CN102140530B - New chikungunya virus fluorescence quantitative polymerase chain reaction detection method and chikungunya virus polymerase chain reaction detection system - Google Patents
New chikungunya virus fluorescence quantitative polymerase chain reaction detection method and chikungunya virus polymerase chain reaction detection system Download PDFInfo
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Abstract
The invention discloses a new chikungunya virus fluorescence quantitative polymerase chain reaction (PCR) detection method and a chikungunya virus PCR detection system. The detection system consists of a primer and a probe, Premix Ex Taq reaction solution and sterilized Tris solution, wherein the primer and probe have good detection specificity. The system has high sensitivity, is very suitable for chikungunya virus and does not have cross reactions with the dengue virus and the like.
Description
Technical field
The invention belongs to field of biological detection, be specifically related to fast qualitative and quantitative detecting method and the Chikungunya virus detection PCR system of Chikungunya virus, a pair of Auele Specific Primer and probe are provided.
Background technology
Chikungunya fever is a kind of acute Natur al foca transmissible disease that is caused by Chikungunya virus (Chikungunya virus), belongs to togavirus, and mosquito is Vector of infection.This disease route of transmission mainly be infected animal (cercopithecus aethiops, baboon, chimpanzee, ox, horse, pig, rabbit etc.) and patient by propagation of sucking blood such as Aedes aegypti, African graceful mosquito, aedes africanus, the graceful mosquitos of brown wing, also can be through respiratory infectious.Chikungunya fever repeatedly betides the ground such as Indonesia, Philippines, Thailand, Vietnam, Burma and India in Africa and Asia.Because population is mobile both at home and abroad, Chikungunya fever also progressively imports China into, and In Xishuangbanna of Yunnan was once found the chikungunya case in 1987, and isolated this virus from its blood.The detection method that is used at present Chikungunya virus has had the nucleic acid amplifications such as ELISA, immunochromatography, IFA (IFA), viral separation, RT-PCR and Real-time PCR, the can yet be regarded as examination criteria of generally acknowledged a kind of more directly perceived, faster a kind of suitable early diagnosis of the method for quantitative fluorescent PCR detection of nucleic acids.
Summary of the invention
The object of the present invention is to provide a kind of Chikungunya virus fluorescence quantitative PCR detection novel method and Chikungunya virus to detect the PCR system, the method can fast qualitative and detection by quantitative Chikungunya virus, comprises that a pair of specificity is higher, highly sensitive, the primer of good reproducibility and probe.
For achieving the above object, Chikungunya virus fluorescence quantitative PCR detection novel method of the present invention, the primer probe that design is adopted in the method is:
| Name | Sequence | Position | Tm℃ | Modification |
| FP | GAACACGTAACAGTGATCCCG | 9999-10019 | 57.8 | |
| RP | GTAATCAAGCGATAGCGTTGG | 10114-10134 | 58.0 | |
| Probe | TGGGAGTACCGTATAAGACTCTAGTCAACAGA | 10028-10059 | 67.0 | CY5/lowa Black |
Further, described Chikungunya virus fluorescence quantitative PCR detection novel method is specially:
1) design primer probe;
2) select fluorescein according to instrument;
3) synthetic primer and probe;
4) preparation positive plasmid standard substance;
5) operation PCR;
6) data analysis;
7) extract viral RNA and carry out the system checking.
Further, described step 2) the selective fluorescent emission group of fluorescein has 6-FAM, TET, HEX, JOE, CY3, CY5, TAMRA, Texas Red in, and the fluorescent quenching group has BHQ-1, BHQ-2, Lowa Black
TMRQ, Lowa Black
TMFQ, Dabcyl.
Further, described step 4) take the gene synthesis mode to make positive plasmid CBG230-29 as positive in: to carry out gene synthetic with prolonging respectively 30～50bp before and after the high conserved region territory bp9999-bp10134 that filters out in the design of primers process, then be cloned in the pMD19-T carrier, be described positive plasmid sample, numbering CBG230-29.
Further, Real-Time PCR reaction is applicable to all quantitative fluorescent PCR reaction instrument described step 5).
Further, described fluorescent quantitation reaction kit comprises SmartCyclerII, ABI real-time PCR system, BioRad PCR in real time detection system, Stratagene quantitative polumerase chain reaction instrument.
Further, best amplification condition is described step 5):
A kind of Chikungunya virus detects the PCR system, it is characterized in that, this system comprise primer or other can with the primer that is equal to of chikungunya full length sequence specific binding and amplification, its similar homology can not surpass 90%, primer sequence is as follows:
A kind of Chikungunya virus detects the PCR system, it is characterized in that, this system comprise primer and can with chikungunya full length sequence specific probe, probe sequence is as follows:
Can with chikungunya full length sequence specific binding and amplification be equal to probe, the fraction of coverage of its site areas can not surpass 50%, its homology can not surpass 70%.
