CN102603875B - 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 detection kit and the method for drug resistant gene.Particularly, the present invention, according to the compound Acinetobacter calcoaceticus bacterial strain of the Bao Man of the OXA-23 transgenation of new screening, has designed the PCR kit for fluorescence quantitative of the compound Acinetobacter calcoaceticus of Bao Man that detects OXA-23 sudden change, the present invention also relates to its detection method.
Background technology
Acinetobacter calcoaceticus is the non-fermentation of a class, strict aerobic gram negative bacilli, is prevalent in occurring in nature.Acinetobacter at least can be divided into 32 gene kind types, and wherein Acinetobacter bauamnnii, Acinetobacter calcoaceticus, acinetobacter calcoaceticus gene kind type 3 and 13TU are difficult to distinguish by conventional phenotypic evaluation, are collectively referred to as the compound Acinetobacter calcoaceticus complex body of Bao Man.
Acinetobacter bauamnnii is modal a kind of gram negative bacilli in acinetobacter, is extensively present in natural water and soil, hospital environment and human body skin, respiratory tract, digestive tube and urogenital tract, and be conditioned pathogen.It is very wide and can long-term surviving that this bacterium distributes in hospital environment, very easily causes the infection of critical patient, therefore often from the samples such as infected patient's blood, urine, fester and respiratory secretions, isolates, and in non-zymocyte, infects and be only second to pseudomonas.
Acinetobacter bauamnnii distributes maximum with ICU lesion in hospital, be secondly patients in respiratory department.Mostly the patient who infects is the patient that gerontal patient, critical illness and Abwehrkraft des Koepers are weak, and uses the patient of various invasive operations and the treatment of life-time service Broad spectrum antibiotics.Again because this bacterium has stronger resistibility to damp and hot ultraviolet ray and chemostefilant, routine disinfection can only suppress its growth and can not kill, and vulnerable or have wound patient may by the hand from medical worker or the halfway medicine equipment of sterilizing with bacterium infect chance more.
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 mechanical ventilationassociatepneumonia pneumonia, urinary tract infections, septicemia, traumatic infection and central nervous system infection etc.Because Acinetobacter bauamnnii has extremely strong adaptive capacity to environment and obtains the ability of exogenous drug resistant gene, thus very easily cause in hospital send out popular.Acinetobacter bauamnnii can pass through the removable gene elements such as plasmid, transposon and integron, obtains the resistance to multiple antibacterials.Complete genome sequencing 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, are assembled, comprise VEB-1, AmpC, multiple Aminoglycosides 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 European MDRAB (ACICU) that clones II of another 1 strain, also send out drug resistant gene island AbaR2.Acinetobacter bauamnnii is integrated the ability of allogenic gene, makes it have the great potential that clinical antibacterials used is produced to resistance.In numerous drug resistant genes, β-lactamase gene is the topmost drug resistant gene of Acinetobacter bauamnnii, comprise that beta-lactamase (belongs to category-A, comprise the 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 (belonging to 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 a little less than gerontal patient, critical illness and Abwehrkraft des Koepers, as the microbiotic of selecting not in time the compound Acinetobacter calcoaceticus sensitivity of Bao Man is treated, by entail dangers to patient's life security.
Summary of the invention
In order to address the above problem, the invention discloses real-time fluorescence quantitative PCR test kit and the method thereof of the compound Acinetobacter calcoaceticus OXA-23 of Bao Man mutator gene.
First the present invention 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 methods to implement:
According to the sequence of the mutant of the D class β-lactamase drug resistant gene OXA-23 newly filtering out, described mutant 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 shown in respectively Sequence NO.4 and Sequence NO.5, and described probe sequence is shown in Sequence NO.6.
Or include the sequence extending to 5 ' end of above-mentioned sequence;
Or the sequence that is greater than 85% with above-mentioned sequence homology.
Described probe 5 ' end mark fluorescent chromophoric group FAM, 3 ' end connects fluorescent quenching group B HQ.Adopt 25 μ l reaction volumes, in each reaction, contain 12.5 μ l universal PC R reaction mixture [TaqMan
universal PCR Master Mix (2 ×)]; The each 10nM of upstream and downstream primer (upper and lower primer sequence is respectively Sequence NO.4 and Sequence NO.5); 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 analyzer and GNS448 drug sensitive plate to carry out drug sensitive test, detect the mutants which had of D class β-lactamase drug resistant gene OXA-23 to antibiotic susceptibility, result shows, described mutants which had Cefotaxime, butylamine OK a karaoke club, Ampicillin Trihydrate, Cephazolin, cefepime, cefoxitin, ceftazime, Cefuroxime sodium, cefuroxime axetil, gentamicin, imipenum, piperacillin/Tazobactam Sodium resistance, 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 for the detection of the compound Acinetobacter calcoaceticus bacterial strain of the Bao Man of OXA-23 transgenation.
