CN102808042A - Method for detecting flavivirus by gene chip method - Google Patents
Method for detecting flavivirus by gene chip method Download PDFInfo
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- CN102808042A CN102808042A CN2012100658981A CN201210065898A CN102808042A CN 102808042 A CN102808042 A CN 102808042A CN 2012100658981 A CN2012100658981 A CN 2012100658981A CN 201210065898 A CN201210065898 A CN 201210065898A CN 102808042 A CN102808042 A CN 102808042A
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- flavivirus
- gene chip
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention discloses a method for detecting flavivirus by a gene chip method. According to the method, sequences of universal primers are designed, namely 5'AACATGATGGGNAAYAGAGAGAA, and 5'GTGTCCCANCCNGCMGTYTCYGC; and specific probes for detecting four flavivirus are designed, namely 5'TGTAATCAAGGACCTATCCACCAAAGAAGGGGG, 5'GTTAACATCCTGCGTGAAGTTGGCACCCGGCCT, 5'GCGTCCAAAAGTTGGGATACATTCTCCGTGACATA and 5'GCACCTAAAAAATTGTCAACATTGGAAGGAGGCC. Aiming at the flavivirus, the defects in the prior art are overcome, and a method for detecting the flavivirus quickly in high flux and in a typing mode is provided.
Description
Technical field
The present invention relates to the virus examination technology, specifically use method for gene chip to detect the method for flavivirus.
Background technology
Flavivirus (Flavircls) is large numbers of single positive chain RNA virus with coating.This viroid is propagated through the arthropods (mosquito, tick, sand fly etc.) of sucking blood and causes infection.Past attempts classifies as arboviruses.Main popular flavivirus has encephalitis b virus, fores encephalitis virus and dengue virus in China.This viroid structural similitude, great majority are the three-dimensional symmetric tunicary RNA viruses of 20 bodies.They can be at arthropods (like mosquito, tick, sand fly etc.) proliferation in vivo, and are not pathogenic to arthropods, give vertebrates or people but can breathe out to sting to infect through insect, cause disease of natural focus.Most virus cause infecting both domestic animals and human, disease of natural focus, be diversified clinical manifestation, mainly comprise encephalitis or encephalomyelitis and systemic infection etc.Because arthropods is a reservoir host is again communication media, its distribution receives geography and climatic influences, so associated diseases has obvious seasonal and region.Flaviviridae (Flaviviridae) comprises 3 Tobamovirus, and promptly Flavivirus (flavivirus), pestivirus (pestivirus) and Hepacivirus (hepacivirus) have and plant virus more 60.
Summary of the invention
To flavivirus, the present invention provides a kind of detection method that can distinguish 4 kinds of flaviviruss simply fast.
The present invention realizes through following technical scheme:
(1) the preparation flavivirus detects gene chip;
(2) the design universal primer multiple flavivirus specific gene fragment that is used to increase;
(3) amplified production and gene chip are hybridized;
(4) with product and chip hybridization, after the hybridization, carry out immunoreation after amplification finishes, judge detected result through the colour-change on the chip.
This method comprises: use this method and detect needed universal primer of flavivirus and supporting with it PCR reaction conditions.Wherein primer sequence is following:
Primer:
SEQ?ID?NO.1：5’AACATGATGGGNAAYAGAGAGAA
SEQ?ID?NO.2：5’GTGTCCCANCCNGCMGTYTCYGC。
Each component composition is following in the nucleic acid amplification reaction system:
The nucleic acid amplification program is:
(1)95℃ 10min
(2)95℃ 30s
(3)55℃ 20s
(4)72℃ 30s
(5) got back to for (2) step, repeat 35 times
(6)72℃ 2min
And preparation detects the used oligonucleotide probe of gene chip:
The yellow fever virus probe
SEQ?ID?NO.3：
5’TGTAATCAAGGACCTATCCACCAAAGAAGGGGGAGGAT
The west nile virus probe
SEQ?ID?NO.4：
5’AAACTGGGTTAACATCCTGCGTGAAGTTGGCACCCGGCCT
The japanese encephalitis virus probe
SEQ?ID?NO.5：
5’TTCAGGCGTCCAAAAGTTGGGATACATTCTCCGTGACATA
The fores encephalitis virus probe
SEQ?ID?NO.6：
5’CTGGCACCTAAAAAATTGTCAACATTGGAAGGAGGCCTCT。
Gene chip preparation method is having the nylon membrane of positive charge (Amersham company for the specific oligonucleotide gene probe point with design; The U.S.) on; The excellent nylon membrane of point carries out crosslinked 5-10min under long-wave ultra violet lamp, the gene fragment that simultaneously 0.2 μ L is marked with the digoxin group is also put on each nylon membrane, as positive control; Distilled water is also put on each nylon membrane, as negative control.
3, the enzyme linked immunological of hybridization and hybridization spot colour developing is carried out according to the specification sheets of digoxin dna marker and detection kit.
