CN102851391A - Human enterovirus four-color fluorescence RT-PCR detection kit and detection method - Google Patents
Human enterovirus four-color fluorescence RT-PCR detection kit and detection method Download PDFInfo
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Abstract
The invention discloses a human enterovirus four-color fluorescence RT-PCR detection kit and a detection method; the detection kit comprises multiple fluorescence RT-PCR reaction mother liquor, AMV reverse transcriptase liquid, Taq DNA polymerase liquid, a positive control, and a negative control; the detection kit is characterized by further comprising an internal control, a multiple 10*PCR reaction buffer, bovine serum albumin with a concentration of 20 mg/ml, various forward primers and reverse primers with a concentration of 100 muM, and various specific probes with a concentration of 50 muM, wherein sequences of the forward primers, reverse primers, specific probes and the internal control are as shown in SEQIDNO. 1, NO. 2, NO.3, NO.4, NO.5, NO.6, NO.7. NO.8, NO.9, NO.10., NO.11., NO.12., and NO.13. The detection kit of the invention has the advantages of high detection sensitivity, good specificity, accuracy, high speed, and no false positivity.
Description
Technical field
The present invention relates to the fluorescent PCR kit in the biological technical field, especially relate to the four look fluorescent RT-PCR detection reagent boxes that comprise internal reference that detect for the general detection of human intestine virus and enterovirus EV 71 type, CoxA16 type somatotype.
Background technology
Enterovirus comprises poliovirus, Coxsackie virus (Coxsackievirus), cause intestinal cells pathology people Orphan virus (enterocytopathic human orphan virus ECHO is called for short Echo virus) and newtype enteroviru totally 71 serotypes.And be a class acute infectious disease by the hand foot mouth disease that enterovirus causes, clinical manifestation is take the fash at the positions such as heating and hand, foot, oral cavity as main clinical characteristics, this sick Acute onset, heating, bleb appears being dispersed in oral mucosa, maculopapule, bleb appear in hand, foot and buttocks, multiplely are born in the preschool children, and be especially the highest with 3 years old following age group sickness rate.This class disease is distributed in Ge Di ﹐ of Shi circle to be had at Perenniporia martius the whole year.
Cause 4 types, 5 types, 7 types, 9 types of the Coxsackie virus that is mainly Picornaviridae (Picornaviriade) enterovirus genus (Coxsackie virus) the A group of hand foot mouth disease etc.; 2 types of Coxsackie B virus group, 5 types; Part Echo virus (ECHO-viruses) and enterovirns type 71.The most common is coxsackievirus A16 (CoxA16) and enterovirns type 71 (EV71), and the two is closely related on genetics, often is polyinfection and alternately infection.China's hand foot mouth disease is popular, and its cause of disease is mainly EV71 type and CoxA16 type.Generally, brothers' mouth of causing of EV71 and CoxA16 is difficult to difference in clinical symptom.
The specificity diagnostic method of enterovirus infection has three kinds, and namely Virus Isolation, serology detect and Protocols in Molecular Biology.Separation and Culture and the serological method of virus, numerous and diverse time-consuming, can't satisfy the needs of processing simultaneously great amount of samples during the viral prevalence.Molecular biology is round pcr particularly, because its test material consumption is few, quick, highly sensitive and have very high specificity, plays an increasingly important role in the diagnosis of virus infection.RT-polymerase chain reaction (RT-PCR) is first the RNA reverse transcription to be become cDNA, then utilizes archaeal dna polymerase in the technology of external rapid amplifying target DNA fragment.The real-time fluorescence RT-PCR technology is on the basis of Conventional RT-PCR, add one or more pairs of specific PCR primers and add simultaneously corresponding specificity fluorescent probe in amplification reaction system, this probe is the oligonucleotide that two ends are marked with respectively fluorescence report group and fluorescent quenching group.When probe was complete, the fluorescent energy that reporter group is launched was absorbed by quenching group, and instrument can't detect signal.And when pcr amplification, probe is with purpose amplified fragments hybridization on the template, probe cut off to the 5 prime excision enzyme activity of 3 ' end because archaeal dna polymerase has 5 ' end, and reporter group is away from quenching group, and its energy can not be absorbed, and namely produces fluorescent signal.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.
The Chinese patent name is called Enterovirus 71 nucleic acid detection kit and detection method (application number 201010105088.5) discloses a kind of Enterovirus 71 nucleic acid detection kit, comprises PCR reaction enzymes, PCR reaction solution and enterovirns type 71 forward primer and reverse primer and specific fluorescent probe; The Chinese patent name is called upstream and downstream primer and the specific probe that enterovirus Cox A 16 nucleic acid fluorescent quantitative RT-PCR detection kit (application number 200810121454.9) discloses a kind of enterovirus COxsackie A16 detection kit; The Chinese patent name is called upstream and downstream primer and the specific probe that enterovirus fluorescence quantitative RT-PCR detecting kit (application number 200810121453.4) discloses a kind of enterovirus.Above-mentioned detection kit has special for detection of corresponding enterovirus, sensitive, fast, advantage easy and simple to handle, but the mentioned reagent box all can't be realized general detection and the enterovirus EV 71 type of enterovirus in same reaction tubes, the somatotype of CoxA16 type detects, lot of experimental data shows simultaneously, at ight soil, procto swab, in the brush,throat sample, the ratio that the PCR inhibition exists is 1.8%～3.4%, show that internal reference has vital role to the existence of monitoring PCR inhibition, and at present the external diagnosis reagent case of existing hand foot mouth disease all do not have internal reference and also have specificity and sensitivity lower, the defectives such as detecting step is loaded down with trivial details need further to improve.
