CN102191338B - 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
- Publication number
- CN102191338B CN102191338B CN2010106054986A CN201010605498A CN102191338B CN 102191338 B CN102191338 B CN 102191338B CN 2010106054986 A CN2010106054986 A CN 2010106054986A CN 201010605498 A CN201010605498 A CN 201010605498A CN 102191338 B CN102191338 B CN 102191338B
- Authority
- CN
- China
- Prior art keywords
- pcr
- encephalitis
- probe
- leather
- primer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 241000700605 Viruses Species 0.000 title claims abstract description 33
- 238000000034 method Methods 0.000 title claims abstract description 31
- 238000001514 detection method Methods 0.000 title claims abstract description 25
- 238000003753 real-time PCR Methods 0.000 title claims abstract description 25
- 238000003752 polymerase chain reaction Methods 0.000 title claims abstract 5
- 208000003152 Yellow Fever Diseases 0.000 title abstract description 8
- 206010012310 Dengue fever Diseases 0.000 title abstract description 7
- 208000001490 Dengue Diseases 0.000 title abstract description 5
- 208000025729 dengue disease Diseases 0.000 title abstract description 5
- 208000006400 Arbovirus Encephalitis Diseases 0.000 title abstract description 4
- 206010052369 Encephalitis lethargica Diseases 0.000 title abstract description 4
- 201000002498 viral encephalitis Diseases 0.000 title abstract description 4
- 239000000523 sample Substances 0.000 claims abstract description 63
- 238000006243 chemical reaction Methods 0.000 claims abstract description 18
- 206010014599 encephalitis Diseases 0.000 claims description 44
- 239000010985 leather Substances 0.000 claims description 31
- 238000013461 design Methods 0.000 claims description 19
- 230000003321 amplification Effects 0.000 claims description 17
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 17
- 239000013612 plasmid Substances 0.000 claims description 12
- 108090000623 proteins and genes Proteins 0.000 claims description 11
- 241000725619 Dengue virus Species 0.000 claims description 10
- UDGUGZTYGWUUSG-UHFFFAOYSA-N 4-[4-[[2,5-dimethoxy-4-[(4-nitrophenyl)diazenyl]phenyl]diazenyl]-n-methylanilino]butanoic acid Chemical compound COC=1C=C(N=NC=2C=CC(=CC=2)N(C)CCCC(O)=O)C(OC)=CC=1N=NC1=CC=C([N+]([O-])=O)C=C1 UDGUGZTYGWUUSG-UHFFFAOYSA-N 0.000 claims description 9
- 241000710772 Yellow fever virus Species 0.000 claims description 9
- 229940051021 yellow-fever virus Drugs 0.000 claims description 9
- MPLHNVLQVRSVEE-UHFFFAOYSA-N texas red Chemical compound [O-]S(=O)(=O)C1=CC(S(Cl)(=O)=O)=CC=C1C(C1=CC=2CCCN3CCCC(C=23)=C1O1)=C2C1=C(CCC1)C3=[N+]1CCCC3=C2 MPLHNVLQVRSVEE-UHFFFAOYSA-N 0.000 claims description 8
- 238000012986 modification Methods 0.000 claims description 7
- 230000004048 modification Effects 0.000 claims description 7
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 claims description 6
- 108020000999 Viral RNA Proteins 0.000 claims description 4
- 238000007405 data analysis Methods 0.000 claims description 4
- 238000010791 quenching Methods 0.000 claims description 4
- 230000000171 quenching effect Effects 0.000 claims description 4
- 239000000126 substance Substances 0.000 claims description 4
- 230000015572 biosynthetic process Effects 0.000 claims description 3
- 238000003786 synthesis reaction Methods 0.000 claims description 3
- WCKQPPQRFNHPRJ-UHFFFAOYSA-N 4-[[4-(dimethylamino)phenyl]diazenyl]benzoic acid Chemical compound C1=CC(N(C)C)=CC=C1N=NC1=CC=C(C(O)=O)C=C1 WCKQPPQRFNHPRJ-UHFFFAOYSA-N 0.000 claims description 2
- BZTDTCNHAFUJOG-UHFFFAOYSA-N 6-carboxyfluorescein Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C11OC(=O)C2=CC=C(C(=O)O)C=C21 BZTDTCNHAFUJOG-UHFFFAOYSA-N 0.000 claims description 2
- MGIODCZGPVDROX-UHFFFAOYSA-N Cy5-bifunctional dye Chemical compound O=C1CCC(=O)N1OC(=O)CCCCCN1C2=CC=C(S(O)(=O)=O)C=C2C(C)(C)C1=CC=CC=CC(C(C1=CC(=CC=C11)S([O-])(=O)=O)(C)C)=[N+]1CCCCCC(=O)ON1C(=O)CCC1=O MGIODCZGPVDROX-UHFFFAOYSA-N 0.000 claims description 2
- 238000011897 real-time detection Methods 0.000 claims description 2
- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 claims description 2
- 238000003556 assay Methods 0.000 claims 7
- 206010061192 Haemorrhagic fever Diseases 0.000 abstract description 3
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 239000007983 Tris buffer Substances 0.000 abstract description 2
- 239000000243 solution Substances 0.000 abstract description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 abstract description 2
