CN102603875A - Fluorescent quantitative polymerase chain reaction (PCR) kit for Acinetobacter baunannii/calcoaceticus complex OXA-23 mutant gene - Google Patents
Fluorescent quantitative polymerase chain reaction (PCR) kit for Acinetobacter baunannii/calcoaceticus complex OXA-23 mutant gene Download PDFInfo
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Abstract
The invention discloses a fluorescent quantitative polymerase chain reaction (PCR) kit and method for an Acinetobacter baunannii/calcoaceticus complex OXA-23 mutant gene. Special oligonucleotide primers and probes are designed according to sequences of the mutant of the newly screened D-type beta-lactamase drug-resistant gene OXA-23 of the Acinetobacter baunannii/calcoaceticus complex. The fluorescent quantitative PCT kit can be used for detecting the Acinetobacter baunannii/calcoaceticus complex.
Description
Technical field
The present invention relates to the detection kit and the method for drug resistant gene.Particularly, the present invention has designed the PCR kit for fluorescence quantitative of the compound Acinetobacter calcoaceticus of Bao Man that detects the OXA-23 sudden change according to the compound Acinetobacter calcoaceticus bacterial strain of Bao Man of the OXA-23 transgenation of new screening, the present invention also relates to its detection method.
Background technology
Acinetobacter calcoaceticus is one type of non-fermentation, strict aerobic gram negative bacilli, is prevalent in occurring in nature.Acinetobacter can be divided into 32 gene kind types at least, and wherein Acinetobacter bauamnnii, Acinetobacter calcoaceticus, acinetobacter calcoaceticus gene kind type 3 and 13TU are difficult to distinguish through conventional phenotypic evaluation, closes to be called the compound Acinetobacter calcoaceticus complex body of Bao Man.
Acinetobacter bauamnnii is modal a kind of gram negative bacilli in the acinetobacter, extensively is present in natural water and soil, hospital environment and human body skin, respiratory tract, digestive tube and the urogenital tract, is conditioned pathogen.This bacterium distributes very wide in hospital environment and can long-term surviving, very easily causes the infection of critical patient, therefore often from being isolated the samples such as the blood of infected patient, urine, fester and respiratory secretions, in non-zymocyte, infects and is only second to pseudomonas.
Acinetobacter bauamnnii distributes maximum with the ICU lesion in hospital, be the Respiratory Medicine patient secondly.Mostly the patient who infects is the patient that gerontal patient, critical illness and Abwehrkraft des Koepers are weak, and the patient who uses various invasive operations and the treatment of life-time service Broad spectrum antibiotics.Again because this bacterium has strong resistibility to damp and hot ultraviolet ray and chemostefilant; Routine disinfection can only suppress its growth and can not kill, and vulnerable or have the patient of wound maybe be by more from the chance of medical worker's the hand or the infectation of bacteria that halfway medicine equipment had of sterilizing.
In recent years, the separation rate of acinetobacter in clinical sample improves year by year, and Acinetobacter bauamnnii accounts for 80%-90% wherein.Acinetobacter bauamnnii can cause respirator associated pneumonia, urinary tract infections, septicemia, traumatic infection and central nervous system infection etc.Because Acinetobacter bauamnnii has extremely strong adaptive capacity to environment and the ability that obtains exogenous drug resistant gene, thus very easily cause in the hospital send out popular.Acinetobacter bauamnnii can pass through removable gene elements such as plasmid, transposon and integron, obtains the resistance to multiple antibacterials.Whole genome sequence analysis to 1 strain Europe clone's I multidrug resistance bacterial strain is found; 45 drug resistant genes on the chromosome segment of the about 86kb of a segment length, have been assembled; Comprise VEB-1, AmpC, multiple aminoglycoside antibacterials modifying enzyme, formed the drug resistant gene island that is called as AbaR1.The gene fragment of this drug resistant gene island and Rhodopseudomonas, Salmonella and escherichia coli has homology, turns out to be acquired external sequence.In the karyomit(e) of the MDRAB (ACICU) of other 1 strain Europe clone's II, also send out drug resistant gene island AbaR2.Acinetobacter bauamnnii is integrated the ability of allogenic gene, it is had clinical used antibacterials are produced chemical sproof great potential.The β-Nei Xiananmei gene is the topmost drug resistant gene of Acinetobacter bauamnnii in numerous drug resistant genes; Comprise that the extended spectrum class (belongs to category-A; Comprise genes such as TEM-type, SHV-type, SIM-type, PER-type), metal enzyme (belong to category-B, comprise IMP and VIM gene), OXA type enzyme (genus D class) etc.OXA-23 type enzyme; Hydrolyzing penicillin and cynnematin, carbapenem; Bacterial strain is produced the antibiotic resistance of imipenum class [Livermore DM; Woodford N.Carbapenemases:a problem in waiting.Curr Opin Microbiol, 2000,3 (5): 489-495.].