Summary of the invention
Technical problem to be solved by this invention provide a kind of general detection that can in same reaction tubes, realize simultaneously human intestine's virus and EV71 hypotype, CoxA16 subtype typing rapid detection high specificity, highly sensitive, reliability strong, without human intestine's virus four look fluorescent RT-PCR detection reagent box and detection methods of false negative result.
The present invention solves the problems of the technologies described above the technical scheme that adopts:
1, enterovirus four look fluorescence RT-PCR test kits (abbreviation test kit), this test kit is on the basis that human intestine's nucleic acid sequence is analyzed, choose the conservative gene sequences Design by the bioinformatics method analysis and go out Auele Specific Primer, utilize Fluorescence PCR assay that enterovirus is detected.In the presence of the primer that designs as follows, probe, in the fluorescent PCR instrument, viral nucleic acid is carried out the RT-PCR amplification, realize the detection to enterovirus.
Specifically, this test kit comprises multi-fluorescence RT-PCR reaction mother liquor, positive reference substance, negative control product, internal reference, AMV reversed transcriptive enzyme, Taq polysaccharase, and wherein multi-fluorescence RT-PCR reaction mother liquor contains multiple 10 * fluorescence RT-PCR reaction buffer, the universal primer of EV, the universal probe of EV, EV71 type primer, EV71 type probe, CoxA16 type primer, CoxA16 type probe, internal reference primer, internal reference probe, bovine serum albumin; Multiple 10 * fluorescence RT-PCR reaction buffer contains Tutofusin tris hydrochloric acid, Repone K, glycerine, four kinds of mononucleotide monomers, magnesium chloride, and wherein the sequence of each primer and probe is as follows,
Design primer, probe according to enterovirus EV 71 type VP1 gene conserved sequence:
The EV71Pf upstream primer: 5 '-CGCAAATGCGTAGAAAGGT-3 ' 19bp;
The EV71Pr downstream primer: 5 '-GGAGCAATTGTGGGACAACT-3 ' 20bp;
The EV71Pb probe: 5 '-FAM-AGCTATTCACCTACATGCGCTTTGATGC-BHQ1-3 ' 28bp;
Design primer, probe according to enterovirus Cox A 16 type VP1 gene conserved sequence:
The CoxA16Pf upstream primer: 5 '-GAACCATCACTCCACACAGGAG-3 ' 22bp;
The CoxA16Pr downstream primer: 5 '-GTACCTGTGGTGGGCATTG-3 ' 19bp;
The CoxA16Pb probe: 5 '-HEX-CAGCCATTGGGAATTTCTTTAGCCGTG-BHQ1-3 ' 27bp;
Design universal primer, probe according to enterovirus EV gene 5 ' non-translational region:
The EVPf upstream primer: 5 '-CCCTGAATGCGGCTAATCC-3 ' 19bp;
The EVPr downstream primer: 5 '-GATTGTCACCATAAGCAGCCA-3 ' 21bp;
The EVPb probe: 5 '-ROX-AACCGACTACTTTGGGTGTCCGTGTTTC-BHQ1-3 ' 28bp;
The internal reference sequence is:
5′-GTGCTCACAC CAGTTGCCGC GGAAAGTATG TGGAATGTTA ACACACCCAC ACCACACCCACACACGTGTT GGATCAATTT CGAGATGCGA GCTGCCAAGC-3′ 100bp；
According to internal reference sequences Design internal reference primer, probe:
The ICPf upstream primer: 5 '-GTGCTCACACCAGTTGCCGC-3 ' 20bp;
The ICPr downstream primer: 5 '-GCTTGGCAGCTCGCATCTCG-3 ' 20bp;
The ICPb probe: 5 '-CY5-ATTGTGTGGGTGTGGTGTGGGTGTGTGC-BHQ2-3 ' 28bp.
Described positive reference substance is the pUCm-T cloning vector of the gene conserved sequence of the primer that is connected with design enterovirus universal, EV71 type, CoxA16 type, probe.
Described negative control product are the DEPC water without the RNA enzyme.
Described internal reference is the pUCm-T cloning vector that is connected with the artificial implementation sequence of internal reference, and the concentration of described internal reference is 10
4Copies/ml.
Described multiple 10 * PCR reaction buffer consists of: Tutofusin tris hydrochloric acid final concentration is that 100mM, Repone K final concentration are that 500mM, glycerine volume ratio are 50%, four kind of mononucleotide monomer (dATP, dGTP, dUTP, dCTP) final concentration is 0.4mM, and the magnesium chloride final concentration is the ultrapure water of 3mM and respective volume.
