CN1948507B - Parotiditis virus fluorencent amplification detection reagent box and detection method - Google Patents
Parotiditis virus fluorencent amplification detection reagent box and detection method Download PDFInfo
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- CN1948507B CN1948507B CN2006100518285A CN200610051828A CN1948507B CN 1948507 B CN1948507 B CN 1948507B CN 2006100518285 A CN2006100518285 A CN 2006100518285A CN 200610051828 A CN200610051828 A CN 200610051828A CN 1948507 B CN1948507 B CN 1948507B
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Abstract
The invention offers detecting reagent boxes for fluorescent augment of mumps virus, which include hemagglutinin gene standards of mumps virus, detecting reagent of fluorescent augment, DNA polymerase and reverse transcriptase. The detecting reagent of fluorescent augment mainly contains buffer solution of one-step RT-PCR, specificity exciters and probes, mixture of deoxidizing triphosphoric acidand nucleoside. The partial sequence of hemagglutinin gene standards of mumps virus is 5'- CTCAAGGACTGTTTGCCTCTTACACCACAACCACCTGCTTTCAAGATACCGGTGATGCTAGTG-3'. The specificity exciters have two sequences: the sequence of upstream exciters is 5'-CTCAAGGACTRTYTGCYTCSTA-3'and the sequence of downstream exciters is 5'-CTCTRGCAT CACCGGTATCTTGAA-3'. The equence of specificity probes is 5'-FAM-ACCACAACCACCTGC-NFQ-MGB-3', in which FAM is reporting fluorescent metakliny, NFQ is non-fluorescent annihilation metakliny, MGB is modifying metakliny. The detecting reagent boxes can detect pathogen nucleic acid directly. At the early stage of mumps disease, specificity gene of mumps virus can be detected, which facilitates early isolation, diagnosis and therapy.
Description
(1) technical field
The present invention relates to a kind of parotiditis virus fluorencent amplification detection kit and detection method.
(2) background technology
Mumps is to be infected by the acute viral that mumps virus causes, this disease transmission is strong, and the four seasons all can be popular, and virus is separated and serological test is the main means of present mumps virus laboratory diagnosis.But because viral disengaging time is long, the requirement condition height, separation rate is low; The serological method susceptibility is low, is not suitable for reasons such as early diagnosis, and these two kinds of methods in use are very restricted.The regular-PCR technology is widely used in the specific detection of virogene, and it has sensitivity, special, advantage fast, needs electrophoresis but exist reaction result to judge, and reaction product is easy to generate and pollutes and cause shortcoming such as false positive.
The fluorescent quantitative PCR technique that grew up in recent years, it has utilized the efficient amplification of PCR to thymus nucleic acid (DNA), the high specific of probe technique and the susceptibility of spectroscopic techniques and quantitative characteristics, not only overcome the deficiency of conventional PCR qualitative detection, and have directly perceived, good reproducibility, high specificity, susceptibility is high and advantage such as easy to operate.
The fluorescent quantitative PCR technique principle: quantitative fluorescent PCR is to utilize the variation of the fluorescence luminous energy that fluorescence dye discharged under the effect of exciting light to come dynamically directly to reflect the variation of pcr amplification product amount.Because of the fluorescent signal variable is directly proportional with the amplified production amount, by enough sensitive automatization quantitative real time PCR Instruments just can by to the collection of fluorescent signal with analyze realize to original template quantitatively.
