CN101121946A - Combination detection gene chip kit for three kinds of genitourinary infection pathogen - Google Patents

Abstract

The invention relates to a combined test gene chip reagent kit for three urogenital tract infection pathogens, pertaining to the field of biotechnology. The gene chip consists of 18 sites, i.e. 5 specific and conservative test-target sequences of each pathogen of chlamydia trachomatis, mycoplasma urealytium and gonorrhoeae are taken as testing sites, and 2 positive control sites and 1 negative control site are added; PCR method is adopted to expand testing-target sites and DNA segments of the control sites to act as a probe and fixed on the peridium slide to achieve a test gene chip. The matrix of the chip is 9X9, each checking site is repeated by 4 times, and arranged evenly. The reagent kit is capable to meet the requirement of all reagents during the testing process. The testing method involves the combination of PCR expansion, molecular hybridization and fluorescent labeling. The invention has the advantages that the testing cost is low, pollution to testing samples is removed, the specificity and flexibility of testing are excellent. The practical clinic application proves that the flexibility of the invention is as high as 97 to 99.7 percent, and 99 to 99.7 percent for specificity.

Description

The combination detection gene chip kit of three kinds of kinds of genitourinary infection pathogen

Technical field:

The present invention relates to a kind of combination detection gene chip kit of three kinds of kinds of genitourinary infection pathogen, belong to biological technical field.

Background technology:

In the urogenital infections venereal disease original, chlamydia trachomatis, Ureaplasma urealyticum, gonococcal infection rate are the highest.According to China relevant department statistics, annual std patient has about 4,000,000 people, and in nearly 10 years with the 20-30% speed increment.Wherein, gonorrhoea is arranged the first, account for 40.72% of venereal disease, nongonococcal urethritis the 3rd accounts for 21.85% of venereal disease, in nongonococcal urethritis, chlamydia trachomatis accounts for 40-50%, and Ureaplasma urealyticum accounts for 20-30%, that is to say, chlamydia trachomatis, Ureaplasma urealyticum, gonococcal infection account for infects the over half of sum, and therefore above-mentioned three kinds of pathogens need to detect simultaneously clinically usually.

Diagnosis is the basis of disease treatment, and current diagnosis to above-mentioned three kinds of pathogens mainly relies on culture method, ELISA and PCR to detect.In these methods, the cultural method cycle is long, and sensitivity and accuracy are low; Immunization method can only could detect after antigen and antibody generation, and the patient lower to gradient of infection can not reach early detection; PCR method exists false positive and the unmanageable problem of false negative, and also there is the inherent defect of simple PCR in fluorescence real-time quantitative PCR, and instrument and correlation detection reagent cost an arm and a leg.

The biochip technology that grew up in recent years has its distinctive advantage at the infectious diseases detection range.The present invention promptly adopt biochip technology succeed in developing chlamydia trachomatis (Chlamydiatrachomatis, Ct); Ureaplasma urealyticum (Ureaplasma urealyticum, Uu); Gonococcus (Neisseria gonorrhoeae, Ng) gene chip united diagnostic reagent box.

Summary of the invention:

The objective of the invention is to overcome the deficiencies in the prior art, a kind of combination detection gene chip kit of three kinds of kinds of genitourinary infection pathogen is provided.

Gene chip kit of the present invention comprises gene chip, detection reagent and detection method 3 parts.

Wherein:

1. gene chip

Gene chip of the present invention is made up of the l8 site, comprise chlamydia trachomatis (Chlamydiatrachomatis, Ct), Ureaplasma urealyticum (Ureaplasma urealyticum, Uu) and gonococcus (Neisseria gonorrhoeae, Ng) 5 special, conservative target sequence sites of each, 2 positive control sites and a negative control site., be printed on the slide glass that wraps quilt as probe with the dna fragmentation of 18 detection site of PCR method amplification with the gene chip sample applying instrument.Matrix during point sample is pressed the probe uniform distribution and is repeated symmetric offset spread.Be that 5 detection site of every kind of pathogen and 5 detection site of other pathogen are alternately distributed, a positive site and 15 detection site are formed 4 * 4 minor matrix, 4 such matrix symmetric offset spread are formed one 9 * 9 large matrix, and a positive control in addition and a negative control are on the symmetry axis of 9 * 9 matrixes.Make the positive site of each minor matrix be positioned on 4 angles of large matrix (seeing accompanying drawing 1) simultaneously.

