CN1436857A - Specificity determination and application of one section of candida albicans DNA sequence - Google Patents

Abstract

The present invention relates to specificity determination and application of one section of candida albicans DNA sequence, and belongs to the field of biomedicine. Through gene data base data search and analysis, the specificity and PCR amplification primer of one section of candida albicans DNA sequence is determined, and the sequence length is 241 bp. By means of PCR amplification and cloning of women's genital tract candida albicans infection sample, the sequence is obtained and sequenced to obtain the result the same as the predicted. Biological informatics and gene chip hybrid analysis proves the specificity of the sequence to candida albicans. The present invention is used as probe and PCR amplification detection sequence for gene chip diagnosis for candida albicans.

Description

The specificity of one section of candida albicans DNA sequence is determined and is used

Technical field:

The specificity that the present invention relates to one section of candida albicans (Candida albicans) dna sequence dna is determined and application, belongs to field of biomedicine technology.

Background technology:

Candida albicans is the pathogen that a kind of woman vagina often infects, 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 of candida albicans 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 Candida albicans.

Technical scheme of the present invention is: the present invention utilizes existing method to determine one section of candida albicans (Candida albicans) specific DNA sequence and the PCR primer thereof of length for 241bp, carries out pcr amplification with primer, and its sequence is as follows:

1?gagaccaact?ggttgggaaa?aacaagattt?acaattataa?tgatttgttt?gtttgtttgc

61?tttatattta?tggtttggta?taagttagat?agtctagttt?gatagtcttg?tttgattgtt

121?tatatatgta?tagatttaat?ggtttgcttt?gttttatgag?tttttttgat?atctggagta

181?gttaaagact?catctttgag?aaataggaga?ccaaacttgt?tgagttacaa?ttgacgtcgc

241?a

Primer sequence is:

1.5’-gagaccaactggttgggaaa-3’

2.5’-tgcgacgtcaattgtaactca-3’

Above-mentioned Candida albicans specific DNA sequence detects sequence as the probe and the pcr amplification of gene chip diagnosis, is used for the clinical diagnosis of Candida albicans.

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. the gene chip hybridization result.

Embodiment:

Be keyword at first with Candida albicans (Candida albicans), the nucleotide sequence of search Candida albicans 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 240 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, among the figure since 47, to 287,241bp is a cloned sequence altogether, all the other sequences are the cloning vector sequence), sequence is 241bp, estimates sequence by the comparison of Blast software, shows with the sequence of estimating is consistent and (sees Fig. 3, change over a by g except that the 79th among the figure, all the other are all consistent with the expectation sequence), show that this sequence can be can be used for Candida albicans 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 Candida albicans, 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, sequence and this sequence homology of having only Candida albicans (Candida albicans), and being to match fully, the sequence homology of one section about 70bp is arranged, also is the sequence of Candida albicans.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 two repetitions among the figure, each repeats to contain 9 kinds of urogenital infections disease pathogen things totally 60 different sites.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 Candida albicans.

The present invention can be used for the clinical detection that PCR method or method for gene chip carry out Candida albicans.Carry out pcr amplification with sequence provided by the invention, obtain product according to PCR method and whether occur judging whether to be candida albicans 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.

Claims (2)

1. the specificity of one section of candida albicans DNA sequence is determined, the length that it is characterized in that this sequence is 241bp, and sequence and PCR primer are as follows:

1?gagaccaact?ggttgggaaa?aacaagattt?acaattataa?tgatttgttt?gtttgtttgc

61?tttatattta?tggtttggta?taagttagat?agtctagttt?gatagtcttg?tttgattgtt

121?tatatatgta?tagatttaat?ggtttgcttt?gttttatgag?tttttttgat?atctggagta

181?gttaaagact?catctttgag?aaataggaga?ccaaacttgt?tgagttacaa?ttgacgtcgc

241?a

Primer sequence is:

1.5’-gagaccaactggttgggaaa-3’

2.5’-tgcgacgtcaattgtaactca-3’

2. the application of one section of candida albicans DNA sequence it is characterized in that this sequence is used for the probe of gene chip diagnosis as the distinguished sequence of Candida albicans, and pcr amplification detects sequence.

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