CN102191338A - Fluorescence quantitative polymerase chain reaction (PCR) new method for detecting multiple viruses of yellow fever, dengue fever and epidemicencephalitis B and multiple virus detection PCR system - Google Patents
Fluorescence quantitative polymerase chain reaction (PCR) new method for detecting multiple viruses of yellow fever, dengue fever and epidemicencephalitis B and multiple virus detection PCR system Download PDFInfo
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Abstract
The invention discloses a fluorescence quantitative polymerase chain reaction (PCR) new method for detecting multiple viruses of yellow fever, dengue fever and epidemicencephalitis B and multiple virus detection PCR system consisting of primers, probes, a Premix Ex Taq reaction solution and a sterilized Tris buffer. With good singularity and high sensitivity, three pairs of primers and probes are very suitable for simultaneously detecting viruses of yellow fever, dengue fever and epidemicencephalitis B. And there is no cross reaction between the primers and probes and several other entomophily hemorrhagic fever viruses, such as Marburg virus and Rift Valley fever virus.
Description
Technical field
The invention belongs to field of biological detection, be specifically related to Huang is warm, step on leather, encephalitis b virus fast qualitative and quantitative detecting method, a pair of Auele Specific Primer and probe respectively are provided.
Background technology
Yellow jack (be commonly called as " yellow Jack ", " black vomitting ", claim America pestilence sometimes again) is a kind of rapid venereal disease viral disease.Once caused some destructive epidemics.To people or person-to-person infection, mosquito is main mediation person to this disease from monkey.The annual whole world has 200,000 yellow jack cases at least, causes 30,000 people's death, and wherein 90% case derives from Africa.Because the high mortality and the strong infectivity of yellow jack have been included into one of quarantinable disease of World Health Organization's regulation.
Epidemic encephalitis type B (epidemicencephalitisB) is by the central nervous system sexually transmitted disease due to the encephalitis b virus of having a liking for nerve, is called for short encephalitis.Propagate through hematophagous buges such as mosquitos, be popular in summer and autumn, pilosity is born in children, is feature with high heat, the disturbance of consciousness, convulsions, respiratory insufficiency and meningeal irritation sign clinically.Part patient leaves serious sequela, and patient with severe symptoms's case fatality rate is higher.Whole world reported cases are counted every year more than 16000, dead 5000 examples.
Dengue virus (Dengue virus) infect cause singapore hemorrhagic fever (Denguefever, DF) or dengue hemorrhagic fever (Dengue haemorrhagic fever, DHF).This disease is popular in the torrid zone, subtropical zone, particularly South East Asia, West Pacific Ocean and Central and South America.China found this disease on Hainan Island and Guangxi and other places discovery is arranged all later in 1978 first in the Foshan.Estimate that according to WHO annual DF number of the infected has 5,000 ten thousand, wherein DHF is about 500,000, and dead more than 20,000 people, compromised population reaches 2,500,000,000.
Three kinds of viruses all belong to flavivirus, the method that detects flavivirus at present has the reverse transcriptase polymerase reaction, SYBR Green Real-time PCR, micropore hybridization, protein chip technology etc., also do not have report to use TaqMan Real-time PCR method to carry out multiple flavivirus and detect simultaneously, and this method undoubtedly more quick and precisely.
Summary of the invention
The object of the present invention is to provide a kind of yellow heat, step on leather, the multiple FLuorescent quantitative PCR of encephalitis B detects novel method and should multiple viral detection PCR system, this method can fast qualitative and detection by quantitative yellow fever virus, dengue virus, encephalitis b virus, comprises that the three pairs of specificitys are higher, highly sensitive, the primer and the probe of good reproducibility.