2. detection reagent

Detection reagent of the present invention is as follows:

Reagent 1:DNA extracts reagent 1XTE

Reagent 2: amplifing reagent comprises all ingredients of pcr amplification process

Reagent 3: the red-label thing, such as dUTP-cy5

Reagent 4: the Green Marker thing, such as dUTP-cy3

Reagent 5:Taq archaeal dna polymerase

Reagent 6: hybridization solution, conventional hybridization solution gets final product

Reagent 7:20XSSC

Reagent 8:10%SDS

Four steam water 0.2ml thin-walled tube

1.5ml centrifuge tube

Filter paper (120mm * 40mm qualitative filter paper)

The consumption of mentioned reagent is determined according to packing specifications.

3. detection method

The present invention gets the secretory product of urogenital tract with conventional clinical method, and extracts DNA with ordinary method.Use at the specific PCR primer amplification that detects target site sequence and control site and detect sample DNA, detect design of primers as shown in Figure 2, add the positive control template in the amplification reaction reagent, and do not add the negative control template, in the time of amplification with the red-label substance markers in amplified production; Parallel work one blank amplified reaction with detection reaction of while, blank is consistent with the agent prescription of detection reaction, just replaces the red fluorescence marker with green fluorescence, and replaces the detection sample DNA with control treatment.The detection amplified production (is seen embodiment) in proportion with the contrast amplified production and is mixed back and chip hybridization.According to whether containing tested pathogen in the hybridization signal judgement sample, or contain the tested pathogen of what sample.

Advantage of the present invention:

1. gene chip diagnosis can detect chlamydia trachomatis, Ureaplasma urealyticum and gonococcus simultaneously, reduces the number of times of detection reaction, reduces and detects cost;

2. can detect 5 target sites of every kind of pathogen simultaneously, reduce the detection false negative that the error that detects because of individual diversity XOR unit point causes effectively.

3. the present invention combines the highly sensitive of PCR and the high specific of hybridization.Add fluorescent mark simultaneously, detection sensitivity reduces 2-3 the order of magnitude than the detectability of PCR method, can reach 10 ¹-10 ²Pathogen cell/ml.

4. because enough volume space are arranged, various contrasts can be set, eliminate systematic error and control and detection system and pollute.

Description of drawings:

Fig. 1 is a dot chart.Among the figure: the positive control site of I, III, the negative site of II, all the other are numbered the detecting position period.In the detection site, C1-C5 represents 5 location points of chlamydia trachomatis inspection, and U1-U5 represents 5 detection site of Ureaplasma urealyticum; N1-N5 represents gonococcal 5 detection site.

Fig. 2 is for detecting the design of primer and probe primer.The inboard is that probe primer, the outside are for detecting primer among the figure.

Embodiment:

Enforcement of the present invention is undertaken by following program:

1. utilize internet web search chlamydia trachomatis, Ureaplasma urealyticum and gonococcal nucleotide sequence, and utilize the online tool on the NIH website to detect the nucleotide sequence that is different from other microorganism in chlamydia trachomatis, Ureaplasma urealyticum and the gonococcus genome, according to the chlamydia trachomatis, Ureaplasma urealyticum and the gonococcus distinguished sequence that obtain, every kind of pathogen has designed the individual detection site more than 10, obtains dna fragmentation by pcr amplification mark clinical sample;

2. chlamydia trachomatis, Ureaplasma urealyticum and the gonococcus specific DNA fragment of clone PCR amplification are determined the exactness of cloned dna sequence as checking order.Make detection chip with about 10 of every kind of pathogen sequences, detect the detectability of each dna fragmentation (target sequence), therefrom select 5 again sequence is detected target site as gene chip by the existing chip detection method.

With the length of pcr amplification relevant detection site and control site be the dna fragmentation of 100-150bp as detection probes, make special-purpose point sample instrument with gene chip and arrange point on the slide glass of bag quilt by the dot matrix of accompanying drawing, promptly become gene chip.The concrete grammar of chip manufacturing is made by existing method.