- 241001115401 Marburgvirus Species 0.000 abstract 1
- 241000713124 Rift Valley fever virus Species 0.000 abstract 1
- 230000010211 insect pollination Effects 0.000 abstract 1
- 201000010099 disease Diseases 0.000 description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 8
- 238000000137 annealing Methods 0.000 description 7
- 238000010790 dilution Methods 0.000 description 7
- 239000012895 dilution Substances 0.000 description 7
- 230000009870 specific binding Effects 0.000 description 6
- 241000001014 Gnathanodon speciosus Species 0.000 description 5
- 235000013862 Narcissus jonquilla Nutrition 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 4
- 241000710831 Flavivirus Species 0.000 description 3
- 108091036078 conserved sequence Proteins 0.000 description 3
- 201000002950 dengue hemorrhagic fever Diseases 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- 230000003612 virological effect Effects 0.000 description 3
- 208000000705 Rift Valley Fever Diseases 0.000 description 2
- 208000009714 Severe Dengue Diseases 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 238000002864 sequence alignment Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 241000282693 Cercopithecidae Species 0.000 description 1
- 206010010904 Convulsion Diseases 0.000 description 1
- 241000256113 Culicidae Species 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 241000255925 Diptera Species 0.000 description 1
- 201000011001 Ebola Hemorrhagic Fever Diseases 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 208000003773 Meningism Diseases 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 208000004756 Respiratory Insufficiency Diseases 0.000 description 1
- 208000019802 Sexually transmitted disease Diseases 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 238000012197 amplification kit Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 230000001010 compromised effect Effects 0.000 description 1
- 230000036461 convulsion Effects 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000009533 lab test Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 201000004193 respiratory failure Diseases 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000012882 sequential analysis Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
Images
Classifications
-
- 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
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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 ", sometimes claim again America pestilence) is a kind of sharply venereal disease viral disease.Once caused some destructive epidemics.This disease is from monkey to the people or person-to-person infection, and mosquito is main mediation person.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.High mortality and strong infectivity due to yellow jack, be 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 of having a liking for due to neural encephalitis b virus, is called for short encephalitis.Propagate through hematophagous buges such as mosquitos, be popular in summer and autumn, multiplely be born in children, take clinically high heat, the disturbance of consciousness, convulsions, respiratory insufficiency and meningeal irritation sign is feature.Some patients were leaves serious sequela, and patient with severe symptoms's case fatality rate is higher.Global Raport case load every year is more than 16000, dead 5000 examples.
Dengue virus (Dengue virus) infects and causes singapore hemorrhagic fever (Denguefever, DF) or dengue hemorrhagic fever (Dengue haemorrhagic fever, DHF).This disease is popular in tropical and subtropical region, particularly South East Asia, West Pacific Ocean and Central and South America.China found this disease first in Foshan in 1978, on Hainan Island and Guangxi and other places, all be found later.According to WHO, estimate, annual DF number of the infected has 5,000 ten thousand, and wherein DHF approximately 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 at present flavivirus 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 new detecting method of encephalitis B and this multiple virus detection PCR system, the method can fast qualitative and detection by quantitative yellow fever virus, dengue virus, encephalitis b virus, comprises three pairs of primer and probes that specificity is higher, highly sensitive, reproducible.
