CN101100694B - Kit for detecting dengue fever virus, and special-purpose amplification primer and probe for the same - Google Patents
Kit for detecting dengue fever virus, and special-purpose amplification primer and probe for the same Download PDFInfo
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- CN101100694B CN101100694B CN2007101430312A CN200710143031A CN101100694B CN 101100694 B CN101100694 B CN 101100694B CN 2007101430312 A CN2007101430312 A CN 2007101430312A CN 200710143031 A CN200710143031 A CN 200710143031A CN 101100694 B CN101100694 B CN 101100694B
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- fever virus
- dengue fever
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
A kit and special alkali inducers for amplification and probes can be used to check out dengue fever virus. The inducers consist of a couple of opposite directions. One is of sequence shown in No.1, another is of sequence shown in No.2, and probes shown in No.3. The process has advantages, e.g. quick action, simple, high sensitivity and specification in detection of dengue fever virus I, II, III and IV.
Description
Technical field
The present invention relates to a kind of test kit and special-purpose amplification primer and probe that detects virus, particularly relate to test kit and the special-purpose amplification primer and the probe of a kind of detection dengue fever virus (DV).
Background technology
Singapore hemorrhagic fever is a kind of acute arboviruses transmissible disease the widest, that number of the infected is maximum that distributes in the world that is popular in the torrid zone, subtropical zone.Singapore hemorrhagic fever is caused that by dengue virus dengue virus is divided into 4 types, promptly steps on leather I II III IV type, and the mat yellow-fever mosquito is propagated vector for it.According to WHO statistics, annual dengue infection number has 5,000 ten thousand, and wherein dengue hemorrhagic fever is about 500,000, and dead the population that is on the hazard reaches 2,500,000,000 more than 20,000 people, and singapore hemorrhagic fever has become the maximum public problem of defending in the whole world.
China occurred once by I II III the singapore hemorrhagic fever epidemic situation that causes of IV type dengue fever virus, but so far China's singapore hemorrhagic fever be still Introduced cases or input cause local transmission.In recent years, the epidemic situation of breaking out that local transmission causes all appearred in Guangdong, Hainan, Guangxi, Fujian etc., and all had every year a plurality of provinces to find the input case.Because singapore hemorrhagic fever does not have specific methods of treatment, does not have effective vaccine to prevent yet, therefore in time find singapore hemorrhagic fever epidemic situation, prevention or reduce local singapore hemorrhagic fever epidemic situation propagation just to become particularly important.In the face of this situation, it is extremely important to develop a kind of diagnostic method fast and effectively.
Up to the present, the detection of dengue fever virus is the most original, classic methods is the specificity virus separation and Culture, but, in real work, be difficult to apply owing to complex operation, the big length consuming time of technical difficulty, requirement for experiment condition height, have serious Biosafety problem.The ELISA method is compared with viral separation and Culture and DNA test, can produce certain false positive.And in the market dengue fever virus the PCR detection kit can only with singapore hemorrhagic fever I II III the IV type detect respectively, consuming time longer, testing process is loaded down with trivial details.
Summary of the invention
The purpose of this invention is to provide a kind of primer special and probe that detects dengue fever virus.
Primer special provided by the present invention is made up of forward primer and reverse primer, the base sequence of described forward primer is shown in SEQIN NO:1, the base sequence of described reverse primer shown in SEQIN NO:2, described application specific probe, its base sequence is shown in SEQIN NO:3.
One end of described probe is connected with fluorescent quenching group TAMRA, and the other end is connected with fluorescence report group FAM.
In common use, the fluorescent quenching group is generally one, is connected 3 ' end of probe, and the fluorescence report group is one also, is connected 5 ' end of probe.Concrete, the fluorescent emission gene of 5 ' end can be 6-Fluoresceincarboxylic acid (its fluorescent emission peak value is at the 518nm place), the fluorescent quenching group of 3 ' end can be 6-carboxyl tetramethylrhodamin (the fluorescent emission peak value is at the 582nm place).
Second purpose of the present invention provides a kind of test kit that dengue fever virus detects that is used for.