The a pair of new primer probe of design of the present invention is set up a kind of quick, responsive, special fluorescence quantifying PCR method in conjunction with SmartCyclerII and is detected Chikungunya virus.Pick out the conservative sequence of Chickungunya virus genome camber by the mode of sequence alignment, at this sequence design pair of primers and a Taqman probe, set up the real-time fluorescence quantitative PCR reaction system, and be optimized, detect sensitivity and the specificity of the method.
Description of drawings
Fig. 1 is the two dimension demonstration figure of amplification curve;
Fig. 2 primer concentration and probe concentration screening figure;
Fig. 3, Fig. 4 are that sensitivity detects schematic diagram;
Fig. 5 is that repeatability detects schematic diagram;
Fig. 6 is canonical plotting.
Embodiment
The principle of work of Chikungunya virus fluorescence quantitative PCR detection novel method of the present invention namely is to utilize the variation qualitative analysis of fluorescent signal, detect in real time the variation of each cyclic amplification product amount in the pcr amplification reaction, by the relation of Ct value and typical curve starting template is carried out quantitative analysis.The invention still further relates to all the elements that above-mentioned detection system comprises.
The method may further comprise the steps:
1) design primer probe;
2) select fluorescein according to instrument;
3) synthetic primer and probe;
4) preparation positive plasmid standard substance;
5) operation PCR;
6) data analysis;
7) extract viral RNA and carry out the system checking.
1, the design of primer probe
According to intending research gene title Chikungunya virus (Chikungunya virus), in genebank, find corresponding complete genome sequence (www.ncbi.nlm.nih.gov), use DNASTAR software to carry out homology analysis and blast sequential analysis, sift out high conservative zone bp88-bp209 as target sequence, adopt primer-design software Primer Premier 5 to carry out the design of primer probe, sequence sees Table 1.
Primer and the probe of table 1 fluorescence quantitative PCR detection Chikungunya virus
| Name | Sequence | Position | Tm℃ | Modification |
| FP | GAACACGTAACAGTGATCCCG | 9999-10019 | 57.8 | |
| RP | GTAATCAAGCGATAGCGTTGG | 10114-10134 | 58.0 | |
| Probe | TGGGAGTACCGTATAAGACTCTAGTCAACAGA | 10028-10059 | 67.0 | CY5/lowa Black |
2, the selection of fluorescein
Use instrument to be the SmartCyclerII of U.S. Cepheid company, the fluorescent emission group adopts the most stable CY5 group of characteristic, and the fluorescent quenching group adopts lowa Black.
3, the synthetic of primer and probe gives precious biotechnology (Dalian) company limited to finish.
4, the present invention takes the gene synthesis mode to make positive plasmid as positive.To carry out gene synthetic with prolonging respectively 30～50bp before and after the high conserved region territory bp88-bp209 that filters out in the design of primers process, then is cloned in the pMD19-T carrier, is our positive plasmid sample, numbering CBG230-29.
5, the preparation of plasmid standard
Synthetic by authorized company and extract positive plasmid sample stoste, recording concentration by the ultramicron ultraviolet spectrophotometer is 670ng/ μ l, calculates according to the copy number calculation formula " copy number concentration (copies/ μ l)=DNA concentration (ng/ μ l) * 6.02 * 1023 (copies/mol)/324 * 2 * (carrier base number+goal gene base number) " of single stranded DNA and learns that the DNA copy number concentration of plasmid stoste is about 2.1 * 10
11Copies/ μ l.Use EASY Dilution (a kind of liquid that is specifically designed to standard substance dilution use, precious biotech firm produces by Dalian) to carry out gradient dilution and finally obtain 1 * 10
0～1 * 10
10The plasmid standard of copies/ μ l.
6, primer and probe dissolved dilution are to desired concn and keep in Dark Place.
Primer generally is diluted to 10 μ m/L, and probe dilution is to 5 μ m/L, and-20 ℃ of refrigerators seal and keep in Dark Place.Dilution institute water is sterilization Tris water (Ph7.0～8.0).
7, Real-Time pcr amplification
The amplification kit of selecting is Premix Ex Taq test kit, available from precious biotechnology (Dalian) company limited.Reaction system component and volume thereof see Table 2.
Table 2
Amplification condition is as follows
8, data analysis
9, extract viral RNA, carry out reverse transcription, gained cDNA uses the aqua sterilisa gradient dilution, replaces positive and carries out detection validation.