For achieving the above object, the yellow heat of the present invention, step on leather, the multiple FLuorescent quantitative PCR new detecting method of encephalitis B, in the 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 new detecting method of encephalitis B, be specially:
1) design primer probe;
2) select fluorescein according to instrument;
3) synthetic primer and probe;
4) prepare the positive plasmid standard substance;
5) operation PCR;
6) data analysis;
7) extract viral RNA and carry out the system checking.
Further, described step 2) in, the selective fluorescent emission group of fluorescein has 6-FAM, TET, HEX, JOE, CY3, CY5, TAMRA, Texas Red, and the fluorescent quenching group has BHQ-1, BHQ-2, Lowa Black
TMRQ, Lowa Black
TMFQ, Dabcyl.
Further, described step 4) in, take the gene synthesis mode to make positive plasmid CBG228-3, CBG231-3, CBG229-1 as positive: by the high conserved region territory bp119-bp238 filtered out in the design of primers process, bp10627-bp10768, extending respectively 5～50bp before and after bp5-bp104, to carry out gene synthetic, then be cloned in the pMD19-T carrier, be described positive plasmid sample, numbering CBG228-3, CBG231-3, CBG229-1.
Further, described step 5), the quantitative fluorescent PCR reaction is applicable to all quantitative fluorescent PCR reaction instrument;
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, described step 5), best amplification condition is:
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 the primer that is equal to of the hot full length sequence specific binding of Huang amplification, its similar homology can not be over 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 with the hot full length sequence specific binding of Huang amplification be equal to probe, the fraction of coverage of its site areas can not surpass 50%, 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 the primer that is equal to of stepping on leather full length sequence specific binding amplification, its similar homology can not be over 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, probe sequence is as follows:
Can with the probe that is equal to of stepping on leather full length sequence specific binding amplification, the fraction of coverage of its site areas can not surpass 50%, 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 the primer that is equal to of encephalitis B full length sequence specific binding amplification, its similar homology can not be over 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 with encephalitis B full length sequence specific binding amplification be equal to probe, the fraction of coverage of its site areas can not surpass 50%, its homology can not surpass 70%.
The present invention designs three pairs of new primer probes, in conjunction with use 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 three kinds of sequences that the viral genome camber is conservative by the mode of sequence alignment, design respectively pair of primers and a Taqman probe on this sequence, set up the real-time fluorescence quantitative PCR reaction system, and be optimized, detect sensitivity and the specificity of the method.
The accompanying drawing explanation
The amplification curve diagram that Fig. 1 is Multiple detection;
The canonical plotting that Fig. 2 is yellow fever virus in Multiple detection;
The canonical plotting that Fig. 3 is encephalitis b virus in Multiple detection;
The typical curve that Fig. 4 is dengue virus in Multiple detection.
Embodiment
The yellow principle of work hot, that step on leather, the multiple FLuorescent quantitative PCR new detecting method of encephalitis B of the present invention is to utilize the variation qualitative analysis of fluorescent signal, detect in real time the variation of each cyclic amplification product amount in pcr amplification reaction, by the relation of Ct value and typical curve, starting template is carried out to quantitative analysis.The invention still further relates to all the elements that above-mentioned detection system comprises.
The method comprises the following steps:
1) design primer probe;
2) select fluorescein according to instrument;
3) synthetic primer and probe;
4) prepare the 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, find corresponding complete genome sequence (www.ncbi.nlm.nih.gov) in genebank, use DNASTAR software to carry out homology analysis and blast sequential analysis, sift out the highly conserved sequence 5 ' UTR119-238 (overall length 120bp) of yellow jack cause of disease, the high conserved sequence bp10627-bp10768 (overall length 141bp) of dengue fever virus, the high conserved sequence bp5-bp104 (overall length 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 is in Table 1. table 2. tables 3.
Primer and the probe of table 1 fluorescence quantitative PCR detection yellow fever virus
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.
Primer and the probe of table 2 fluorescence quantitative PCR detection dengue virus
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.
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 that instrument is 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, synthetic precious biotechnology (Dalian) company limited that gives of primer and probe completes.