For achieving the above object, the yellow heat of the present invention, step on leather, the multiple FLuorescent quantitative PCR detection of encephalitis B novel method, in this method:
1) first pair of primer probe of design detection yellow fever virus is:
| Name | Sequence | Position | Tm℃ | Modification |
| FP | ATGTCTGGTCGTAAAGCTCAGG | 119-140 | 58.7 | ? |
| RP | AGGTCCAGGTCTGTTTCCAAT | 218-238 | 57.6 | ? |
| Probe | TCAATATGGTACGACGAGGAGTTCGC | 156-181 | 67.7 | FAM/BHQ-1 |
2) second pair of primer probe of design detection dengue virus is:
| Name | Sequence | Position | Tm℃ | Modification |
| FP | GCTGGAAGGACTAGAGGTTAGAG | 10627-10649 | 56.6 | ? |
| RP | GATTCAACAGCACCATTCCAT | 10748-10768 | 57.2 | ? |
| Probe | AAACAGCATATTGACGCTGGGAAAGA | 10670-10695 | 67.4 | Texas?Red/BHQ-2 |
3) the 3rd pair of primer probe of design detection encephalitis b virus is:
| Name | Sequence | Strand | Position | Tm℃ | Modification |
| FP | GTTTATCTGTGTGAACTTCTTGGC | forward | 5-28 | 57.9 | ? |
| RP | TTTAGTCATGGTTATCTTCCGTTC | reverse | 81-104 | 57.7 | ? |
| Rrobe | AGTATCGTTGAGAAGAATCGAGAGATTAGTGC | reverse | 31-62 | 67.8 | CY3/BHQ-2 |
Further, described yellow heat, step on leather, the multiple FLuorescent quantitative PCR detection of encephalitis B novel method, be 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, take the gene synthesis mode to make positive plasmid CBG228-3, CBG231-3, CBG229-1 as positive in the described step 4): with the high conserved region territory bp119-bp238 that filters out in the design of primers process, bp10627-bp10768, prolonging 5～50bp before and after the bp5-bp104 respectively, to carry out gene synthetic, be cloned into then in the pMD19-T carrier, be described positive plasmid sample, numbering CBG228-3, CBG231-3, CBG229-1.
Further, the quantitative fluorescent PCR reaction is applicable to all quantitative fluorescent PCR reaction instrument in the described step 5);
Further, described quantitative fluorescent PCR reaction instrument 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 in the described step 5):
A kind of yellow heat, step on leather, the multiple virus of encephalitis B detects the PCR system, this system comprise that primer or other can combine with the hot full length sequence specificity of Huang and increase be equal to primer, its similar homology can not be above 90%, primer sequence is as follows:
A kind of yellow heat, step on leather, the multiple virus detection of encephalitis B PCR system, this system comprise primer and can with the hot full length sequence specific probe of Huang, probe sequence is as follows:
Can combine the probe that is equal to of also amplification with the hot full length sequence specificity of Huang, the fraction of coverage of its site areas can not surpass 50%, and its homology can not surpass 70%.
A kind of yellow heat, step on leather, the multiple virus of encephalitis B detects the PCR system, this system comprise primer or other can with step on leather full length sequence specificity combine and increase be equal to primer, its similar homology can not be above 90%, primer sequence is as follows:
A kind of yellow heat, step on leather, the multiple virus of encephalitis B detects the PCR system, this system comprises primer and can remove from office the full length sequence specific probe with stepping on that probe sequence is as follows:
Can combine the probe that is equal to that also increases with stepping on leather full length sequence specificity, the fraction of coverage of its site areas can not surpass 50%, and its homology can not surpass 70%.
A kind of yellow heat, step on leather, the multiple virus of encephalitis B detects the PCR system, this system comprise that primer or other can combine with encephalitis B full length sequence specificity and increase be equal to primer, its similar homology can not be above 90%, primer sequence is as follows:
A kind of yellow heat, step on leather, the multiple virus detection of encephalitis B PCR system, this system comprise primer and can with encephalitis B full length sequence specific probe, probe sequence is as follows:
Can combine the probe that is equal to of also amplification with encephalitis B full length sequence specificity, the fraction of coverage of its site areas can not surpass 50%, and its homology can not surpass 70%.
The present invention designs three pairs of new primer probes, in conjunction with utilization SmartCyclerII set up a kind of fast, responsive, special fluorescence quantifying PCR method detects yellow heat simultaneously, steps on leather, encephalitis b virus.Pick out the conservative sequence of three kinds of viral genome camber by the mode of sequence alignment, on this sequence, design a pair of primer and a Taqman probe respectively, set up the real-time fluorescence quantitative PCR reaction system, and be optimized, detect the sensitivity and the specificity of this method.