Dengue fever virus detection kit provided by the present invention comprises above-mentioned detection dengue fever virus universal primer and specific probe.
Use for convenience, dengue fever virus detection kit provided by the present invention also comprises and contains dengue fever virus gene PCR amplifing reagent, and dengue fever virus RNA standard substance.
Described dengue fever virus gene PCR amplifing reagent comprises Probe RT-PCR Master Mix, Probe RT-PCRBuffer.
The 3rd purpose of the present invention provides a kind of method of utilizing the mentioned reagent box that dengue fever virus is detected.
The method of detection dengue fever virus provided by the present invention, be to be template with the total RNA in the extract in testing sample serum or the blood plasma, cDNA is synthesized in reverse transcription, is template with this cDNA again, carries out fluorescence quantitative PCR detection with the test kit of above-mentioned detection dengue fever virus.
The present invention is directed to that existing dengue fever virus detection method is time-consuming, the shortcoming of effort, provide a kind of easy to operate, specificity is strong, the test kit of sensitivity higher detection dengue fever virus.Be applicable to present discovery singapore hemorrhagic fever I II III the detection and quantitatively of IV all types virus, can be widely used in the monitoring and the diagnosis of dengue virus infection, have important application value.(wherein I type patient is 45,21 of II type patients, 19 of I type patients to utilize 103 singapore hemorrhagic fever patients that the inventive method provides Guangdong Province inspection and quarantine bureau, 18 of IV type type patients) detect, the result shows that positive rate is 99%, and the recall rate that method of the present invention is described is very high.
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment.
Embodiment 1, the primer special that detects dengue fever virus and the design of probe
According to ncbi database singapore hemorrhagic fever I～IV C-type virus C gene sequencing, to be respectively the conserved sequence of EF032590, NC_001474, DQ401690, AF326573 for Genbank number, find out the identical sequence in its conserved sequence district through the contrast of ClustalW software, there is not homology sequence through NCBI-Blast analysis and other viruses and microorganism, and at this zone design probe.
Its primer special is made up of forward primer and reverse primer, and the base sequence of described forward primer is shown in SEQ IN NO:1, and the base sequence of described reverse primer is shown in SEQIN NO:2.The base sequence of application specific probe is shown in SEQIN NO:3.
Embodiment 2, with test kit of the present invention sample to be tested is carried out fluorescent quantitation and detect
1, reaction solution preparation
Reaction solution is 18 μ l for every part, contain following component in every part of reaction solution: 10 μ l Probe RT-PCR Master Mix, 0.2 μ l Probe RT-PCR Buffer, 1 μ l upstream primer (10pmol/ μ l), 1 μ l1.0 μ M downstream primer (10pmol/ μ l), 0.25 μ l probe (10pmol/ μ l), DEPC water 5.55 μ l.
2, sample collecting
Serum: extract 2 milliliters in person under inspection personnel venous blood with disposable sterilized injector, inject aseptic dry glass tube, room temperature (22～25 ℃) is placed 30～60min blood specimen can spontaneous complete aggegation separate out serum, or direct usage level whizzer, centrifugal 5 minutes of 1500rpm; Draw upper serum, it is standby to be transferred to 1.5ml sterilization centrifuge tube.
Blood plasma: extract 2 milliliters in person under inspection personnel venous blood with disposable sterilized injector, injection contains the Glass tubing of EDTA (disodium ethylene diamine tetraacetate) or sodium citrate anticoagulant, putting upside down Glass tubing immediately gently mixes 5～10 times, make the abundant mixing of antithrombotics and venous blood, can isolate blood plasma behind 5～10min, it is standby to be transferred to 1.5ml sterilization centrifuge tube.