4, the present invention takes the gene synthesis mode to make positive plasmid as positive.By extending respectively 30～50bp before and after the high conserved region territory that filters out in the design of primers process, to carry out gene synthetic, then is cloned in the pMD19-T carrier, is our positive plasmid sample, numbering CBG228-3, CBG231-3, CBG229-1.
5, the preparation of plasmid standard
The positive plasmid sample stoste of by authorized company, synthesizing and extracting, 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, 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, calculate 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 respectively 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 sterilizing Tris water (Ph7.0～8.0).
7, Real-Time pcr amplification
The amplification kit of selecting is Premix Ex Taq test kit, purchased from precious biotechnology (Dalian) company limited.Reaction system component and volume thereof are in Table 4.
(M means Marburg to table 4.; E means the Ebola)
Amplification condition is as follows
8, data analysis
9, extract viral RNA, carry out reverse transcription, gained cDNA is used the aqua sterilisa gradient dilution, replaces positive and carries out detection validation.
The optimization of primer concentration and probe concentration
1. at first dilute each primer probe standby to lower concentration, primer is diluted to 10 μ m/L, and probe is to 5 μ m/L;
2. at first to the upstream and downstream primer (10 μ m/L) and the 0.5 μ L Taqman probe (5 μ m/L) that add respectively tri-kinds of viruses of 0.5 μ L in 25 μ L systems, 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.Result demonstration CY3 and Texas Red fluorescent value are all very little, be starkly lower than FAM fluorescence, show in three kinds of simultaneous situations of fluorophor, CY3 and Texas Red luminous efficiency are quite low, for not affecting positive judgement, the pre-add-on that improves CY3 and Texas Red probe primer, be convenient to data and 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 large 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, again move PCR.The luminous value of three kinds of fluorescence still can not be fully balanced as a result.Consider, fluorescence difference is inevitable for this reason, and too much primer and probe also can affect the reaction efficiency of system, therefore, without too being entangled with the excessive fluorescent signal of difference, only need stable detection to arrive goal gene, obtains stable Ct value and gets final product.Therefore, determine that in 25 μ L multiple fluorescence quantitative PCR reaction systems, yellow fever virus primer concentration and probe concentration is 80 * 40nm/L, encephalitis primer concentration and probe concentration is 400 * 200nm/L, steps on the same encephalitis of leather primer concentration and probe concentration.
The selection of annealing temperature
Under perfect condition, annealing temperature is enough low, to guarantee primer, with aim sequence, effectively anneals, and wants enough high, to reduce non-specific binding simultaneously.When being set, annealing temperature carries out as follows: using estimate during primer lower than design 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 strongest minimum temperature of Ct value simultaneously of fluorescent signal is 56 ℃, finally determines that optimum annealing temperature is 56 ℃.
Repeatability detects
By the DNA profiling quantitative fluorescent PCR of each concentration gradient of Fig. 2 .3.4., produced comparatively approaching Ct value in 2 to 3 parallel laboratory tests of each gradient, it is 0.99 upper and lower that reaction relation conefficient r-squared can reach, and repeatability is better.
The specificity check
Detect with yellow heat, step on the viruses such as Marburg that leather, encephalitis b virus be all entomophila hemorrhagic fever cause of disease, Rift Valley fever by set up method, result is all negative.
Show that this designed primer probe specificity is fine.
The foundation of typical curve
The quality of system can reflect by amplification efficiency e, and the e value more levels off to 1, illustrates that amplification efficiency more approaches 100%, and reaction system is better; E<1, illustrate that the system amplification efficiency is lower, and system needs to optimize; E>1, contain inhibition in the explanation system, needs to reduce the interference of even getting rid of inhibition.Amplification efficiency optimum range generally acknowledged in this field is between 0.8-1.2.Adopt respectively gradient template separately to be increased, 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 use 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 three kinds of sequences that the viral genome camber is conservative by the mode of sequence alignment, design respectively pair of primers and a Taqman probe on this sequence, 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 yellow heat, steps on leather, encephalitis b virus detects simultaneously, with other several entomophila hemorrhagic fever viruss as Marburg, Rift Valley fever no cross reaction.