The appearance of the compound Acinetobacter calcoaceticus of multidrug resistance Bao Man; Bring great difficulty to hospital infection control and clinical treatment; Because mostly the patient who infects is the patient that gerontal patient, critical illness and Abwehrkraft des Koepers are weak; Treat like the compound Acinetobacter calcoaceticus sensitive antibiotics of untimely selection Bao Man, with entail dangers to patient's life security.
Summary of the invention
In order to address the above problem, the invention discloses the real-time fluorescence quantitative PCR test kit and the method thereof of the compound Acinetobacter calcoaceticus OXA-23 of Bao Man mutator gene.
The present invention at first discloses the real-time fluorescence quantitative PCR test kit of the compound Acinetobacter calcoaceticus OXA-23 of Bao Man mutator gene.Test kit of the present invention comprises Auele Specific Primer, specific probe, standard DNA template and other conventional quantitative fluorescent PCR reagent.Test kit of the present invention can adopt following method to implement:
According to the sequence of the two mutants of the D class β-Nei Xiananmei drug resistant gene OXA-23 that newly filters out, said two mutants nucleotides sequence is classified Sequence NO.3 as, and its coded protein amino acid sequence is Sequence NO.7.According to Sequence NO.3 designing probe and upstream and downstream primer.The sequence of described upstream and downstream primer is seen Sequence NO.4 and Sequence NO.5 respectively, and described probe sequence is seen Sequence NO.6.
The sequence that perhaps includes above-mentioned sequence to 5 ' end prolongation;
Perhaps with above-mentioned sequence homology greater than 85% sequence.
Said probe 5 ' end mark fluorescent chromophoric group FAM, 3 ' end connects fluorescent quenching group B HQ.Adopt 25 μ l reaction volumes, contain 12.5 μ l universal PC R reaction mixtures [TaqMan
Universal PCR Master Mix (2 *)] in each reaction; Each 10nM of upstream and downstream primer (primer sequence is respectively Sequence NO.4 and Sequence NO.5 up and down); 400nM probe (probe sequence is Sequence NO.6); 2 μ l dna profilings; Mend sterilized water to 25 μ l.After reaction conditions is 95 ℃ of 10min, circulate 45 times with 95 ℃ of 40s and 60 ℃ of 1min.
The present invention adopts VITEK32 Bacteria Identification appearance and GNS448 drug sensitive plate to carry out drug sensitive test; The mutants which had that detects D class β-Nei Xiananmei drug resistant gene OXA-23 is to antibiotic susceptibility; The result shows; Said mutants which had is to cefotaxime, butylamine OK a karaoke club, Ampicillin Trihydrate, Cephazolin, cefepime, cefoxitin, ceftazime, Cefuroxime sodium, cefuroxime axetil, qingfengmeisu qiong, imipenum, piperacillin/Tazobactam Sodium resistance, and is comparatively responsive to cefoperazone/sulbactam and levofloxacin.
The invention provides the application of the real-time fluorescence quantitative PCR test kit that detects the compound Acinetobacter calcoaceticus OXA-23 of Bao Man mutator gene.PCR kit for fluorescence quantitative of the present invention can be used for the detection of the compound Acinetobacter calcoaceticus bacterial strain of Bao Man of OXA-23 transgenation.
Description of drawings
Fig. 1. the corresponding loop parameter (Ct value) of each concentration of standard plasmid.Curve is 10 from a left side to representing standard plasmid concentration again successively among the figure
8Copies/ μ l, 10
7Copies/ μ l, 10
6Copies/ μ l, 10
5Copies/ μ l, 10
4Copies/ μ l, 10
3Copies/ μ l, 10
2Quantitative fluorescent PCR curve during copies/ μ l.
Fig. 2 .OXA-23 mutator gene and OXA-23 gene order comparison figure.The site of trilateral indication is the mutational site.