Description of drawings
Fig. 1 is the amplification curve diagram of multiple detection;
Fig. 2 is the canonical plotting of yellow fever virus in the multiple detection;
Fig. 3 is the canonical plotting of encephalitis b virus in the multiple detection;
Fig. 4 is the typical curve of dengue virus in the multiple detection.
Embodiment
The yellow principle of work hot, that step on leather, the multiple FLuorescent quantitative PCR detection of encephalitis B novel method of the present invention promptly is to utilize the variation qualitative analysis of fluorescent signal, detect the variation of each cyclic amplification product amount in the pcr amplification reaction in real time, starting template is carried out quantitative analysis by the relation of Ct value and typical curve.The invention still further relates to all the elements that above-mentioned detection architecture comprises.
This 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 the yellow heat of research gene title, step on leather, encephalitis B, in genebank, find corresponding complete genome sequence (www.ncbi.nlm.nih.gov), utilization DNASTAR software carries out homology analysis and blast sequential analysis, sift out the highly conserved sequence 5 ' UTR119-238 (length overall 120bp) of yellow jack cause of disease, the high conserved sequence bp10627-bp10768 (length overall 141bp) of dengue fever virus, the high conserved sequence bp5-bp104 (length overall 100bp) of encephalitis b virus is as the testing goal gene, adopt primer-design software Primer Premier 5 to carry out the design of primer probe, sequence sees Table 1. table 2. tables 3.
The primer and the probe of table 1 fluorescence quantitative PCR detection yellow fever virus
| Name | Sequence | Position | Tm℃ | Modification |
| FP | ATGTCTGGTCGTAAAGCTCAGG | 119-140 | 58.7 | ? |
| RP | AGGTCCAGGTCTGTTTCCAAT | 218-238 | 57.6 | ? |
| Probe | TCAATATGGTACGACGAGGAGTTCGC | 156-181 | 67.7 | FAM/BHQ-1 |
Annotate: primer and the probe nucleic acid site on the yellow fever virus genome is based on the strain sequence that the GenBank sequence number is NC_002031.
The primer and the probe of table 2 fluorescence quantitative PCR detection dengue virus
| Name | Sequence | Position | Tm℃ | Modification |
| FP | GCTGGAAGGACTAGAGGTTAGAG | 10627-10649 | 56.6 | ? |
| RP | GATTCAACAGCACCATTCCAT | 10748-10768 | 57.2 | ? |
| Probe | AAACAGCATATTGACGCTGGGAAAGA | 10670-10695 | 67.4 | Texas?Red/BHQ-2 |
Annotate: primer and the probe nucleic acid site on the dengue virus genome is based on the strain sequence that the GenBank sequence number is NC_001474.
The primer and the probe of table 3 fluorescence quantitative PCR detection encephalitis b virus
| Name | Sequence | Position | Tm℃ | Modification |
| FP | GTTTATCTGTGTGAACTTCTTGGC | 5-28 | 57.9 | ? |
| RP | TTTAGTCATGGTTATCTTCCGTTC | 81-104 | 57.7 | ? |
| Rrobe | AGTATCGTTGAGAAGAATCGAGAGATTAGTGC | 31-62 | 67.8 | CY3/BHQ-2 |
Annotate: primer and the probe nucleic acid site on the encephalitis b virus genome is based on the strain sequence that the GenBank sequence number is NC_001437.
2, the selection of fluorescein
Use the SmartCyclerII of instrument as U.S. Cepheid company, yellow fever virus fluorescent emission group adopts the most stable FAM group of characteristic, and the fluorescent quenching group adopts BHQ-1; Dengue virus fluorescent emission group adopts Texas Red, and the fluorescent quenching group adopts BHQ-2; Encephalitis b virus adopts CY3/BHQ-2, and the three uses different fluorescent signals to show difference.
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 30～50bp before and after the high conserved region territory that filters out in the design of primers process respectively, is cloned into then in the pMD19-T carrier, is our positive plasmid sample, numbering CBG228-3, CBG231-3, CBG229-1.
5, the preparation of plasmid standard
Synthetic by authorized company and extract positive plasmid sample stoste, record concentration by the ultramicron ultraviolet spectrophotometer and be respectively yellow hot 310ng/ μ l, step on leather 299ng/ μ l, encephalitis 301ng/ μ l, calculate 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 learn that the DNA copy number concentration of amaril grain stoste is about 1 * 10
11Copies/ μ l; Step on leather and be about 9.6 * 10
10Copies/ μ l, encephalitis is about 1 * 10 equally
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 respectively 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 for use is a Premix Ex Taq test kit, available from precious biotechnology (Dalian) company limited.Reaction system component and volume thereof see Table 4.