Described multi-fluorescence RT-PCR reaction mother liquor preparation: multiple 10 * PCR reaction buffer 5ul; The bovine serum albumin 1ul of concentration 20mg/ml; Concentration is each 0.4ul of primer EVPf, EVPr, EV71Pf and EV71Pr of 100 μ M; Concentration is primer CoxA16Pf and each 0.5ul of CoxA16Pr of 100 μ M; Concentration is 50 μ M probe EVPb, EV71Pb and each 0.3ul of CoxA16Pb; Concentration is primer I CPf and each 0.2ul of ICPr of 100 μ M; Concentration is the probe I CPb 0.1ul of 50 μ M; Without RNA enzyme DEPC water 31.1ul, mix rear in refrigerator-20 ℃ preservation.
2, human intestine's virus four look fluorescent RT-PCR method for detecting
(1) sample preparation
Gather patient suspected's throat swab, bleb liquid, cerebrospinal fluid or human intestine's virus separation and Culture thing, the commercialization sampling tube (friendly Kang Jiye, MT0301-1) of putting into rapidly the virus preservation liquid that 1-2ml is housed uses as testing sample;
(2) extraction of viral nucleic acid RNA
Extract test kit QIAGEN Viral RNA Mini Extraction Kit (QIAGEN, CAT:52904) operation in strict accordance with commercialization nucleic acid RNA, from clinical samples or viral separation and Culture thing, extract viral RNA; The concentration that every duplicate samples all adds 10ul is 10
4The internal reference of copies/ml participates in the extraction of nucleic acid samples synchronously;
(3) multi-fluorescence RT-PCR reaction solution preparation
Sample nucleic acid, 10ul positive reference substance and the 10ul negative control product of getting the 10ul extraction add respectively 40ul multi-fluorescence RT-PCR reaction solution and carry out multi-fluorescence RT-PCR amplification to corresponding PCR pipes, and wherein multi-fluorescence RT-PCR reaction solution is formulated as follows: multi-fluorescence RT-PCR reaction mother liquor 39ul; The AMV reversed transcriptive enzyme 0.4ul of concentration 5U/ul; The Taq archaeal dna polymerase 0.6ul of concentration 5U/ul;
(4) four look fluorescence RT-PCR augmentation detection
Carry out at four look quantitative real time PCR Instruments, the probe in detecting pattern is set to: the FAM passage for detection of EV71 type, HEX passage for detection of CoxA16 type, ROX passage for detection of EV universal and CY5 passage for detection of internal reference, reaction conditions: 50 ℃ of 1 circulations in 30 minutes; 95 ℃ of 1 circulations in 3 minutes; 95 ℃ 5 seconds, 55 ℃ 40 seconds, circulate 40 times; After setting is finished, preserve file, working procedure;
(4) Analysis of test results
The internal reference amplification curve is normal, presses following rule judgment result:
S shape amplification curve occurs, the Ct value is less than 37 positive results; Amplification curve is arranged but not S-shaped and threshold value surpasses 40 or the negative result of S shape amplification curve do not occur; S shape amplification curve occurs, but Ct value is greater than 37, less than 40 be suspicious result, to suspicious result, answers repeated experiments, if S shape amplification curve appears in repeated experiments, negative control can be judged as the positive less than pollution.
Described multi-fluorescence RT-PCR reaction mother liquor preparation: multiple 10 * PCR reaction buffer 5ul; The bovine serum albumin 1ul of concentration 20mg/ml; Concentration is each 0.4ul of primer EVPf, EVPr, EV71Pf and EV71Pr of 100 μ M; Concentration is primer CoxA16Pf and each 0.5ul of CoxA16Pr of 100 μ M; Concentration is 50 μ M probe EVPb, EV71Pb and each 0.3ul of CoxA16Pb; Concentration is primer I CPf and each 0.2ul of ICPr of 100 μ M; Concentration is the probe I CPb 0.1ul of 50 μ M; RNA enzyme DEPC water 31.1ul mixes rear in refrigerator-20 ℃ preservation.
Described multiple 10 * PCR reaction buffer consists of: Tutofusin tris hydrochloric acid final concentration is that 100mM, Repone K final concentration are that 500mM, glycerine volume ratio are 50%, four kind of mononucleotide monomer (dATP, dGTP, dUTP, dGTP) final concentration is 0.4mM, and the magnesium chloride final concentration is the ultrapure water of 3mM and respective volume.
Compared with prior art, the invention has the advantages that human enterovirus four look fluorescence PCR detection reagent kit and the detection methods of the present invention, because this test kit has been introduced for the enterovirus EV 71 type, the specificity amplification primer that coxsackievirus A16 and enterovirus universal nucleic acid sequence are designed and four look fluorescent probes, can be simultaneously the general detection of enterovirus and enterovirns type 71 and coxsackievirus A16 being carried out somatotype detects, thereby realized detecting simultaneously in the sample purpose of three kinds of viruses, production cost and testing cost had both been saved, improve again detection efficiency and shortened the time, in general, the judgement from the processing sample to the result only needed about 2 hours; And the more important thing is and introduced internal reference, can effectively monitor the inhibition in the sample, can also avoid operate miss and pcr amplification product to pollute the false negative that causes, have better sensitivity and specificity so that detect, thereby avoided the not high problem of other detection method specificitys.
