CN100365124C - Determination and application of hepatitis C virus specific cDNA sequence - Google Patents
Determination and application of hepatitis C virus specific cDNA sequence Download PDFInfo
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- CN100365124C CN100365124C CNB2005100110873A CN200510011087A CN100365124C CN 100365124 C CN100365124 C CN 100365124C CN B2005100110873 A CNB2005100110873 A CN B2005100110873A CN 200510011087 A CN200510011087 A CN 200510011087A CN 100365124 C CN100365124 C CN 100365124C
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Abstract
The present invention relates to the determination and the application of a cDNA sequence specific to hepatitis C viruses, which belongs to the technical field of biomedicine. In the present invention, the cDNA sequence specific to hepatitis C viruses and a PCR amplification primer are determined through the search and the analysis of gene data base information, and the length of the sequence is 222 bp. The sequence is obtained by the PCR amplification and the cloning of a human genital tract specimen infected by hepatitis C viruses, and sequence test proves that the sequence is consistent with the expected sequence. The sequence is analyzed by bioinformatical methods and gene chip hybridization, and results confirm that the sequence is specific to hepatitis C viruses. The cDNA sequence specific to hepatitis C viruses can be used as a probe sequence of a gene chip for diagnosing hepatitis C viruses; through the PCR amplification of the primer sequence and after the primer sequence is labeled, the cDNA sequence is used for detecting hybridization or directly used for PCR diagnosis. A method in a clinical detection technology established by the present invention has the advantages of rapidness, exactness, easy operation, low cost, etc.
Description
Technical field:
The present invention relates to the application of one section hepatitis C virus (Hepatitis C virus) specificity cDNA sequence, belong to field of biomedicine technology.
Background technology:
Hepatitis C is a kind of serious communicate illness that is caused by blood transfusion or blood products, can cause serious consequences such as hepar damnification and liver cancer, diagnosis accurately is to carry out the foundation of treatment in time and reliably, can only mainly rely on serology and immunological method to the diagnosis of hepatitis C at present.PCR and gene chip diagnosis are a kind of rapid and precise detection methods.And the diagnostic method of this class depends on the special nucleotide sequence of acquisition.
Summary of the invention:
The invention reside in by information biology and Protocols in Molecular Biology, determined one section hepatitis C virus specific nucleotide sequence and PCR primer thereof.This sequence can be used for setting up gene chip with corresponding PCR primer and PCR method is carried out the diagnosis of hepatitis C virus.
Technical scheme of the present invention is: the present invention has determined according to a conventional method that by ncbi database length is special cDNA sequence of one section hepatitis C virus and the PCR primer thereof of 222bp, carries out pcr amplification with primer, and its sequence is as follows:
caagcaccct atcaggcagt accacaaggc ctttcgcgac ccaacactac tcggctagtg 60
atctcgcggg ggcacgccca aatttctggg tattgagcgg gttattccaa gaaaggaccc 120
ggtcacccca gcgattccgg tgtactcacc ggttccgcag accactatgg ctctcccggg 180
agggggggtc ctggaggctg cacgacactc gtactaacgc ca 222
Primer sequence is:
1.5’-caagcaccctatctggcagt-3’
2.5’-tggcgttagtacgagtgtcg-3’
The special cDNA sequence of above-mentioned hepatitis C virus can be used as the probe of gene chip diagnosis, the detection kit that reaches the clinical hepatitis C of pcr amplification and related products exploitation or is directly used in the application that PCR diagnoses.
The employed method of setting up with the present invention of clinical detection technique has quick, accurate, easy and simple to handle, low cost and other advantages.
Description of drawings:
Fig. 1 is the pcr amplification collection of illustrative plates of clinical samples.1 road is the standard molecular weight mark; 2,3,4, negative sample does not have amplified production; 5 positive sample, the amplified production size is consistent with expectation.
Fig. 2 is a sequencer map.Wherein Fig. 2 is the sequence map of measuring.
Fig. 3 is with the sequencing result and the comparative analysis of estimating sequence of Blast software to pcr amplification product.