Fluorescence labeling method commonly used can simply be divided into two big classes: 1, non-special detection-double-stranded DNA interpolation type fluorescence dye; 2, the single-minded detection of extension increasing sequence-mainly refer to fluorescent probe and primer probe, fluorescently-labeled probe has three classes: (1) molecular beacon probe; (2) the two probes of hybridization; (3) Taqman double-tagging probe.When adding a pair of primer, add a specific fluorescent probe when widely used TaqMan probe method is meant pcr amplification in addition, this probe only with template specificity combine, its binding site is between two primers.5 of probe ' end be marked with the fluorescence report group (Reporter, R), as FAM, VIC etc., 3 ' end be marked with the fluorescent quenching group (Quencher, Q).When probe is complete, 5 ' end reporter group through the light source for instrument excited fluorescent just in time by in-plant 3 ' end fluorophor cancellation, instrument detecting less than 5 ' end reporter group institute excited fluorescent signal (emission wavelength of 5 ' end fluorophor just in time is the absorbing wavelength of 3 ' end fluorophor in other words, thus energy be absorbed be delivered to 3 ' end fluorophor and send other fluorescence).Carrying out along with PCR, the Taq enzyme runs in the chain extension process and template bonded probe, (this activity is a double-stranded specific to its 5 '-3 ' 5 prime excision enzyme activity, the free single-stranded probe is unaffected) will cut probe, discharging 5 ' end reporter group is free in the reaction system, away from 3 ' and the shielding of end fluorescent quenching group, 5 ' end reporter group institute's fluorescent signal emitted that is stimulated just can be detected by probe.That is to say DNA chain of every amplification, just have a fluorescence molecule to form, realized that the accumulation of fluorescent signal and PCR product form fully synchronously.From instrument detecting go out the minimum cycle number that fluorescent value goes out the peak (cycle threshold, Ct) with detect viral nucleic acid amount logarithmic value and be linear negative correlation.Just can calculate primary template amount according to the Ct value in the quantitative fluorescent PCR reaction.
(3) summary of the invention
The present invention is exactly according to above-mentioned principle, and design is fit to Auele Specific Primer and the specific probe of mumps virus, and goal of the invention is to provide gene quick detection kit and the detection method of a kind of mumps virus.
For reaching goal of the invention the technical solution used in the present invention be:
A kind of parotiditis virus fluorencent amplification detection kit, comprise mumps virus hemagglutinin gene standard substance, amplified fluorescence detection reagent and archaeal dna polymerase and reversed transcriptive enzyme, described amplified fluorescence detection reagent mainly comprises single stage method RT-PCR damping fluid, specificity amplification primer and specific probe, deoxidation nucleoside triphosphate mixture, described mumps virus hemagglutinin gene standard substance partial sequence is: 5 '-CTCAAGGACTGTTTFGCCTCTTACACCACAACCACCTGCTTTCAAGATACCGGTGA TGCTAGTG-3 ', described specificity amplification primer is: upstream primer sequence 5 '-CTCAAGGACTRTYTGCYTCSTA-3 ', downstream primer sequence 5 '-CTCTRGCATCACCGGTATCTTGAA-3 ', described specific probe sequence is: 5 "-FAM-ACCACAACCACCTGC-NFQ-MGB-3 '; wherein FAM is the report fluorophor; NFQ is non-fluorescent quenching group; itself do not produce fluorescence; can reduce the intensity of PCR reaction fluorescence background signal; MGB is a modification group, the probe annealing temperature can be improved about 10 ℃.
Described amplified fluorescence detection reagent main component is as follows:
Single stage method RT-PCR damping fluid final concentration is 1 *
Specific amplification upstream primer 0.6～1.0 μ M
Specific amplification downstream primer 0.6～1.0 μ M
Specific probe 0.2～0.5 μ M
Deoxidation nucleoside triphosphate mixture 400～800 μ M
Surplus is a DEPC water.
Above concentration is the final concentration of each material in reaction system.Deoxidation nucleoside triphosphate mixture is the mixing of dATP, dTTP, dCTP, dGTP.Described DEPC water refers to that diethypyrocarbonate (diethylpyrocarbonate) handled and through the MiliQ of high temperature, autoclave sterilization level pure water.
Described single stage method RT-PCR damping fluid final concentration is 1 *, represent that promptly each component final concentration in reaction solution is identical with each concentration of component among 1 * RT-PCR buffer in the damping fluid, each component final concentration is as follows: KCl50mM, Tris-HCl 10mM, TritonX-100 0.1%, MgCl
21.5mM.Selecting volume among the present invention for use is 5 * RT-PCR buffer of reaction solution cumulative volume 20%.
