CN104630329A - Mycoplasma pneumonia 23S rRNA 2063 locus A:G mutation detection specific primer and probe - Google Patents
Mycoplasma pneumonia 23S rRNA 2063 locus A:G mutation detection specific primer and probe Download PDFInfo
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- CN104630329A CN104630329A CN201310550380.1A CN201310550380A CN104630329A CN 104630329 A CN104630329 A CN 104630329A CN 201310550380 A CN201310550380 A CN 201310550380A CN 104630329 A CN104630329 A CN 104630329A
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Abstract
The invention discloses a specific primer and probe for detecting A:G mutation on the mycoplasma pneumonia 23S rRNA 2063 locus. The primer sequence comprises an upstream primer MPF sequence: 5'TGTAACCNTCTCTTGNCTGTCT3' and a downstream primer MPR sequence: 5'CGATTNCTCCTACCTNTTCTCTA3'. The probe 2063P sequence is 5'FAMCGGGGTCNTCNCGTCCCBHQ13'.
Description
Technical field
The present invention be a kind of for detect mycoplasma pneumoniae 23S rRNA 2063 site A:G suddenly change Auele Specific Primer and probe.
Background technology
Mycoplasma pneumoniae (
mycoplasma pneumoniaemP) be a kind of common respiratory pathogen, usually slight upper respiratory tract infection is caused, as having a sore throat, pharyngolaryngitis and trachitis, its pneumonia caused accounts for 50% of non-bacterial pneumonia, also can cause extrapulmonary complication, involve one or more system, organ, as skin, gi tract, cardiovascular, skeletal muscle and kidney, maincenter and peripheral nervous system pathology time serious, can be caused, even dead.Propagated with aerosol form by the spittle, remain in nose, larynx, tracheae and sputum, close contact may cause breaking out of mycoplasma pneumoniae.Mycoplasma pneumoniae is acellular wall, therefore, as insensitive in penicillin etc. on the microbiotic affecting Cell wall synthesis, but, on affecting the microbiotic of Bacterioprotein biosynthesis as the sensitivity such as Macrolide, quinolones.Macrolide is the choice drug for the treatment of mycoplasma pneumoniae infection, the part between the transpeptidase center of mycoplasma pneumoniae rrna 50S subunit and peptide output channel can be incorporated into, the extension of peptide chain is suppressed by mechanical blockage passage, thus reach the object of arrestin synthesis, its binding site is made up of the Nucleotide of 23S rRNA structural domain V, and wherein 2063 sites are main integral parts.In recent years, along with widely using of macrolide antibiotics, clinically isolate Resistant strain, the existence of prompting mycoplasma pneumoniae resistance phenomenon.Research both domestic and external shows, produces the origination point sudden change in 23S rRNA sequence of the mycoplasma pneumoniae of resistance, and wherein, modal is the sudden change of 2063 site A to G.
At present, clinically to the inspection of mycoplasma pneumoniae resistance, mainly rely on separation and the drug sensitive experiment of mycoplasma pneumoniae, because mycoplasma pneumoniae is separated comparatively difficulty, so sensitivity is lower, and takes time and effort, be unfavorable for timely direction of medication usage.The Fluorescence PCR assay that the present invention adopts, easy and simple to handle, detect rapidly, recall rate is high, the clear and definite infection whether having resistance mycoplasma pneumoniae is got final product by single test, be conducive to diagnosing in time and choose reasonable treatment plan disease, compared with traditional separation and Culture detection technique, improve detector efficiency, reduce loss.The examination of high efficient and reliable can be carried out various clinical sample, and the clinical state of an illness and medication effect are monitored, instruct rational use of drug.
Summary of the invention
The object of the present invention is to provide a kind of for detect mycoplasma pneumoniae 23S rRNA 2063 site A:G suddenly change primer and probe.
Based on above-mentioned purpose, the present invention by the following technical solutions.
For detecting Auele Specific Primer that mycoplasma pneumoniae 23S rRNA 2063 site A:G suddenlys change and probe sequence comprises: upstream primer MPF sequence is 5 ' TGT AAC CNT CTC TTG NCT GTC T 3 ', downstream primer MPR sequence be 5 ' CGA TTN CTC CTA CCT NTT CTC TA 3 ' and probe 2063P sequence is 5 ' FAM CGG GGT CNT CNC GTC CC BHQ1 3 '.
