CN1267564C - Determination of one section urea degraded mycoplasmos DNA sequence (5) specificity and its application - Google Patents

Abstract

The present invention relates to specificity determination of a urea degraded mycoplasma DNA sequence (5) and the use thereof, which belongs to the field of biomedical technology. The specificity of a section of urea degraded mycoplasma DNA sequence (5) and a PCR amplification primer are determined by the data search and analysis of a gene data base of the present invention, and the sequence length is 288 bp. The urea degraded mycoplasma infection specimen of a human being reproduction duct is processed by PCR amplification and cloned to obtain the sequence, and a test proves that the sequence is coincident with the estimated sequence. Results of the bioinformatics method analysis and gene chip hybridization analysis prove that the sequence has specificity for the urea degraded mycoplasma. The present invention used as probes for diagnosing gene chips and sequences for detecting PCR amplification is used for diagnosing the urea degraded mycoplasma. The method used by the clinical detection technology established by the present invention has the advantages of quickness, accuracy, easy operation, low cost, etc.

Description

The specific DNA sequences of segment of ureaplasma and application thereof

Technical field:

The present invention relates to specific DNA sequences and the application thereof of segment of ureaplasma (Ureaplasma urealyticum), belong to field of biomedicine technology.

Background technology:

Ureaplasma 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 segment of ureaplasma 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 detection of ureaplasma.

Technical scheme of the present invention is: the present invention utilizes existing method to determine segment of ureaplasma specific DNA sequence and the PCR primer thereof of length for 288bp, carries out pcr amplification with primer, and its sequence is as follows:

caccaccagc?accttctgta?tgataagcat?gaattgttcg?ccctttcata?gctgcaattg 60

tatgttctac?aaatccagct?tcatttaatg?tatctgtatg?aatagcaaca?gctacatcag 120

ttttatcagc?aactgttaat?gctaaatcaa?tcgcatttcc?tgttgcccct?cagtcttcat 180

ggattttaag?tccacaagct?ccagcagcaa?tttgctcaaa?aattggatct?tccatacctt 240

gacctttagc?taaaaaaccg?gcattaattg?gtaatccatc?agctgctt 288

Primer sequence is:

A.5’-aagcagctgatggattaccaa-3’

B.5’-caccaccagcaccttctgta-3’

The specific DNA sequence of above-mentioned ureaplasma is used to detect the probe sequence of the gene chip of ureaplasma, and carries out pcr amplification by above-mentioned primer sequence, hybridizes behind the mark to detect or be directly used in PCR and detect.

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) is a Blast Search Results in the gene database.

Fig. 5 is the gene chip hybridization result.

Embodiment:

Be keyword at first with the ureaplasma, the nucleotide sequence of search ureaplasma 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 290 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 345,288bp is a cloned sequence altogether, all the other sequences are the cloning vector sequence), sequence is 288bp, estimate sequence by the comparison of Blast software, showing that sequence with expectation is consistent (sees Fig. 3, among the figure the 23rd, 29,32,83,101,109,179,209,221,254 and 263 become g by a, the 38th, 53,68,191 and 200 become a by t, the the 41st and 215 becomes t by c, the 50th, 77,125,203,206 become t by a, the 182nd, 207 become a by g, and the 185th becomes c by t, the 260th becomes t by g, all the other are all consistent with the expectation sequence), show that this sequence can be can be used for ureaplasma 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 ureaplasma, and do not have homology [to see that Fig. 4, Fig. 4 continue (1), Fig. 4 continuous (2) with other species sequence, search result shows among the figure, have only plasmid dna sequence and this sequence homology of ureaplasma, 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 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 ureaplasma.

The present invention can be used for the clinical detection that PCR method or method for gene chip carry out ureaplasma.Carry out pcr amplification with sequence provided by the invention, obtain product according to PCR method and whether occur judging whether to infect into ureaplasma 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 UJ-5 sequence table

The specification sheets sequence table

SEQUENCE?LISTING

＜110〉basic biochip development corporation, Ltd. is wrapped up in Kunming

＜120〉segment of ureaplasma DNA sequence (5) is specific determines and application

<130>

<140>02133341.6

<141>2002-06-20

<160>1

<170>PatentIn?version?3.1

<210>1

<211>288

<212>DNA

＜213〉ureaplasma (Ureaplasma urealyticum)

<220>

<221>gene

<222>(1)..(288)

<223>

<400>1

caccaccagc?accttctgta?tgataagcat?gaattgttcg?ccctttcata?gctgcaattg 60

tatgttctac?aaatccagct?tcatttaatg?tatctgtatg?aatagcaaca?gctacatcag 120

ttttatcagc?aactgttaat?gctaaatcaa?tcgcatttcc?tgttgcccct?cagtcttcat 180

ggattttaag?tccacaagct?ccagcagcaa?tttgctcaaa?aattggatct?tccatacctt 240

gacctttagc?taaaaaaccg?gcattaattg?gtaatccatc?agctgctt 288

Claims (2)

1, the specific DNA sequences of segment of ureaplasma is characterized in that the length of this sequence is 288bp, and sequence is as follows:

caccaccagc?accttctgta?tgataagcat?gaattgttcg?ccctttcata?gctgcaattg 60

tatgttctac?aaatccagct?tcatttaatg?tatctgtatg?aatagcaaca?gctacatcag 120

ttttatcagc?aactgttaat?gctaaatcaa?tcgcatttcc?tgttgcccct?cagtcttcat 180

ggattttaag?tccacaagct?ccagcagcaa?tttgctcaaa?aattggatct?tccatacctt 240

gacctttagc?taaaaaaccg?gcattaattg?gtaatccatc?agctgctt 288

Amplification PCR primer is:

A.5’-aagcagctgatggattaccaa-3’

B.5’-caccaccagcaccttctgta-3’

2, the application of the specific DNA sequences of the said segment of ureaplasma of claim 1, it is characterized in that this sequence probe sequence as the gene chip that is used to detect ureaplasma, and carry out pcr amplification by above-mentioned primer sequence, hybridize behind the mark and detect or be directly used in PCR and detect.

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