4. when using, adopt with the essentially identical method of existing gene chip detection method and carry out,, handle difference to some extent early stage because the sample source is different.Main program is as follows:

(1) sampling:

Clinical sample: by the secretory product of existing clinical method district urogenital tract.(1) gets urogenital tract secretory product with cotton swab by the standard clinical sampling method, secretory product under the wash-out in reagent 1, cotton swab tube wall extruding stem, discarded cotton swab (must through sterilising treatment), 4 ℃ of preservation of elutriant standby (detection indifference in the week).

(2) get the sample 40ul that previous step prepares, add in the 0.2ml thin-walled tube, put into the PCR instrument and carry out thermal cycling according to following program with the 2ul positive control dna.Thermal circulation parameters is as follows: 65 ℃ 30 seconds; 8 ℃ 30 seconds; 65 ℃ 90 seconds; 97 ℃ 180 seconds; 8 ℃ 60 seconds; 65 ℃ 180 seconds; 97 ℃ 60 seconds; 65 ℃ 60 seconds; Circulate 2 times.80 ℃ kept 5 minutes.

(3) 4 ℃ of preservations are standby as the clinical detection template centrifugal 30 seconds for 10000rpm.

(4) when carrying out aforesaid operations, get 40ul 1XTE and 2ul positive control dna adding 0.2ml thin-walled tube and clinical sample and carry out above-mentioned (2) and the operation of (3) step simultaneously, obtain the negative control template.

(5) pcr amplification and mark

Get 3.5ul clinical sample DNA, end mark has the detection primer of all detection site and the control site of red fluorescence, and amplification system is 12.5ul, carries out pcr amplification and mark in the usual way.Do blank reaction simultaneously, and use the green fluorescence mark.

(6) get blank amplification-marked product 3ul and 10ul hybridization solution and add in the clinical sample amplification-mark pipe, fully mixing all drips the chip matrix position behind prehybridization; Covered.Build hybridizing box, put into 42 ℃ of thermostat container hybridization 4 hours.

(7) judgement of hybridization signal:

1). the unit point signal is judged

If certain detects (F635-B635)/B635 〉=2 for certain unit point of point, then this unit point is judged to the dUTP-Cy5 signal positive, otherwise negative; If certain detects (F532-B532)/B532 〉=2 for certain unit point of point, then be somebody's turn to do to point is judged to the dUTP-Cy3 signal positive, on the contrary negative.

2) detecting validity judges

If a. in the unit point of control site I, dUTP-Cy5 and dUTP-Cy3 are male unit point number n 〉=2

If b. in the unit point of control site III, dUTP-Cy5 and dUTP-Cy3 are male unit point number n 〉=2

If c. in the unit point of control site II, unit point number n 〉=2 that dUTP-Cy5 and dUTP-Cy3 are all negative

Satisfy above-mentioned a, b, c condition simultaneously, this time detects effectively; Otherwise it is invalid.

3). the detection site unit point pollutes to be judged

If the dUTP-Cy3 of certain unit point in the detection site is positive, the unit point of this detection site is judged to pollution.

4) the detection site unit point is positive judges

If certain unit point in the detection site does not pollute, and this unit point dUTP-Cy5 is positive, this unit point is judged to the positive, on the contrary the feminine gender of being judged to; If certain unit point in the detection site has pollution, this unit point is invalid.

5). detection site is positive to be judged

If in 4 unit points of certain detection site, positive number of sites 〉=2 judge that then this detection site is positive, otherwise negative.

6) pathogen is positive judges

If in 5 detection site of certain pathogen, positive detection number of sites 〉=1 judges that then this pathogen is positive, otherwise negative.

Test kit of the present invention is through the clinical examination of Kunming 3 relevant section office of tame front three hospital, and sensitivity reaches 97-99.7%; Specificity reaches 99-99.7%.