Also being added with final concentration in the described amplified fluorescence detection reagent is the RNA enzyme inhibitors RNasin of 0.8 unit of activity/μ L.The RNasin nucleic acid inhibitor is by the Promega research and development, and the RNA enzyme with wide spectrum suppresses vigor, is used to remove the RNA enzyme and pollutes.The RNasin nucleic acid inhibitor is the albumen of 1 50kD size, and it and RNA enzyme are pressed combination in 1: 1 with non covalent bond, and suppress the RNA enzyme activity, and binding constant is 10～14.The unit of activity of RNasin nucleic acid inhibitor is defined as and suppresses 5ngRNase A hydrolysis 2`, the amount of the inhibitor that 50% vigor of the single phosphoric acid cyclisation of 3`-cytidine is used.
Also be added with the DTT of final concentration 5mM in the described amplified fluorescence detection reagent.
Described deoxidation nucleoside triphosphate mixture is dATP, dTTP, dCTP, dGTP amount of substance 1: 1: 1: 1 mixture.
Described archaeal dna polymerase consumption is 0.5～5 enzyme activity unit/reaction, is selected from one of following: 1. Ampli TaqR archaeal dna polymerase; 2. rTth archaeal dna polymerase; 3. rTth archaeal dna polymerase XL.
Described reversed transcriptive enzyme consumption is 6～300 enzyme activity unit/reactions, is selected from one of following: 1. M-MuLV reversed transcriptive enzyme; 2. AMV reversed transcriptive enzyme.
Concrete, described amplified fluorescence detection reagent is composed as follows:
Single stage method RT-PCR buffer final concentration is 1 *
DTT 5mM
The RNA enzyme suppresses RNasin 0.8 unit of activity/μ L
Specific amplification upstream primer 0.88 μ M
Specific amplification downstream primer 0.88 μ M
Specific probe 0.28 μ M
Deoxidation nucleoside triphosphate mixture is:
DATP, dTTP, each 200 μ M of dCTP, dGTP
Surplus is a DEPC water.
A kind of parotiditis virus fluorencent amplification detection method, described detection method is contrast with mumps virus hemagglutinin gene standard substance, and the gargarism that adopts patient to be measured is an analyzing samples, and described method steps is as follows:
(1) gets gargarism and extract viral RNA;
(2) fluorescent PCR augmentation detection: get amplified fluorescence detection reagent and reaction required archaeal dna polymerase and reversed transcriptive enzyme and be made into reaction solution, the RNA that adds standard substance and analyzing samples carries out amplified reaction respectively, and reaction tubes places the quantitative fluorescence PCR instrument to carry out fluoroscopic examination;
(3) interpretation of result: select fluoroscopic examination model F AM fluorescence, baseline adjustment is got 3～15 round-robin fluorescent signals; The threshold setting principle is with the vertex of threshold line just above normal negative control; The fluorescence growth curve surpasses threshold line, and is good logarithmic growth, is judged as the positive;
Described mumps virus hemagglutinin gene standard substance partial sequence is: 5 '-CTCAAGGACTGTTTGCCTCTTACACCACAACCACCTGCTTTCAAGATACCGGTGAT GCTAGTG-3 ', described specificity amplification primer is: upstream primer sequence 5 '-CTC AAG GAC TRT YTG CYT CST A-3 ', downstream primer sequence 5 '-CTC TRG CAT CAC CGG TAT CTT GAA-3 ', described specific probe sequence is 5 '-FAM-ACCACAACCACCTGC-NFQ-MGB-3 ', wherein FAM is the report fluorophor, NFQ is non-fluorescent quenching group, and MGB is a modification group.Concrete, described method is got quantitative fluorescence augmentation detection reagent preparation reaction solution, and per 25 μ L are composed as follows for reaction system:
5×RT-PCR?buffer 5μL
DTT 5mM
RNA enzyme inhibitors RNasin 0.8 unit/μ L
Specific amplification upstream primer 0.88 μ M
Specific amplification downstream primer 0.88 μ M
Specific probe 0.28 μ M
Deoxidation nucleoside triphosphate mixture is:
DATP, dTTP, each 200 μ M of dCTP, dGTP
2.5 enzyme activity unit/the reactions of Ampli TaqR archaeal dna polymerase
50 enzyme activity unit/the reactions of AMV reversed transcriptive enzyme
Analyzing samples viral RNA 5 μ L
DEPC water complements to 25 μ L;
The PCR cycling condition is set to 45 ℃ of 30min reverse transcriptions, 94 ℃ of sex change 2min, and with 95 ℃ of 15s, 40 circulations of 60 ℃ of annealing 1min amplifications are carried out the single-point fluoroscopic examination at 60 ℃.The annealing temperature optimum range is 60～50 ℃, and the annealing time optimum range is 30sec～1min.