In sum, a kind of four look fluorescence PCR detection reagent kits for quick real-time quantitative detection human intestine virus of the present invention, Detecting susceptibility is high, and specificity is good, reduce the false positive rate of conventional pcr amplification, can also effectively solve the easy pollution problem of conventional PCR; Have the Noncompetitive internal comparison system, reliability is strong, without false negative result, can realize enterovirus EV 71 type, CoxA16 type and enterovirus universal fast, are accurately reached specific detection simultaneously.
Description of drawings
Fig. 1 is the human enterovirus four look fluorescence RT-PCR amplification figure of the present invention.
Embodiment
Embodiment is described in further detail the present invention below in conjunction with accompanying drawing.
Specific embodiment one
Enterovirus four look fluorescence RT-PCR test kits
1, the RT-PCR test kit forms
This test kit comprises multi-fluorescence RT-PCR reaction mother liquor, positive reference substance, negative control product, internal reference, AMV reversed transcriptive enzyme, Taq polysaccharase.Wherein multi-fluorescence RT-PCR reaction mother liquor contains multiple 10 * fluorescence RT-PCR reaction buffer, the universal primer of EV, EV universal probes, EV71 type primer, EV71 type probe, CoxA16 type primer, CoxA16 type probe, internal reference primer, internal reference probe, bovine serum albumin; Multiple 10 * fluorescence RT-PCR reaction buffer contains Tutofusin tris hydrochloric acid, Repone K, glycerine, four kinds of mononucleotide monomers, magnesium chloride.
Wherein multi-fluorescence RT-PCR reaction mother liquor is formulated as follows (being depicted as corresponding material concentration in the bracket):
Multiple 10 * PCR reaction buffer 5ul
Bovine serum albumin (20mg/ml) 1ul
Each 0.4ul of EVPf and EVPr (100 μ M)
EV71Pf and EV71r(100 μ M) each 0.4ul
CoxA16Pf and CoxA16Pr(100 μ M) each 0.5ul
EVPb, EV71Pb and CoxA16Pb(50 μ M) each 0.3ul
ICPf and ICPr(100 μ M) each 0.2ul
ICPb（50μM） 0.1ul
Without RNA enzyme DEPC water 31.1ul
Preparation mixes rear in refrigerator-20 ℃ preservation.
Wherein multiple 10 * PCR reaction buffer consists of: Tutofusin tris hydrochloric acid final concentration be 100mM, Repone K final concentration be 500mM, glycerine volume ratio be the volume of 50%(glycerine be multiple 10 * PCR reaction buffer volume 50%), four kinds of mononucleotide monomers (dATP, dGTP, dUTP, dGTP) final concentration is 0.4mM, the magnesium chloride final concentration is the ultrapure water of 3mM and respective volume.
The negative control product are the DEPC water without the RNA enzyme, and positive reference substance is the pUCm-T cloning vector plasmids of the gene conserved sequence of the primer that contains enterovirus universal, EV71 type, CoxA16 type, probe; Internal reference is the pUCm-T cloning vector that is connected with the artificial implementation sequence of internal reference, and internal reference concentration is 10
4Copies/ml.Test kit of the present invention is stored in-20 ℃, reduces multigelation as far as possible.
2, primer, probe design and synthetic
Download all enterovirus EV 71 types from GenBank, the gene order of coxsackievirus A16 and other each subtype virus of enterovirus, repeatedly compare through bioinformatic analysis, select such as lower area as amplification enterovirus EV 71 type, the specificity target area of coxsackievirus A16 and enterovirus universal, use the software design such as Primer Premier and Oligo and screen the EV universal primer, the general fluorescent probe of EV, EV71 type primer, EV71 type probe, CoxA16 type primer, CoxA16 type probe, internal reference primer and internal reference probe, the internal reference sequence adopts the Perl software design:
Enterovirus EV 71 type primer, probe:
Upstream primer EV71Pf:5 '-CGCAAATGCGTAGAAAGGT-3 ' 19bp;
Downstream primer EV71Pr:5 '-GGAGCAATTGTGGGACAACT-3 ' 20bp;
Probe EV71Pb:5 '-FAM-AGCTATTCACCTACATGCGCTTTGATGC-BHQ1-3 ' 28bp
Enterovirus Cox A 16 type primer, probe:
Upstream primer CoxA16Pf:5 '-GAACCATCACTCCACACAGGAG-3 ' 22bp;
Downstream primer CoxA16Pr:5 '-GTACCTGTGGTGGGCATTG-3 ' 19bp
Probe CoxA16Pb:5 '-HEX-CAGCCATTGGGAATTTCTTTAGCCGTG-BHQ1-3 ' 27bp
Enterovirus universal primer, probe:
Upstream primer EVPf:5 '-CCCTGAATGCGGCTAATCC-3 ' 19bp
Downstream primer EVPr:5 '-GATTGTCACCATAAGCAGCCA-3 ' 21bp
Probe EVPb:5 '-ROX-AACCGACTACTTTGGGTGTCCGTGTTTC-BHQ1-3 ' 28bp
The internal reference sequence:
5′-GTGCTCACAC CAGTTGCCGC GGAAAGTATG TGGAATGTTA ACACACCCAC ACCACACCCA CACACGTGTT GGATCAATTT CGAGATGCGA GCTGCCAAGC-3′ 100bp
Internal reference primer, probe:
ICPf：5′-GTGCTCACACCAGTTGCCGC-3′ 20bp
ICPr：5′-GCTTGGCAGCTCGCATCTCG-3′ 20bp
ICPb：5′-CY5-ATTGTGTGGGTGTGGTGTGGGTGTGTGC-BHQ2-3′ 28bp。
Above-mentioned enterovirus EV 71 type fluorescent probe 5 ' end flag F AM fluorescence report group, enterovirus Cox A 16 type fluorescent probe 5 ' end mark HEX fluorescence report group, enterovirus universal fluorescent probe 5 ' end mark JOE fluorescence report group, interior mark fluorescent probe 5 ' end mark CY5 fluorescence report group, enterovirus EV 71 type fluorescent probe 3 ' end, enterovirus Cox A 16 type fluorescent probe 3 ' end and enterovirus universal fluorescent probe 3 ' end is mark BHQ1 fluorescent quenching group respectively, internal reference fluorescent probe 3 ' end mark BHQ2 fluorescent quenching group.