The selection of threshold value
In the process of the various parameter optimizations of quantitative fluorescent PCR, the Ct value is the reference factor of most critical.The method of calculating the Ct value has in conjunction with two kinds of method of intersection and quafric curve Peak Intensity Method.We will react the threshold line that presents in the amplification curve diagram and drag up and down, para-curve in conjunction with the amplification rate velocity of variation is observed, when the corresponding Ct value of the intersection point of threshold line and amplification curve is passed through the peak value of quafric curve just, the threshold value of this moment is optimal threshold, and the Ct value also is Ct value the most accurately.With reference to figure 1, determine that the body series optimal threshold is 30.
The optimization of primer concentration and probe concentration
1. at first dilute the primer probe for subsequent use to lower concentration, primer is diluted to 10 μ m/L, and probe is to 5 μ m/L;
2. add 0.5 μ L, 1 μ L to each system respectively and diluted upstream and downstream primer (10 μ m/L), 0.5 μ L, 1 μ L Taqman probe (5 μ m/L) move PCR simultaneously, and other components of system are pressed table 2 preparation;
3. the preferred optimum response concentration combination of relative method, the result is such as Fig. 2. shown in: two suite lines are respectively 0.5 μ L primer probe, amplification curve diagram under the 1 μ L primer probe combinations, and the fluorescent signal that the former can detect is about about 100, and the Ct value is larger; Latter's fluorescent signal is also little near 400, Ct value.According to the little principle of the high Ct value of fluorescent value, we determine to add in the system 1 μ L upstream and downstream primer, and 1 μ L probe is best, i.e. primer probe final concentration 400 * 200nmol/L.
The selection of annealing temperature
Under the perfect condition, annealing temperature is enough low, effectively anneals with aim sequence to guarantee primer, wants simultaneously enough high, to reduce non-specific binding.Following carrying out when annealing temperature is set: be lower than that design estimates during primer Tm5 ℃ as initial annealing temperature, take 2 ℃ as increment, progressively improve annealing temperature.5 groups of temperature of initial setting: 52 ℃, 54 ℃, 56 ℃, 58 ℃, 60 ℃.All the purpose fragment be can detect under five groups of annealing temperatures, this primer and probe better performances proved, adaptable wider range.But the minimum temperature of the strongest while Ct of fluorescent signal value is 54 ℃, determines that finally optimum annealing temperature is 54 ℃.
The mensuration of sensitivity
Detect gradient template, Fig. 3 according to the reaction system of having optimized and response procedures. be 1e
3, 1e
2, 1e
1, 1e
0The detected result figure of four gradient templates, Fig. 4. be Fig. 3. middle 1e
0The dna profiling of copies/ μ l detects figure, obviously 1e
0Have to an amplification curve in six parallel tests of copies/ μ l, think that lowest detectable limit can only reach tens level, 1e
1The dilution DNA of copies/ μ l.
Repeatability detects
Fig. 5 is 1e
8～1e
4The dna profiling quantitative fluorescent PCR of five concentration gradients of copies/ μ l, each gradient have all been done 2 to 3 parallel laboratory tests, and the amplification curve that each gradient template produces among the figure has all produced the Ct value that very approaches, and repeatability is very good.
The specificity check
Detect the dengue virus that similar communication media and clinical manifestation are arranged to Chikungunya virus with the method for setting up, the result is negative.Show that this designed primer probe specificity is very high.
The foundation of typical curve
The quality of system can reflect by amplification efficiency e, and the e value more levels off to 1, amplification efficiency is described more near 100%, and reaction system is better; E＜1 illustrates that the system amplification efficiency is lower, and system needs to optimize; Inhibition is contained in the explanation system in e＞1, needs to reduce even get rid of the interference of inhibition.The amplification efficiency optimum range of generally acknowledging in this field is between 0.8-1.2.Adopt 1e
8～1e
4Copies/ μ l template increases, and the gained typical curve is Fig. 6, the amplification function
Y=-0.305X+13.561, amplification efficiency e=10-k-1=0.1018=101.8%.(k＝-0.305)
Purpose of the present invention is exactly a pair of new primer probe of design, sets up a kind of quick, responsive, special fluorescence quantifying PCR method in conjunction with SmartCyclerII and detects Chikungunya virus.Pick out the conservative sequence of Chickungunya virus genome camber by the mode of sequence alignment, at this sequence design pair of primers and a Taqman probe, set up the real-time fluorescence quantitative PCR reaction system, and be optimized, detect sensitivity and the specificity of the method.Detection method of the present invention is highly suitable for Chikungunya virus, with no cross reactions such as dengue fever viruss.
