CN1676612A - Method for detecting and identifying mycoplasma - Google Patents

Abstract

This invention is a MP testing and identifying method, including distill DNA of samples to be tested, use marked MP general lead to expand the DNA of pending tested samples in PCR method, hybridize the PCR expanded products with the MP species-specific probe markedly located on the nylon film, wash the un-hybridized PCR expanded products on the nylon film, display the hybridizing results and etc, and meanwhile, this invention also provides the MP general lead, the MP species-specific probe and the reagent boxes of testing and identifying MP. The probe of this invention is delicate, and the testing and identifying are fast sensitive and the cost is low.

Description

A kind of method of identifying mycoplasma that detects

Technical field

The present invention relates to the detection authentication method of microorganism, particularly the detection authentication method of mycoplasma is specifically related to use new mycoplasma specific specificity probe to detect and identify ten kinds of common cell contaminations and pathogenic mycoplasma in conjunction with reverse line dot blot (Reverse Line Blot Hybridisation) method.

Background technology

Mycoplasma contamination is a common problem during cell culture experiments, pharmaceutical industry and biotechnological formulation are produced, and it can cause the failure and the biological product downgrade of testing.In the report of different experiments chamber, there is mycoplasma contamination in the cell cultures of 15%-85%.Pollute in the mycoplasma at present known 20 various kinds of cell, mycoplasma arginini (Mycoplasma arginin), mycoplasma fermentans (Mycoplasma fermentans), mycoplasma hyorhinis (Mycoplasma hyorhinis), Mycoplasma orale (Mycoplasma orale) and Lai Shi acholeplasma (Acholeplasma laidlawii) are modal.In cell cultures, the mycoplasma contamination more than 95% is by due to this five mycoplasma species pollution.Genital tract mycoplasma (Mycoplasmagenitalium), mycoplasma hominis (Mycoplasma hominis), small urea substance (Ureaplasma parvum), ureaplasma urealyticum (Ureaplasma urealyticum) and mycoplasma pneumoniae (Mycoplasma pneumoniae) are five kinds of clinical common pathogenic mycoplasmas.Genital tract mycoplasma, mycoplasma hominis, small urea substance and ureaplasma urealyticum parasitize human body urinary tract and reproductive tract, and they can cause nongonococcal urethritis, cervicitis, a series of diseases such as infertile and sterile clinically.Mycoplasma pneumoniae is one of pathogenic agent that causes human especially children Streptococcus.Because the small and poor growth of mycoplasma form, their infection are difficult in clinical and laboratory by early diagnosis, evaluation.And mycoplasma is of a great variety, about kind more than 120.Traditional mycoplasma diagnostic method mainly contains mycoplasma cultivation, mycoplasma DNA fluorescent dye, making nucleic acid molecular hybridization and the special poly synthase of mycoplasma kind chain type amplification methods such as (PCR).PCR method is present the most frequently used diagnostic method (reference Kong, F. wait 2001.Species-specific PCR for identification of commoncontaminant mollicutes in cell culture.Appl.Environ.Microbiol.67:3195-3200), but use PCR to detect, identify multiple mycoplasma exists length consuming time, repeating step shortcoming how.None detects multiple mycoplasma fast, simultaneously at present, and the method sensitive, that specificity is high and cost is low.

Summary of the invention

The purpose of this invention is to provide a kind of new detection and identify the method for mycoplasma, make it have to detect simultaneously and identify multiple mycoplasma, and have fast, sensitive, characteristics such as specificity is high and cost is low, to overcome the deficiencies in the prior art.

The method of mycoplasma is identified in detection provided by the invention, comprises the following steps:

(1) DNA of extraction testing sample;

(2) use is through the mycoplasma universal primer of mark, with the DNA of PCR method amplification testing sample;

(3) the mycoplasma specific specificity probe hybridization on nylon membrane with pcr amplification product and telltale mark;

(4) demonstration and differentiation results of hybridization.

Realize technical scheme of the present invention, comprise specific probe and the reverse line dot blot of designing diagnosis mycoplasma kind.

In Mycoplasma, the nucleotide sequence of their 16S-23S rRNA transcribed spacer is nucleotide sequence (the reference Harasawa of at present known tool mycoplasma difference between species, R. wait .2000.Comparison of the 16S-23S rRNAintergenic spacer regions among strains of the Mycoplasma mycoides cluster, and reassessmentof the taxonomic position of Mycoplasma sp.bovine group 7.Int.J.Syst.Evol.Microbiol.50 Pt3:1325-1329).In the domestic and foreign literature report, still nobody is at the specific probe of this zone design diagnosis mycoplasma kind at present.