Accompanying drawing explanation
Fig. 1. the standard plasmid corresponding loop parameter of each concentration (Ct value).In figure, curve is from a left side to representing successively that again standard plasmid concentration is 10
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 when copies/ μ l.
Fig. 2 .OXA-23 mutator gene and OXA-23 gene order comparison chart.The site of trilateral indication is mutational site.
The protein amino acid sequence of Fig. 3 .OXA-23 mutator gene coding and the protein amino acid sequence comparison chart of OXA-23 genes encoding.The site of trilateral indication is mutational site.
Embodiment
Determining of the new sequence of embodiment 1: Bao Man compound Acinetobacter calcoaceticus drug resistant gene OXA-23
1. bacterium source: we have collected compound Acinetobacter calcoaceticus 32 strains to the Bao Man of microbiotic height resistance (especially to imipenem-resistant) from year May in October, 2009 to 2011.Described 32 strain Specimen origin patients' Yu sputum.
2.OXA-23 design of primers: the present invention has designed the Auele Specific Primer of a pair of D class β-lactamase drug resistant gene OXA-23, and the sequence of described primer is shown in SEQ ID NO:1 and SEQ ID NO:2.
The amplification of 3.OXA-23 gene: take the compound Acinetobacter calcoaceticus lysate of above-mentioned 32 strain Bao Man as template, carry out pcr amplification, amplification system is (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 instrument reaction conditions is as follows:
Denaturation: 95 ℃ of 5min
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: PCR product is done to agarose electrophoresis, cut object band under ultraviolet lamp, utilize the quick glue of QIANGEN to reclaim test kit and reclaim PCR product.The product of recovery is served to Hai Shenggong to be measured.
5. sequencing result and BLASTn compare, only find that wherein 1 strain is numbered the bacterial strain OXA-23 group positive of AB37, determine similar to OXA-23 gene, its nucleotide sequence is shown in SEQ ID NO:3, protein sequence is shown in SEQ ID NO:7, described nucleotide mutant site is shown in Fig. 2, and Fig. 3 is seen in the coded protein mutant site of described 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 obtaining according to above-described embodiment 1.Adopt ABI primer Express 2.0 software design probes and primer (above sea base Kanggong department is synthetic).Upstream primer sequence is shown in that SEQ ID NO:4 and downstream primer sequence be shown in SEQ ID NO:5, and probe sequence is shown in SEQ ID NO:6.Described probe 5 ' end mark fluorescent chromophoric group FAM, 3 ' end connects fluorescent quenching group B HQ.
2. standard DNA template preparation
Adopt upstream and downstream primer pair described in previous step to go out 200bp fragment take the bacterial strain of above-mentioned AB37 as template amplification, PCR product purification (QIAgen, German) and be connected to PGM-T cloning vector after be transformed into DH5 α competent cell.After special primer screening positive clone, extract plasmid DNA, this plasmid DNA is standard DNA template, for this area researchist is tested and can directly be obtained its sequence information by molecules according to primer and the described template of design, repeats no more herein.Adopt the quantitative plasmid DNA of NanoDrop ND-1000 nucleic acid quantification instrument, make standard plasmid concentration range 10
8～10
2copies/ μ l, as the standard substance of described fluorescence quantitative kit.
3. the dNTP of fluorescence quantitative PCR reaction solution: 10mmol/L, the MgCl of 25mmol/L
2.
4. quantitative fluorescent PCR 10xBuffer:500mM KCl, 100mM Tris-HCl, 1%Triton X-100.
5. negative quality control standard product: 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), the each 10nM of upstream and downstream primer (upper and lower primer sequence is respectively Sequence NO.4 and Sequence NO.5); 400nM probe (probe sequence is Sequence NO.6), 2 μ l DNA profilings, adds ddH
2o to 25 microlitre.Response procedures is 95 ℃ of 10min denaturations, connects 45 circulations: 95 ℃ of 40s, 60 ℃ of 1min.Sterilized water is for negative control.