Claims (7)
1. the non-diagnostic assays method of Chikungunya virus quantitative fluorescent PCR is characterized in that, the primer probe that design is adopted in the method is:
2. the non-diagnostic assays method of Chikungunya virus quantitative fluorescent PCR as claimed in claim 1 is characterized in that, the method is specially:
1) design primer probe;
2) select fluorescein according to instrument;
3) synthetic primer and probe;
4) preparation positive plasmid standard substance;
5) operation PCR;
6) data analysis;
7) extract viral RNA and carry out the system checking.
3. the non-diagnostic assays method of Chikungunya virus quantitative fluorescent PCR as claimed in claim 2, it is characterized in that, described step 2) the selective fluorescent emission group of fluorescein has 6-FAM, TET, HEX, JOE, CY3, CY5, TAMRA, Texas Red in, and the fluorescent quenching group has BHQ-1, BHQ-2, Lowa Black
TMRQ, Lowa Black
TMFQ, Dabcyl.
4. the non-diagnostic assays method of Chikungunya virus quantitative fluorescent PCR as claimed in claim 2, it is characterized in that, described step 4) take the gene synthesis mode to make positive plasmid CBG230-29 as positive in: to carry out gene synthetic with prolonging respectively 30～50bp before and after the high conserved region territory bp9999-bp10134 that filters out in the design of primers process, then be cloned in the pMD19-T carrier, be described positive plasmid sample, numbering CBG230-29.
5. the non-diagnostic assays method of Chikungunya virus quantitative fluorescent PCR as claimed in claim 2 is characterized in that described step 5) in PCR reaction be applicable to all quantitative fluorescent PCRs reaction instrument.
6. the non-diagnostic assays method of the Chikungunya virus quantitative fluorescent PCR described in the claim 5, it is characterized in that, described fluorescent quantitation reaction instrument comprises SmartCyclerII, ABI real-time PCR system, BioRad PCR in real time detection system, Stratagene quantitative polumerase chain reaction instrument.
7. the non-diagnostic assays method of Chikungunya virus quantitative fluorescent PCR as claimed in claim 1 is characterized in that described step 5) in best amplification condition be:
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| CN102268489A (en) * | 2011-08-23 | 2011-12-07 | 中国检验检疫科学研究院 | Non-diagnostic method for detecting chikungunya virus nucleic acid through nanogold fluorescence quantitative PCR (Polymerase Chain Reaction) |
| CN102443051B (en) * | 2011-09-30 | 2017-07-18 | 中国人民解放军广州军区疾病预防控制中心 | A kind of recombinant antigen protein, kit and its application for detecting chikungunya virus |
| CN102776297B (en) * | 2012-08-09 | 2014-04-23 | 中山大学达安基因股份有限公司 | Reagent kit for distinguishing and detecting dengue virus/yellow fever virus/west nile virus/chikungunya virus |
| CN104513865B (en) * | 2014-11-25 | 2016-02-24 | 扬州大学 | Reverse transcription PCR detects test kit and the detection method thereof of Chikungunya virus |
| CN104745692A (en) * | 2015-03-10 | 2015-07-01 | 普生（天津）科技有限公司 | Chikungunya virus sequence |
| CN105256058B (en) * | 2015-11-20 | 2019-03-26 | 浙江省疾病预防控制中心 | A kind of constant-temperature amplification detection kit and detection method of chikungunya fever virus |
| CN111826463A (en) * | 2019-12-31 | 2020-10-27 | 深圳市人民医院 | Primer probe combination and kit for detecting five important arthropod/rodent-borne viruses and application of primer probe combination and kit |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101270394A (en) * | 2008-05-05 | 2008-09-24 | 广东出入境检验检疫局检验检疫技术中心 | Chikungunya virus testing method |
Non-Patent Citations (4)
| Title |
|---|
| Carolyn J. Edwards et al..Molecular diagnosis and analysis of Chikungunya virus.《Journal of Clinical Virology》.2007,第39卷(第4期),271-275. |
| Molecular diagnosis and analysis of Chikungunya virus;Carolyn J. Edwards et al.;《Journal of Clinical Virology》;20070831;第39卷(第4期);271-275 * |
| 黄吉城等.基孔肯雅病毒的实时荧光RT - PCR检测方法研究.《中国卫生检验杂志》.2008,第18卷(第9期),1721-1723. |
| 黄吉城等.基孔肯雅病毒的实时荧光RT- PCR检测方法研究.《中国卫生检验杂志》.2008,第18卷(第9期),1721-1723. * |
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