(M represents Marburg to table 4.; E represents the Ebola)
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 optimization of primer concentration and probe concentration
1. it is standby to lower concentration at first to dilute each primer probe, and primer is diluted to 10 μ m/L, and probe is to 5 μ m/L;
2. the upstream and downstream primer (10 μ m/L) and the 0.5 μ L Taqman probe (5 μ m/L) that at first in 25 μ L systems, add three kinds of viruses of 0.5 μ L respectively, template respectively adds 1 μ L, Premix Ex Taq reaction solution still adds 12.5 μ L, and residual volume is supplied with aqua sterilisa.Operation PCR.The result shows that CY3 and Texas Red fluorescent value are all very little, be starkly lower than FAM fluorescence, show under three kinds of simultaneous situations of fluorophor, CY3 and Texas Red luminous efficiency are quite low, for not influencing positive judgement, the pre-add-on that improves CY3 and Texas Red probe primer is convenient to data read to improve its luminous efficiency.
3. encephalitis, the primer probe add-on of stepping on leather are doubled, i.e. 1 μ L, the primer probe amount of yellow heat is constant simultaneously, other as above, operation PCR.Effect increases, but still has very big fluorescent value difference.
4. reduce the add-on of yellow fever virus primer probe, reduce to 0.2 μ L, encephalitis, dengue virus all continue to add 1 μ L, move PCR once more.The luminous value of three kinds of fluorescence still can not be balanced fully as a result.Consider that for this reason fluorescence difference is inevitable, too much primer and probe also can influence the reaction efficiency of system, so need not too to be entangled with the excessive fluorescent signal of difference, only need and can stable detection arrive goal gene, obtain stable Ct value and get final product.Therefore, determine that yellow fever virus primer concentration and probe concentration is 80 * 40nm/L in the 25 μ L multiple fluorescence quantitative PCR reaction systems, encephalitis primer concentration and probe concentration is 400 * 200nm/L then, steps on the same encephalitis of leather primer concentration and probe concentration.
The selection of annealing temperature
Under the perfect condition, annealing temperature is enough low, effectively anneals with aim sequence to guarantee primer, wants enough high simultaneously, 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, with 2 ℃ be 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 56 ℃, determines that finally optimum annealing temperature is 56 ℃.
Repeatability detects
By the dna profiling quantitative fluorescent PCR of each concentration gradient of Fig. 2 .3.4., all produced comparatively approaching Ct value in 2 to 3 parallel laboratory tests of each gradient, reaction relation conefficient r-squared can both reach about in the of 0.99, and repeatability is better.
The specificity check
Detect with yellow heat, step on viruses such as Marburg that leather, encephalitis b virus be all entomophila hemorrhagic fever cause of disease, Rift Valley fever with the method set up, the result is all negative.
Show that this designed primer probe specificity is fine.
The foundation of typical curve
The quality of system can reflect that the e value levels off to 1 more by amplification efficiency e, amplification efficiency is described more near 100%, and reaction system is good more; 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 respectively separately that gradient template increases, the gained typical curve is Fig. 2. Fig. 3. and Fig. 4, amplification function
Yellow heat: Y=-0.295X+11.636, e=10
-k-1=0.97=97%.(k＝-0.295)
Encephalitis: Y=-0.296X+12.667, e=10
-k-1=0.98=98%.(k＝-0.296)
Step on leather: Y=-0.283X+11.958, e=10
-k-1=0.92=92%.(k＝-0.283)
Purpose of the present invention is exactly three pairs of new primer probes of design, in conjunction with utilization SmartCyclerII set up a kind of fast, responsive, special fluorescence quantifying PCR method detect simultaneously yellow hot, step on leather, encephalitis b virus.Pick out the conservative sequence of three kinds of viral genome camber by the mode of sequence alignment, on this sequence, design a pair of primer and a Taqman probe respectively, set up the real-time fluorescence quantitative PCR reaction system, and be optimized, detect the sensitivity and the specificity of this method.Detection method of the present invention is highly suitable for yellow heat, steps on leather, encephalitis b virus detects simultaneously, with other several entomophila hemorrhagic fever viruss such as Marburg, Rift Valley fever no cross reaction.
