CN1238521C - Determination of a section of man-shaped mycoplasma DNA sequence (4) specificity and its use - Google Patents
Determination of a section of man-shaped mycoplasma DNA sequence (4) specificity and its use Download PDFInfo
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- CN1238521C CN1238521C CN 02133331 CN02133331A CN1238521C CN 1238521 C CN1238521 C CN 1238521C CN 02133331 CN02133331 CN 02133331 CN 02133331 A CN02133331 A CN 02133331A CN 1238521 C CN1238521 C CN 1238521C
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Abstract
The present invention relates to determination for the specificity of a section of human mycoplasma DNA sequence (4), and the application thereof, which belongs to the technical field of biomedicine. In the present invention, the data of the gene database is searched and analyzed to determine the specificity of a section of human mycoplasma DNA sequence and a PCR amplification primer, wherein the length of the sequence is 202 bp; the sequence is obtained by PCR amplification and clone of the human genital tract human mycoplasma infection specimen, and the sequence is proved to be consistent to the preestimated sequence; the specificity of the sequence to human mycoplasma is verified by the analysis of a bioinformatics method and the analysis of gene chip hybridization. The gene chip diagnosis probe and the PCR amplification and detection sequence of the present invention can be used for human mycoplasma diagnosis, and the used method of the clinical detection technology established by the present invention has the advantages of quickness, accuracy, convenient operation, low cost, etc.
Description
Technical field:
The present invention relates to the application of one section mycoplasma hominis (Mycoplasma hominis) dna sequence dna (4), belong to field of biomedicine technology.
Background technology:
Mycoplasma hominis is the pathogen that a kind of human reproduction's device often infects, and can cause many urogenital tract diseases, and 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 mycoplasma hominis 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 mycoplasma hominis.
Technical scheme of the present invention is: the present invention utilizes existing method to determine one section mycoplasma hominis specific DNA sequence (4) and the PCR primer thereof of length for 202bp, carries out pcr amplification with primer, and its sequence is as follows:
ttcacctgtc?acaccatcaa?atctatatgc?gttgaagttt?ggatcacgtg?ggtcatcaaa 60
gctcttaact?gtaactttaa?ttcttgggtc?tagttcagaa?aaaacgtttt?taactttttc 120
tcatgcatta?atgtatagtt?gtggagcatt?taaatatcta?aatcctactt?caaattcaat 180
tgtttgtttt?tcgggatcgg?ta 202
Primer sequence is:
1.5’-taccgatcccgaaaaacaaa-3’
2.5’-ttcacctgtcacaccatcaaa-3’
The specific DNA sequence of above-mentioned mycoplasma hominis (4) is used to diagnose the probe sequence of the gene chip of mycoplasma hominis, and carries out pcr amplification by above-mentioned primer sequence, hybridizes behind the mark to 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 are continuous to be Blast Search Results in the gene database.
Fig. 5 is the gene chip hybridization result.
Embodiment:
Be keyword at first with the mycoplasma hominis, the nucleotide sequence of search mycoplasma hominis 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 1Kb.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 with estimate that big or small consistent (see Fig. 1, sequence number is among the figure: 1. standard molecular weight DNA, 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 clinical negative sample does not have amplified production; 3. clinical positive sample, amplified production is very pure, and size about 200 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 55, to 256,202bp is a cloned sequence altogether, all the other sequences are the cloning vector sequence), sequence is 202bp, estimates sequence by the comparison of Blast software, show with the sequence of estimating is consistent and (see Fig. 3, remove the 90th among the figure and change over a by g, the 104th and 132 are changed over outside the c by a, and all the other are all consistent with the expectation sequence), show that this sequence can be can be used for mycoplasma hominis PCR 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 mycoplasma hominis, and do not have homology (to see that Fig. 4, Fig. 4 are continuous with other species sequence, search result shows among the figure, have only plasmid dna sequence and this sequence homology of mycoplasma hominis, and be to match fully.And other sequence has only the short sequence about 40 characters, because these sequences do not have primer to match therewith, so can not be detected in detection).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 four 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 mycoplasma hominis.