Specific embodiment two
Human intestine's virus four look fluorescent RT-PCR method for detecting, each detection all should be set up positive control and negative control
1, sample preparation:
Gather patient suspected's throat swab, bleb liquid, cerebrospinal fluid or human intestine's virus separation and Culture thing, the commercialization sampling tube (friendly Kang Jiye, MT0301-1) of putting into rapidly the virus preservation liquid that 1-2ml is housed uses as testing sample;
2, the extraction of viral nucleic acid RNA:
Extract test kit QIAGEN Viral RNA Mini Extraction Kit (QIAGEN, CAT:52904) operation in strict accordance with commercialization nucleic acid RNA, from clinical samples or viral separation and Culture thing, extract viral RNA; Every duplicate samples all adds the 10ul internal reference, and (concentration is 10
4Copies/ml) participate in synchronously the extraction of nucleic acid samples;
3, RT-PCR reaction system preparation
Sample nucleic acid, 10ul positive reference substance and the 10ul negative control product of getting the 10ul extraction add respectively 40ul multi-fluorescence RT-PCR reaction solution and carry out multi-fluorescence RT-PCR amplification to corresponding PCR pipes, and wherein multi-fluorescence RT-PCR reaction solution is formulated as follows: multi-fluorescence RT-PCR reaction mother liquor 39ul; The AMV reversed transcriptive enzyme 0.4ul of concentration 5U/ul; The Taq archaeal dna polymerase 0.6ul of concentration 5U/ul;
Wherein multi-fluorescence RT-PCR reaction mother liquor preparation: multiple 10 * PCR reaction buffer 5ul; The bovine serum albumin 1ul of concentration 20mg/ml; Concentration is each 0.4ul of primer EVPf, EVPr, EV71Pf and EV71Pr of 100 μ M; Concentration is primer CoxA16Pf and each 0.5ul of CoxA16Pr of 100 μ M; Concentration is 50 μ M probe EVPb, EV71Pb and each 0.3ul of CoxA16Pb; Concentration is primer I CPf and each 0.2ul of ICPr of 100 μ M; Concentration is the probe I CPb 0.1ul of 50 μ M; RNA enzyme DEPC water 31.1ul mixes rear in refrigerator-20 ℃ preservation;
Wherein multiple 10 * PCR reaction buffer consists of: Tutofusin tris hydrochloric acid final concentration is that 100mM, Repone K final concentration are that 500mM, glycerine volume ratio are 50%, four kind of mononucleotide monomer (dATP, dGTP, dUTP, dGTP) final concentration is 0.4mM, and the magnesium chloride final concentration is the ultrapure water of 3mM and respective volume;
4, four look fluorescence RT-PCR augmentation detection
Carry out at four look quantitative real time PCR Instruments, the probe in detecting pattern is set to: the FAM passage for detection of EV71 type, HEX passage for detection of CoxA16 type, ROX passage for detection of EV universal and CY5 passage for detection of internal reference, reaction conditions: 50 ℃ of 1 circulations in 30 minutes; 95 ℃ of 1 circulations in 3 minutes; 95 ℃ 5 seconds, 55 ℃ 40 seconds, circulate 40 times; After setting is finished, preserve file, working procedure;
5, Analysis of test results
The internal reference amplification curve is normal, presses following rule judgment result:
S shape amplification curve occurs, the Ct value is less than 37 positive results; S shape amplification curve do not occur or amplification curve is arranged but not S-shaped and threshold value surpasses 40 negative results; S shape amplification curve occurs, but Ct value is greater than 37, less than 40 be suspicious result, to suspicious result, answers repeated experiments, if S shape amplification curve appears in repeated experiments, negative control can be judged as the positive less than pollution.
Concrete type is identified and carried out according to following table: table 1 detected result is judged
Internal reference is unusual without amplification curve or amplification curve, then need to sample re-start nucleic acid extraction or with sample with after carrying out nucleic acid extraction after the rnase-free DEPC water dilution, carry out augmentation detection.