Digoxigenin labeled PCR product and oligonucleotide probe hybridization method are following:
Prehybridization
Prehybridization solution is preheating to 53 ℃ earlier, puts into the prehybridization bag to the nylon membrane of clicking and entering oligonucleotide probe, adds prehybridization solution, seals mouth, in 53 ℃ of prehybridization 30min.
The sex change of pcr amplification product
The PCR product of DIG mark is heated to 95 ℃, and 10min inserts ice bath rapidly.
The target DNA molecule of digoxigenin labeled and nylon membrane hybridization
Put into hybridization bag to the nylon membrane that prehybridization is good, add the PCR product 10 μ L of the DIG mark of sex change, add 1mL hybridization solution (the Dig Easy Hyb solution of Roche company) again, seal mouth.53 ℃ of hybridization 1hr, gentleness is shaken.
The enzyme linked immunological colour developing of hybridization positive spots
The enzyme of hybridization spot connects the immunity colour developing to carry out according to Roche company digoxin dna marker and detection kit specification sheets.
Interpretation as a result
The positive results of hybridization of mazarine spot on the nylon membrane shows the specific sequence that probe has detected in the sample to be contained.
This primer sequence and probe sequence are with reference to the NS5 gene in the multiple flavivirus sequence among the GenBank.
The present invention is with specific chip detection flavivirus, and reaction back visual observation is not found false positive and false-negative result.
Compared with prior art, the invention has the beneficial effects as follows: compare traditional Physiology and biochemistry detection method, be applicable to that based on method for gene chip the high throughput testing flavivirus can once detect multiple flavivirus.Improved efficient greatly and practiced thrift the time, avoided cultivating repeatedly, saved time; This authentication method does not receive the influence of culture condition and bacterium physiological status, and is more accurate than the Physiology and biochemistry authentication method.
Description of drawings
Fig. 1 gene chip mode chart; P series is 3 parallel positive reference points; N series is 3 parallel negative reference points, and 1 point is 3 parallel yellow fever virus probe points, and 2 point are 3 parallel west nile virus probe points; 3 point are 3 parallel japanese encephalitis virus probe points, and 4 point are 3 parallel fores encephalitis virus probe points.
Embodiment
Below in conjunction with accompanying drawing and embodiment the present invention is described in further detail.
Sample: certain testing sample.
1. sample preparation
The sample mixing is handled.
2. nucleic acid extracting
Sample thief is resuspended in PBS, uses RNA extraction agent box to require to carry out the RNA extracting according to specification sheets.
3.cDNA preparation
Total RNA is used RNA rt test kit, and requiring rt RNA according to specification sheets is cDNA.
4.PCR amplification
In the isothermal duplication system in the reaction system each component composition following
Each component composition is following in the nucleic acid amplification reaction system:
The nucleic acid amplification program is:
(1)95℃ 10min
(2)95℃ 30s
(3)55℃ 20s
(4)72℃ 30s
(5) got back to for (2) step, repeat 35 times
(6)72℃ 2min
5. prehybridization
Prehybridization solution is preheating to 53 ℃ earlier, puts into the prehybridization bag to the nylon membrane of clicking and entering oligonucleotide probe, adds prehybridization solution, seals mouth, in 53 ℃ of prehybridization 30min.
The sex change of pcr amplification product
The PCR product of DIG mark is heated to 95 ℃, and 10min inserts ice bath rapidly.
The target DNA molecule of digoxigenin labeled and nylon membrane hybridization
Put into hybridization bag to the nylon membrane that prehybridization is good, add the PCR product 10 μ L of the DIG mark of sex change, add 1mL hybridization solution (the Dig Easy Hyb solution of Rochc company) again, seal mouth.53 ℃ of hybridization 1hr, gentleness is shaken.
The enzyme linked immunological colour developing of hybridization positive spots
The enzyme of hybridization spot connects the immunity colour developing to carry out according to Roche company digoxin dna marker and detection kit specification sheets.
Interpretation as a result
The positive results of hybridization of mazarine spot that positive reference and flavivirus probe appear on the nylon membrane shows the specific sequence that probe has detected in the sample to be contained, and sample contains flavivirus nucleic acid.
Sample: certain testing sample.
1. sample preparation
The sample mixing is handled.
2. nucleic acid extracting
Sample thief is resuspended in PBS, uses RNA extraction agent box to require to carry out the RNA extracting according to specification sheets.
3.cDNA preparation
Total RNA is used RNA rt test kit, and requiring rt RNA according to specification sheets is cDNA.
4.PCR amplification
In the isothermal duplication system in the reaction system each component composition following
Each component composition is following in the nucleic acid amplification reaction system:
The nucleic acid amplification program is:
(1)95℃ 10min
(2)95℃ 30s
(3)55℃ 20s
(4)72℃ 30s
(5) got back to for (2) step, repeat 35 times
(6)72℃ 2min
5. prehybridization
Prehybridization solution is preheating to 53 ℃ earlier, puts into the prehybridization bag to the nylon membrane of clicking and entering oligonucleotide probe, adds prehybridization solution, seals mouth, in 53 ℃ of prehybridization 30min.