The present invention can be used for the clinical detection that PCR method or method for gene chip carry out mycoplasma hominis.Carry out pcr amplification with sequence provided by the invention, obtain product according to PCR method and whether occur judging whether to be human-like mycoplasma infection 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 Mh-4 sequence table
The specification sheets sequence table
SEQUENCE?LISTING
＜110〉basic biochip development corporation, Ltd. is wrapped up in Kunming
＜120〉one section mycoplasma hominis dna sequence dna (4) is specific determines and application
<130>
<140>02133331.9
<141>2002-06-20
<160>1
<170>PatentIn?version?3.1
<210>1
<211>202
<212>DNA
＜213〉mycoplasma hominis (Mycoplasma horninis)
<220>
<221>gene
<222>(1)..(202)
<223>
<400>1
ttcacctgtc?acaccatcaa?atctatatgc?gttgaagttt?ggatcacgtg?ggtcatcaaa 60
gctcttaact?gtaactttaa?ttcttgggtc?tagttcagaa?aaaacgtttt?taactttttc 120
tcatgcatta?atgtatagtt?gtggagcatt?taaatatcta?aatcctactt?caaattcaat 180
tgtttgtttt?tcgggatcgg?ta 202
Claims (1)
1, the application of one section mycoplasma hominis dna sequence dna (4), the length of this sequence is 202bp, sequence is:
ttcacctgtc?acaccatcaa?atctatatgc?gttgaagttt?ggatcacgtg?ggtcatcaaa 60
gctcttaact?gtaactttaa?ttcttgggtc?tagttcagaa?aaaacgtttt?taactttttc 120
tcatgcatta?atgtatagtt?gtggagcatt?taaatatcta?aatcctactt?caaattcaat 180
tgtttgtttt?tcgggatcgg?ta 202
PCR thing sequence is:
1.5’-taccgatcccgaaaaacaaa-3’
2.5’-ttcacctgtcacaccatcaaa-3’
It is characterized in that: this sequence is used to diagnose the probe sequence of the gene chip of mycoplasma hominis, and carries out pcr amplification by above-mentioned primer sequence, hybridizes behind the mark to detect or be directly used in the PCR diagnosis.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02133331 CN1238521C (en) | 2002-06-20 | 2002-06-20 | Determination of a section of man-shaped mycoplasma DNA sequence (4) specificity and its use |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02133331 CN1238521C (en) | 2002-06-20 | 2002-06-20 | Determination of a section of man-shaped mycoplasma DNA sequence (4) specificity and its use |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1492052A CN1492052A (en) | 2004-04-28 |
| CN1238521C true CN1238521C (en) | 2006-01-25 |
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| CN 02133331 Expired - Fee Related CN1238521C (en) | 2002-06-20 | 2002-06-20 | Determination of a section of man-shaped mycoplasma DNA sequence (4) specificity and its use |
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Owner name: KUNMING HUANJI BIO-CHIP INDUSTRIAL CO., LTD. Free format text: FORMER OWNER: KUNMING YUNDA BIOLOGY + CHEMICAL SCIENCE + TECHNOLOGY CO., LTD. Effective date: 20120813 |
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Free format text: CORRECT: ADDRESS; FROM: 650118 KUNMING, YUNNAN PROVINCE TO: 650000 KUNMING, YUNNAN PROVINCE |
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Effective date of registration: 20120813 Address after: 650000, Yunnan, Kunming Province opened the district information industry base, Yunnan returnees Pioneer Park 4, 3, building 301, 302 Patentee after: Kunming Huanji Biological Chip Industry Co., Ltd. Address before: 650118, No. 9, North District, Kunming hi tech Zone, Yunnan, Yunnan Province University Science Park A2-507 Patentee before: Kunming Yunda Biology & Chemical Science & Technology Co., Ltd. |
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Granted publication date: 20060125 Termination date: 20120620 |