3, the Trizol method is extracted dengue fever virus RNA
(1) gets above-mentioned serum of 200ul or plasma sample, negative control, each 1.5ml of positive control in sterilization eppendorf pipe;
(2) add 600ul Trizol, the thermal agitation mixing left standstill 10 minutes, added the 200ul chloroform again, put upside down mixing;
(3) the centrifugal 15min of 13000rpm;
(4) when the 3rd step centrifugal fast ends, other gets same a plurality of eppendorf pipe, the Virahol of adding 400ul-20 degree precooling;
(5) get the 3rd step centrifugal supernatant (will be drawn to the middle white layer scarcely, when the 3rd centrifugal end of step was taken outward, pipe tried not) and transfer in the pipe of the 4th step preparation, put upside down mixing;
(6) the centrifugal 15min of 13000rpm removes supernatant gently; On thieving paper, be stained with dry liquids as far as possible;
(7) add 600ul75% ethanol, put upside down for several times to wash remaining Virahol;
(8) the centrifugal 15min of 13000rpm removes supernatant gently, is stained with dry liquids on thieving paper as far as possible;
(9) the centrifugal 10sec of 4000rpm is thrown to the bottom with the remaining liquid of tube wall, blots drying at room temperature 2～3min (can not overdrying, prevent that next step RNA from not dissolving) with micro-rifle head;
(10) add 20ul DEPE water (water behind the pure water high pressure of adding depc is DEPE water), mixing dissolving RNA gently.The centrifugal 5sec of 2000rpm preserves standby (using in best 2 hours, in order to avoid the RNA degraded) on ice.
4, the preparation of standard substance
Standard substance comprise: negative quality control product, critical positive quality control product and strong positive quality control product.Can also can prepare as follows with the standard substance that provide in the test kit of the present invention.
With above-mentioned serum or the blood plasma of determining to contain dengue fever virus RNA that extracts with Tri zol method, detect quality and the concentration of RNA and carry out reverse transcription reaction through the nucleic acid-protein uv analyzer, contain 1 * RT Buffer, 0.5mmol/LdNTPs, 1 μ mol/L specificity downstream primer in the reaction system, RNase inhibitor 10U, RT enzyme AMV4U, 37 ℃ were reacted 1 hour down, at 65 ℃ of following 15 minutes deactivation reversed transcriptive enzymes, obtain the cDNA template again; Get above-mentioned cDNA template 2 μ l, add PCR reaction solution 23 μ l, contain 1XPCRBuffer, 25mmol/LMgCl in the PCR reaction solution
2, 0.2mmol/LdNTPs, TaqDNA polysaccharase 4U and each 0.5pmol/L of DV upstream and downstream primer, carry out following circulation in PT-200 amplification instrument: 94 ℃ of pre-sex change 4min, 94 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 30s circulates 30 times, last 72 ℃ of extension 10min.The PCR product is through 1.5% agarose electrophoresis, glue reclaims test kit and reclaims PCR electrophoresis product, with be connected 12h～16h under 4 ℃ in the pGEM-T carrier, there is PCR glue to reclaim product 3 μ l in the ligation, connect damping fluid 5 μ l, T carrier 1 μ l and T4 ligase enzyme 1 μ l, connect product transformed competence colibacillus bacillus coli DH 5 alpha (E.Coli, DH5 α), coat on 1.5% agar plate that contains X-gal and IPTG and tool amicillin resistance, be inverted cultivation 12h～16h for 37 ℃ and to plate, grow blue hickie, sterilization toothpick picking hickie, place and contain 50mg/ml: the 5ml LB liquid nutrient medium of penbritin, 37 ℃ of shaking table 200rpm jolt and spend the night; Get 1.6ml overnight culture extracting plasmid, the EcoRI enzyme is cut preliminary evaluation, positive colony carries out sequencing analysis, the positive strain that filters out is further increased, the extracting plasmid, the PstI enzyme is cut and is made plasmid linearization, with t7 rna polymerase external cDNA that transcribes under 37 ℃,: digest 15min with DNaseI again,, use the accurately quantitative (METHOD FOR CONTINUOUS DETERMINATION 4 times of nucleic acid-protein uv analyzer behind the purifying with 0.2mol/L EDTA termination reaction, each 4 pipes that repeat, get average), and carry out 10 times of serial dilutions, obtain concentration and be respectively 10
8, 10
7, 10
6, 10
5, copies/ml 4 standard substance, both positive quantitative reference material.