Oppositely the line dot blot is the hybridization authentication method that is based upon on DNA poly synthase chain type amplification (PCR) basis.During the branch kind that this method is applied to tuberculosis is the earliest identified (reference Aranaz .1996.Spoligotyping ofMycobacterium biovis strains from cattleand other animals:a tool for epidemiology oftuberculosis.J.Clin.Microbiol.34:2734-2740. such as A).Its advantage is to carry out sensitive, fast branch kind, the somatotype evaluation of microorganism.Therefore we design the new specific specificity probe of mycoplasma and in conjunction with reverse line dot blot method, set up a kind of new, rapid detection and identify multiple mycoplasma method, for laboratory and clinical position provide strong help, to remedy the deficiencies in the prior art.

At the specific probe of the design of mycoplasma 16S-23S rRNA transcribed spacer, synthetic different mycoplasma kinds, 5 ' terminal amino acid mark is marked at specific probe on the nylon membrane.Design, synthesize the 16S-23S rRNA transcribed spacer consensus primer of aforementioned common ten mycoplasma species again, 5 ' end biotin labeling.Applying marking has ten kinds of common mycoplasma 16S-23SrRNA transcribed spacer DNA of consensus primer amplification of vitamin H.The PCR product is combined with the mycoplasma specific specificity probe of telltale mark on nylon membrane.On nylon membrane, add photographic developer, the vitamin H combination of mark on photographic developer and the PCR product, fluorescence excitation makes the exposure of x exographX, and if any stain, ecbatic is positive, and identifies the mycoplasma kind by the stain that different positions on the egative film manifests; As not having stain, ecbatic is negative.

The method of mycoplasma is identified in detection provided by the invention, and energy is responsive, rapid detection is identified mycoplasma, can detect, identify multiple mycoplasma in 2 day time simultaneously.The susceptibility of this method and specificity are not less than mycoplasma PCR detection method commonly used at present, and once can detect nearly 45 samples simultaneously, and the nylon membrane of probe mark can use about 20 times repeatedly, promptly the nylon membrane of a probe mark can enough detect 900 samples, thereby reduces the detection cost of each sample greatly.

Another task of the present invention provides a kind of test kit of identifying mycoplasma that detects, and it is characterized in that it contains:

Container;

The universal primer of the mycoplasma 16S-23SrRNA transcribed spacer DNA that is used to increase provided by the invention;

At least a mycoplasma specific specificity probe with described base sequence of SEQ ID NO:5 to SEQ ID NO:24 in the sequence table;

And operation instruction.

This test kit also can contain PCR and react needed reagent.

Description of drawings

Fig. 1 is detection of mycoplasma authentication method egative film exposure colour developing result of the present invention.

Embodiment

Embodiment:

The instrument that uses has :-20 ℃ and 4 ℃ of refrigerators, normal temperature supercentrifuge (Eppendorf company, Germany), PCR instrument (Eppendorf company, Germany), Miniblotter (Immunetic company, the U.S.), hybridization case (Thermo company, the U.S.), magazine.

The reagent that uses has: Luo Shi respiratory tract specimen dna extracts test kit (Luo Shi diagnostic reagent company, the U.S.), Taq enzyme, dNTP, 10 * PCR buffer, ultra-clean water, 0.5M NAHCO ₃(PH8.4), 0.1M NaOH, 16%w/v 1Ethyl-3-(3 Dimethylamino-propyl) carbodiimide (EDAC) (Sigma company, the U.S.), 20 * SSPE (0.2MNa ₂HPO ₄, 3.6M NaCl, 20mMEDTA, PH7.4), 2 * SSPE/0.1%SDS, 2 * SSPE/0.5%SDS, 2 * SSPE, 1%SDS, 20mM EDTA, Biodyne C nylon membrane (Immunetic company, the U.S.), Streptavidin-peroxidase conjugate (Luo Shi diagnostic reagent company, the U.S.), Enhancedchemiluminescence (ECL) photographic developer (Amersham company, Britain), X-ray sheet (Amersham company, Britain).

