CN101660002A - HCV genotyping DNA microarray chip - Google Patents
HCV genotyping DNA microarray chip Download PDFInfo
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- CN101660002A CN101660002A CN200810198010A CN200810198010A CN101660002A CN 101660002 A CN101660002 A CN 101660002A CN 200810198010 A CN200810198010 A CN 200810198010A CN 200810198010 A CN200810198010 A CN 200810198010A CN 101660002 A CN101660002 A CN 101660002A
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Abstract
The invention relates to a DNA microarray chip, in particular to a hepatitis C virus (HCV) genotyping DNA microarray chip. A solid-phase carrier substrate is used, and an oligonucleotide probe is designed in view of different genotypes of HCV so as to prepare the DNA microarray chip; and when matched with a PCR primer and other components, the DNA microarray chip can quickly and accurately classify the types of the hepatitis C virus in a blood sample.
Description
Technical field
The present invention relates to a kind of dna microarray chip, particularly relate to hepatitis C virus (HCV) gene typing DNA micro-array chip.The present invention adopts the solid phase carrier substrate, at HCV different genotype design oligonucleotides probe, is prepared into the dna microarray chip, is used for quick and precisely the blood sample hepatitis C virus being carried out somatotype and detects.
Background technology
Hepatitis C is serious harm human health, popular far-ranging transmissible disease, its pathogenic agent be hepatitis C virus (hepatitis C virus, HCV).At present, there are 1.7 hundred million people's HCV infection of surpassing in the whole world, and wherein has every year the 100000 routine HCV patients of surpassing to develop into liver cancer, and then digestive tract hemorrhage and ascites occur.According to WHO annual report in 2002, in calendar year 2001, chronic hepatopathy causes that 1,400 ten thousand example is dead, comprise 79.6 ten thousand examples by liver cirrhosis cause, 61.6 ten thousand examples cause by primary hepatocarcinoma.
HCV is a positivity single stranded RNA flavivirus, comprises about 9,400 Nucleotide.The HCV genome has an independent open reading frame, gene structure is made up of 5 '-NCR-C-E1-E2-NS1-NS2-NS3-NS4-NS5-N-CR-3 ', coding one has 3,010 amino acid whose polyprotein body is divided into virus replication and virion and forms necessary structure and Nonstructural Protein after the translation.HCV has variability highly, and (its aberration rate is up to 1.4-1.9 * 10
-3/ Nucleotide/year) and itself have the feature of negative selective action, high sudden change is because replicative enzyme does not have correct functioning; Easy mistake property RDRP (error-prone RDRP, reasons such as existence EP-RDRP) and positive selective action.Show as and itself have negative selective action: range gene structure and function difference in the viral genome, some region height is conservative.According to the isolating HCV in different areas, the whole world all or part of genomic phylogenetic analysis of homophyletic not, HCV can be divided into 1～6 type totally six types, the various hypotype of dividing again at present, the HCV hypotype is above 50, wherein modal is 1a, 1b, 2a, 2b, 3a, 6a differs 31-34% between the various nucleotide sequence, it is about 30% that aminoacid sequence differs, and differ about 20-23% between the hypotype sequence.There are several HCV virus strain mainly to be separated, are named as HCV 7,8 from South East Asia, 9,10,11 types, except HCV10a type (being put into the HCV3 type at present), since the similarity on its phyletic evolution, all the other various HCV 6 types that are put into.
Studies show that the susceptibility to interferon therapy between the different types of HCV differs, particularly 1 type is particularly poor than other type susceptibility.The infectious hepatitis of different type HCV, moderate that it is acute, chronic and severe, posthepatitic cirrhosis and primary hepatocarcinoma incidence all have difference, and the clinical state of an illness of HCV genotype and hepatitis C patients and severity thereof are closely related.
At present the HCV methods of genotyping is mainly contained: restricted length polymorphism (RFLP), the type specificity primer PCR, type specificity probe nucleic acid hybridization analysis, RDB, direct method such as order-checking, but there is complex operation mostly in these methods, detection time is long, flux is low, also exists sensitivity low, the not high shortcoming of specificity.The present invention adopts biochip technology, because this technology can be fixed in extremely a large amount of HCV typing probes on the upholder simultaneously, be prepared into the HCV gene typing DNA micro-array chip, so this chip can disposablely carry out accurate somatotype to the HCV in a plurality of samples, thereby has solved the deficiency of traditional method.
Summary of the invention
Technical problem solved by the invention is, overcomes the defective and the deficiency of prior art, makes the dna microarray chip with high-throughput, high accuracy, can disposablely carry out accurate somatotype to HCV in a plurality of samples and detect.