Beneficial effect of the present invention is mainly reflected in:
1, early diagnosis: PCR can directly detect pathogen nucleic acid, and is early stage in parotitis morbidity, just can detect and whether contain the mumps virus specific gene, for isolating early, make a definite diagnosis and treatment being provided convenience.
2, sampling is simple and convenient: the desirable latent period or the early stage parotitis patient respiratory road sample of falling ill detect.
3, compare with traditional gene amplification technology, detection method provided by the invention is time saving and energy saving, can finish detection in 2～3 hours.
4, highly sensitive, owing to adopted specific gene amplification and specific gene probe hybridization bonded double technique, the sensitivity of diagnosis is higher.
5, adopt the computer real-time monitoring technology, in the experiment process, can judge whether to contain virogene, and the judgement of experimental result is accurately convenient.
6, can realize the quantitative analysis of viral nucleic acid, utilize the known copy number, contain the mumps virus gene recombination plasmid and be standard substance production standard curve, can carry out quantitative analysis the viral level in the type specimen.
(4) description of drawings
Fig. 1 is mumps virus TaqMan-MGB fluorescence quantifying PCR method standard substance amplification curve diagrams, and from left to right: the viral copy number of amplification is followed successively by 10
7, 10
6, 10
5, 10
4, 10
3, 10
2, 10;
Fig. 2 is a mumps virus TaqMan-MGB fluorescence quantifying PCR method typical curve;
Fig. 3 is the circulation ratio of fluorescence quantitative RT-RCR to the mumps virus detection of nucleic acids, and from left to right: the viral copy number of amplification is followed successively by 10
6, 10
5, 10
4
Fig. 4 is the detection of TaqMan-MGB fluorescence quantitative RT-RCR to the mumps virus clinical samples,
A is the positive internal reference nucleic acid of mumps virus among the figure, and B is that the mumps virus positive contrasts nucleic acid outward.
(5) embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Embodiment 1:
TaqMan-MGB detection method step is as follows:
(1) extracts RNA
Get gargarism 200ul and extract viral RNA (1 μ g/ μ L) with the Reansy Mini Kit of German QIAGEN company.
(2) fluorescent PCR amplification
Get quantitative fluorescence augmentation detection reagent preparation reaction solution, reaction system is per 25 μ L, comprising:
5×RT-PCR?buffer 5μL
DTT 5mM
RNA enzyme inhibitors RNasin 0.8 unit/μ L
Specific amplification upstream primer 0.88 μ M
Specific amplification downstream primer 0.88 μ M
Specific probe 0.28 μ M
Deoxidation nucleoside triphosphate mixture is:
DATP, dTTP, each 200 μ M of dCTP, dGTP
2.5 enzyme activity unit/the reactions of Ampli TaqR archaeal dna polymerase
50 enzyme activity unit/the reactions of AMV reversed transcriptive enzyme
Analyzing samples viral RNA (1 μ g/ μ L) 5 μ L
DEPC water complements to 25 μ L;
The PCR cycling condition is set to 45 ℃ of 30min reverse transcriptions, 94 ℃ of sex change 2min, and with 95 ℃ of 15s, 40 circulations of 60 ℃ of annealing 1min amplifications are carried out the single-point fluoroscopic examination at 60 ℃.