Specific embodiment three
Detection kit sensitivity, specificity analyses
1, experiment material virus strain
Target strain group: enterovirus EV 71 type strain, coxsackievirus A16 strain, CA group 1 type, CA group 11 types, CA group 13 types, Coxsackie B virus group 3 types, enterovirus EV68 type, Echo virus 5 types, Echo virus 13 types; Non-target strain group: rotavirus, norovirus I type and II type, H1N1virus, Influenza B virus, above-mentioned strain be available from U.S. culture presevation administrative center, or separated by the disease prevention and control center, Ningbo and to preserve.
2, detected result
Adopt enterovirus four look fluorescence RT-PCR test kits that above-mentioned strain is detected, the enterovirus EV 71 type strain of target strain group, coxsackievirus A16 strain, CA group 1 type, CA group 11 types, CA group 13 types, Coxsackie B virus group 3 types, enterovirus EV68 type, Echo virus 5 types, Echo virus 13 types etc. detect all positive, and the EV71 type is two positive at FAM passage and ROX passage, and the CoxA16 type is two positive at HEX passage and ROX passage.But not the detected results such as the rotavirus of target strain group, norovirus I type and II type, H1N1virus, Influenza B virus are all negative, and specificity is 100%(Fig. 1); Reach 50 copies with detection sensitivity behind the plasmid positive control doubling dilution that makes up.
This figure mainly is test kit specific detection experimental result diagram, detection comprises that the target strain group of enterovirus EV 71 type strain, coxsackievirus A16 strain etc. is all positive, detects to comprise that the non-target strain group of H1N1virus, Influenza B virus etc. is all negative.
Specific embodiment four
Human intestine's virus four look fluorescent RT-PCR detection reagent boxes detect doubtful enterovirus infection patient throat swab sample 196 examples, and each this liquid 140ul that takes a sample carries out viral nucleic acid and extracts.
1, test kit forms shown in above-mentioned embodiment one
2, the using method of test kit
Each detection all should be set up positive control and negative control.
2.1 the extraction of viral nucleic acid RNA:
Extract test kit QIAGEN Viral RNA Mini Extraction Kit (QIAGEN, CAT:52904) operation in strict accordance with commercialization nucleic acid RNA, extract viral RNA from clinical samples; Every duplicate samples all adds the 10ul internal reference, and (concentration is 10
4Copies/ml) participate in synchronously the extraction of sample nucleic acid;
2.2 four look fluorescence RT-PCR augmentation detection (every part of 50 ul systems):
Get sample nucleic acid, positive reference substance, negative control product that 10ul extracts, add respectively 40ul multi-fluorescence RT-PCR reaction solution and to corresponding PCR pipe, carry out the amplification of four look fluorescence RT-PCRs,
A. the preparation of four look fluorescence RT-PCR reaction solutions:
Multi-fluorescence RT-PCR reaction mother liquor 39ul
AMV reversed transcriptive enzyme (5U/ul) 0.4ul
Taq archaeal dna polymerase (5U/ul) 0.6ul
Sample nucleic acid (or negative control product or positive reference substance) 10ul
B. four look fluorescence RT-PCR response procedures
Carry out cumulative volume 50ul at four look quantitative real time PCR Instruments; The probe in detecting pattern is set to: the FAM passage for detection of EV71 type, HEX passage for detection of CoxA16 type, ROX passage for detection of EV universal and CY5 passage for detection of internal reference; Reaction conditions: 50 ℃ of 1 circulations in 30 minutes; 95 ℃ of 1 circulations in 3 minutes; 95 ℃ 5 seconds, 55 ℃ 40 seconds, circulate 40 times; After setting is finished, preserve file, working procedure;
(3) Analysis of test results
Adopt which kind of infection of Ct value judgement sample:
The ROX passage is the universal detection of HEV, and the CY5 passage is that internal reference samples normal and Ct value≤37 have 127 examples, is judged to the enterovirus positive, internal reference is normal but the sample of Ct value＞37 has 9 examples, through duplicate detection, 6 routine sample Ct value≤37 are arranged, be judged as the enterovirus positive;
The FAM passage is that the EV71 type detects, and the CY5 passage is that internal reference samples normal and Ct value≤37 have 71 examples, and it is positive to be judged as the EV71 type, and internal reference is normal but the sample of Ct value＞37 has 3 examples, through duplicate detection, 2 routine sample Ct value≤37 is arranged, and it is positive to be judged as the EV71 type;
The HEX passage is that the CoxA16 type detects, and the CY5 passage is that internal reference samples normal and Ct value≤37 have 21 examples, and it is positive to be judged as the CoxA16 type, internal reference is normal but the sample of Ct value＞37 has 5 examples, through duplicate detection, 3 routine sample Ct value≤37 are arranged, be judged as the CoxA16 positive;
Three passages all negative and CY5 passage are that the normal sample of internal reference has 52 examples, are judged as non-enterovirus;
Three passages all negative but CY5 passage are internal reference unusual sample has 11 examples, by again detecting or the dilution of DEPC water, the Ct value of the ROX passage of 11 routine samples is equal＜and 37, be judged as the enterovirus positive.