Therefore, the objective of the invention is to prepare a kind of hepatitis C virus (HCV) gene typing DNA micro-array chip, carry out the HCV needs of somatotype quick and precisely to satisfy a large amount of blood samples.
For this reason, the present invention is by the following technical solutions: adopt the solid phase carrier substrate, the specific oligonucleotide probe at the design of HCV different genotype is prepared into the dna microarray chip, be equipped with the RT-PCR system again, make every chip can disposablely detect the HCV genotype of a plurality of samples simultaneously.
Embodiment preferred according to the present invention, at other gene order of HCV different shaped, design one cover specific oligonucleotide probe (seeing Table 1), adopt Linomat device people, probe is printed on the specific region of a slide, every slide is printed 10 microarray matrixes, so just makes the dna microarray chip, and a chip can detect 8 increments originally.
Another embodiment preferred according to the present invention is equipped with other necessary component again on the basis of above-mentioned dna microarray chip, as PCR primer (seeing Table 2), promptly form complete product.When actual detected, get 8 parts of human gene group DNA's solution, adopt CY3 labeled primer and asymmetric PCR method, amplify the HCV genome C Y3 mark target fragment that may exist.Get one of dna microarray chip, add 8 kinds of PCR product 2ul and hybridization solution 8ul respectively at 8 hybridization regions, add with reference to product solution in positive and negative control area simultaneously, chip is placed in the hybridization groove of automatic hybridization washing instrument, 50 ℃ of hybridization temperatures, hybridization time 40 minutes dries up automatically.Take out slide, put into GenePix 4100A scanner and scan, operational analysis software carries out the view data conversion process to scan image signal, and analyzes generation sample HCV type result.
Another embodiment preferred according to the present invention, the sheet base carrier of dna microarray chip can be the carriers that slide, nylon membrane or other can adhesion probes.
Table 1 sequence oligonucleotide probe and the type that is detected
| Title | Sequence (5 '----3 ') | Sequence number | The gene type |
| ??H1 | ??ATGCCTGGAGATTTGGG | ??SEQ?ID?NO：1 | ??1 |
| ??H2 | ??GCGAGACTGCTAGCCG | ??SEQ?ID?NO：2 | ??1 |
| ??H3 | ??CGAGACCGCTAGCCGAG | ??SEQ?ID?NO：3 | ??1 |
| ??H4 | ??GTAGCGTTGGGTTGCGAA | ??SEQ?ID?NO：4 | ??2 |
| ??H5 | ??TCCTTTCTTGGATAAACCCA | ??SEQ?ID?NO：5 | ??2 |
| ??H6 | ??CCGGGAAGACTGGGTCCT | ??SEQ?ID?NO：6 | ??2 |
| ??H7 | ??CCCACTCTATGCCCGGCCA | ??SEQ?ID?NO：7 | ??2 |
| ??H8 | ??CCGCGAGATCACTAGCCG | ??SEQ?ID?NO：8 | ??3 |
| ??H9 | ??AATCGCTGGGGTGACCGGG | ??SEQ?ID?NO：9 | ??3 |
| ??H10 | ??CCGCTCAATGCCCGGAAA | ??SEQ?ID?NO：10 | ??3 |
| ??H11 | ??ACCGGGTCCTTTCCATTGG | ??SEQ?ID?NO：11 | ??6 |
| ??H12 | ??ATCAACCCGCTCAAT | ??SEQ?ID?NO：12 | ??6 |
| ??H13 | ??GATCAACCCGCTCAAT | ??SEQ?ID?NO：13 | ??6 |
| ??H14 | ??TACTGCCTGATAGGGTGC | ??SEQ?ID?NO：14 | ??1-6 |
Table 2 primer sequence
| Title | Sequence (5 '----3 ') | Sequence number | Function |
| The RT primer | ??TCATGGTGCACGGTCTACGAGAC | ??SEQ?ID?NO：15 | Reverse transcriptase primer |
| ??F | ??CTAGCCATGGCGTTAGTATGAGT | ??SEQ?ID?NO：16 | Upstream primer |
| ??R | ??ACTCGCAAGCACCCTATCAGGCA | ??SEQ?ID?NO：17 | Downstream primer, the CY3 mark |
Description of drawings
Fig. 1 HCV gene typing DNA micro-array chip
1 chip overall appearance (synoptic diagram)
2 hybridization regions and probe microarray (synoptic diagram)
Fig. 2 HCV micro probe array is arranged synoptic diagram
Wherein: the positive contrast probe of HP; The negative contrast probe of HN
Figure 31 type HCV Virus Sample detected result scintigram (synoptic diagram)
Figure 42 type HCV Virus Sample detected result scintigram (synoptic diagram)
Figure 53 type HCV Virus Sample detected result scintigram (synoptic diagram)
Figure 66 type HCV Virus Sample detected result scintigram (synoptic diagram)
Embodiment
The following example is intended to illustrate rather than limit the present invention.
