyme water, 10 * PCR damping fluid, 25mM Mg
2+, 10mM dNTPs.
6. according to the described detection kit of claim 1, it is characterized in that described enzyme mixed solution comprises: RNA enzyme inhibitors, Taq enzyme, reversed transcriptive enzyme, 10 * damping fluid, remove RNA enzyme water.
7. according to the described detection kit of claim 6, it is characterized in that: said reversed transcriptive enzyme is the M-MLV reversed transcriptive enzyme; Said Taq enzyme is the warm start enzyme.
8. according to the described detection kit of claim 1, it is characterized in that: also contain enterovirus Cox A 16/EV71 type/universal positive reference article.
9. the said test kit of claim 1 detects the application in enterovirus Cox A 16 type, EV71 type or other enterovirus reagent in preparation.
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| CN102559939B (en) * | 2012-03-29 | 2013-11-27 | 东南大学 | Monotube double one-step RT-PCR (Reverse Transcription-Polymerase Chain Reaction) primers for identifying enterovirus (EV) 71 and non-EV71 and application of primers |
| CN102888469A (en) * | 2012-10-15 | 2013-01-23 | 西安建筑科技大学 | Method for detecting content of human enteroviruses and non-human enteroviruses in environmental water |
| CN108753768B (en) * | 2014-06-03 | 2021-06-11 | 深圳市晋百慧生物有限公司 | Nucleic acid for detecting enterovirus and application thereof |
| CN105483288A (en) * | 2015-12-29 | 2016-04-13 | 杭州迪安生物技术有限公司 | Hand-foot-mouth disease virus quick detection kit and application thereof |
| CN105483289B (en) * | 2015-12-29 | 2019-10-08 | 深圳澳东检验检测科技有限公司 | A kind of detection kit and reaction system of the triple direct fluorescence RT-PCRs of enterovirus |
| CN105624331A (en) * | 2016-01-13 | 2016-06-01 | 江苏和创生物科技有限公司 | Fluorescent PCR detection kit for intestinal viruses |
| CN107529559B (en) * | 2017-08-31 | 2021-02-19 | 南京美宁康诚生物科技有限公司 | Multiple fluorescence PCR detection kit for hand-foot-and-mouth disease virus and application |
| CN108424977B (en) * | 2018-02-09 | 2021-06-25 | 上海伯杰医疗科技有限公司 | Primer probe for enterovirus typing identification and identification method |
| CN109355432A (en) * | 2018-11-22 | 2019-02-19 | 重庆高圣生物医药有限责任公司 | Enterovirus EV 71 type isothermal reverse transcription recombinase polymeric enzymatic amplification primer, detection method and kit |
| CN109609689B (en) * | 2018-12-18 | 2019-10-08 | 北京卓诚惠生生物科技股份有限公司 | For detecting the nucleic acid reagent, kit and system of enterovirus |
| CN111748648A (en) * | 2019-12-24 | 2020-10-09 | 深圳市人民医院 | Kit for detecting five infant hand-foot-and-mouth disease series viruses and application thereof |
| CN112111609A (en) * | 2020-10-29 | 2020-12-22 | 上海伯杰医疗科技有限公司 | Universal nucleic acid detection kit for enteroviruses |
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