Finally, detect positive 144 examples of enterovirus in the 196 routine doubtful enterovirus infection patient oropharyngeal swab specimens, positive 73 examples of EV71 type wherein, positive 24 examples of CoxA16 type, recall rate 100%.
Specific embodiment five
With aforesaid method other 46 parts of doubtful enterovirus infection patient bleb liquid samples are detected, wherein detect enterovirus 18 examples, positive 8 examples of EV71 type wherein, positive 2 examples of CoxA16 type, recall rate 100%.
The present invention is described in conjunction with most preferred embodiment, yet after having read foregoing of the present invention, those skilled in the art can make various changes or modifications the present invention, and these equivalent form of values fall within the application's appended claims limited range equally.
Claims (8)
1. human intestine virus four look fluorescent RT-PCR detection reagent boxes, comprise multi-fluorescence RT-PCR reaction mother liquor, AMV reversed transcriptive enzyme liquid, Taq archaeal dna polymerase liquid, positive reference substance, negative control product, it is characterized in that: comprise that also internal reference, described multi-fluorescence RT-PCR reaction mother liquor comprise multiple 10 * PCR reaction buffer, each upstream and downstream primer of bovine serum albumin bletilla and each specific probe, wherein said each upstream and downstream primer sequence, described specific probe sequence and internal reference sequence are as follows:
Enterovirus EV 71 type upstream primer EV71Pf shown in SEQ ID NO.1,5 '-CGCAAATGCGTAGAAAGGT-3 ';
Enterovirus EV 71 type downstream primer EV71Pr shown in SEQ ID NO.2,5 '-GGAGCAATTGTGGGACAACT-3 ';
Enterovirus EV 71 type fluorescent probe EV71Pb shown in SEQ ID NO.3,
5′-FAM-AGCTATTCACCTACATGCGCTTTGATGC-BHQ1 -3′；
Enterovirus Cox A 16 type upstream primer CoxA16Pf shown in SEQ ID NO.4,5 '-GAACCATCACTCCACACAGGAG-3 ';
Enterovirus Cox A 16 type downstream primer CoxA16Pr shown in SEQ ID NO.5,5 '-GTACCTGTGGTGGGCATTG-3 ';
Enterovirus Cox A 16 type fluorescent probe CoxA16Pb shown in SEQ ID NO.6,
5′-HEX- CAGCCATTGGGAATTTCTTTAGCCGTG-BHQ1- 3′；
Enterovirus universal upstream primer EVPf shown in SEQ ID NO.7,5 '-CCCTGAATGCGGCTAATCC-3 ';
Enterovirus universal downstream primer EVPr shown in SEQ ID NO.8,5 '-GATTGTCACCATAAGCAGCCA-3 ';
Enterovirus universal fluorescent probe VPb shown in SEQ ID NO.9,
5′-ROX-AACCGACTACTTTGGGTGTCCGTGTTTC-BHQ1- 3′；
The internal reference sequence is shown in SEQ ID NO.10:
5′-GTGCTCACAC CAGTTGCCGC GGAAAGTATG TGGAATGTTA ACACACCCAC ACCACACCCA CACACGTGTT GGATCAATTT CGAGATGCGA GCTGCCAAGC-3′；
Internal reference upstream primer ICPf shown in SEQ ID NO.11,5 '-GATTAGTATGTCGGCTGCAGGTA-3 ';
Internal reference downstream primer ICPr shown in SEQ ID NO.12,5 '-CAAGGCTCATAGCGCGTATC-3 ';
Internal reference fluorescent probe CPb shown in SEQ ID NO.13,5 '-CY5-CCGGTACATCCGACGGAGCGTC – BHQ2-3 '.
2. human intestine's virus four look fluorescent RT-PCR detection reagent boxes according to claim 1 is characterized in that described multi-fluorescence RT-PCR reaction mother liquor is formulated as follows: multiple 10 * PCR reaction buffer 5ul; The bovine serum albumin 1ul of concentration 20mg/ml; Concentration is each 0.4ul of primer EVPf, EVPr, EV71Pf and EV71Pr of 100 μ M; Concentration is primer CoxA16Pf and each 0.5ul of CoxA16Pr of 100 μ M; Concentration is 50 μ M probe EVPb, EV71Pb and each 0.3ul of CoxA16Pb; Concentration is primer I CPf and each 0.2ul of ICPr of 100 μ M; Concentration is the probe I CPb 0.1ul of 50 μ M; RNA enzyme DEPC water 31.1ul mixes rear in refrigerator-20 ℃ preservation.
3. human intestine according to claim 2 virus four look fluorescent RT-PCR detection reagent boxes, it is characterized in that described multiple 10 * PCR reaction buffer consists of: Tutofusin tris hydrochloric acid final concentration is that 100mM, Repone K final concentration are that 500mM, glycerine volume ratio are 50%, four kind of mononucleotide monomer final concentration is 0.4mM, and the magnesium chloride final concentration is the ultrapure water of 3mM and respective volume.