The protein amino acid sequence comparison figure of Fig. 3 .OXA-23 mutator gene encoded protein matter aminoacid sequence and OXA-23 genes encoding.The site of trilateral indication is the mutational site.
Embodiment
Embodiment 1: the confirming of the new sequence of Bao Man compound Acinetobacter calcoaceticus drug resistant gene OXA-23
1. bacterium source: we have collected compound Acinetobacter calcoaceticus 32 strains of Bao Man to microbiotic height resistance (especially to the imipenum resistance) from year May in October, 2009 to 2011.Said 32 strain samples derive from patient's sputum.
2.OXA-23 design of primers: the present invention has designed the Auele Specific Primer of a pair of D class β-Nei Xiananmei drug resistant gene OXA-23, and the sequence of said primer is seen SEQ ID NO:1 and SEQ ID NO:2.
3.OXA-23 the amplification of gene: with the compound Acinetobacter calcoaceticus lysate of above-mentioned 32 strain Bao Man is template; Carry out pcr amplification, amplification system is (a 25ul system): template DNA (bacterial lysate) 2ul, primer 1 and 2 each 2ul; TaqE:0.25ul (0.5U); 10XBuffer 2.5ul, dNTP 2ul uses ddH
2O complements to 25ul.PCR appearance reaction conditions is following:
Sex change in advance: 95 ℃ of 5min
The circulation (35): sex change: 94 ℃ 30 seconds
Annealing: 55 ℃ 30 seconds
Extend: 72 ℃ 30 seconds
Extend: 72 ℃ of 5min
4.OXA-23 gene amplification product reclaims: the PCR product is done agarose electrophoresis, under uv lamp, downcut the purpose band, utilize the quick glue of QIANGEN to reclaim test kit and reclaim the PCR product.The product that reclaims is served Hai Shenggong to be measured.
5. sequencing result and BLASTn comparison; Only find that wherein 1 strain is numbered the bacterial strain OXA-23 crowd positive of AB37; Definite similar with the OXA-23 gene, its nucleotide sequence is seen SEQ ID NO:3, and protein sequence is seen SEQ ID NO:7; Said nucleotide mutant site is seen Fig. 2, and Fig. 3 is seen in the coded protein mutant site of said nucleotide sequence.
Embodiment 2: detect the real-time fluorescence quantitative PCR test kit preparation of the new sequence of OXA-23
1. design of primers and probe are synthetic
The SEQ ID NO:3 sequence that obtains according to the foregoing description 1.Adopt ABI primer Express 2.0 software design probes and primer (it is synthetic to go up sea base Kanggong department).The upstream primer sequence sees that SEQ ID NO:4 and downstream primer sequence see SEQ ID NO:5, and probe sequence is seen SEQ ID NO:6.Said probe 5 ' end mark fluorescent chromophoric group FAM, 3 ' end connects fluorescent quenching group B HQ.
2. standard DNA template preparation
Adopting last described upstream and downstream primer of a step is that template amplification goes out the 200bp fragment to the bacterial strain with above-mentioned AB37, and (QIAgen German) and after being connected to the PGM-T cloning vector is transformed into DH5 α competent cell to the PCR product purification.Extract DNA behind the special primer screening positive clone, this DNA is the standard DNA template, for this area researchist can directly obtain its sequence information according to designed primer and said template through the molecules experiment, repeats no more here.Adopt the quantitative DNA of NanoDrop ND-1000 nucleic acid quantification appearance, make the standard plasmid concentration range 10
8～10
2Copies/ μ l is as the standard substance of said fluorescence quantitative kit.
3. the MgCl of the dNTP of fluorescence quantitative PCR reaction solution: 10mmol/L, 25mmol/L
2
4. quantitative fluorescent PCR 10xBuffer:500mM KCl, 100mM Tris-HCl, 1%Triton X-100.
5. negative quality control standard article: aseptic double-distilled water.
6. quantitative fluorescent PCR reaction conditions
Quantitative real time PCR Instrument is Line-Gene K (rich day of Hangzhou; China); 25 microlitre reaction systems comprise: 12.5 μ l, 2 * TaqMan Universal PCR Master Mix (American AB I company), each 10nM of upstream and downstream primer (primer sequence is respectively Sequence NO.4 and Sequence NO.5 up and down); 400nM probe (probe sequence is Sequence NO.6), adds ddH at 2 μ l dna profilings
2O to 25 microlitre.Response procedures is 95 ℃ of preparatory sex change of 10min, connects 45 circulations: 95 ℃ of 40s, 60 ℃ of 1min.Sterilized water is used for negative control.
