CN103820557B - Mycoplasma pneumoniae resistance diagnostic kit - Google Patents
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Abstract
The present invention discloses a kind of mycoplasma pneumoniae resistance diagnostic kit.The kit includes upstream primer sequence P1：5'TCCAGGTACGGGTGAAGACA'3；Downstream primer sequence P2：5'GTCCTGATCAATATTAAGCTACAGTAAAGCT'3；Probe sequence T1：5'ACGGAAAGACCCC'3, VIC are marked；Probe sequence T2：5'CGGGAAGACCCC'3, FAM are marked.Mycoplasma pneumoniae resistance diagnostic kit provided by the invention identified the genotype in MP2063 sites by establishing quantitative fluorescent PCR allele discrimination method, judges its drug resistance while quick diagnosis, and detection sensitivity is preferable.
Description
Technical field
It is resistance to more specifically to a kind of mycoplasma pneumoniae of real-time quantitative the present invention relates to a kind of PCR detection kit
Medicine diagnostic kit.
Background technology
Mycoplasma pneumoniae（Mycoplasma pneumoniae, referred to as MP）It is that children and adolescents and adult upper and lower exhale
Inhale the common causative of road infection.23%~50% preschool child and teenager's community acquired pneumonia are caused by MP infection
, popular cycle is 3~7 years.Mycoplasma pneumoniae pneumonia easily recurrence or protracted course of disease, patient with severe symptoms often merges the outer symptom of lung, right
Health hazard is serious.
MP is acellular wall construction, it can only clinically select to suppress or influence the medicine of its protein and nucleic acid synthesis, it is such as big
Cyclic lactone class antibiotic, tetracycline antibiotics, carbostyril antibiotic etc..But due to the adverse reaction of other antibiotic, control
Treat children MP and infect preferred macrolide antibiotics.Since 2001 find to the MP of macrolide antibiotics resistance,
MP resistant rates raise year by year and Epidemic Scope gradually expands, and existing multiple countries are separated to MP persisters.According to document report
Road, Japanese MP resistant rates in 2007 are up to more than 40%, and South Korea children inpatient MP resistant rates are 61.3%, and Italy is
26%, France is 9.8%, and Germany is 3%.Chinese MP resistances situation is then more severe, in MP clinical separation strains in more than 92% big ring
Esters antibiotics resistance.MP resistance mechanism is mainly large ribosomal subunit 23S rRNA gene mutation and ribosomal protein
L4, L22 gene mutation, the point mutation in wherein 2063 sites and 2064 sites in 23S rRNA domains V areas are occupied an leading position.I
2063A-G mutation account for more than 97% in the resistance MP that state finds.
At present, the MP diagnostic methods clinically commonly used are MP cultures, Serologic detection and PCR.Time-consuming for MP cultures, positive
Rate is low, and the monitoring of drug resistance usually requires in vitro culture and carries out drug sensitive test, determines minimal inhibitory concentration（MIC）.Serology
Detection method is simple and easy to do, but needs patient's convalescent serum to compare, and acute stage diagnosis effect is bad.Conventional PCR method has
There is the advantages of quick, positive rate is high, but the problem of false positive be present.Quantitative fluorescent PCR is with high specificity, quantitative accurate, repetition
The advantages that property is good and developed rapidly, but it can not judge pathogen drug resistance, limited to the directive significance of clinical treatment.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of mycoplasma pneumoniae resistance diagnostic kit.The kit
The genotype in MP2063 sites can be identified, in diagnosis by establishing quantitative fluorescent PCR allele discrimination method
Judge its drug resistance simultaneously.
To realize above-mentioned goal of the invention, the present invention uses following technical schemes：
A kind of mycoplasma pneumoniae resistance diagnostic kit, including：
Upstream primer sequence P1：5'TCCAGGTACGGGTGAAGACA'3；
Downstream primer sequence P2：5'GTCCTGATCAATATTAAGCTACAGTAAAGCT'3；
Probe sequence T1：5'ACGGAAAGACCCC'3, VIC are marked；
Probe sequence T2：5'CGGGAAGACCCC'3, FAM are marked.
Mycoplasma pneumoniae resistance diagnostic kit provided by the invention is by establishing quantitative fluorescent PCR allele discriminating side
Method, the genotype in MP2063 sites is identified, its drug resistance is judged while quick diagnosis, it is clinical real so as to instruct
Trample.The detection sensitivity of the diagnostic kit is preferable, and potential applicability in clinical practice is huge.