By above-mentioned reaction solution prescription, the RNA that adds standard substance and analyzing samples prepares reaction solution respectively, carries out amplified reaction, and reaction tubes places quantitative real time PCR Instrument to carry out fluoroscopic examination; The standard substance amplification curve is seen Fig. 1, obtains typical curve and sees Fig. 2, selects viral copy number to be followed successively by 10
6, 10
5, 10
4The standard samples of/reaction carries out quantitative RT-PCR and detects, and each sample repeats to do five times, the results are shown in Figure 3 and table 1, and the Ct value variation coefficient of each concentration 5 secondary response is all less than 1%.Mumps virus clinical samples amplification curve is seen Fig. 4;
Table 1: fluorescence quantitative RT-RCR detects the circulation ratio result of mumps virus
(3) interpretation of result: select fluoroscopic examination model F AM fluorescence, baseline adjustment is got 3～15 round-robin fluorescent signals; The threshold setting principle is with the vertex of threshold line just above normal negative control; The fluorescence growth curve surpasses threshold line, and is good logarithmic growth, is judged as the positive;
Experiment shows that the Ct value variation coefficient of 5 reaction repeated of each standard nucleic acid concentration illustrates that all less than 1% the reproduction of this method is well better to the circulation ratio of mumps virus detection of nucleic acids by fluorescence quantitative RT-RCR.
Utilize this TaqMan-MGB fluorescence quantifying PCR method that some areas, Zhejiang Province doubtful mumps epidemic situation sample is detected for 35 parts, and compare with common RT-PCR method, the result shows, positive 27 parts of fluorescence quantitative PCR detection, regular-PCR detects positive 18 parts, and positive 7 parts of virus culture.The sample that RT-PCR and virus culture are positive, fluorescence quantitative RT-RCR detects also all positive, and coincidence rate is fine.
Sequence table .ST25
SEQUENCE?LISTING
＜110〉Zhejiang Polytechnical University
＜120〉a kind of parotiditis virus fluorencent amplification detection kit and detection method
<130>
<160>4
<170>PatentIn?version?3.2
<210>1
<211>63
<212>DNA
<213>Paramyxovirus
<400>1
ctcaaggact?gtttgcctct?tacaccacaa?ccacctgctt?tcaagatacc?ggtgatgcta 60
gtg 63
<210>2
<211>22
<212>DNA
<213>Unknown
<220>
＜223〉artificial sequence
<400>2
ctcaaggact?rtytgcytcs?ta 22
<210>3
<211>24
<212>DNA
<213>Unknown
<220>
＜223〉artificial sequence
<400>3
ctctrgcatc?accggtatct?tgaa 24
<210>4
<211>15
<212>DNA
<213>Unknown
<220>
＜223〉artificial sequence
<400>4
accacaacca?cctgc 15
Claims (8)
1. parotiditis virus fluorencent amplification detection kit, comprise mumps virus hemagglutinin gene standard substance, amplified fluorescence detection reagent and archaeal dna polymerase and reversed transcriptive enzyme, described amplified fluorescence detection reagent mainly comprises single stage method RT-PCR damping fluid, specificity amplification primer and specific probe, deoxidation nucleoside triphosphate mixture, it is characterized in that: described mumps virus hemagglutinin gene standard substance partial sequence is: 5 '-CTCAAGGACTGTTTGCCTCTTACACCACAACCACCTGCTTTCAAGATACCGGTGAT GCTAGTG-3 ', described specificity amplification primer is: upstream primer sequence 5 '-CTCAAGGACTRTYTGCYTCSTA-3 ', downstream primer sequence 5 '-CTCTRGCATCACCGGTATCTTGAA-3 ', described specific probe sequence is: 5 '-FAM-ACCACAACCACCTGC-NFQ-MGB-3 ', wherein FAM is the report fluorophor, NFQ is non-fluorescent quenching group, and MGB is a modification group.
2. parotiditis virus fluorencent amplification detection kit as claimed in claim 1 is characterized in that described amplified fluorescence detection reagent main component is as follows:
Single stage method RT-PCR damping fluid final concentration is 1 *
Specific amplification upstream primer 0.6～1.0 μ M
Specific amplification downstream primer 0.6～1.0 μ M
Specific probe 0.2～0.5 μ M
Deoxidation nucleoside triphosphate mixture 400～800 μ M
Surplus is a DEPC water.