Detect the target site sequence

Organization?Applicant

----------------------

Street: No. 9 tunnels, North Area, high and new technology industrial development zone, Kunming, university science and technology park, Yunnan Province A2-507-508

City: Kunming

State: Yunnan Province

Country: China

PostalCode：650106

PhoneNumber：0871-8314895

FaxNumber：0871-8314895

EmailAddress：

＜110〉OrganizationName: Kunming Yunda Biochemical Technology Co., Ltd

Application?Project

-------------------

<120>Title：

<130>AppFileReference：

<140>CurrentAppNumber：

<141>CurrentFilingDate：

Sequence

--------

＜213〉OrganismName: chlamydia trachomatis

<400>PreSequenceString：

ggaggtaaac?gctcctctga?agtcttaagc?ttggagatta?gtcagatttg?tttccaacaa 60

gctaccattt?ctttctccca?gcttaagaac?cgtcagacag?aaaagaggat?tattataact 120

tatcctcaga?agtttatgca?ctttctacaa?gagtacatcg?gtcaacgaag?aggttttgtc 180

ttcgtaactc?gctccggaaa?aatg 204

<212>Type：DNA

<211>Length：204

SequenceName: chlamydia trachomatis detection site 1

SequenceDescription：

Sequence

--------

＜213〉OrganismName: chlamydia trachomatis

<400>PreSequenceString：

tatctttttc?tggcggcact?gcgtccttgt?taaagccaga?caagatgtcc?tgtagcaatt 60

aatggcctga?ggaatgtctt?gcaagtaaaa?gcagccttaa?tcgagaacat?gtcgttcaat 120

gctcgagacc?ataacaaaat?tttcttattc?tcgtaaagtt?gataagaaaa?acgttcgtcc 180

caggaagaag?cccttaagcg?cctcctgatt?tagc 214

<212>Type：DNA

<211>Length：214

SequenceName: chlamydia trachomatis detection site 2

SequenceDescription：

Sequence

--------

＜213〉OrganismName: chlamydia trachomatis

<400>PreSequenceString：

caaagattcg?ctgtggtcaa?gagaaatagc?ctttatcaag?gtttccgata?aatccagaat 60

ctctaaagaa?acaagaaagt?taatcccaga?cgcataattt?tttctagtta?gatgagataa 120

agtagataac?caaatttccg?acgcgtcccc?aaaagttaag?aacaatctac?ttttatggaa 180

agccatcgag?cccattttct?ta 202

<212>Type：DNA

<211>Length：202

SequenceName: chlamydia trachomatis detection site 3

SequenceDescription：

Sequence

--------

＜213〉OrganismName: chlamydia trachomatis

<400>PreSequenceString：

catcggttgg?agaaaatcgt?caaaatgaaa?tagcagatat?atctagaacc?ttaagaggtt 60

tagcctcaga?gctaaacatt?cctatagttt?gtttatccca?actatctaga?aaagttgggg 120

atagagcaaa?taaagttccc?atgctttcag?atttgcgaga?cagcggtcaa?atagagcaag 180

acgcagatgt?ga 192

<212>Type：DNA

<211>Length：192

SequenceName: chlamydia trachomatis detection site 4

SequenceDescription：

Sequence

--------

＜213〉OrganismName: chlamydia trachomatis

<400>PreSequenceString：

atctcccaaa?ttggctcaaa?atgggatggt?agaagttata?ggtcttgatt?ttctttcatc 60

tcattaccat?gcattagcag?ctatccaaag?attactgacc?gcaacgaatt?acaaggggaa 120

cacaaaaggg?gttgttttat?ccagagaatc?aaatagtttt?caatttgaag?gatggatacc 180

aagaatccg 189

<212>Type：DNA

<211>Length：189

SequenceName: chlamydia trachomatis detection site 5

SequenceDescription：

Sequence

--------

＜213〉OrganismName: Ureaplasma urealyticum

<400>PreSequenceString：

tggggaccgt?cctatacaag?ttggatcaca?ttttcacttg?tttgaagtga?atagtgcatt 60

agtatttttt?gatgaaaaag?gaaatgaaga?taaagaacgc?aaagttgctt?atggacgacg 120

tttcgatatt?ccatcaggta?ctgctattcg?ttttgaacca?ggagataaaa?aagaagtttc 180

aattattgat?ttagccggaa?cacg 204

<212>Type：DNA

<211>Length：204

SequenceName: Ureaplasma urealyticum detection site 1