Claims (10)
1. yellow heat, step on leather, the multiple FLuorescent quantitative PCR detection of encephalitis B novel method, it is characterized in that, in this method:
1) first pair of primer probe of design detection yellow fever virus is:
2) second pair of primer probe of design detection dengue virus is:
3) the 3rd pair of primer probe of design detection encephalitis b virus is:
2. yellow heat as claimed in claim 1, step on leather, the multiple FLuorescent quantitative PCR detection of encephalitis B novel method, it is characterized in that this 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. yellow heat as claimed in claim 2, step on leather, the multiple FLuorescent quantitative PCR detection of encephalitis B novel method, 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. yellow heat as claimed in claim 2, step on leather, the multiple FLuorescent quantitative PCR of encephalitis B detects novel method, it is characterized in that, take the gene synthesis mode to make positive plasmid CBG228-3 in the described step 4), CBG231-3, CBG229-1 is as positive: with the high conserved region territory bp119-bp238 that filters out in the design of primers process, bp10627-bp10768, prolonging 5～50bp before and after the bp5-bp104 respectively, to carry out gene synthetic, be cloned into then in the pMD19-T carrier, be described positive plasmid sample, numbering CBG228-3, CBG231-3, CBG229-1.
A yellow heat, step on leather, the multiple virus detection of encephalitis B PCR system, it is characterized in that, this system comprises that primer or other can combine the primer that is equal to of also amplification with the hot full length sequence specificity of Huang, and its similar homology can not surpass 90%, and primer sequence is as follows:
A yellow heat, step on leather, the multiple virus detection of encephalitis B PCR system, it is characterized in that, this system comprise primer and can with the hot full length sequence specific probe of Huang, probe sequence is as follows:
Can combine the probe that is equal to of also amplification with the hot full length sequence specificity of Huang, the fraction of coverage of its site areas can not surpass 50%, and its homology can not surpass 70%.
A yellow heat, step on leather, the multiple virus detection of encephalitis B PCR system, it is characterized in that, this system comprises that primer or other can combine the primer that is equal to that also increases with stepping on leather full length sequence specificity, and its similar homology can not surpass 90%, and primer sequence is as follows:
A yellow heat, step on leather, the multiple virus of encephalitis B detects the PCR system, it is characterized in that, this system comprises primer and can remove from office the full length sequence specific probe with stepping on that probe sequence is as follows:
Can combine the probe that is equal to that also increases with stepping on leather full length sequence specificity, the fraction of coverage of its site areas can not surpass 50%, and its homology can not surpass 70%.
A yellow heat, step on leather, the multiple virus detection of encephalitis B PCR system, it is characterized in that, this system comprises that primer or other can combine the primer that is equal to of also amplification with encephalitis B full length sequence specificity, and its similar homology can not surpass 90%, and primer sequence is as follows:
A yellow heat, step on leather, the multiple virus detection of encephalitis B PCR system, it is characterized in that, this system comprise primer and can with encephalitis B full length sequence specific probe, probe sequence is as follows:
Can combine the probe that is equal to of also amplification with encephalitis B full length sequence specificity, the fraction of coverage of its site areas can not surpass 50%, and its homology can not surpass 70%.
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-
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- 2010-12-24 CN CN2010106054986A patent/CN102191338B/en not_active Expired - Fee Related
Non-Patent Citations (4)
| Title |
|---|
| DAY-YU CHAO ET AL.: "Development of Multiplex Real-Time Reverse Transcriptase PCR Assays for Detecting Eight Medically Important Flaviviruses in Mosquitoes", 《JOURNAL OF CLINICAL MICROBIOLOGY》 * |
| J. DYER ET AL.: "A multiplexed TaqMan assay for the detection of arthropod-borne flaviviruses", 《JOURNAL OF VIROLOGICAL METHODS》 * |
| 师永霞等: "实时荧光RT-PCR技术在黄热病快速检测中的应用", 《现代预防医学》 * |
| 王军军等: "4株蚊媒病毒多重RT-PCR快速检测方法的建立", 《热带医学杂志》 * |
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