The sex change of pcr amplification product
The PCR product of DIG mark is heated to 95 ℃, and 10min inserts ice bath rapidly.
The target DNA molecule of digoxigenin labeled and nylon membrane hybridization
Put into hybridization bag to the nylon membrane that prehybridization is good, add the PCR product 10 μ L of the DIG mark of sex change, add 1mL hybridization solution (the Dig Easy Hyb solution of Roche company) again, seal mouth.53 ℃ of hybridization 1hr, gentleness is shaken.
The enzyme of the enzyme linked immunological colour developing hybridization spot of hybridization positive spots connects the immunity colour developing to carry out according to Roche company digoxin dna marker and detection kit specification sheets.
Interpretation as a result
The positive results of hybridization of mazarine spot that positive reference and japanese encephalitis virus probe appear on the nylon membrane shows the specific sequence that probe has detected in the sample to be contained, and sample contains japanese encephalitis virus nucleic acid.
Claims (5)
1. a method of utilizing method for gene chip to detect flavivirus is characterized in that, comprises following two primers:
SEQ?ID?NO.1：5’AACATGATGGGNAAYAGAGAGAA
SEQ?ID?NO.2：5’GTGTCCCANCCNGCMGTYTCYGC。
2. a kind of method of utilizing method for gene chip to detect flavivirus according to claim 1 is characterized in that, comprises following four probes:
The yellow fever virus probe
SEQ?ID?NO.3：5’TGTAATCAAGGACCTATCCACCAAAGAAGGGGGAGGAT
The west nile virus probe
SEQ?ID?NO.4：5’AAACTGGGTTAACATCCTGCGTGAAGTTGGCACCCGGCCT
The japanese encephalitis virus probe
SEQ?ID?NO.5：5’TTCAGGCGTCCAAAAGTTGGGATACATTCTCCGTGACATA
The fores encephalitis virus probe
SEQ?ID?NO.6：5C?TGGCACCTAAAAAATTGTCAACATTGGAAGGAGGCCTCT。
3. a kind of method of utilizing method for gene chip to detect flavivirus according to claim 1 is characterized in that each component composition is following in the nucleic acid amplification reaction system:
4. a kind of method of utilizing method for gene chip to detect flavivirus according to claim 1 is characterized in that the nucleic acid amplification program is:
(1)95℃ 10min
(2)95℃ 30s
(3)55℃ 20s
(4)72℃ 30s
(5) got back to for (2) step, repeat 35 times
(6)72℃ 2min。
5. a kind of method of utilizing method for gene chip to detect flavivirus according to claim 1 is characterized in that the PCR product of mark and the hybridization temperature of chip are 53 ℃.
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| CN2012100658981A CN102808042A (en) | 2012-03-14 | 2012-03-14 | Method for detecting flavivirus by gene chip method |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102796827A (en) * | 2011-05-24 | 2012-11-28 | 上海透景生命科技有限公司 | Method and kit for detecting multiple encephalitis related viruses |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004092412A2 (en) * | 2003-03-31 | 2004-10-28 | Roche Diagnostics Gmbh | Compositions and methods for detecting certain flaviviruses, including members of the japanese encephalitis virus serogroup |
| CN101349701A (en) * | 2008-09-09 | 2009-01-21 | 深圳市检验检疫科学研究院 | Protein chip for sironi virus detection and preparation method thereof |
| CN101781687A (en) * | 2009-01-19 | 2010-07-21 | 中国人民解放军军事医学科学院微生物流行病研究所 | Flavivirus-detecting gene chip probe and gene chip detection method |
-
2012
- 2012-03-14 CN CN2012100658981A patent/CN102808042A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004092412A2 (en) * | 2003-03-31 | 2004-10-28 | Roche Diagnostics Gmbh | Compositions and methods for detecting certain flaviviruses, including members of the japanese encephalitis virus serogroup |
| CN101349701A (en) * | 2008-09-09 | 2009-01-21 | 深圳市检验检疫科学研究院 | Protein chip for sironi virus detection and preparation method thereof |
| CN101781687A (en) * | 2009-01-19 | 2010-07-21 | 中国人民解放军军事医学科学院微生物流行病研究所 | Flavivirus-detecting gene chip probe and gene chip detection method |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102796827A (en) * | 2011-05-24 | 2012-11-28 | 上海透景生命科技有限公司 | Method and kit for detecting multiple encephalitis related viruses |
| CN102796827B (en) * | 2011-05-24 | 2016-11-09 | 上海透景生命科技股份有限公司 | A kind of method detecting multiple encephalitis correlated virus and test kit |
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Application publication date: 20121205 |