SequenceDescription：

Sequence

--------

＜213〉OrganismName: Ureaplasma urealyticum

<400>PreSequenceString：

ctgctgttgc?gtttgcttta?ttagctatgc?atttaaaaat?agatttaaaa?actgctttat 60

atacgcatct?ttactctaca?gttgctgcat?taacgcaaaa?ctgtgtacgt?gcaattccat 120

taggacaagt?taagggacaa?aaaataattt?atcaacttaa?acatgtttat?tttgatgata 180

ttgttaataa?agtctttaca?ttggatttta?aaacagattt?ttgcaaaaat?ataccaggcc 240

ttgaaattgc?acaaatggaa?cacga 265

<212>Type：DNA

<211>Length：265

SequenceName: Ureaplasma urealyticum detection site 2

SequenceDescription：

Sequence

--------

＜213〉OrganismName: Ureaplasma urealyticum

<400>PreSequenceString：

tcgtagccaa?acaattgcag?ctgaagactt?attgcacgat?atgggtgcaa?tttcaattat 60

gtcatcagat?acattagcta?tgggacgtat?tggcgaagtt?gcaactcgta?catgacaaat 120

ggctcacaaa?atgaaagcac?aatttggatc?attaaaaggt?gatagtgaat?tcagtgataa 180

caatcgtgta?aagcgttata?tttctaaata?taeaattaac?ccagctattg?cacatggtgt 240

tgattcttat?attggttcac?tagaagttgg?taaattagct?gatattgttg?cttgagaacc 300

taaattcttt?ggtgcaaaac?ctt 323

<212>Type：DNA

<211>Length：323

SequenceName: Ureaplasma urealyticum detection site 3

SequenceDescription：

Sequence

--------

＜213〉OrganismName: Ureaplasma urealyticum

<400>PreSequenceString：

cctcaaacat?ttgatgctgc?tgttgactac?aacgacttag?aaaactgatt?agaacaacca 60

gctgctgaat?tagctaagaa?attaaagaaa?actgcaaacg?gtaaatacgt?acttgatgca 120

gaacctctaa?cagaagctcc?attagcacaa?agatacttct?tattctaatt?cttgaattat 180

tttgatttag?taattcaatt?tccaactaca?tttaaaagaa?gcgaggtata?aatcttgact 240

gtatttaaag?aaattttagg?taacattact?gacatcgaaa?atgttgaaag?ttaccaaatt 300

gagaacattc?atttaacaag?cgacgacg 328

<212>Type：DNA

<211>Length：328

SequenceName: Ureaplasma urealyticum detection site 4

SequenceDescription：

Sequence

--------

＜213〉OrganismName: Ureaplasma urealyticum

<400>PreSequenceString：

aaattgctgc?tggagcttgt?ggacttaaaa?tccatgaaga?ctgaggggca?acaggaaatg 60

cgattgattt?agcattaaca?gttgctgata?aaactgatgt?agctgttgct?attcatacag 120

atacattaaa?tgaagctgga?tttgtagaac?atacaattgc?agctatgaaa?gggcgaacaa 180

ttcatgctta?tcatacagaa?ggtgctggtg?g 211

<212>Type：DNA

<211>Length：211

SequenceName: Ureaplasma urealyticum detection site 5

SequenceDescription：

Sequence

--------

＜213〉OrganismName: gonococcus inspection

<400>PreSequenceString：

tgctaaattg?cggattggaa?aattttgaaa?taagaacccc?tatcgggctg?ttatctgatt 60

agggttattt?tgacatagtt?gtatcatctt?aaaaacaagc?atacaatctg?caaatcttag 120

acaaagcaaa?acccccgcca?aacgccaatc?tgcacggggg?tttcgagata?caacatgagc 180

caattataca?cccaacccg 199

<212>Type：DNA

<211>Length：199

SequenceName: gonococcus detects target site 1

SequenceDescription：

Sequence

--------

＜213〉OrganismName: gonococcus inspection

<400>PreSequenceString：

tgcaccctag?acctgttgga?aaccaagacc?aaaggtttga?cactggaaaa?ttgcccagtc 60

gagaacagca?aagcaacgcg?ggtatgcgta?gcgaccgaga?agcgaatgct?ggacgcgtta 120

gcggaattag?agagcaatca?cgcagcaatc?gagcagcgaa?tgatgaaagc?cttaacgcac 180

ttgggcgaaa?ggttggcaga?gctag 205

<212>Type：DNA

<211>Length：205

SequenceName: gonococcus detects target site 2

SequenceDescription：

Sequence

--------

＜213〉OrganismName: gonococcus inspection

<400>PreSequenceString：

ggatgtggcg?gtttgtattt?gggctttcat?caagctggtt?gtgaaaccgt?ttgggcgaac 60