Operation steps:

One, uses Luo Shi respiratory tract specimen dna to extract test kit and extract DNA

With testing sample at normal temperatures with 13000 rev/mins centrifugal 10 minutes, remove supernatant liquor, keep 10 μ l liquid, add 500 μ l washing lotions (extracting test kit by Luo Shi respiratory tract specimen dna is equipped with), mixing; Again with 13000 rev/mins centrifugal 10 minutes, remove supernatant liquor, add 50 μ l, hatched 45 minutes at 60 ℃, add 50 μ l balance liquids (extracting test kit by Luo Shi respiratory tract specimen dna is equipped with) at last, mixing is stored in-20 ℃ with DNA extraction liquid, and is standby.

Two, use nest-type PRC amplification mycoplasma 16S-23S rRNA transcribed spacer

The present invention has designed two pairs of universal primers, i.e. SPS1, SPA2; SPS2, SPA1, ten mycoplasma species 16S-23SrRNA transcribed spacers increase.SPS1, SPA2 are outer primer; SPS2, SPA1 are inner primer.5 ' the end of SPS2 and SPA1 gives biotin labeling, as using carboxyl fourth two inferior carboxylic acid amide esters (biotinyl N hydroxy succinimide ester, BNHS) mark.This primer is synthetic by Sigma company, and its sequence is as follows:

Outer primer

SPS1：????5’-CCCTACGRGAACGTGGGGVTGGAWYACCTCCT-3’(SEQ?ID?NO：1)

SPA2：????5’-CSDDGCWTWTCGSWGDTWRDYVCGTCCTTCATCG-3’(SEQ?IDNO：4)

Inner primer

SPS2：????5’-CGRGAACGTGGGGVTGGAWYACCTCCTTTC-3’(SEQ?ID?NO：2)

SPA1：????5’-CGTCCTTCATCGMCTNTYYDGWSCCAAGGCATYCAC-3’(SEQ?IDNO：3)

The PCR scheme is: the condition of increase for the first time sex change, annealing, extension be respectively 94 ℃ 10 seconds, 65 ℃ 10 seconds, 74 ℃ 1 minute, 30 circulations; The condition of sex change, annealing, extension of increasing for the second time be respectively 94 ℃ 10 seconds, 70 ℃ 10 seconds, 74 ℃ 1 minute, 30 circulations.Get 20 μ l pcr amplification products and be used for reverse line dot blot experiment.

Three, oppositely line dot blot experiment

(1) label probe

The ten mycoplasma species specific specificity probe sequences that use are as follows, and 5 ' end gives hexylamino mark, and is synthetic by Sigma company:

Mycoplasma fermentans 1A 5 '-CCC ATA AAA AAG CCA CAT AAC-3 ' (SEQ ID NO:5)

Mycoplasma fermentans 2S 5 '-CAT CAT AAC AAA CTA TAA CAA TAG GAA-3 ' (SEQ ID

NO：6)

Mycoplasma arginini 1A 5 '-AAA GAA CAA ATT GAG AGA TAG GTC-3 ' (SEQ ID NO:

7)

Mycoplasma arginini 2S 5 '-CAA TAG GTC TTA TAC TAC TAT TAA ACA AGA T-3 ' (SEQ

ID?NO：8)

Mycoplasma orale 1A 5 '-GAA TAT TGG GCC ATT AAC TAT TT-3 ' (SEQ ID NO:9)

Mycoplasma orale 2S 5 '-CAA TAG GTC AAA AAT ACT TAT ACG TAA-3 ' (SEQ ID

NO：10)

Mycoplasma hyorhinis 1A 5 '-CTA GAC ACG AAT CGA TTA TGT AAT-3 ' (SEQ ID NO:

11)

Mycoplasma hyorhinis 2S 5 '-AAC GAT CTT TTT TAT AAC CGA G-3 ' (SEQ ID NO:12)

Mycoplasma hominis 1A 5 '-CAA AGA ACC GAG AGA TAA ATC T-3 ' (SEQ ID NO:13)

Mycoplasma hominis 2S 5 '-CAA TAG GTC ATA CAA TTA ACA AAA C-3 ' (SEQ ID

NO：14)

Mycoplasma pneumoniae 1A 5 '-ACC GAT AAA TAA ATG GAT TTT G-3 ' (SEQ ID NO:15)

Mycoplasma pneumoniae 2S 5 '-ACA TTT CCG CTT CTT TCA A-3 ' (SEQ ID NO:16)

Genital tract mycoplasma 1A 5 '-CAC CGA AAA AAT TAA TGG G-3 ' (SEQ ID NO:17)