3. parotiditis virus fluorencent amplification detection kit as claimed in claim 2 is characterized in that also being added with in the described amplified fluorescence detection reagent RNA enzyme inhibitors RNasin that final concentration is 0.8 unit of activity/μ L.
4. parotiditis virus fluorencent amplification detection kit as claimed in claim 2 is characterized in that also being added with in the described amplified fluorescence detection reagent DTT of final concentration 5mM.
5. parotiditis virus fluorencent amplification detection kit as claimed in claim 1 is characterized in that described deoxidation nucleoside triphosphate mixture is dATP, dTTP, dCTP, dGTP amount of substance 1: 1: 1: 1 mixture.
6. parotiditis virus fluorencent amplification detection kit as claimed in claim 1 is characterized in that described archaeal dna polymerase consumption is 0.5～5 enzyme activity unit/reaction, is selected from one of following: 1. Ampli TaqR archaeal dna polymerase; 2. rTth archaeal dna polymerase; 3. rTth archaeal dna polymerase XL.
7. parotiditis virus fluorencent amplification detection kit as claimed in claim 1 is characterized in that described reversed transcriptive enzyme consumption is 6～300 enzyme activity unit/reactions, is selected from one of following: 1. M-MuLV reversed transcriptive enzyme; 2. AMV reversed transcriptive enzyme.
8. parotiditis virus fluorencent amplification detection kit as claimed in claim 1 is characterized in that described amplified fluorescence detection reagent is composed as follows:
Single stage method RT-PCR buffer final concentration is 1 *
DTT 5mM
The RNA enzyme suppresses RNasin 0.8 unit of activity/μ L
Specific amplification upstream primer 0.88 μ M
Specific amplification downstream primer 0.88 μ M
Specific probe 0.28 μ M
Deoxidation nucleoside triphosphate mixture is:
DATP, dTTP, each 200 μ M of dCTP, dGTP
Surplus is a DEPC water.
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| CN104593523A (en) * | 2014-12-04 | 2015-05-06 | 湖北永邦医疗科技有限公司 | Primer, probe and kit all used for detecting mumps virus |
| CN114836574B (en) * | 2021-11-10 | 2023-06-16 | 江汉大学 | MNP (MNP) marking site of mumps virus, primer composition, kit and application of MNP marking site |
Citations (3)
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| US20020064795A1 (en) * | 2000-11-30 | 2002-05-30 | Koji Hashimoto | Nucleic acid detection method and apparatus, and vessel for detecting nucleic acid |
| CN1641355A (en) * | 2004-12-20 | 2005-07-20 | 山东省医药生物技术研究中心 | Protein chip for detecting blood and cerebro spinal fluid pathogen antibody, and its preparing method and use |
| CN2771864Y (en) * | 2005-03-22 | 2006-04-12 | 山东省医药生物技术研究中心 | Protein chip for detecting blood cerebrospinal fluid pathogenic antibody |
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2006
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020064795A1 (en) * | 2000-11-30 | 2002-05-30 | Koji Hashimoto | Nucleic acid detection method and apparatus, and vessel for detecting nucleic acid |
| CN1641355A (en) * | 2004-12-20 | 2005-07-20 | 山东省医药生物技术研究中心 | Protein chip for detecting blood and cerebro spinal fluid pathogen antibody, and its preparing method and use |
| CN2771864Y (en) * | 2005-03-22 | 2006-04-12 | 山东省医药生物技术研究中心 | Protein chip for detecting blood cerebrospinal fluid pathogenic antibody |
Non-Patent Citations (4)
| Title |
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| 严菊英等.RT-PCR快速检测腮腺炎病毒.中国公共卫生19 3.2003,19(3),347-349. * |
| 李敏红等.多重逆转录-聚合酶链反应检测麻疹 风疹 流行性腮腺炎病毒基因的研究.中国计划免疫11 1.2005,11(1),9-11. |
| 李敏红等.多重逆转录-聚合酶链反应检测麻疹 风疹 流行性腮腺炎病毒基因的研究.中国计划免疫11 1.2005,11(1),9-11. * |
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