Claims (7)
- Yellow heat, step on leather, the non-diagnostic assays method of the multiple FLuorescent quantitative PCR of encephalitis B, it is characterized in that, in the 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 - Yellow heat as claimed in claim 1, step on leather, the non-diagnostic assays method of the multiple FLuorescent quantitative PCR of encephalitis B, it is characterized in that, the method is specially:1) design primer probe;2) select fluorescein according to instrument;3) synthetic primer and probe;4) prepare the positive plasmid standard substance;5) operation PCR;6) data analysis;7) extract viral RNA and carry out the system checking.
- Yellow heat as claimed in claim 2, step on leather, the non-diagnostic assays method of the multiple FLuorescent quantitative PCR of encephalitis B, it is characterized in that, described step 2) in, the selective fluorescent emission group of fluorescein has 6-FAM, TET, HEX, JOE, CY3, CY5, TAMRA, Texas Red, and the fluorescent quenching group has BHQ-1, BHQ-2, Lowa Black TMRQ, Lowa Black TMFQ, Dabcyl.
- 4. as claimed in claim 2 yellow hot, step on leather, the non-diagnostic assays method of the multiple FLuorescent quantitative PCR of encephalitis B, it is characterized in that, described step 4) in, take the gene synthesis mode to make positive plasmid CBG228-3, CBG231-3, CBG229-1 is as positive: by the high conserved region territory bp119-bp238 filtered out in the design of primers process, bp10627-bp10768, extending respectively 5～50bp before and after bp5-bp104, to carry out gene synthetic, then be cloned in the pMD19-T carrier, be described positive plasmid sample, numbering CBG228-3, CBG231-3, CBG229-1.
- Yellow heat as claimed in claim 2, step on leather, the non-diagnostic assays method of the multiple FLuorescent quantitative PCR of encephalitis B, it is characterized in that described step 5) in the quantitative fluorescent PCR reaction be applicable to all quantitative fluorescent PCRs and react instrument.
- Yellow heat as claimed in claim 5, step on leather, the non-diagnostic assays method of the multiple FLuorescent quantitative PCR of encephalitis B, it is characterized in that, 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.
- Yellow heat as claimed in claim 2, step on leather, the non-diagnostic assays method of the multiple FLuorescent quantitative PCR of encephalitis B, it is characterized in that described step 5) in best amplification condition be:
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010106054986A CN102191338B (en) | 2010-12-24 | 2010-12-24 | 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 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010106054986A CN102191338B (en) | 2010-12-24 | 2010-12-24 | 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 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102191338A CN102191338A (en) | 2011-09-21 |
| CN102191338B true CN102191338B (en) | 2013-12-04 |
Family
ID=44600203
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010106054986A Expired - Fee Related CN102191338B (en) | 2010-12-24 | 2010-12-24 | 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 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102191338B (en) |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102399905B (en) * | 2011-11-04 | 2013-08-21 | 重庆大学 | Oligonucleotide primers for detecting arbo encephalitis viruses and detection method thereof |
| WO2013080307A1 (en) * | 2011-11-29 | 2013-06-06 | 株式会社 東芝 | Primer set for amplifying mosquito-borne virus, assay kit for detecting mosquito-borne virus, and detection method using said primer set and said assay kit |
| CN102424866A (en) * | 2012-01-10 | 2012-04-25 | 中华人民共和国江苏出入境检验检疫局 | Fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction) kit for rapidly detecting XHF (Xinjiang hemorrhagic fever) viruses |
| CN105950759B (en) * | 2016-06-23 | 2019-12-24 | 中国检验检疫科学研究院 | Kit for simultaneously detecting four pathogenic bacteria and non-diagnostic detection method thereof |
| CN106282416B (en) * | 2016-10-08 | 2018-05-25 | 广东省实验动物监测所 | A kind of the multi-fluorescence immunoassay primer, kit and method of 5 kinds of fowl immunosupress cause of diseases of quick differentiation |
| CN106755573A (en) * | 2016-12-07 | 2017-05-31 | 深圳澳东检验检测科技有限公司 | Zika virus, dengue fever virus, the RT PCR detection methods of chikungunya fever virus, primer and probe and kit |