Brief description of the drawings
Fig. 1 is MP type strains FH, persister 307 and structure standard plasmid PCR results.Wherein, 1 is MP type strains FH, 2
It is sensitive 2063A plasmids for persister 307,3,4 be resistance 2063G plasmids, and N is negative control, and P is positive control；M is
100bp DNA Ladder。
Fig. 2 is real-time fluorescence quantitative PCR standard curve.
Fig. 3 shows that real-time fluorescence quantitative PCR identifies the diagnostic value of 2063 allele.
Embodiment
The present invention is expanded on further with reference to specific embodiment.Experimental method used in following embodiments is such as without spy
Different explanation, is conventional method.Material used, reagent etc. in following embodiments, unless otherwise specified, commercially
Obtain.It should be understood that these embodiments are only illustrative of the invention and is not intended to limit the scope of the invention.In the following example not
The experimental method of actual conditions is indicated, generally according to normal condition, or according to the condition proposed by manufacturer.
Mycoplasma pneumoniae（MP）It is a kind of common causative for causing children Streptococcus and extrapulmonary complication.Children MP feels at present
The primary treatment medicine of dye is macrolide antibiotics.Recent year, international clinically more and more it is separated to resistance to big ring
The MP strains of lactone antibiotic, main resistance mechanism are the 23S rRNA of large ribosomal subunit encoding gene mutation.In China,
The persister of report more than 97% is mutated for 2063A → G at present.From 2000 for the first time report real-time quantitative PCR diagnosis MP with
Come, the existing real-time quantitative PCR diagnostic method research largely for different extension increasing sequences occurs, and many commercial reagent also occurs
Box.But these methods can only provide positive or negative result, it is impossible to obtain resistance information.Therefore, the present invention is for causing MP resistance to
2063 A → G of medicine point mutation, primer and probe is designed, establish the kit of MP resistances diagnosis one-step method.This is deployed below
Detailed specific description.
1 materials and methods
1.1 sample
1.1.1MP type strain
MP type strains FH（ATCC15531）, mycoplasma hominis（ATCC27813）For the attached Beijing friendship of the Capital University of Medical Sciences
Febris nosocomialis band Institute for Medical Research laboratory storage；Pyriform mycoplasma, myoplasna penetranses, mycoplasma genitalium are defended by Southeast China University is public
Raw institute professor Wang Bei present.
1.1.2MP clinical separation strain
MP clinical separation strains are isolated from going to a doctor in the attached Beijing friendship of the Capital University of Medical Sciences for 2 months 2010~2 months 2011
Pediatric hospital（Inpatient department and outpatient service）, Beijing Chaoyang Hospital Attached to Capital Medical Univ.'s paediatrics（Inpatient department）43 Diagnosis of Suspected Pneumonia
The respiratory tract oropharyngeal swab specimen of children with mycoplasma pneumoniae pneumonia.The 12 parts of collections of Nasopharyngeal swabs sample are gone to a doctor big in Beijing from December, 2012
Learn the 3rd doubtful MPP of pediatric hospital case.
1.1.3 bacterial strain is compared
Pseudomonas aeruginosa（ATCC27853）, EHEC（ATCC25922）, enterobacter cloacae（ATCC700323）, it is golden yellow
Color staphylococcus（ATCC25922）, the primary clinical separation strain of kerekou pneumonia（1705）By the attached Beijing friendship doctor of the Capital University of Medical Sciences
Clinical laboratory of institute presents.
1.2 reagent
Calf serum, yeast extract, glucose, ampicillin, IPTG, X-Gal, agarose, tryptone are purchased
From Beijing ring Ya Taike biologies Co., Ltd；2 × Tap plus PCR Green Mix are purchased from Takara companies；pGEM-T
Easy Vector Systems II are purchased from Promega companies；QIAamp DNA minikit DNA extraction kits are purchased from
Qiagen companies；PCR primer QIAquick Gel Extraction Kit and plasmid purification kit are purchased from Beijing CoWin Bioscience Co., Ltd.；
Quantitative fluorescent PCR Genotyping mix are purchased from American AB I companies.
1.3 quantitative fluorescent PCR
1.3.1DNA template extraction
Culture MP, the 20000g for taking 200 μ L logarithmic phases to grow, centrifuge 20min；200 μ L PBS precipitations are resuspended；QIAamp
DNA minikit extract DNA profiling.The sterile picking colony of oese is into 200 μ L PBS, QIAampDNA minikit extractions
DNA profiling.The sterilized cotton swabs for extracting oropharyngeal swab specimen are soaked with MP nutrient solutions, cotton swab is discarded after repeatedly rinsing extruding, will
Soak centrifuges 10min with 20000g, the μ L of mark-on present treatment liquid 50 after abandoning supernatant, and fully mixes, and puts 100 DEG C of boiling water
Middle water-bath 10min, this solution are standby as DNA profiling.