gatttctccc?attgggcttg?cgaaagtttc?cgtaaaaata?tcggcgatgt?catcgtagaa 120

ggtgatattg?aacaaattaa?tccgaatgat?ccaactattc?ccgattgcga?catcatttta 180

ggcggattcc?caa 193

<212>Type：DNA

<211>Length：193

SequenceName: gonococcus detects target site 3

SequenceDescription：

Sequence

--------

＜213〉OrganismName: gonococcus inspection

<400>PreSequenceString：

tttttgggat?agcacggaac?gtacttatga?aactatgttg?tatttgaacg?gtaagcctgc 60

acagattttg?caggcagata?ttcaaagtac?tattcataag?ctaggcaaaa?atatccaaga 120

agttgaaaga?ccaagtaaat?ttattgaaca?taatagccat?ttagaaaatt?gtttgggtgt 180

tcagaaaatt?gcaccagaac?aattcggcaa?tt 212

<212>Type：DNA

<211>Length：212

SequenceName: gonococcus detects target site 4

SequenceDescription：

Sequence

--------

＜213〉OrganismName: gonococcus inspection

<400>PreSequenceString：

acagcaggtc?aggccatatc?caatattcca?caaaatgcca?gtaataatga?attactgaag 60

atcagcgata?aaacacgccg?tatgttggaa?ttaattcctg?aaggtggaaa?ttttaccgat 120

attcctaaag?atcatccttt?atatgtgaaa?ggtatgatta?gccacgttta?tcgtcgtatg 180

catcgga 187

<212>Type：DNA

<211>Length：187

SequenceName: gonococcus detects target site 5

SequenceDescription：

Sequence

--------

＜213〉OrganismName: people

<400>PreSequenceString：

ggcatcctca?ccctgaagta?ccccactgag?cactgcatca?tcaccaactg?ggaagacatg 60

gagaagatct?ggcaccacac?cttctacaat?gagttgtggc?tcccaaggag?caccctgtgc 120

tattgaccaa?ggtccccctg?gaccccaagg?ccaaccatga?gaagatagcc?cagatcatgt 180

ttgagacctt?caacacccc 199

<212>Type：DNA

<211>Length：199

SequenceName: control test site I

SequenceDescription：

Sequence

--------

＜213〉OrganismName: plant

<400>PreSequenceString：

ctaccacata?acgcctgcaa?tgtgacaccc?tacctattca?ctagtgtgcc?tcttcccaca 60

cgctttccac?ccgtactgct?cacagcttta?agaaccagaa?caaatgagta?atattagtgt 120

cggttcatgg?ctaaaaccag?cactgatgta?catgaccaca?tatgtcaaat?gctgcttcta 180

ggcatgaccc?gctcttac 198

<212>Type：DNA

<211>Length：198

SequenceName: control test site II

SequenceDescription：

Sequence

--------

＜213〉OrganismName: plant

<400>PreSequenceString：

gctgccaaga?tatcagtgtc?cttggtttcg?tactccgggg?tgtagtaagc?caatttataa 60

tccttaacac?cagctttgaa?tccaacactt?gctttagttt?ctgtttgtgg?tgcatacgtc 120

cctccctaca?actcatgaat?taagaattct?cacaacgaca?gggtctactc?gatatggatt 180

aggcgtaaat?gaaacctttg?caa 203

<212>Type：DNA

<211>Length：203

SequenceName: control test site III

SequenceDescription：

Claims (1)

1. the combination detection gene chip kit of three kinds of kinds of genitourinary infection pathogen, comprise gene chip, detection reagent and detection method, it is characterized in that gene chip of the present invention is made up of 18 sites, that is: 5 special, conservative detection target sequences selecting chlamydia trachomatis, Ureaplasma urealyticum and every kind of pathogen of gonococcus add 2 positive control site and 1 negative control site simultaneously as detection site; Dna fragmentation with PCR method augmentation detection target site and control site is made the detection gene chip as probe stationary on the slide glass of bag quilt, detect in the clinical sample whether have pathogen by the gene chip detection mode then.

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