Fig. 4-A, Fig. 4-B, Fig. 4-C, Fig. 4-D are Blast Search Results in the gene database.
Fig. 5 is the gene chip hybridization result.
Embodiment:
1, with the hepatitis C virus keyword at first, the nucleotide sequence of search hepatitis C virus in gene database.Obtain the closer nucleotide sequence of several relations altogether, with these nucleotide sequences with the nucleotide sequence (nr) in the online use software of the online Blast of the NCBI comparison gene database, discharge nonspecific nucleotide sequence, obtained the specific nucleotide sequence of hepatitis C.
2, with this section sequences Design surplus 30 to the PCR primer, extract RNA and reverse transcription becomes cDNA with clinical samples, carry out pcr amplification, screen a pair of sequence and the primer that is fit to carry out pcr amplification, product that pcr amplification obtains and the big or small consistent (see figure 1) of expectation; Adopt clinical negative sample as template, do not have amplified production; Adopt clinical positive sample as template, can obtain very pure amplified production, size about 222 is with the big or small consistent (see figure 1) of expectation.
3, pcr amplification product is cloned with T-vector, and checks order (sequencing result is seen Fig. 2), compares by Blast software and estimate sequence, the consistent (see figure 3) of sequence of demonstration and expectation; Sequence is with the DNA/cDNA sequence of the Blast muca gene database of NCBI website, and this sequence is only consistent with the cDNA sequence of hepatitis C virus, and does not have the homology (see figure 4) with other species sequence.
4, with behind this sequence mark as probe with contain the gene chip hybridization of 15 gene locuss of 3 kinds of blood-borne class disease infection pathogens, have only sequence of the present invention that hybridization signal is arranged therewith, and other all sequences amixia signal (see figure 5) therewith, each detects gene locus among the figure four repetitions, and chip has 15 different gene locuss and 3 control site.Results of hybridization shows to have only this sequence that special signal is arranged, and other equal amixia signal shows that this sequence is the distinguished sequence of hepatitis C virus.
The contriver develops or is directly used in the application of PCR diagnosis with the special cDNA sequence of above-mentioned hepatitis C virus as the probe of gene chip diagnosis, the detection kit that reaches the clinical hepatitis C of pcr amplification and related products.
The hepatitis C blood sample that 96 examples that the contriver uses Yunnan Province San Jia front three hospital to provide are made a definite diagnosis through the various clinical detection method is verified total positives coincidence rate 100% with the method that gene chip detects to this sequence of the present invention; Adopt 20 kinds of pathogens and micro-biological samples beyond the hepatitis C virus this sequence to be verified total negative match-rate 100% with the method that gene chip detects.
Practical application shows: the employed method of setting up with the present invention of clinical detection technique truly has quick, accurate, easy and simple to handle, low cost and other advantages.
SEQUENCE LISTING
＜110〉Yunnan University
＜120〉one section hepatitis C virus detects determining of cDNA sequence and application thereof
<130>
<140>
<141>
<160>1
<170>PatentIn version 3.1
<210>1
<211>222
<212>DNA
＜213〉hepatitis C virus (Hepatitis C virus)
<220>
<221>gene
<222>(1)..(222)
<223>
<400>1
caagcaccct atcaggcagt accacaaggc ctttcgcgac ccaacactac tcggctagtg 60
atctcgcggg ggcacgccca aatttctggg tattgagcgg gttattccaa gaaaggaccc 120
ggtcacccca gcgattccgg tgtactcacc ggttccgcag accactatgg ctctcccggg 180
agggggggtc ctggaggctg cacgacactc gtactaacgc ca 222
Claims (1)
1. one section special cDNA sequence of hepatitis C virus detects the PCR of hepatitis c virus gene or the application in the chip related products in preparation, and wherein, this sequence length is that 222bp, cDNA sequence are:
caagcaccct atcaggcagt accacaaggc ctttcgcgac ccaacactac tcggctagtg 60
atctcgcggg ggcacgccca aatttctggg tattgagcgg gttattccaa gaaaggaccc 120
ggtcacccca gcgattccgg tgtactcacc ggttccgcag accactatgg ctctcccggg 180
agggggggtc ctggaggctg cacgacactc gtactaacgc ca 222
Primer sequence is:
Primer 1:5 '-caagcaccctatctggcagt-3 '
Primer 2: 5 '-tggcgttagtacgagtgtcg-3 '.