1.3.2 primer sequence and probe sequence
According to MP type strain M129 sequences（NC_000912.1）Design upstream and downstream primer and probe in 23S RNA mutational sites；
Upstream primer sequence P1：5'TCCAGGTACGGGTGAAGACA'3（SEQ ID NO.1）, downstream primer sequence P2：5'
GTCCTGATCAATATTAAGCTACAGTAAAGCT'3（SEQ IDNO.2）, probe sequence T1：5'ACGGAAAGACCCC'3
（SEQ ID NO.3）, VIC marks；T2：5'CGGGAAGACCCC'3（SEQ ID NO.4）, FAM marks.Primer sequence and probe
Sequence synthesizes by Invitrogen trade Co., Ltd.
1.3.3PCR
Reaction system：2 × buffer solution 10.0 μ L, 10 μm of ol/L primer P31.0 μ L, 10 μm of ol/L primer P41.0 μ L, mould
Plate DNA1.0 μ L, ddH2O7.0 μ L.
Reaction condition：93 DEG C of denaturation 2min；95 DEG C of 1min, 50 DEG C of 1min, 72 DEG C of 1min, 35 circulations；72 DEG C of extensions
5min。
Quantitative fluorescent PCR product is reclaimed using glue reclaim kit, is cloned using T Easy carriers linked system, 4 DEG C of companies
Take over night, transformed competence colibacillus cell, blue hickie screening.6 white colonies of random picking, using lysate as template, with original glimmering
Fluorescent Quantitative PCR reaction system is expanded, and 2% agarose gel electrophoresis identification recon selects 2 positive colonies and send Invitrogen
Trade Co., Ltd is sequenced；Sequence analysis is carried out using Blast functions.It is standby to select the consistent clone's extraction plasmid of sequencing result
With.
1.3.4 real-time fluorescence quantitative PCR
Reaction system：2 × buffer solution 10.0 μ L, 40 × Assay Mix（Containing P1, P2, T1, T2）0.5 μ L, DNA profiling（It is wild
Raw type 2063A or saltant type 2063G standard plasmids）1.0 μ L, ddH2O7.0 μ L, the μ L of cumulative volume 20.
Reaction condition：95℃1min；92 DEG C of 15s, 60 DEG C of 1min, 40 circulations；When collecting before expanding, expanding respectively, expand
Fluorescence signal after increasing, standard curve is obtained using the SDS softwares of ABI companies quantitative real time PCR Instrument 7500.Positive plasmid from
600000 to 6 carry out 10 times of doubling dilutions.Specificity experiments are carried out as template using different strains DNA, detect real-time fluorescence
The selectivity of primer and probe in quantitative PCR reaction system.43 plants of loci gene types of MP clinical separation strains 2063 and Nasopharyngeal swabs
The identification of the loci gene type of sample 2063 is completed under this reaction system.
1.3.523S rRNA nest-type PRCs 23S rRNA nest-type PRCs primer is outer sleeve downstream：5′
GGTCCTAAGGTAGCGAAATT3′（SEQ ID NO.5）；Overcoat downstream：5′CAGTTACCAATTAGAACAGC3′（SEQ ID
NO.6）；Interior sleeve downstream P3：5′CCTAGTCGGGTAAATTCCGT3′（SEQ ID NO.7）；Inner sleeve downstream P4：5′
CCAAGGGTAGTATTCCACCT3′（SEQ ID NO.8）（Referring to Li Jing, Cui Feifei, Xin Deli paper《Childrens respiratory tract lung
Scorching mycoplasma infection resistance present situation》, it is published in《Applied Clinical Pediatrics magazine》2009,24（22）：1717~1719.）, two wheel productions
Wu Song Invitrogens trade Co., Ltd is sequenced.Using 23S rRNA nest-type PRCs sequencing results as control, real time fluorescent quantitative is calculated
PCR identifies the susceptibilitys of 2063 loci gene types, specificity, positive predictive value, negative predictive value etc..
2 results
2.1 structure standard plasmids
With MP type strains FH（Sensitive strain, 2063A）With clinical separation strain 317（Persister, 2063G）For DNA profiling, carry out
PCR is expanded, and amplified fragments are located at 250bp right positions, in the same size with the purpose fragment 244bp of design（See Fig. 1）.Will be above-mentioned
PCR primer is connected to transformed competence colibacillus cell after T-EASY, and coated plate contains ITPG, X-gal and ampicillin selectivity flat board,
The bacterium colony of white is selected to enter performing PCR identification, obtained clip size is with being expected unanimously（See Fig. 1）.Plasmid order-checking result is shown, quick
It is A to feel the site of 2063A plasmids 2063 with MP type strains FH, and the site of resistance 2063G plasmids 2063 is G, with MP type strains FH not
Unanimously.
