CN1495266A - Definition of human papillomavirus-6 DNA squence (5) speicificity and its application - Google Patents
Definition of human papillomavirus-6 DNA squence (5) speicificity and its application Download PDFInfo
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- CN1495266A CN1495266A CNA021334072A CN02133407A CN1495266A CN 1495266 A CN1495266 A CN 1495266A CN A021334072 A CNA021334072 A CN A021334072A CN 02133407 A CN02133407 A CN 02133407A CN 1495266 A CN1495266 A CN 1495266A
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- sequence
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- human papillomavirus
- pcr amplification
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Abstract
The present invention relates to the definition of specificity of a tract of human papillomavirus-6 DNA sequence (5) and its application. It utilizes the search and analysis of gene data base material to define the specificity of a tract of human papillomavirus-6 DNA sequence (5) and PCR amplification primer, and the length of said sequence is 214 bp. It utilizes the PCR amplification and clone of human genital duct human papillomavirus-6 infection specimen to obtain said sequence, and the sequencing shows that the sequence is identical to the predefined sequence, and the results of the biological informatic method analysis and gene chip hybridization analysis show the specificity of said sequence. Said invention can be used as probe of gene chip diagnosis and PCR amplification detection sequence for diagnosing human papillomavirus-6.
Description
Technical field:
The present invention relates to the specific definite and application of one section human-like papilloma virus (human papillomavirua)-6DNA sequence (5), belong to field of biomedicine technology.
Background technology:
Human-like papilloma virus (human papillomavirua) the-the 6th, the virus that a kind of urinary system often infects.Diagnosis accurately is to carry out the foundation of treatment immediately and reliably, can only diagnose according to clinical indication 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 human-like papilloma virus (human papillomavirua)-6 specific nucleotide sequences 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 human-like papilloma virus (human papillomavirua)-6.
Technical scheme of the present invention is: the present invention utilizes existing method to determine one section human-like papilloma virus (human papillomavirua)-6 specific DNA sequence (5) and the PCR primers thereof of length for 214bp, carries out pcr amplification with primer, and its sequence is as follows:
caaaagggaa?tggatttgga?aatgtaaatg?ttgttactct?agtatgtaaa?tacttatatt?60
tatcttcttt?agtaatatct?atgttggacg?ttactagcag?aggtggacat?ttaattaatg?120
tcaatgcttt?atgctttctg?tcaatactca?taggattacc?atctaacaaa?tttctcatat?180
atgtatccat?atatatccaa?catggctgtg?ttgc 214
Primer sequence is:
1.5’-gcaacacagccatgttggat-3’
2.5’-caaaagggaatggatttgga-3’
The specific DNA sequence (5) of above-mentioned human-like papilloma virus (human papillomavirua)-6 is used to diagnose the probe sequence of the gene chip of human-like papilloma virus-6, and carry out pcr amplification by above-mentioned primer sequence, hybridize behind the mark and detect or be directly used in the PCR diagnosis.
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.
Fig. 2, Fig. 2 are continuous to be sequencer map.Wherein Fig. 2 be sequence, Fig. 2 of measuring continuous be the primitive sequencer collection of illustrative plates.
Fig. 3 is with the sequencing result and the comparative analysis of estimating sequence of Blast software to pcr amplification product.
Fig. 4, Fig. 4 continue (1), Fig. 4 continuous (2), Fig. 4 continuous (3) is a Blast Search Results in the gene database.
Fig. 5 is the gene chip hybridization result.