Genital tract mycoplasma 2S 5 '-AAG AAT GTT TTT GAA CAG TTC TTT-3 ' (SEQ ID NO:

18)

Ureaplasma urealyticum 1A 5 '-GGC TAA TAT TCA CAT GGA TTT TTA TA-3 ' (SEQ ID

NO：19)

Ureaplasma urealyticum 2S 5 '-AAA TAT TTC AAA AGT TCA TAT GGT C-3 ' (SEQ ID

NO：20)

Lai Shi acholeplasma 1A 5 '-AAG TGT TAG TTA GCC TTT CTC CT-3 ' (SEQ ID NO:21)

Lai Shi acholeplasma 2S 5 '-AAA TGA TGT CTG AAA AGA AAT AAG-3 ' (SEQ ID NO:

22)

Small urea substance 1A 5 '-GGC TTA TAT TCA TAT GGA TTT TAA TA-3 ' (SEQ ID

NO：23)

Small urea substance 2S 5 '-TAT TTT TAA AAA TTC ATA TGG TCG-3 ' (SEQ ID NO:

24)

1. use 0.5M NaHCO ₃(PH8.4) dilute above-mentioned ten mycoplasma species specific specificity probes respectively to suitable concentration, (dilution and definite method of suitable concentration are referring to Vinje J, Koopmans MP.Simultaneous detectionand genotyping of " Norwalk-like viruses " by oligonucleotide array ina reverse line blot hybridization format.J Clin Microbiol.2000Jul; 38 (7): 2595-601.);

2. Biodyne C nylon membrane is cut to 15 * 15 centimetres of sizes;

3. under the room temperature, 16%w/v 1 Ethyl-3-(3 Dimethylamino-propyl) carbodiimide (EDAC) was hatched the BiodyneC nylon membrane 10 minutes;

4. rinsed with deionized water Biodyne C nylon membrane is 1 minute;

5. Biodyne C nylon membrane is put into Miniblotter, lid is tight, absorbs the nylon membrane surface liquid;

6. the mycoplasma probe of ten kinds of oneself dilutions is respectively got 150 μ l and add successively respectively in the different slots of Miniblotter groove, hatched under the room temperature 5 minutes;

7. absorb the nylon membrane surface liquid;

8. open Miniblotter, take out nylon membrane, nylon membrane is put into 0.1M NaOH liquid deactivation 9 minutes;

9.100ml 2 * SSPE rinsing Biodyne C nylon membrane 5 minutes;

10. used 60 ℃ of 2 * SSPE/0.5%SDS liquid rinsing Biodyne C nylon membranes 5 minutes;

11. at room temperature cleaned Biodyne C nylon membrane 20 minutes with 200ml 20mM EDTA liquid;

12. Biodyne C nylon membrane is put into clean plastics bag, and sealing is stored in 4 ℃ of refrigerators, and is standby.

(2) reverse line dot blot

1. 20 μ l pcr amplification products of each sample and 150 μ l, 2 * SSPE/0.1%SDS mixing are added in the EP pipe, be heated to 100 ℃ 10 minutes, the EP pipe is positioned on ice fast;

2. with the Biodyne C nylon membrane of 250ml 60 ℃ of 2 * SSPE/0.1%SDS liquid rinsing label probe 5 minutes;

3. the Biodyne C nylon membrane after will washing is positioned among the Miniblotter, absorbs the nylon membrane surface liquid;

4. in the Miniblotter groove, add 150 μ l pcr amplification products respectively successively and mix liquid (its direction is vertical with probe direction), put into 60 ℃ of hybridization casees hybridization 60 minutes with 2 * SSPE/0.1%SDS;

5. absorb Biodyne C nylon membrane surface liquid;

6. from Miniblotter, take out Biodyne C nylon membrane.Wash Biodyne C nylon membrane twice with 60 ℃ of 2 * SSPE/0.5%SDS liquid of 250ml, each 10 minutes;

7. the Biodyne C nylon membrane after will washing was put into cylinder, adds 10ml 2 * SSPE/0.5%SDS and 1/5000Streptavidin-peroxidase conjugate mixed solution, 42 ℃ of hybridization 45 minutes;

8. wash Biodyne C nylon membrane twice with 42 ℃ of 2 * SSPE/0.5%SDS liquid of 250ml, each 10 minutes;

9. wash Biodyne C nylon membrane twice with 250ml 2 * SSPE liquid under the room temperature, each 5 minutes;

10. add 20ml Enhanced chemiluminescence (ECL) photographic developer on Biodyne C nylon membrane, incubated at room 1 minute;

11. Biodyne C nylon membrane is put into magazine, in magazine, put into the X-ray sheet, exposed 5 minutes, develop a film observations: it is positive that the correspondent probe zone manifests the black round dot, illustrated and the corresponding mycoplasma of probe; If the correspondent probe zone does not manifest look then be negative, illustrating does not have and the corresponding mycoplasma of probe.