2.2 standard curve
The R of sensitive 2063A plasmid controls curve2For 0.9979, slope is -3.4893, amplification efficiency 93%；Resistance
The R of 2063G plasmid control curves2For 0.9979, slope is -3.7262, amplification efficiency 86%.It is quick under identical copies number
Ct values corresponding to 2063A plasmids and resistance 2063G plasmids are felt very close to reappearance is preferable（See Fig. 2）.Sensitive 2063A plasmids and
The Monitoring lower-cut of resistance 2063G plasmids be respectively and 6 and 60, sensitive 2063A plasmid copy numbers be 6~6 × 106, genotype is
2063A；Resistance 2063G plasmid copy numbers are 60~6 × 106, genotype 2063G.
2.3 specificity experiments
The Ct values of persister are 24.69 in MP clinical separation strains, genotype 2063G；MP type strains FH Ct values are
26.05, genotype 2063A.In addition to mycoplasma genitalium, mycoplasma hominis, pyriform mycoplasma, myoplasna penetranses and green pus bar
Bacterium, EHEC, enterobacter cloacae, staphylococcus aureus, klebsiella pneumoniae clinical separation strain do not expand,
The Ct values of mycoplasma genitalium are 26.15, genotype 2063A.
2.443 plants of loci gene types of clinical separation strain 2063
43 plants of clinical separation strains carry out real-time fluorescence quantitative PCR and expanded, and Ct values are 26.82~34.25, copy number
For 2.05 × 104~2.95 × 106；Wherein 6 plant of 2063 site is A, is sensitive strain, and 37 plant of 2063 site is G, is persister, MP
Resistant rate is 86.05%.Compared with 23S rRNA nest-type PRC sequencing results, real-time fluorescence quantitative PCR identifies 2063 allele
Susceptibility, specificity, positive predictive value, negative predictive value be 100%（See Fig. 3）.
The loci gene type of 2.5 Nasopharyngeal swabs sample 2063
2 parts of progress real-time fluorescence quantitative PCRs do not expand in 12 parts of Nasopharyngeal swabs samples, 6 in remaining 10 parts of sample
2063 sites of part are A, are sensitive strain, and 3 plant of 2063 site is G, are persister, and 1 part of sample finds 2063A and 2063G positions simultaneously
Point.Nasopharyngeal swabs sample MP positive rates are 83.33%（10/12）, resistant rate 33.33%（4/12）.
The present invention uses MP type strain FH, using the clear and definite clinical separation strain 307 of Antibiotic Resistance as template, builds responsive type
2063A plasmids, using drug-resistant type 2063G plasmids as positive criteria product, draw standard curve.The minimum detection of amphitypy is respectively 6 Hes
60 copy numbers.Drug-resistant type standard items compared with responsive type order of magnitude lower, consider reason may be 2063 G hydrogen bonds fractures need compared with
High energy.So in identical annealing temperature, the efficiency of open chain containing G is low compared with A, therefore it is poor compared with A to detect effect.So according to difference
It is necessary that genotype structure standard curve carries out nucleic acid quantification again.Specificity experiments result shows that mycoplasma genitalium MG has expansion
Increase, genotype 2063A.MG is a kind of pathogenic pathogen that spreads through sex intercourse that has for being colonized in urogenital tract, first choice treatment
Medicine is macrolides, quinolone and tetracycline antibiotics.Sequence analysis is carried out to MG, finds 2063A positions MG sequences nearby
Row reach 99% with type strain M129 uniformity.Because of the limit in detection site region, fluorescence quantification PCR primer and probe experimental design
System, the intersection are inevitable.But serology or 16s RNA PCR sectors point can be passed through.
43 plants of clinical separation strains are detected using the kit, 6 plants are sensitive strain, and 37 plants are persisters.By 43 plants of clinics point
Display result is sequenced after traditional 23S rRNA nest-type PRCs from strain and the present invention is completely the same.It can thus be seen that for clinic
Separation strains, 2063 genotype can be quick and precisely identified using the kit, distinguish resistance and sensitive strain, so as to simplify operation
Flow, reduce labor intensity.
The kit is applied to clinical samples, and it is 88.33% to find MP positive rates.With batch sample 23S rRNA nidos
PCR detects the positive.Inventor is by analysis, it is believed that is probably that oropharyngeal swab specimen DNA extract solutions are mixed with a large amount of impurity, simply boils
Boiling method can not fully erased PCR mortifiers, cause real-time quantitative PCR efficiency decline, recall rate reduce；Or suffered from due to indivedual
Caused by person's carrying capacity is relatively low, it may be considered that optimization DNA samples extracting method uses ripe DNA extraction kit.