4. human intestine according to claim 1 virus four look fluorescent RT-PCR detection reagent boxes is characterized in that: described positive reference substance is the pUCm-T cloning vector of the gene conserved sequence of the primer that is connected with design enterovirus universal, EV71 type, CoxA16 type, probe; Described negative control product are the DEPC water without the RNA enzyme, and described internal reference is the pUCm-T cloning vector that is connected with the artificial implementation sequence of internal reference, and the concentration of described internal reference is 10
4Copies/ml.
5. the detection method of human intestine according to claim 1 virus four look fluorescence RT-PCRs is characterized in that may further comprise the steps:
(1) sample preparation:
Gather patient suspected's throat swab, bleb liquid, cerebrospinal fluid or human intestine's virus separation and Culture thing, the sampling tube of putting into rapidly the virus preservation liquid that 1-2ml is housed uses as testing sample;
(2) extraction of viral nucleic acid RNA:
Extract test kit QIAGEN Viral RNA Mini Extraction Kit operation in strict accordance with commercialization nucleic acid RNA, from clinical samples or viral separation and Culture thing, extract viral RNA; The concentration that every duplicate samples all adds 10ul is 10
4The internal reference of copies/ml participates in the extraction of sample nucleic acid synchronously;
(3) four look fluorescence RT-PCR augmentation detection
Sample nucleic acid, 10ul positive reference substance and the 10ul negative control product of getting the 10ul extraction add respectively 40ul multi-fluorescence RT-PCR reaction solution and carry out multi-fluorescence RT-PCR amplification to corresponding PCR pipes, four look fluorescence RT-PCR response procedures carry out at four look quantitative real time PCR Instruments, the probe in detecting pattern arranges: the FAM passage for detection of EV71 type, HEX passage for detection of CoxA16 type, ROX passage for detection of EV universal and CY5 passage for detection of internal reference, reaction conditions: 50 ℃ of 1 circulations in 30 minutes; 95 ℃ of 1 circulations in 3 minutes; 95 ℃ 5 seconds, 55 ℃ 40 seconds, circulate 40 times; Being formulated as follows of RT-PCR reaction solution wherein: multi-fluorescence RT-PCR reaction mother liquor 39ul, concentration are the AMV reversed transcriptive enzyme 0.4ul of 5U/ul, Taq archaeal dna polymerase 0.6ul, the RNA nucleic acid samples 10ul that concentration is 5U/ul;
(4) Analysis of test results
The internal reference amplification curve is normal, and by following rule judgment result: S shape amplification curve occurs, the Ct value is less than 37 positive results; Amplification curve is arranged but not S-shaped and threshold value surpasses 40 or the negative result of S shape amplification curve do not occur; S shape amplification curve occurs, but Ct value is greater than 37, less than 40 be suspicious result, to suspicious result, answers repeated experiments, if S shape amplification curve appears in repeated experiments, negative control can be judged as the positive less than pollution.
6. the detection method of human intestine's virus four look fluorescence RT-PCRs according to claim 5 is characterized in that described multi-fluorescence RT-PCR reaction mother liquor is formulated as follows: multiple 10 * PCR reaction buffer 5ul; The bovine serum albumin 1ul of concentration 20mg/ml; Concentration is each 0.4ul of primer EVPf, EVPr, EV71Pf and EV71Pr of 100 μ M; Concentration is primer CoxA16Pf and each 0.5ul of CoxA16Pr of 100 μ M; Concentration is 50 μ M probe EVPb, EV71Pb and each 0.3ul of CoxA16Pb; Concentration is primer I CPf and each 0.2ul of ICPr of 100 μ M; Concentration is the probe I CPb 0.1ul of 50 μ M; RNA enzyme DEPC water 31.1ul mixes rear in refrigerator-20 ℃ preservation.
7. the detection method of human intestine according to claim 6 virus four look fluorescence RT-PCRs is characterized in that:
Enterovirus EV 71 type upstream primer such as SEQ ID NO.1, its downstream primer shown in SEQ ID NO.2,
Enterovirus EV 71 type fluorescent probe is shown in SEQ ID NO.3;
Enterovirus Cox A 16 type upstream primer such as SEQ ID NO.4, its downstream primer shown in SEQ ID NO.5,
Enterovirus Cox A 16 type fluorescent probe is shown in SEQ ID NO.6;
The enterovirus universal upstream primer shown in SEQ ID NO.7, its downstream primer shown in SEQ ID NO.8,
The enterovirus universal fluorescent probe is shown in SEQ ID NO.9; The internal reference sequence shown in SEQ ID NO.10,
The internal reference upstream primer shown in SEQ ID NO.11, its downstream primer shown in SEQ ID NO.12,
The internal reference fluorescent probe is shown in SEQ ID NO.13.
8. human intestine according to claim 6 virus four look fluorescent RT-PCR method for detecting, it is characterized in that described multiple 10 * PCR reaction buffer consists of: Tutofusin tris hydrochloric acid final concentration is that 100mM, Repone K final concentration are that 500mM, glycerine volume ratio are 50%, four kind of mononucleotide monomer final concentration is 0.4mM, and the magnesium chloride final concentration is the ultrapure water of 3mM and respective volume.
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