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| Application Number | Priority Date | Filing Date | Title |
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| CNB2005100110873A CN100365124C (en) | 2005-10-27 | 2005-10-27 | Determination and application of hepatitis C virus specific cDNA sequence |
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| CNB2005100110873A CN100365124C (en) | 2005-10-27 | 2005-10-27 | Determination and application of hepatitis C virus specific cDNA sequence |
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| CN1789423A CN1789423A (en) | 2006-06-21 |
| CN100365124C true CN100365124C (en) | 2008-01-30 |
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1087915A (en) * | 1992-07-16 | 1994-06-15 | 东燃株式会社 | Be used for the antigen peptide of hepatitis C virus classification, contain the medicine box of described peptide and the method for classifying with described peptide |
| JPH08187096A (en) * | 1994-11-18 | 1996-07-23 | Tonen Corp | Judging method of genotype of c-type hepatitis virus and pcr primer set used in the method |
| WO1999067376A1 (en) * | 1998-06-25 | 1999-12-29 | Institut Pasteur | Exhaustive analysis of viral protein interactions by two-hybrid screens and selection of correctly folded viral interacting polypeptides |
| WO2000075338A2 (en) * | 1999-06-04 | 2000-12-14 | The Government Of The United States Of America As Represented By The Secretary, Department Of Health And Human Services | CLONED GENONE OF INFECTIOUS HEPATITIS C VIRUS OF GENOTYPE 2a AND USES THEREOF |
| WO2003077729A2 (en) * | 2002-03-11 | 2003-09-25 | Carol Holland-Staley | Methods and compositions for identifying and characterizing hepatitis c |
| CN1547588A (en) * | 2001-01-11 | 2004-11-17 | Purified hepatitis C virus envelope proteins for diagnostic and therapeutic use | |
| CN1622828A (en) * | 2001-12-18 | 2005-06-01 | 基因创新有限公司 | Purified hepatitis c virus envelope proteins for diagnostic and therapeutic use |
-
2005
- 2005-10-27 CN CNB2005100110873A patent/CN100365124C/en not_active Expired - Fee Related
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1087915A (en) * | 1992-07-16 | 1994-06-15 | 东燃株式会社 | Be used for the antigen peptide of hepatitis C virus classification, contain the medicine box of described peptide and the method for classifying with described peptide |
| JPH08187096A (en) * | 1994-11-18 | 1996-07-23 | Tonen Corp | Judging method of genotype of c-type hepatitis virus and pcr primer set used in the method |
| WO1999067376A1 (en) * | 1998-06-25 | 1999-12-29 | Institut Pasteur | Exhaustive analysis of viral protein interactions by two-hybrid screens and selection of correctly folded viral interacting polypeptides |
| WO2000075338A2 (en) * | 1999-06-04 | 2000-12-14 | The Government Of The United States Of America As Represented By The Secretary, Department Of Health And Human Services | CLONED GENONE OF INFECTIOUS HEPATITIS C VIRUS OF GENOTYPE 2a AND USES THEREOF |
| CN1547588A (en) * | 2001-01-11 | 2004-11-17 | Purified hepatitis C virus envelope proteins for diagnostic and therapeutic use | |
| CN1622828A (en) * | 2001-12-18 | 2005-06-01 | 基因创新有限公司 | Purified hepatitis c virus envelope proteins for diagnostic and therapeutic use |
| WO2003077729A2 (en) * | 2002-03-11 | 2003-09-25 | Carol Holland-Staley | Methods and compositions for identifying and characterizing hepatitis c |
Non-Patent Citations (2)
| Title |
|---|
| 5'非翻译区在丙型肝炎病毒基因诊断和基因治疗中的应用. 李勇年等.中华传染病杂志，第16卷第4期. 1998 * |
| DQ 073355. Madhavi.C et al.NCBI. 2005 * |
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| CN1789423A (en) | 2006-06-21 |
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