Of the invention that 2063A and 2063G heterozygous genotypes are detected in same sample for the first time, illustrating can in same patient
There can be sensitive and persister simultaneously.And common Chao Shi PCR sequencings can cause dominant strain gene to be largely amplified, sequencing result
Generally only illustrate dominant microflora genotype, it is impossible to react the actual phenotype for infecting MP of patient strictly according to the facts.The achievement in research of the present invention shows
Show that MP can produce drug resistance under macrolide antibiotics pressure in vitro, or may also be that MP have 2063A → G in environment
Low-level mutation, simply antibiotic usage kill sensitive strain when, persister just turns into dominant microflora.
In summary, the present invention tentatively establishes a kind of quick diagnosis MP infection and judges the mycoplasma pneumoniae of drug-resistant phenotype
Resistance diagnostic kit.The kit detection sensitivity is preferable, but 2063A with 2063G genotype detections lower limit is different, 2063G
Compared with 2063A order of magnitude lower, only have with mycoplasma genitalium and intersect.Optimizing reaction system should be continued and expanding clinical practice,
Effect is assessed.It is of the invention that Sensitivity and Specificity is preferable compared with traditional 23S rRNA Chao Shi PCR sequence measurements,
Potential applicability in clinical practice is huge.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
Member, under the premise without departing from the principles of the invention, can also make some improvements and modifications, these improvements and modifications also should be regarded as
Protection scope of the present invention.
SEQUENCE LISTING
<110>Beijing Friendship Hospital Attached to Capital Medical Univ.
<120>Mycoplasma pneumoniae resistance diagnostic kit
<130>/
<160>8
<170>PatentIn version3.5
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tccaggtacg ggtgaagaca20
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<213>It is artificial synthesized
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gtcctgatca atattaagct acagtaaagc t31
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<213>It is artificial synthesized
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acggaaagac ccc13
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<213>It is artificial synthesized
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<213>It is artificial synthesized
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Claims (1)
- A kind of 1. mycoplasma pneumoniae resistance diagnostic kit, it is characterised in that including：Upstream primer sequence P1：5'TCCAGGTACGGGTGAAGACA'3；Downstream primer sequence P2：5'GTCCTGATCAATATTAAGCTACAGTAAAGCT'3；Probe sequence T1：5'ACGGAAAGACCCC'3, VIC are marked；Probe sequence T2：5'CGGGAAGACCCC'3, FAM are marked.
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| CN201310526965 | 2013-10-30 | ||
| CN201410073883.9A CN103820557B (en) | 2013-10-30 | 2014-02-28 | Mycoplasma pneumoniae resistance diagnostic kit |
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Citations (2)
| 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 |
| CN104630329A (en) * | 2013-11-08 | 2015-05-20 | 江苏默乐生物科技有限公司 | Mycoplasma pneumonia 23S rRNA 2063 locus A:G mutation detection specific primer and probe |
-
2014
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Patent Citations (2)
| 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 |
| CN104630329A (en) * | 2013-11-08 | 2015-05-20 | 江苏默乐生物科技有限公司 | Mycoplasma pneumonia 23S rRNA 2063 locus A:G mutation detection specific primer and probe |
Non-Patent Citations (5)
| Title |
|---|
| Detection of Macrolide Resistance in Mycoplasma pneumoniae by Real-Time PCR and High-Resolution Melt Analysis;Bernard J. Wolff,et al;《ANTIMICROBIAL AGENTS AND CHEMOTHERAPY》;20081031;第52卷(第10期);3542–3549 * |
| Mycoplasma pneumoniae M129 strain M129;ATCC 29342 23s ribosomal RNA,complete sequence;Dendekar,T.;《Genbank NR_077056》;20130525 * |
| 儿童呼吸道肺炎支原体感染耐药现状;李靖等;《实用儿科临床杂志》;20091130;第24卷(第22期);1717-1728 * |
| 实时定量PCR检测肺炎支原体2063位点点突变的方法学研究;李静宜等;《中国全科医学》;20131110;第16卷(第11B期);545-548 * |
| 实时定量PCR检测肺炎支原体2063点突变的方法建立;李静宜;《中华医学会第十次全国临床微生物学术年会暨第九届全球华人临床微生物与感染症学术论坛暨2013年浙江省医学微生物与免疫学及医学病毒学学术年会论文汇编》;20130905;125 * |
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