CN105803109A - Method for rapid detection of yellow fever virus by using isothermal nucleic acid amplification technology - Google Patents
Method for rapid detection of yellow fever virus by using isothermal nucleic acid amplification technology Download PDFInfo
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- CN105803109A CN105803109A CN201410796592.2A CN201410796592A CN105803109A CN 105803109 A CN105803109 A CN 105803109A CN 201410796592 A CN201410796592 A CN 201410796592A CN 105803109 A CN105803109 A CN 105803109A
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Abstract
The invention discloses a method for rapid detection of yellow fever virus by using isothermal nucleic acid amplification technology. The detection method comprises a recombinase mediation of an isothermal nucleic acid amplification system, a reaction buffer and detection of universal primers by yellow fever virus. The method is carried out under isothermal conditions (35-45 DEG C) within 10-20 minutes, so as to achieve amplification on yellow fever virus RNA. The detection method provided by the present invention has the advantages of high sensitivity, good specificity, simple and rapid operation and low quality requirements on the detection material, facilitates clinical applications, and is applicable to rapid detection of yellow fever virus.
Description
Technical field
The invention belongs to molecular Biological Detection field, be specifically related to a kind of method utilizing isothermal amplification quickly to detect yellow fever virus.
Background technology
Yellow fever (YellowFever, YF) it is a kind of by the acute viral hemorrhagic disease of infected killing propagation, mosquito is primary vehicle, occurs mainly in Africa and South America, and the same plague, cholera are listed as the infectious disease of three big international quarantines, monitoring.Estimate according to World Health Organization (WHO) (WHO), the annual people infecting yellow fever has 200,000 examples, 30,000 people are wherein had to die from yellow fever, in past 20 years, due to the impact of the factors such as immunity degradation, disafforestation, urbanization, movement of population and climate change that disease is infected by crowd, yellow fever cases number is increase trend.Yellow fever is not had specific treatment method.Treat according only to symptom, it is therefore an objective to mitigation symptoms, alleviate the sense of discomfort of patient.Although China there is no the report that yellow fever is popular or makes a definite diagnosis at present, but increasing along with China's reform and opening-up and foreign exchanges, yellow fever probably comes in and goes out China with Migrant women or medium, and therefore, the quarantine of yellow fever is particularly important.
Yellow fever virus (Yellowfevervirus, YF) it is the pathogen causing yellow fever, belong to flaviviridae (Flaviridae) Flavivirus (Flavivirus), it is a kind of RNA viruses, at present the detection method of yellow fever virus is broadly divided into the methods such as virus purification cultivation, serodiagnosis and molecular biology.
In general, virus purification is cultivated the most reliable, but requires that the bio-safety grade of laboratory is high, it is necessary to carrying out at BSL-3 laboratory, and the time of Virus Isolation is longer, wastes time and energy, detection sensitivity is not high yet.
Serodiagnosis mainly includes neutralization test, hemagglutination inhibition test and complement fixation test etc., and that currently mainly applies has elisa (ELISA), indirect immunofluorescence analysis (IFA) etc..But owing to there is serological cross reaction between banzi virus, affecting the correctness of diagnostic result, specificity is not strong.
Molecular biology is compared with virus purification cultivation, serodiagnosis, there is the advantages such as high specificity, highly sensitive and detection time is short, become one of modal method of Pathogen test at present, particularly PCR (polymerasechainreaction) technology, can carry out rapid amplifying to trace sample.Yellow fever virus is RNA viruses, when carrying out pcr amplification, need to be that template produces cDNA through reverse transcription by RNA, genes of interest is obtained for template carries out expanding again with cDNA, molecular biology method mainly includes RT-PCR and real-time fluorescence RT-PCR, but both approaches must complete in laboratory, the requirement of instrument is higher and need technical professional to have operated, and unwind owing to PCR reaction is divided into, anneal and extend three phases and only have the extension stage to carry out DNA cloning, its amplification efficiency and speed are greatly affected, consuming time longer, these factors strongly limit its application in grass-roots unit and Site Detection.
Due to the demand that deficiency and the yellow fever virus of existing malaria yellow fever virus detection method quickly detect on the spot, therefore research one can be applied in basic unit, can be highly desirable to by the plasmodial amplification method of simple and quick detection.
Except carrying out DNA detection, after reaction system adds reverse transcriptase, detection can also be proceeded by from template ribonucleic acid by isothermal nucleic acid amplification (RT-RAA) method of reverse transcription-recombinase-mediated, a reverse transcription and isothermal duplication step at the same temperature completes, and greatly enhances the sensitivity of detection and saves the response time.And break away from the constraint of instrument, it is possible to carry out in a non-laboratory environment, it is possible to reach the purpose of Site Detection；Additionally, it is not the method is easy to operate, personnel requirement is high.Therefore the method has highly application value, and is not only suitable for research work, it is also possible to carry out conventional and Emergent detection instrument as the grass-roots unit related personnel that appointed condition is relatively poor.