The design of embodiment 1:HCV gene typing DNA micro-array chip PCR primer and probe
5 ' UTR district design RT and PCR primer adopt asymmetric RT-PCR system in the HCV genome, the needed target fragment of amplification somatotype; In the target fragment scope, design 3 of 1 type probes, 4 of 2 type probes, 3 of 3 type probes, 1 of three of 6 type probes and general probe.
The preparation of embodiment 2:HCV gene typing DNA micro-array chip
Adopt the automatic point sample instrument of gene chip,, make dna microarray 14 typing probes and 2 specific regions that contrast probe points system to slide.
(1) point sample probe solution: in probe dilution to 5 * SSC, 0.05%SDS solution, final concentration is 30 μ M;
(2) point sample matrix: on slide, each probe laterally repeats 2 points in hybridization region with probe points, and every row 8 points totally 4 are gone 32 points (referring to accompanying drawing 1); Hybridization region is divided into 10 districts, can detect 8 parts simultaneously
Sample (9,10 hybridization regions are the check plot).
Embodiment 3: use product of the present invention to detect 8 parts of clinical samples
Get 8 parts of clinical samples and positive and negative and contrast each portion, extract RNA,, carry out pcr amplification, obtain DNA hybridization template according to RT-PCR reagent operation instructions.
Adopt HCV gene typing DNA micro-array chip of the present invention, above sample is carried out disposable detection.
Get a micro-array chip, get 10 holes that 10 each 2ul of pipe PCR product are added to chip respectively, (5 * SSC) 8ul, chip are put in the groove of automatic hybridization washing instrument, set 50 ℃ of hybridization temperatures, hybridization time 40 minutes to add hybridization solution in each hole again.The washing and air-dry automatically of hybridization back.
Adopt GenePix 4100A scanner, the scanning hybridization signal obtains the results of hybridization scintigram, and image transitions is become data.
Adopt analysis software, above data analysis is converted into the gene type of each sample, simultaneously,, contrast as detected result with 8 these dna sequencings of increment.The result shows, 8 these detected results of increment conform to fully with the sample sequencing result (seeing Table 3).
Embodiment has illustrated that product of the present invention is detecting the advantage (8 samples of one-time detection) aspect the flux and the reliability (detected result conforms to fully with the sample sequencing result) of detection accuracy aspect.
The comparison of this detected result of table 3 and sample sequencing result
Sequence table
＜110〉Da
＜120〉HCV gene typing DNA micro-array chip
<140>
<141>
<160>17
<210>1
<211>17
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, to react probe as chip hybridization.
<400>1
atgcctggagatttggg
<210>2
<211>16
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, to react probe as chip hybridization.
<400>2
gcgagactgctagccg
<210>3
<211>17
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, to react probe as chip hybridization.
<400>3
cgagaccgctagccgag
<210>4
<211>18
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, to react probe as chip hybridization.
<400>4
gtagcgttgggttgcgaa
<210>5
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, to react probe as chip hybridization.
<400>5
tcctttcttggataaaccca
<210>6
<211>18
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, to react probe as chip hybridization.
<400>6
ccgggaagactgggtcct
<210>7
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, to react probe as chip hybridization.
<400>7
cccactctatgcccggcca
<210>8
<211>18
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, to react probe as chip hybridization.
<400>8
ccgcgagatcactagccg
<210>9
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, to react probe as chip hybridization.
<400>9
aatcgctggggtgaccggg
<210>10
<211>18
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, to react probe as chip hybridization.
<400>10
ccgctcaatgcccggaaa
<210>11
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, to react probe as chip hybridization.
<400>11
accgggtcctttccattgg
<210>12
<211>15
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, to react probe as chip hybridization.
<400>12
atcaacccgctcaat
<210>13
<211>16
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, to react probe as chip hybridization.
<400>13
gatcaacccgctcaat
<210>14
<211>18
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, to react probe as chip hybridization.
<400>14
tactgcctgatagggtgc
<210>15
<211>23
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, to be used as the PCR primer.
<400>15
tcatggtgcacggtctacgagac
<210>16
<211>23
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, to be used as the PCR primer.
<400>16
ctagccatggcgttagtatgagt
<210>17
<211>23
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, to be used as the PCR primer.