Biodyne C nylon membrane is washed twice with 80 ℃ of 250ml 1%SDS, each 30 minutes, cleaned Biodyne C nylon membrane 15 minutes with 200ml 20mMEDTA liquid again, Biodyne C nylon membrane is put into clean plastics bag, sealing is stored in 4 ℃ of refrigerators, can get time use ready.

Detect 19 strain mycoplasma ATCC type strains with the reverse line dot blot of mycoplasma of the present invention method, comprise M.hyorhinisATCC 17981, mycoplasma fermentans ATCC 19989, Lai Shi acholeplasma ATCC 23206, mycoplasma pneumoniae ATCC 29342 (M129), small urea substance of mycoplasma genitalium ATCC 33530,14 strains and ureaplasma urealyticum ATCC type strain.Also detected simultaneously respectively from Australian and the isolating mycoplasma arginini in Beijing, Mycoplasma orale, each strain of mycoplasma hominis.The result shows: use the reverse line dot blot of mycoplasma method can accurately detect 19 strain mycoplasma ATCC type strains and mycoplasma arginini, Mycoplasma orale, each strain isolated of mycoplasma hominis.(see accompanying drawing, in the accompanying drawings,, only show that small urea substance is with serotype 3 and ureaplasma urealyticum serotype 8 detected results) for small urea substance and ureaplasma urealyticum.

Detect 180 clinical samples simultaneously with mycoplasma kind PCR with the reverse line dot blot of mycoplasma of the present invention method, comprise 80 cell culture samples, 86 uropoiesis, genital secretion sample, 14 respiratory secretions samples (table 1, table 2), relatively two kinds of method detected results.The result shows: in clinical sample detects, be consistent with mycoplasma specific specificity PCR with detected result with the reverse line dot blot of mycoplasma of the present invention detection method.

Use two kinds of methods to detect the mycoplasma result relatively in table 1 cell culture

Species title and routine number	Reverse line dot blot detected result	Specific specificity PCR detected result
			Mycoplasma fermentans 13 routine mycoplasma arginini 24 routine Mycoplasma orale 9 routine mycoplasma hyorhinis 16 routine mycoplasma fermentanses and mycoplasma arginini 5 routine mycoplasma fermentanses, mycoplasma arginini and Mycoplasma orale 2 routine mycoplasma argininis and Mycoplasma orale 4 routine Mycoplasma orales and mycoplasma hyorhinis 7 examples amount to	Positive 80 examples of positive 7 examples of positive 4 examples of positive 2 examples of positive 5 examples of positive 16 examples of positive 9 examples of positive 24 examples of 13 examples are positive	Positive 80 examples of positive 7 examples of positive 4 examples of positive 2 examples of positive 5 examples of positive 16 examples of positive 9 examples of positive 24 examples of 13 examples are positive

Use two kinds of methods to detect the mycoplasma result relatively in table 2 uropoiesis, reproductive tract, the respiratory secretions sample

Species title and routine number	Reverse line dot blot detected result	Specific specificity PCR detected result
			Mycoplasma pneumoniae 14 examples	14 examples are positive	14 examples are positive

Genital tract mycoplasma 6 routine small urea substance 60 routine ureaplasma urealyticum 20 examples amount to

Positive 100 examples of positive 20 examples of positive 60 examples of 6 examples are positive

Positive 100 examples of positive 20 examples of positive 60 examples of 6 examples are positive

The result who detects clinical sample by mycoplasma specific specificity PCR detection method and the reverse line dot blot of mycoplasma method more as can be seen, detection and evaluation mycoplasma that the inventive method and designed mycoplasma specific specificity probe can be sensitive, special.The reverse line dot blot of mycoplasma of the present invention method susceptibility is identical with specificity with mycoplasma specific specificity PCR detection method, the advantage of this method is that (2 days) detect multiple mycoplasma simultaneously at short notice, PCR method then needs repeated multiple times could determine each mycoplasma, illustrate the present invention be a kind of have to detect simultaneously identify multiple mycoplasma, and have fast, sensitive, specificity is high and cost is low detection of mycoplasma method.