The experiment of embodiment 3 vitro detection
1. susceptibility detects
The standard DNA template is carried out 10 times of gradient dilutions, make the standard plasmid concentration range 10
8～10
2Copies/ μ l is as the standard substance of fluorescence quantitative kit.The quantitative fluorescent PCR reaction conditions; 25 microlitre reaction systems: 12.5 μ l, 2 * TaqMan Universal PCR Master Mix (American AB I company); Each 10nM of upstream and downstream primer (primer sequence is respectively Sequence NO.4 and Sequence NO.5 up and down); 400nM probe (probe sequence is Sequence NO.6), adds ddH at the standard DNA template of 2 μ l gradient dilutions
2O to 25 microlitre.Response procedures is 95 ℃ of preparatory sex change of 10min, connects 45 circulations: 95 ℃ of 40s, 60 ℃ of 1min.Sterilized water is used for negative control.
Detected result is seen Fig. 1, and sensitive analysis shows that minimum can detect the 100copies/ reaction.This experiment batch interior repetition 3 times repeated 3 times to analyze its repeatability between batch.The repeatability result shows in batch interior CV%＜5%, CV%＜10% between batch.Explain that this test kit has good repeatability.
2. specific detection
1) negative control bacterial strain: streptococcus pneumoniae, streptococcus aureus, Proteus mirabilis, pseudomonas putida, the compound Acinetobacter calcoaceticus of Bao Man, above bacterial strain are all from Chinese microorganism strains preservation management committee.
2) positive control strain: be 4 clones of the compound Acinetobacter calcoaceticus of Bao Man of the isolating OXA-23 transgenation in this chamber.
3) the PCR reaction system is: 12.5 μ l, 2 * TaqMan Universal PCR Master Mix (American AB I company); Each 10nM of upstream and downstream primer (primer sequence is respectively Sequence NO.4 and Sequence NO.5 up and down); 400nM probe (probe sequence is Sequence NO.6), adds ddH at each bacterial classification DNA extraction liquid of 2 μ l and standard DNA plasmid
2O to 25 microlitre.Response procedures is 95 ℃ of preparatory sex change of 10min, connects 45 circulations: 95 ℃ of 40s, 60 ℃ of 1min.
The specificity interpretation of result shows, has only 4 clones of the compound Acinetobacter calcoaceticus of Bao Man of the isolating OXA-23 transgenation in this chamber positive, and other sample is all negative, so this test kit detection specificity reaches 100%.
The drug sensitivity assay of the compound Acinetobacter calcoaceticus drug resistant gene of embodiment 3 Bao Man OXA-23 new mutant body bacterial strain
The present invention adopts VITEK32 Bacteria Identification appearance and GNS448 drug sensitive plate to carry out drug sensitive test; The mutants which had that detects D class β-Nei Xiananmei drug resistant gene OXA-23 is to antibiotic susceptibility; With Pseudomonas aeruginosa (ATCC 27853) and escherichia coli (ATCC25922) as susceptibility Quality Control bacterial strain; Result such as following table are carried out in result's judgement by the CLSI2000 standard:
The result shows; Said mutants which had is to cefotaxime, butylamine OK a karaoke club, Ampicillin Trihydrate, Cephazolin, cefepime, cefoxitin, ceftazime, Cefuroxime sodium, cefuroxime axetil, qingfengmeisu qiong, imipenum, piperacillin/Tazobactam Sodium resistance, and is comparatively responsive to cefoperazone/sulbactam and levofloxacin.
Claims (7)
1. OXA-23 mutain, its sequence is shown in SEQ ID NO:7.
2. coding claim 1 said proteic Nucleotide.
3. Nucleotide according to claim 2 is characterized in that: its sequence is shown in Sequence NO.3.
4. the real-time fluorescence quantitative PCR test kit of the compound Acinetobacter calcoaceticus OXA-23 of the Bao Man mutator gene of a test right requirement 2 or 3 comprises Auele Specific Primer and specific probe, and said Auele Specific Primer comprises upstream primer and downstream primer:
Its sequence of upstream primer is Sequence NO.4, perhaps comprises the sequence to 5 ' end prolongation of Sequence NO.4 sequence;
Its sequence of downstream primer is Sequence NO.5, perhaps comprises the sequence to 5 ' end prolongation of Sequence NO.5 sequence;
Its sequence of said specific probe is Sequence NO.6, perhaps comprises the sequence to 5 ' or 3 ' end prolongation of Sequence NO.6 sequence.