Concrete principle of the present invention is the Auele Specific Primer and the probe that utilize a pair target nucleic acid sequence, adopts Real-Time Fluorescent Quantitative PCR Technique, is realized the amplification of target nucleic acid sequence segment by PCR.The probe used is for distinguishing the oligonucleotide of mark fluorescent reporter group (R) and fluorescent quenching group (Q) in two ends.When probe is complete, the fluorescence that reporter group sends is quenched group absorptions, in pcr amplification process, the fluorescent probe enzyme of specific combination in target nucleotide segment is cut degraded by 5 ' end 5 prime excision enzyme activity of archaeal dna polymerase, the fluorescent signal of reporter group can be detected, the change of fluorescent signal amount is directly proportional to amplified production amount, thus can be judged the existence of sample to be tested target nucleotide sequence by fluorescence power.
Accompanying drawing explanation
Fig. 1 is the fluorescent PCR amplification figure utilizing primer pair MPF/MPR and probe 2063P to detect mycoplasma pneumoniae 23S rRNA 2063 site A:G sudden change positive sample.
Embodiment
1. the design of primer and probe: by comparing analysis to all known mycoplasma pneumoniae 23S rDNA sequences respectively, selecting 2063 place, site sections, designing multipair primer and probe, about primer length is generally 20 bases.Optimum primer probe sequence combination is as follows:
Upstream primer MPF:5 ' TGT AAC CNT CTC TTG NCT GTC T 3 '
Downstream primer MPR:5 ' CGA TTN CTC CTA CCT NTT CTC TA 3 '
Probe 2063P: 5 ' FAM CGG GGT CNT CNC GTC CC BHQ1 3 '.
2. the optimization of reaction system: utilize the resistance mycoplasma pneumoniae deactivation liquid of clinical separation as measuring samples, with magnetic bead cracking process extracting mycoplasma pneumoniae nucleic acid, be stored in-80 DEG C after packing.
The optimization of 2.1 primer concentrations is when in reaction system, other condition is identical, mycoplasma pneumoniae primer concentration is done multiple proportions serial dilution from 0.1 μm of ol/L to 1.6 μm of ol/L respectively, by the com-parison and analysis to test-results, determine that best primer concentration is 0.2 μm of ol/L.
The optimization of 2.2 concentration and probe concentration is when in reaction system, other condition is identical, mycoplasma pneumoniae abrupt climatic change concentration and probe concentration is done multiple proportions serial dilution from 0.1 μm of ol/L to 0.5 μm of ol/L respectively, by the com-parison and analysis to test-results, determine that best concentration and probe concentration is 0.16 μm of ol/L.
Utilize above-mentioned primer and probe to carry out the foundation of reaction system, finally determine that the real-time fluorescence PCR reaction system that the mycoplasma pneumoniae 23S rRNA 2063 site A:G adopted suddenlys change is 25 μ L.The configuration carrying out reaction solution is described according to PCR kit for fluorescence quantitative.
3. the selection of instrument sense channel
The reporter fluorescence group that the fluorescence detection channel selected should mark with probe is consistent, specifically arranges according to instrument working instructions.
4. PCR reaction conditions is as follows
50 DEG C of 2min carry out UNG enzyme reaction; 95 DEG C of 3min, 1 circulation; 94 DEG C of 10s, 62 DEG C of 40s, 40 circulations, collect fluorescence 62 DEG C of 40s stages.
5. Analysis of test results
If containing the mycoplasma pneumoniae that generation 2063 site A:G suddenlys change in sample to be checked, then show positive amplification curve, its detection sensitivity can reach 1000 copies/mL, if there is no the mycoplasma pneumoniae that 2063 site A:G suddenly change in sample to be checked, then show negative amplification curve, namely without amplified signal, above-mentioned primer pair and probe is pointed out to have good specificity and sensitivity.
The invention has the advantages that:
(1) detection sensitivity of primer provided by the invention and probe can reach 1000 copies/mL, illustrates that its sensitivity is good.
(2) primer provided by the invention and the sample of probe to the mycoplasma pneumoniae do not suddenlyd change containing 2063 site A:G do not have detection signal, and its high specificity is described.
(3) owing to the present invention be directed to 2063A:G mutational site, carry out program and system optimization, avoid the appearance of false negative and false positive results.