Sequence table

＜110〉Wuhan City No.1 Hospital

＜120〉the detection authentication method of a mycoplasma species

<130>

<160>24

<170>PatentIn?version?3.1

<210>1

<211>32

<212>DNA

＜213〉synthetic primer

<400>1

ccctacgrga?acgtggggvt?ggawyacctc?ct????????????????????????????????????32

<210>2

<211>30

<212>DNA

＜213〉synthetic primer

<400>2

cgrgaacgtg?gggvtggawy?acctcctttc???????????????????????????????????????30

<210>3

<211>36

<212>DNA

＜213〉synthetic primer

<220>

<221>misc_feature

<222>(16)

＜223〉n=a or g or c or t

<400>3

cgtccttcat?cgmctntyyd?gwsccaaggc?atycac????????????????????????????????36

<210>4

<211>34

<212>DNA

＜213〉synthetic primer

<400>4

csddgcwtwt?cgswgdtwrd?yvcgtccttc?atcg??????????????????????????????????34

<210>5

<211>21

<212>DNA

＜213〉mycoplasma fermentans (Mycoplasma fermentans)

<400>5

cccataaaaa?agccacataa?c????????????????????????????????????????????????21

<210>6

<211>52

<212>DNA

＜213〉mycoplasma fermentans (Mycoplasma fermentans)

<400>6

catcataaca?aactataaca?ataggaa??????????????????????????????????????????27

<210>7

<211>24

<212>DNA

＜213〉mycoplasma arginini (Mycoplasma arginini)

<400>7

aaagaacaaa?ttgagagata?ggtc?????????????????????????????????????????????24

<210>8

<211>31

<212>DNA

＜213〉mycoplasma arginini (Mycoplasma arginini)

<400>8

caataggtct?tatactacta?ttaaacaaga?t?????????????????????????????????????31

<210>9

<211>23

<212>DNA

＜213〉Mycoplasma orale (Mycoplasma orale)

<400>9

gaatattggg?ccattaacta?ttt??????????????????????????????????????????????23

<210>10

<211>27

<212>DNA

＜213〉Mycoplasma orale (Mycoplasma orale)

<400>10

caataggtca?aaaatactta?tacgtaa??????????????????????????????????????????27

<210>11

<211>24

<212>DNA

＜213〉mycoplasma hyorhinis (Mycoplasma hyorhinis)

<400>11

ctagacacga?atcgattatg?taat?????????????????????????????????????????????24

<210>12

<211>22

<212>DNA

＜213〉mycoplasma hyorhinis (Mycoplasma hyorhinis)

<400>12

aacgatcttt?tttataaccg?ag???????????????????????????????????????????????22

<210>13

<211>22

<212>DNA

＜213〉mycoplasma hominis (Mycoplasma hominis)

<400>13

caaagaaccg?agagataaat?ct???????????????????????????????????????????????22

<210>14

<211>25

<212>DNA

＜213〉mycoplasma hominis (Mycoplasma hominis)

<400>14

caataggtca?tacaattaac?aaaac????????????????????????????????????????????25

<210>15

<211>25

<212>DNA

＜213〉mycoplasma pneumoniae (Mycoplasma pneumoniae)

<400>15

accgataaat?aaatggattt?tg???????????????????????????????????????????????22

<210>16

<211>19

<212>DNA

＜213〉mycoplasma pneumoniae (Mycoplasma pneumoniae)

<400>16

acatttccgc?ttctttcaa???????????????????????????????????????????????????19

<210>17

<211>19

<212>DNA

＜213〉genital tract mycoplasma (Mycoplasma genitalium)

<400>17

caccgaaaaa?attaatggg???????????????????????????????????????????????????19

<210>18

<211>24

<212>DNA

＜213〉genital tract mycoplasma (Mycoplasma genitalium)

<400>18

aagaatgttt?ttgaacagtt?cttt?????????????????????????????????????????????24

<210>19

<211>26

<212>DNA

＜213〉ureaplasma urealyticum (Ureaplasma urealyticum)

<400>19

ggctaatatt?cacatggatt?tttata???????????????????????????????????????????26

<210>20

<211>25

<212>DNA

＜213〉ureaplasma urealyticum (Ureaplasma urealyticum)