Negative quality control product prepares with normal virus-free infection human serum.Strong positive quality control product and critical positive quality control product be will preparation standard substance RNA join in the normal virus-free infection human serum and prepare.
In the present invention, the setting of standard substance and the kind of standard substance and gradient can be adjusted according to concrete practical situation, if just carry out qualitative detection to whether infecting the singapore hemorrhagic fever disease, also can consider without standard substance.
5, fluorescent quantitation detects
Getting the extract and the concentration that obtain in the step 3 is 10
8, 10
7, 10
6, 10
5, copies/ml each 2 μ l of 4 standard substance, add the reaction solution 18 μ l of step 1, establish blank pipe and negative control simultaneously, increase; Amplification and other reaction tubes amplifications that will contain testing sample serum or blood plasma extract reaction tubes compare, and draw the content that whether contains DV RNA and DV RNA in testing sample serum or the blood plasma.Above-mentioned amplification is to carry out in Roche fluorescent quantitation detector, and concrete reaction conditions is: 50 ℃ of 20min (* 1), 95 ℃ of 10min (* 1), 94 ℃ of 15s, 59 ℃ of 20s, 72 ℃ of 30s (* 45).
In order to verify the reliability of test kit detected result of the present invention, the fluorescent PCR reaction product is carried out agarose electrophoresis, detected result is consistent with the purpose clip size, the PCR reaction product is checked order, analyze through NCBI-Blast, the result shows that the product sequence is the dengue fever virus gene order.
The testing process of utilizing test kit of the present invention to carry out can be finished with Luo Shi LightCycler instrument, and specific operation process is as follows:
(1) application of sample
Get sample (comprising sample, negative quality control product, critical positive quality control product and strong positive quality control product) supernatant liquor 2 μ l and positive quantitatively reference material 2 μ l behind the reverse transcription, add respectively in the special-purpose kapillary, covering PCR reaction lid (notes: when the lid reaction is covered, it is vertical that the hand power thrusts is wanted, in order to avoid kapillary fracture) 4000rpm is centrifugal 3 minutes, inserts in order and puts into instrument after circular chuck was inverted for 20 seconds.
(2) interpretation of result
Reaction finishes automatic preservation the in back and detects data file, and adjusting fluorescent reporter numerical value (Fluorescence) is F1/F2, clicks Quantification and enters assay surface.Select Fit Points on the Analysis hurdle, Proportional is selected on the BaselineAdjustment hurdle, regulates noise margin to more than the baseline noise, requires under Step3:Analysis r numerical value between-1.0～-0.97.
The unknown sample numerical value (C) that the instrument automatic analyser is calculated below the record Calculated Concentration.
(3) quality control
Negative quality control product: the Ct value is blank;
Positive quality control product: the S-type curve of growth curve, and the quantitative reference value of strong positive quality control product is 5.0 * 10
5IU/ml～5.0 * 10
7The IU/ml scope, the quantitative reference value of critical positive quality control product is 2.0 * 10
2IU/ml～5.0 * 10
4The IU/ml scope; (numerical value that reference value is calculated for the instrument automatic analyser)
Positive quantitatively reference material: the S-type curve of growth curve, Ct value＜37, and 0.97≤| r|≤1;
Need more than to require in once testing, satisfying simultaneously, otherwise this experiment is invalid, need carry out again.
Determining of embodiment 3, test kit sensitivity of the present invention and linearity range
1, test kit sensitivity of the present invention determines
To the RNA of step 4 preparation, through the dilution of 10 multiple proportions example, minimum concentration is 1.0 * 10IU/ml, detects 1.0 * 10 through step 5
3What IU/ml concentration was above can detect fluorescent signal, determines that therefore this test kit is 1.0 * 10 to the sensitivity of handling the back sample
3IU/ml.