5. test kit according to claim 4 is characterized in that, said test kit can be used to contain the selection of the early stage antibacterials of disease that the compound Acinetobacter calcoaceticus of Bao Man of OXA-23 mutator gene causes.
6. test kit according to claim 5 is characterized in that, the preferred levofloxacin of described antibacterials.
7. claim 2 or 3 described Nucleotide detect the purposes in the test kit of the compound Acinetobacter calcoaceticus bacterial strain of Bao Man of OXA-23 sudden change in preparation.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105349694A (en) * | 2015-12-24 | 2016-02-24 | 重庆医科大学 | Primers, kit and method for detecting drug resistance of acinetobacter baumannii |
| EP2948549A4 (en) * | 2013-05-21 | 2016-12-07 | Ind And Acad Coop Found Myongji Univ | Primers and kits for colony multiplex pcr for the detection of class a, b, c, and d b-lactamase genes and methods of using thereof |
| CN106282354A (en) * | 2016-08-26 | 2017-01-04 | 扬州大学 | The detection primer of a kind of acinetobacter lwoffii and fluorescent quantitative PCR detection method |
| EP3392350A1 (en) * | 2012-12-31 | 2018-10-24 | Fundación para el Fomento de la Investigación Sanitaria y Biomedica de la comunitat Valenciana | Primers for detection and typing of carbapenemase producing bacterial strains, and detection method and kit |
| CN109266658A (en) * | 2018-10-17 | 2019-01-25 | 昆明理工大学 | The specific gene and its primer of a kind of Acinetobacter bauamnnii and application |
| CN109628620A (en) * | 2019-01-22 | 2019-04-16 | 南方医科大学南方医院 | Primer, method and the kit of the detection of complete sequence fluorescent PCR OXA-23 family and OXA-51 family gene type |
-
2011
- 2011-12-26 CN CN201110442340.6A patent/CN102603875B/en not_active Expired - Fee Related
Non-Patent Citations (3)
| Title |
|---|
| 吕美艳等: "161株鲍曼不动杆菌OXA-23基因组介导的耐药性和同源性分析", 《中国卫生检验杂志》 * |
| 周敏等: "鲍曼不动杆菌OXA-23型碳青霉烯酶相关耐药基因分析", 《南京医学大学学报（自然科学版）》 * |
| 段樱等: "PCR检测鲍曼不动杆菌的耐碳青霉烯OXA－23基因", 《广东医学》 * |
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| EP3392350A1 (en) * | 2012-12-31 | 2018-10-24 | Fundación para el Fomento de la Investigación Sanitaria y Biomedica de la comunitat Valenciana | Primers for detection and typing of carbapenemase producing bacterial strains, and detection method and kit |
| EP2948549A4 (en) * | 2013-05-21 | 2016-12-07 | Ind And Acad Coop Found Myongji Univ | Primers and kits for colony multiplex pcr for the detection of class a, b, c, and d b-lactamase genes and methods of using thereof |
| CN105349694A (en) * | 2015-12-24 | 2016-02-24 | 重庆医科大学 | Primers, kit and method for detecting drug resistance of acinetobacter baumannii |
| CN105349694B (en) * | 2015-12-24 | 2020-09-18 | 重庆医科大学 | Primers, kit and method for detecting drug resistance of acinetobacter baumannii |
| CN106282354A (en) * | 2016-08-26 | 2017-01-04 | 扬州大学 | The detection primer of a kind of acinetobacter lwoffii and fluorescent quantitative PCR detection method |
| CN106282354B (en) * | 2016-08-26 | 2021-03-12 | 扬州大学 | Detection primer and fluorescent quantitative PCR detection method for acinetobacter iwoffii |
| CN109266658A (en) * | 2018-10-17 | 2019-01-25 | 昆明理工大学 | The specific gene and its primer of a kind of Acinetobacter bauamnnii and application |
| CN109628620A (en) * | 2019-01-22 | 2019-04-16 | 南方医科大学南方医院 | Primer, method and the kit of the detection of complete sequence fluorescent PCR OXA-23 family and OXA-51 family gene type |
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