Summary of the invention
It is an object of the invention to provide a kind of method utilizing isothermal amplification quickly to detect yellow fever virus, having main steps that of this detection method first carries out yellow fever virus RNA extraction, yellow fever virus RNA is joined in the isothermal nucleic acid amplification system containing special primer and probe and expand, under isothermy (35-45 DEG C), within 10-20 minute, amplified production can be detected.
The concrete technical scheme of the present invention is as follows:
1. obtain yellow fever virus RNA, the primer of design yellow fever virus and probe, set up yellow fever virus isothermal nucleic acid amplification system, yellow fever virus RNA is joined in amplification system, add reaction buffer, under isothermy (35-45 DEG C), within 10-20 minute, namely can detect that amplified production.
2., according to the method described in above-mentioned 1, set up yellow fever virus isothermal nucleic acid amplification system, as follows:
3., according to the isothermal nucleic acid amplification system described in above-mentioned 1,2, its state is Powdered after lyophilization.
4., according to utilizing, described in above-mentioned 1, the method that isothermal amplification quickly detects yellow fever virus, wherein said reaction buffer is final concentration of 6% (w/v), and molecular weight is the Aqueous Solutions of Polyethylene Glycol of 35000.
5., according to utilizing, described in above-mentioned 1, the method that isothermal amplification quickly detects yellow fever virus, reverse transcriptase wherein used is M-MLV reverse transcriptase.
6., according to the primer sets described in above-mentioned 1,2, its primer sequence is:
Forward primer sequence: 5 '-AAATCCTGKGTGCTAATTGAGGTGYATTGG-3 ',
Reverse primer sequences: 5 '-ACATDWTCTGGTCARTTCTCTGCTAATCGC-3 '.
7., according to the isothermal nucleic acid amplification system described in above-mentioned 1,2, its probe sequence is:
5’-GCAAATCGAGTTGCTAGGCAATAAACACATTTGGATTAATTTTRATCGTTC-3。
8. according to the fluorescence isothermal nucleic acid amplification method described in above-mentioned 1,2, it is characterised in that the fluorescent probe used is the probe that fluorescence/quencher is modified, and the fluorophor used is FAM fluorophor.
9. according to utilizing, described in above-mentioned 1, the method that isothermal amplification quickly detects yellow fever virus, its=the Optimum Isothermal condition of amplification is 39 DEG C.
10. according to utilizing, described in above-mentioned 1, the method that isothermal amplification quickly detects yellow fever virus, it is characterised in that the Best Times of amplification is 15 minutes.
11. utilize, according to above-mentioned 1-10, the method that isothermal amplification quickly detects yellow fever virus, wherein yellow fever virus RNA is joined in isothermal nucleic acid amplification system, system volume can be any one volume in 25 μ l, 50 μ l, 100 μ l or other volume, optimal volume is 50 μ l, reaction system adds yellow fever virus RNA, putting in the instrument that can detect FAM fluorescence, 39 DEG C are reacted 15 minutes, the amplification situation of yellow fever virus RNA namely be can be observed.
Method provided by the present invention, is more suitably applied to the detection of a large amount of sample, operates more simple, requires lower to the technology of operator.The detection time is short, and can detect large batch of sample simultaneously, has good specificity, sensitivity and stability, it is easy to popularization and application on a large scale, has wide market prospect and bigger economical, societal benefits.
Accompanying drawing explanation
Fig. 1 is that the isothermal amplification that utilizes using the present invention quickly detects the real-time fluorescence result figure of yellow fever virus.
Detailed description of the invention
It is described in further detail the present invention by embodiment below, but present disclosure is not limited thereto.
Embodiment one:
1, sample source and yellow fever virus RNA extract
The yellow fever live vaccine (Beijing Tiantan Bio-pharmaceuticals) that virus sample system is provided by Zhejiang International Travel Healthcare Center, RNA extracts and adopts maturation to extract test kit AM1836 (ambion, lifetechnologies), extraction equipment is KINGFISHER Full automatic instrument for extracting nucleic acid (Thermo).
2, primer and design
Design following yellow fever virus primer and probe, and student on commission work biological engineering (Shanghai) limited company synthesize:
Forward primer sequence: 5 '-AAATCCTGKGTGCTAATTGAGGTGYATTGG-3 ',
Reverse primer sequences: 5 '-ACATDWTCTGGTCARTTCTCTGCTAATCGC-3 '.Probe sequence:
5’-GCAAATCGAGTTGCTAGGCAATAAACACATTTGGATTAATTTTRATCGTTC-3。
3, preparation amplification system
200 μ L centrifuge tubes carry out isothermal nucleic acid amplification system preparation (volume is 50 μ L) by following proportioning:
The amplification system prepared above negative pressure lyophilization in freezer dryer is become powdered amplification system.