2, test kit linearity range of the present invention determines
To RNA concentration is 1.0 * 10～1.0 * 10
10The standard substance of IU/ml detect, and find that concentration is less than 1.0 * 10
3IU/ml and greater than 1.0 * 10
8The not S-type curve of the growth curve of IU/ml, within the linearity range that detects, as seen, the linearity range of test kit of the present invention is not 1.0 * 10
3～1.0 * 10
8IU/ml.
(1) if not S-type curve of growth curve or Ct value are blank, the DV RNA total content of then declaring sample is less than limit of detection.
(2) if S-type curve of growth curve and Ct value＜40, then judgement by the following method:
If the C＜1.000E+03 of sample then declares sample DV RNA total content＜1.0 * 10
3IU/ml;
If the 1.000E+03≤C≤1.000E+08 of sample then declares sample DV RNA total content=C IU/ml;
If the C of sample〉1.000E+08, then sample DV RNA total content〉1.0 * 10
8IU/ml.The accurate quantification result can detect the diluted sample after extracting to linearity range more if desired.
Sequence table
<160>3
<210>1
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>1
<210>2
<211>19
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>2
<210>3
<211>21
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>3
Claims (4)
1. a test kit that detects dengue fever virus comprises the application specific probe that is used to detect the primer special of dengue fever virus and is used to detect dengue fever virus;
The described primer special that is used to detect dengue fever virus is made up of forward primer and reverse primer, and the base sequence of described forward primer is shown in SEQ IN NO:1, and the base sequence of described reverse primer is shown in SEQ IN NO:2;
The described application specific probe that is used to detect dengue fever virus, its base sequence is shown in SEQ IN NO:3;
Described 3 ' the end that is used to detect the application specific probe of dengue fever virus is connected with a cancellation fluorophor TAMRA, and 5 ' end is connected with a report fluorophor FAM.
2. test kit according to claim 1 is characterized in that: described test kit also comprises dengue fever virus gene PCR amplifing reagent.
3. test kit according to claim 2 is characterized in that: described pcr amplification reagent comprises ProbeRT-PCR Master Mix and Probe RT-PCR Buffer.
4. according to claim 2 or 3 described test kits, it is characterized in that: described test kit also comprises dengue fever virus RNA standard substance.
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| CN2007101430312A CN101100694B (en) | 2007-08-21 | 2007-08-21 | Kit for detecting dengue fever virus, and special-purpose amplification primer and probe for the same |
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| CN2007101430312A CN101100694B (en) | 2007-08-21 | 2007-08-21 | Kit for detecting dengue fever virus, and special-purpose amplification primer and probe for the same |
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| CN101100694A CN101100694A (en) | 2008-01-09 |
| CN101100694B true CN101100694B (en) | 2011-08-17 |
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103025893A (en) * | 2010-07-29 | 2013-04-03 | 彼格泰格私人有限公司 | Probes and primers for detection of dengue |
| CN102277447B (en) * | 2011-05-27 | 2012-11-28 | 江苏硕世生物科技有限公司 | Dual fluorescence PCR detection kit for dengue virus I/II |
| CN102808041A (en) * | 2012-03-14 | 2012-12-05 | 中华人民共和国天津出入境检验检疫局 | Method for detecting dengue by using gene chip method |
| CN102643930A (en) * | 2012-04-05 | 2012-08-22 | 中华人民共和国大榭出入境检验检疫局 | Consensus-degenerate hybridoligonucleotide primer (CODEHOP) reverse transcription-polymerase chain reaction (RT-PCR) reagent and method for detecting flavivirus viruses |
| CN104450971A (en) * | 2014-12-25 | 2015-03-25 | 中国医学科学院医学生物学研究所 | Absolute quantitative detection method of gene copy numbers of II type and III type dengus viruses |
| CN110894552A (en) * | 2019-11-08 | 2020-03-20 | 珠海国际旅行卫生保健中心(拱北海关口岸门诊部) | Primer, probe, kit and RT-iPCR method for detecting dengue virus |
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|---|---|---|---|---|
| WO1998049351A1 (en) * | 1997-04-28 | 1998-11-05 | University Of Massachusetts | Methods and reagents for rapid diagnosis of dengue virus infection |
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