| CN109628637B (en) * | 2018-09-11 | 2022-09-23 | 山东国际旅行卫生保健中心 | Method for detecting entomovirus based on hyperbranched rolling circle amplification nucleic acid test strip |
| CN116064952B (en) * | 2022-09-29 | 2023-08-08 | 首都医科大学附属北京友谊医院 | Double-gene primer probe set for detecting yellow fever virus, kit and application |
| CN116987826A (en) * | 2023-09-28 | 2023-11-03 | 广州达安基因股份有限公司 | Group of molecular targets for detecting hemorrhagic fever pathogens and application thereof |
-
2010
- 2010-12-24 CN CN2010106054986A patent/CN102191338B/en not_active Expired - Fee Related
Non-Patent Citations (6)
| Title |
|---|
| 4株蚊媒病毒多重RT-PCR快速检测方法的建立;王军军等;《热带医学杂志》;20100831;第10卷(第8期);918-920 * |
| 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》.2007,第45卷(第2期),584–589. * |
| J. Dyer et al..A multiplexed TaqMan assay for the detection of arthropod-borne flaviviruses.《Journal of Virological Methods》.2007,第145卷9-13. * |
| 实时荧光RT-PCR技术在黄热病快速检测中的应用;师永霞等;《现代预防医学》;20100225;第37卷(第4期);715-718 * |
| 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》.2010,第37卷(第4期),715-718. |
| 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》.2010,第10卷(第8期),918-920. |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102191338A (en) | 2011-09-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102191338B (en) | 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 | |
| CN106119413B (en) | AIDS virus multiple fluorescence PCR detection kit and detection method | |
| CN110760620A (en) | Classical swine fever virus and African classical swine fever virus dual-fluorescence PCR detection reagent, kit and detection method | |
| CN111074000A (en) | Triple fluorescence quantitative PCR detection material and kit for distinguishing ASFV wild strain and double-gene deletion strain | |
| Huang et al. | Molecular and epidemiological study of enterovirus D68 in Taiwan | |
| CN102140530B (en) | New chikungunya virus fluorescence quantitative polymerase chain reaction detection method and chikungunya virus polymerase chain reaction detection system | |
| CN102140529A (en) | New dengue virus fluorescence quantitative polymerase chain reaction detection method and dengue virus polymerase chain reaction detection system | |
| WO2013116039A1 (en) | Amplification primers and probes for detection of hiv-1 | |
| CN102140533B (en) | Marburg and Ebola dual-virus fluorescent quantitative PCR (Polymerase Chain Reaction) detection method and dual-virus detection PCR system | |
| CN102534031A (en) | High-specificity kit for detecting deafness predisposing genes | |
| Yang et al. | RT-LAMP assay for rapid detection of the R203M mutation in SARS-CoV-2 Delta variant | |
| CN102140532A (en) | Novel Ebola virus fluorescent quantitative PCR (Polymerase Chain Reaction) detection method and system | |
| WO2023279042A2 (en) | Compositions and methods for detection of severe acute respiratory syndrome coronavirus 2 variants | |
| CN102140528B (en) | New fluorescence quantitative polymerase chain reaction (PCR) detection method for rift valley fever virus and rift valley fever virus detection PCR system | |
| CN102277446B (en) | Novel fluorescence quantitative PCR (Polymerase Chain Reaction) detection method for yellow fever viruses and yellow fever virus detection PCR system | |
| CN103484568B (en) | Method and kit for dual fluorescence quantitative PCR detection of grass carp reovirus types I and II | |
| CN102140531B (en) | Novel Marburg virus fluorescent quantitative PCR (Polymerase Chain Reaction) detection method and Marburg virus PCR detection system | |
| CN104293932A (en) | Method for detecting HLA-B * 5801 allele based on real-time fluorescence PCR | |
| CN101676406A (en) | Primer for coxsackie virus A16 nucleic acid detection, probe and kit | |
| CN102943116B (en) | Gene detection kit for Thailand type alpha-thalassemia | |
| CN109897914B (en) | Primer for simultaneously detecting YFV, RVFV, WNV and DENV four viruses and detection method thereof | |
| CN101122563A (en) | Hepatitis E virus fluorescent quantitative PCR detection method | |
| CN113755568B (en) | Primer probe, kit and application for detecting alpha globin gene copy number by utilizing microdroplet digital PCR | |
| Lan et al. | Molecular epidemiology of the 2005 enterovirus 71 outbreak in central Taiwan | |
| CN102534030A (en) | Kit for jointly detecting four deafness predisposing genes and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20131204 |