Claims (2)
1. one kind for detecting and identify the specific primer sequence that mycoplasma pneumoniae 23S rRNA 2063 site A:G suddenlys change, it is characterized in that described primer sequence to comprise upstream primer MPF sequence be 5 ' TGT AAC CNT CTC TTG NCT GTC T 3 ', downstream primer MPR sequence to be 5 ' CGA TTN CTC CTA CCT NTT CTC TA 3 '.
2., for detecting and identify the specific probe sequence that mycoplasma pneumoniae 23S rRNA 2063 site A:G suddenlys change, it is characterized in that described probe sequence is 5 ' FAM CGG GGT CNT CNC GTC CC BHQ1 3 '.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103820557A (en) * | 2013-10-30 | 2014-05-28 | 首都医科大学附属北京友谊医院 | MP (Mycoplasma Pneumoniae) drug resistance diagnostic kit |
| CN106191239A (en) * | 2016-07-11 | 2016-12-07 | 宁波基内生物技术有限公司 | A kind of real-time PCR detection mycoplasma pneumoniae nucleic acid and the primer of medicament-resistant mutation, probe, method and test kit |
| CN107254524A (en) * | 2017-06-22 | 2017-10-17 | 中国人民解放军第三〇七医院 | A kind of method and kit of Rapid nucleic acid detection mycoplasma pneumoniae and medicament-resistant mutation |
| CN110325639A (en) * | 2017-02-22 | 2019-10-11 | 株式会社友华 | Have probe, its design method and its application that false positive inhibits function |
| WO2021075655A1 (en) * | 2019-10-18 | 2021-04-22 | 고려대학교 산학협력단 | Primer and probe for simultaneous detection of mycoplasma pneumoniae and mycoplasma pneumoniae mutation, and uses thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102002522A (en) * | 2009-11-10 | 2011-04-06 | 复旦大学附属华山医院 | Method for detecting resistant mutant of mycoplasma pneumoniae |
-
2013
- 2013-11-08 CN CN201310550380.1A patent/CN104630329A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102002522A (en) * | 2009-11-10 | 2011-04-06 | 复旦大学附属华山医院 | Method for detecting resistant mutant of mycoplasma pneumoniae |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103820557A (en) * | 2013-10-30 | 2014-05-28 | 首都医科大学附属北京友谊医院 | MP (Mycoplasma Pneumoniae) drug resistance diagnostic kit |
| CN103820557B (en) * | 2013-10-30 | 2018-03-16 | 首都医科大学附属北京友谊医院 | Mycoplasma pneumoniae resistance diagnostic kit |
| CN106191239A (en) * | 2016-07-11 | 2016-12-07 | 宁波基内生物技术有限公司 | A kind of real-time PCR detection mycoplasma pneumoniae nucleic acid and the primer of medicament-resistant mutation, probe, method and test kit |
| CN110325639A (en) * | 2017-02-22 | 2019-10-11 | 株式会社友华 | Have probe, its design method and its application that false positive inhibits function |
| CN107254524A (en) * | 2017-06-22 | 2017-10-17 | 中国人民解放军第三〇七医院 | A kind of method and kit of Rapid nucleic acid detection mycoplasma pneumoniae and medicament-resistant mutation |
| WO2021075655A1 (en) * | 2019-10-18 | 2021-04-22 | 고려대학교 산학협력단 | Primer and probe for simultaneous detection of mycoplasma pneumoniae and mycoplasma pneumoniae mutation, and uses thereof |
| KR20210046888A (en) * | 2019-10-18 | 2021-04-29 | 고려대학교 산학협력단 | Primers and probes for simultaneous detecting Mycoplasma pneumoniae and M. pneumoniae mutants and use thereof |
| KR102250376B1 (en) * | 2019-10-18 | 2021-05-12 | 고려대학교 산학협력단 | Primers and probes for simultaneous detecting Mycoplasma pneumoniae and M. pneumoniae mutants and use thereof |
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Address after: 225300 building R19, road, Taizhou hi tech Development Zone, Zhejiang Province, China Applicant after: JIANGSU MOLE BIOSCIENCE CO., LTD. Address before: 225300, R19 building, No. 1, drug city road, Hailing District, Jiangsu, Taizhou Applicant before: JIANGSU MOLE BIOSCIENCE CO., LTD. |
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Application publication date: 20150520 |