<400>17
actcgcaagcaccctatcaggca
Claims (5)
1, a kind of dna microarray chip, adopt the dna microarray chip technology, HCV is carried out genotype tests, it is characterized in that one group of HCV different genotype specific oligonucleotide probe, point is on carrier, make the dna microarray chip, be equipped with RT-PCR primer and other component again, form complete test kit.
2, dna microarray chip according to claim 1, its feature also be wherein the to increase sequence of specificity RT-PCR primer of HCV gene is respectively:
RT primer 5 '-TCATGGTGCACGGTCTACGAGAC-3 '
Upstream primer 5 '-CTAGCCATGGCGTTAGTATGAGT-3 '
Downstream primer 5 '-ACTCGCAAGCACCCTATCAGGCA-3 '.
3, dna microarray chip according to claim 1, its feature also is wherein to adopt 14 typing probes, and point is made dna microarray on carrier, be used for and sample amplification template hybridization, and the sequence of 14 oligonucleotide probes is respectively:
H1??5’-ATGCCTGGAGATTTGGG-3’
H2??5’-GCGAGACTGCTAGCCG-3’
H3??5’-CGAGACCGCTAGCCGAG-3’
H4??5’-GTAGCGTTGGGTTGCGAA-3’
H5??5’-TCCTTTCTTGGATAAACCCA-3’
H6??5’-CCGGGAAGACTGGGTCCT-3’
H7??5’-CCCACTCTATGCCCGGCCA-3’
H8??5’-CCGCGAGATCACTAGCCG-3’
H9??5’-AATCGCTGGGGTGACCGGG-3’
H10?5’-CCGCTCAATGCCCGGAAA-3’
H11?5’-ACCGGGTCCTTTCCATTGG-3’
H12?5’-ATCAACCCGCTCAAT-3’
H13?5’-GATCAACCCGCTCAAT-3’
H14?5’-TACTGCCTGATAGGGTGC-3’。
4, dna microarray chip according to claim 1, its feature are that also the carrier of oligonucleotide probe point sample wherein can be the carrier that slide, nylon membrane or other can the set probes.
5, dna microarray chip according to claim 1 is characterized in that also being to be mainly used in the hepatitis C virus in the blood sample is carried out the somatotype detection.
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| CN200810198010A CN101660002A (en) | 2008-08-27 | 2008-08-27 | HCV genotyping DNA microarray chip |
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| CN200810198010A CN101660002A (en) | 2008-08-27 | 2008-08-27 | HCV genotyping DNA microarray chip |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559930A (en) * | 2012-01-16 | 2012-07-11 | 中山大学达安基因股份有限公司 | Kit of detecting hepahtis C virus by fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction) |
| CN103710464A (en) * | 2013-12-30 | 2014-04-09 | 湖南圣湘生物科技有限公司 | HCV (Hepatitis c virus) genotype detection kit |
| CN103966363A (en) * | 2014-05-16 | 2014-08-06 | 福州泰普生物科学有限公司 | Hepatitis C virus (HCV) genotyping kit |
| CN104263851A (en) * | 2014-07-15 | 2015-01-07 | 周有良 | Hepatitis C virus genetic typing chip and typing method |
| CN108018376A (en) * | 2016-10-31 | 2018-05-11 | 深圳华大基因股份有限公司 | A kind of probe and method for hepatitis C virus parting and drug resistance site primer |
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2008
- 2008-08-27 CN CN200810198010A patent/CN101660002A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559930A (en) * | 2012-01-16 | 2012-07-11 | 中山大学达安基因股份有限公司 | Kit of detecting hepahtis C virus by fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction) |
| CN103710464A (en) * | 2013-12-30 | 2014-04-09 | 湖南圣湘生物科技有限公司 | HCV (Hepatitis c virus) genotype detection kit |
| CN103966363A (en) * | 2014-05-16 | 2014-08-06 | 福州泰普生物科学有限公司 | Hepatitis C virus (HCV) genotyping kit |
| CN103966363B (en) * | 2014-05-16 | 2016-05-04 | 福州泰普生物科学有限公司 | A kind of kit of HCV gene typing |
| CN104263851A (en) * | 2014-07-15 | 2015-01-07 | 周有良 | Hepatitis C virus genetic typing chip and typing method |
| CN104263851B (en) * | 2014-07-15 | 2017-01-11 | 周有良 | Hepatitis C virus genetic typing chip and typing method |
| CN108018376A (en) * | 2016-10-31 | 2018-05-11 | 深圳华大基因股份有限公司 | A kind of probe and method for hepatitis C virus parting and drug resistance site primer |
| CN108018376B (en) * | 2016-10-31 | 2020-11-03 | 深圳华大因源医药科技有限公司 | Probe and method for hepatitis C virus typing and drug-resistant site detection |
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