Embodiment:
Be keyword at first with human-like papilloma virus (human papillomavirua)-6, the nucleotide sequence of the human-like papilloma virus of search (human papillomavirua)-6 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 6Kb.With this section sequences Design surplus 20 to the PCR primer, extract DNA with clinical samples, carry out pcr amplification, screen a pair of sequence and the primer that is fit to carry out pcr amplification, the product that pcr amplification obtains is consistent with the expectation size, and (see Fig. 1, sequence number 1 is standard molecular weight DNA among the figure, the molecular weight size is respectively from top to bottom: 622,527,404,309,242/238,217,201,190,180,160+160,147+147,123; 2,4,5 is clinical negative sample, does not have amplified production; 3 is clinical positive sample, and amplified production is very pure, and size is about 210, and is consistent with the expectation size.The collection of illustrative plates result shows with the special amplification clinical sample of the energy of the primer among the present invention), clone with T-vector then, and the order-checking (sequencer address is seen Fig. 2, Fig. 2 is continuous, among the figure since 58, to 271,214bp is a cloned sequence altogether, all the other sequences are the cloning vector sequence), sequence is 214 to estimate sequence by Blast software comparison, shows that sequence with expectation is consistent (to see Fig. 3, remove the 62nd among the figure and change over c by a, the 71st changes over g by a, and the 89th is changed over outside the t by c, and all the other are all consistent with the expectation sequence), show that this sequence can be can be used for human-like papilloma virus (human papillomavirua)-6PCR and detect by specific amplification.Sequence is with DNA (the comprising cDNA) sequence of the Blast muca gene database of NCBI website, this sequence is only consistent with the sequence of human-like papilloma virus (human papillomavirua)-6, and do not have homology [to see that Fig. 4, Fig. 4 continue (1), Fig. 4 continuous (2), Fig. 4 continuous (3) with other species sequence, search result shows among the figure, have only plasmid dna sequence and this sequence homology of human-like papilloma virus (human papillomavirua)-6, and be to match fully.And other sequence, owing to do not have primer to match with it, so in detection, can not be detected].With behind this sequence mark as probe with contain the gene chip hybridization of 45 gene locuss of 9 kinds of kinds of genitourinary infection pathogen, have only sequence of the present invention that hybridization signal is arranged therewith, and other all sequences therewith the amixia signal (see Fig. 5, be 4 repetitions among the figure, each repeats to contain 9 kinds of urogenital infections disease pathogen things totally 45 different sites and 4 control site.Results of hybridization shows to have only this sequence that special signal is arranged, and other equal amixia signal), show that this sequence is the distinguished sequence of human-like papilloma virus (human papillomavirua)-6.
The present invention can be used for the clinical detection that PCR method or method for gene chip carry out human-like papilloma virus (humanpapillomavirua)-6.Carry out pcr amplification with sequence provided by the invention, obtain product according to PCR method and whether occur judging whether to infect into human-like papilloma virus (human papillomavirua)-6 with size.An also available sequence is a probe, as a detection site in the gene chip, then with the amplification of the PCR primer sequence among the present invention clinical samples, and hybridizes detection behind the mark.
The specification sheets sequence table
＜110〉Kunming Huanji Biochip Development Co., Ltd
＜120〉one section human-like papilloma virus-6DNA sequence (5) is specific definite
<140>
<141>
<160>1
<210>1
<211>214
<212>DNA
＜213〉human-like papilloma virus (human papillomavirua)-6
<220>
<221>gene
<222>(1)…(214)
<400>1
caaaagggaa?tggatttgga?aatgtaaatg?ttgttactct?agtatgtaaa?tacttatatt?60
tatcttcttt?agtaatatct?atgttggacg?ttactagcag?aggtggacat?ttaattaatg?120
tcaatgcttt?atgctttctg?tcaatactca?taggattacc?atctaacaaa?tttctcatat?180
atgtatccat?atatatccaa?catggctgtg?ttgc 214
Claims (2)
1. one section human-like papilloma virus-6DNA sequence (5) is specific definite, and the length that it is characterized in that this sequence is 214bp, carries out pcr amplification with primer, and its sequence is as follows:
caaaagggaa?tggatttgga?aatgtaaatg?ttgttactct?agtatgtaaa?tacttatatt?60
tatcttcttt?agtaatatct?atgttggacg?ttactagcag?aggtggacat?ttaattaatg?120
tcaatgcttt?atgctttctg?tcaatactca?taggattacc?atctaacaaa?tttctcatat?180
atgtatccat?atatatccaa?catggctgtg?ttgc 214
Primer sequence is:
1.5’-gcaacacagccatgttggat-3’
2.5’-caaaagggaatggatttgga-3’
2. the specific application of one section human-like papilloma virus-6 specific DNA sequence (5), it is characterized in that this sequence is used to diagnose the probe sequence of the gene chip of human-like papilloma virus-6, and carry out pcr amplification by above-mentioned primer sequence, hybridize behind the mark and detect or be directly used in the PCR diagnosis.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA021334072A CN1495266A (en) | 2002-07-02 | 2002-07-02 | Definition of human papillomavirus-6 DNA squence (5) speicificity and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA021334072A CN1495266A (en) | 2002-07-02 | 2002-07-02 | Definition of human papillomavirus-6 DNA squence (5) speicificity and its application |
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| Publication Number | Publication Date |
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| CN1495266A true CN1495266A (en) | 2004-05-12 |
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| CNA021334072A Pending CN1495266A (en) | 2002-07-02 | 2002-07-02 | Definition of human papillomavirus-6 DNA squence (5) speicificity and its application |
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2002
- 2002-07-02 CN CNA021334072A patent/CN1495266A/en active Pending
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