Embodiment 3 vitro detection experiments
1. susceptibility detects
Standard DNA template is carried out to 10 times of gradient dilutions, make standard plasmid concentration range 10
8～10
2copies/ μ l, as the standard substance of fluorescence quantitative kit.Quantitative fluorescent PCR reaction conditions, 25 microlitre reaction systems: 12.5 μ l 2 × TaqMan Universal PCR Master Mix (American AB I company), the each 10nM of upstream and downstream primer (upper and lower primer sequence is respectively Sequence NO.4 and Sequence NO.5), 400nM probe (probe sequence is Sequence NO.6), the standard DNA template of 2 μ l gradient dilutions, adds ddH
2o to 25 microlitre.Response procedures is 95 ℃ of 10min denaturations, connects 45 circulations: 95 ℃ of 40s, 60 ℃ of 1min.Sterilized water is for negative control.
Detected result is shown in Fig. 1, and sensitive analysis shows that minimum can detect 100copies/ reaction.This experiment batch interior repetition 3 times, repeats 3 times to analyze its repeatability between batch.Repeatability result shows in crowd interior CV% < 5%, CV% < 10% between crowd.Illustrate 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 is all from Chinese microorganism strains preservation management committee.
2) positive control strain: 4 clones of the compound Acinetobacter calcoaceticus of Bao Man of the OXA-23 transgenation separating for this chamber.
3) PCR reaction system is: 12.5 μ l 2 × TaqMan Universal PCR Master Mix (American AB I company), the each 10nM of upstream and downstream primer (upper and lower primer sequence is respectively Sequence NO.4 and Sequence NO.5), 400nM probe (probe sequence is Sequence NO.6), 2 μ l each bacterial classification DNA extraction liquid and standard DNA plasmid, adds ddH
2o to 25 microlitre.Response procedures is 95 ℃ of 10min denaturations, connects 45 circulations: 95 ℃ of 40s, 60 ℃ of 1min.
Specificity interpretation of result shows, only has 4 clones of the compound Acinetobacter calcoaceticus of Bao Man of the OXA-23 transgenation that this chamber separates 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 analyzer and GNS448 drug sensitive plate to carry out drug sensitive test, detect the mutants which had of D class β-lactamase drug resistant gene OXA-23 to antibiotic susceptibility, with Pseudomonas aeruginosa (ATCC 27853) and escherichia coli (ATCC25922) as susceptibility Quality Control bacterial strain, result judges by CLSI2000 standard is undertaken, and result is as following table:
Result shows, described mutants which had Cefotaxime, butylamine OK a karaoke club, Ampicillin Trihydrate, Cephazolin, cefepime, cefoxitin, ceftazime, Cefuroxime sodium, cefuroxime axetil, gentamicin, imipenum, piperacillin/Tazobactam Sodium resistance, comparatively responsive to cefoperazone/Sulbactam and levofloxacin.
Claims (6)
1. an OXA-23 mutain, its sequence is as shown in SEQ ID NO:7.
2. the Nucleotide of albumen described in coding claim 1.
3. Nucleotide according to claim 2, is characterized in that: its sequence is as shown in Sequence NO.3.
4. a real-time fluorescence quantitative PCR test kit for the compound Acinetobacter calcoaceticus OXA-23 of the Bao Man mutator gene of test right requirement 2 or 3, comprises Auele Specific Primer and specific probe, and described Auele Specific Primer comprises upstream primer and downstream primer:
Described upstream primer sequence is shown in Sequence NO.4;
Described downstream primer sequence is shown in Sequence NO.5;
Described specific probe sequence is shown in Sequence NO.6.
5. test kit according to claim 4, is characterized in that, the selection of the early stage antibacterials of disease that described test kit causes for the compound Acinetobacter calcoaceticus of Bao Man that contains OXA-23 mutator gene.
6. test kit according to claim 5, is characterized in that, described antibacterials are levofloxacin.
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| CN105073992B (en) * | 2013-05-21 | 2018-11-30 | 明知大学产学协力团 | For detecting the primer and kit and its application method of the bacterium colony multiplex PCR of A class, B class, C class and D class beta-lactam enzyme gene |
| 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 |
| CN106282354B (en) * | 2016-08-26 | 2021-03-12 | 扬州大学 | Detection primer and fluorescent quantitative PCR detection method for acinetobacter iwoffii |
| CN109266658B (en) * | 2018-10-17 | 2022-08-19 | 昆明理工大学 | Acinetobacter baumannii specific gene and primer and application thereof |
| CN109628620B (en) * | 2019-01-22 | 2023-05-05 | 南方医科大学南方医院 | Primers, method and kit for detecting OXA-23 family and OXA-51 family genotypes by full-sequence fluorescent PCR |
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