4, yellow fever virus RNA detection
In centrifuge tube, add final concentration of 6% (w/v's), molecular weight is that system is dissolved as reaction buffer and become 49 μ l by the Polyethylene Glycol of 35000 again, add the 1 μ L yellow fever virus RNA prepared, mix homogeneously, brief centrifugation, put in the instrument that can detect FAM fluorescence, react 15 minutes at 39 DEG C.(note: for guaranteeing experiment accuracy, the system being not added with template is set as negative control).Result shows, starts the fluorescence signal of display amplification after 6 minutes.As shown in Figure 1.Repeat above example, identical amplification fluorescent signal can be obtained, reproducible.
Above example illustrates that the isothermal amplification that utilizes using the present invention can quickly detect yellow fever virus, and easy and simple to handle, the time used reduces significantly, it is not necessary to large-scale instrument and equipment, it is adaptable to screen on a large scale.Detection method sensitivity provided by the present invention is high, specificity is good, easy and simple to handle, rapid and test material prescription is low, it is simple to application clinically, and the method is applicable to the quick detection to yellow fever virus.
The foregoing is only the preferably enforcement example of the present invention, not in order to limit the present invention, all within the spirit and principles in the present invention, any amendment of making, equivalent replacement, improvement etc., should be included within protection scope of the present invention.
Claims (11)
1. one kind utilizes the method that isothermal amplification quickly detects yellow fever virus, this detection method includes isothermal nucleic acid amplification system, primer sets, probe and reaction buffer, amplification carries out at isothermy 35-45 DEG C, namely can detect that yellow fever virus nucleic acid in 10-20 minute.
2. the method utilizing isothermal amplification quickly to detect yellow fever virus according to claim 1, it is characterised in that described isothermal nucleic acid amplification system and each constituent content are shown below:
3. the isothermal nucleic acid amplification system according to claim 1,2, it is characterised in that described system status is Powdered after lyophilization.
4. the method utilizing isothermal amplification quickly to detect yellow fever virus according to claim 1, it is characterised in that described reaction buffer is final concentration of 6% (w/v), and molecular weight is the Aqueous Solutions of Polyethylene Glycol of 35000.
5. the method utilizing isothermal amplification quickly to detect yellow fever virus according to claim 1, it is characterised in that reverse transcriptase used is M-MLV reverse transcriptase.
6. the primer sets according to claim 1,2, it is characterised in that described primer sequence is:
Forward primer sequence: 5 '-AAATCCTGKGTGCTAATTGAGGTGYATTGG-3 ',
Reverse primer sequences: 5 '-ACATDWTCTGGTCARTTCTCTGCTAATCGC-3 '.
7. the isothermal nucleic acid amplification system according to claim 1,2, it is characterised in that described probe sequence is:
5’GCAAATCGAGTTGCTAGGCAATAAACACATTTGGATTAATTTTRATCGTTC-3。
8. fluorescence isothermal nucleic acid amplification method according to claim 1, it is characterised in that the fluorescent probe used is the probe that fluorescence/quencher is modified, and the fluorophor used is FAM fluorophor.
9. the method utilizing isothermal amplification quickly to detect yellow fever virus according to claim 1, it is characterised in that the Optimum Isothermal condition of amplification is 39 DEG C.
10. the method utilizing isothermal amplification quickly to detect yellow fever virus according to claim 1, it is characterised in that the Best Times of amplification is 15 minutes.
11. utilize, according to claim 1-10, the method that isothermal amplification quickly detects yellow fever virus, it is characterized in that, system is dissolved by available reaction buffer again, system volume can be any one volume in 25 μ l, 50 μ l, 100 μ l or other volume, optimal volume is 50 μ l, and reaction system adds yellow fever virus RNA, puts in the instrument that can detect FAM fluorescence, 39 DEG C are reacted 15 minutes, the amplification situation of yellow fever virus RNA namely be can be observed.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106544449A (en) * | 2017-01-12 | 2017-03-29 | 中国人民解放军军事医学科学院微生物流行病研究所 | A kind of yellow fever virus detection kit and its application based on RPA technologies |
| CN106701940A (en) * | 2016-12-23 | 2017-05-24 | 江苏省血吸虫病防治研究所 | Method for detecting DNA of schistosoma through fluorescent probe |
-
2014
- 2014-12-16 CN CN201410796592.2A patent/CN105803109A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| CAMILLE ESCADAFAL等: "Rapid Molecular Assays for the Detection of Yellow Fever Virus in Low-Resource Settings", 《PLOS NEGLECTED TROPICAL DISEASES》 * |
| 吕蓓等: "用重组酶介导扩增技术快速扩增核酸", 《中国科学：生命科学》 * |
| 郑伟等: "重组酶介导扩增方法快速检测黄热病毒", 《中国卫生检验杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106701940A (en) * | 2016-12-23 | 2017-05-24 | 江苏省血吸虫病防治研究所 | Method for detecting DNA of schistosoma through fluorescent probe |
| CN106544449A (en) * | 2017-01-12 | 2017-03-29 | 中国人民解放军军事医学科学院微生物流行病研究所 | A kind of yellow fever virus detection kit and its application based on RPA technologies |
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Application publication date: 20160727 |