<400>20

aaatatttca?aaagttcata?tggtc????????????????????????????????????????????25

<210>21

<211>23

<212>DNA

＜213〉Lai Shi acholeplasma (Acholeplasma laidlawii)

<400>21

aagtgttagt?tagcctttct?cct??????????????????????????????????????????????23

<210>22

<211>24

<212>DNA

＜213〉Lai Shi acholeplasma (Acholeplasma laidlawii)

<400>22

aaatgatgtc?tgaaaagaaa?taag?????????????????????????????????????????????24

<210>23

<211>26

<212>DNA

＜213〉small urea substance (Ureaplasma parvum)

<400>23

ggcttatatt?catatggatt?ttaata???????????????????????????????????????????26

<210>24

<211>24

<212>DNA

＜213〉small urea substance (Ureaplasma parvum)

<400>24

tatttttaaa?aattcatatg?gtcg?????????????????????????????????????????????24

Claims (10)

1. the detection authentication method of a mycoplasma species is characterized in that comprising the following steps:

(1) DNA of extraction testing sample;

(2) use is through the mycoplasma universal primer of mark, with the DNA of PCR method amplification testing sample;

(3) the mycoplasma specific specificity probe hybridization on nylon membrane with pcr amplification product and telltale mark;

(4) pcr amplification product of not hybridizing on the flush away nylon membrane;

(5) show results of hybridization.

2. the detection authentication method of mycoplasma according to claim 1 is characterized in that described mycoplasma specific specificity probe is to have a kind of in the probe of the described base sequence of SEQ ID NO:5 to SEQ ID NO:24 in the sequence table or several or all.

3. the detection authentication method of mycoplasma according to claim 1 is characterized in that described mycoplasma universal primer comprises:

Outer primer:

SPS1：5’-CCCTACGRGAACGTGGGGVTGGAWYACCTCCT-3’(SEQ?ID?NO：1)

SPA2：5’-CSDDGCWTWTCGSWGDTWRDYVCGTCCTTCATCG-3’(SEQ?IDNO：4)；

Inner primer:

SPS2：5’-CGRGAACGTGGGGVTGGAWYACCTCCTTTC-3’(SEQ?ID?NO：2)

SPA1：5’-CGTCCTTCATCGMCTNTYYDGWSCCAAGGCATYCAC-3’(SEQ?IDNO：3)。

4. the detection authentication method of mycoplasma according to claim 1 is characterized in that with the described mycoplasma universal primer of biotin labeling.

5. the detection authentication method of mycoplasma according to claim 1 is characterized in that the method for described demonstration results of hybridization is: on nylon membrane, add photographic developer, and the vitamin H combination of mark on photographic developer and the PCR product, fluorescence excitation makes the exposure of x exographX.

6. be used to detect the gene probe of identifying mycoplasma, it is characterized in that having the arbitrary base sequence in the described base sequence of SEQ ID NO:5 to SEQID NO:24 in the sequence table.

7. be used to the to increase universal primer of mycoplasma 16S-23SrRNA transcribed spacer DNA is characterized in that comprising outer primer SPS1, the outer primer SPA2 with described base sequence of SEQ ID NO:4 in the sequence table with described base sequence of SEQ ID NO:1 in the sequence table, has the inner primer SPS2 of the described base sequence of SEQ ID NO:2 in the sequence table and has the inner primer SPA1 of the described base sequence of SEQ ID NO:3 in the sequence table.

8. the detection authentication method of mycoplasma according to claim 1 is characterized in that the PCR reaction conditions is: the condition of increase for the first time sex change, annealing, extension be respectively 94 ℃ 10 seconds, 65 ℃ 10 seconds, 74 ℃ 1 minute, 30 circulations; The condition of sex change, annealing, extension of increasing for the second time be respectively 94 ℃ 10 seconds, 70 ℃ 10 seconds, 74 ℃ 1 minute, 30 circulations.

9. detect to identify mycoplasma detection kits for one kind, it is characterized in that it contains:

Container;

The universal primer of mycoplasma 16S-23SrRNA transcribed spacer DNA is used to increase;

At least a mycoplasma specific specificity probe with described base sequence of SEQ ID NO:5 to SEQ ID NO:24 in the sequence table;

And operation instruction.

10, mycoplasma detection kits is identified in detection according to claim 9, it is characterized in that this test kit also contains PCR and reacts needed reagent.