CN101519688A - Detection method for drug resistant genes of Klebsiella pneumoniae against belta-lactamase - Google Patents
Detection method for drug resistant genes of Klebsiella pneumoniae against belta-lactamase Download PDFInfo
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Abstract
The invention discloses a detection method for drug resistant genes of Klebsiella pneumoniae against belta-lactamase, pertaining to the technical field of gene detection methods. The detection method comprises the following steps: 1. Klebsiella pneumoniae strains of inpatients are selected and sampling for backup is carried out after strain identification is carried out to all strains using bacteria identifying apparatus; 2. the sensitivity of antibacterials is determined by adopting the micro dilution method so as to judge the sensitivity of antibiotic; and 3. drug resistant genes are detected by adopting a PCR method and belta-lactamase drug resistant genes are checked and DNA positive strains are selected for carrying out sequencing analysis. The detection method finds the drug resistant genes resulting in the resistance of Klebsiella pneumoniae to belta lactam drugs by carrying out a systematical and complete research on the drug resistant mechanism of the Klebsiella pneumoniae, thus having important value for instructing the clinical and reasonable use of antibacterials, being capable of reducing the infection rate in a hospital, reducing mortality rate and saving social medical resources, and having good social and economic benefits.
Description
Technical field
The present invention relates to a kind of gene tester, especially relate to a kind of Klebsiella Pneumoniae β-Nei Xiananmei drug resistant gene detection method.
Background technology
The history of antibacterials development is the history that mankind and bacterium are struggled.The human new drug of constantly developing, bacterium is then resisted with antibacterials mutually by the structure that change is controlled oneself.The abuse of antibacterials causes the resistant organism ratio increasing, and drug-resistant intensity is more and more higher, the speed that the speed of new drug development produces not as good as bacterial drug resistance far away.Antibacterials abuses be systems exist worldwide problem, China is particularly serious.According to statistics, the ratio of use antibacterials is occupied the patient's of institute 60%-70% among China inpatient, only is 20% in the U.S..The abuse of antibacterials has directly caused resistant organism to increase year by year, and the ward infection incidence constantly rises.If do not strengthen standard, rational use of drug, in case it is popular the drug-fast bacteria infection illness outbreak to occur, the difficult situation that will occur pasting medical help.Therefore, research bacterial resistance mechanism and rule thereof just seem very urgent and necessary.
Klebsiella Pneumoniae is distributed widely in nature, healthy people's skin, enteron aisle and respiratory tract.Under the normal circumstances, Klebsiella Pneumoniae does not infect any tissue of human body.Elderly patients' hypoimmunity, long-term a large amount of the use under antibacterials or the traumatic medical care precess situations such as (as tracheotomy, intraurethral cannula and kidney dialysis etc.) easily causes autogenous infection.The most common in infecting due to the Klebsiella Pneumoniae with respiratory tract infection, urinary tract infection, microbemia etc.Klebsiella Pneumoniae is 8.0% at the separation rate of clinical samples, and its resistant rate is 6.1%-85.7%.In recent years, can tolerate the multiclass antibacterials from the clinical Klebsiella Pneumoniae that is separated to: comprise penicillins, cephalosporins, aminoglycoside, fluoroquinolones, paraxin, sulfa drugs etc., multiple antibiotic resistant strain occurred., further investigate at Klebsiella Pneumoniae molecule resistance mechanism and the relation of infecting for this reason, significant to the popular situation of drug resistant gene of understanding this area Klebsiella Pneumoniae, the guiding that instructs clinical rational use antibacterials, research and development new drug etc.
Summary of the invention
For solving the problems of the technologies described above, the invention provides a kind of Klebsiella Pneumoniae to β-Nei Xiananmei drug resistant gene detection method, be intended to use modern molecular biology technique and modern information technologies and carry out bacterial resistance mechanism and the research of drug-fast genes involved, disclose Klebsiella Pneumoniae beta-lactam class antibacterials are produced the resistance reason, reach the purpose of quick diagnosis drug-resistance of bacteria, the popular situation of clear and definite this area Klebsiella Pneumoniae drug resistant gene rationally uses antibacterials that foundation is provided for instructing clinical study.
The technical scheme that the present invention takes for achieving the above object is as follows, and a kind of Klebsiella Pneumoniae may further comprise the steps β-Nei Xiananmei drug resistant gene detection method:
1, select inpatient's Klebsiella Pneumoniae bacterial strain, all strains examined identifies that through the Bacteria Identification instrument bacterial classification post-sampling is standby.
2, adopt micro-dilution method to measure the susceptibility of antibacterials, carry out antibiotics sensitivity and judge.
3, adopt the PCR method to detect drug resistant gene, check the β-Nei Xiananmei drug resistant gene and choose the positive strain sequencing analysis of DNA.
1., template preparation
Choose the pure culture bacterium colony and insert in the 0.5ml centrifuge tube (in preset 200ng/ml Proteinase K solution 200 μ l), 56 ℃ of water-baths 2 hours change 95 ℃ of water-baths 10 minutes, add Chelex-10040 μ l, centrifugal (15000rpm) 30 seconds.Supernatant liquor is the template liquid of gene test, and-20 ℃ of refrigerators are preserved standby.
2., the detection of gene
Adopt polymerase chain reaction (PCR) method, various target gene pcr amplification systems are: every reaction system P
1, P
2Each 0.5 μ M of primer, each mM of dNTPs200, KCl 10mM, (NH
4)
2SO
48mM, MgCl
22mM, Tris-HCl (pH9.0) 10mM, NP
400.5%, BSA 0.02% (wt/vol), Taq DNA pol 1U.Total reaction volume 20 μ l (wherein template liquid 5 μ l).Pcr amplification product〉the 500bp thermal circulation parameters is: 93 ℃ of pre-sex change 2min, 93 ℃ of 60s → 55 ℃ 60s → 72 ℃ of 60s circulated for 35 cycles then, and last 72 ℃ extend to 5min.Pcr amplification product＜500bp thermal circulation parameters is: 93 ℃ of pre-sex change 2min, and 93 ℃ of 30s → 55 ℃ 30s → 72 ℃ of 60s circulated for 35 cycles then, and last 72 ℃ extend to 5min.Product the purpose band suitable with the positive control molecule occur and is judged to the positive through 1% agarose gel electrophoresis.
Preferably, select following β-Nei Xiananmei drug resistant gene TEM, SHV, LEN, OKP, CTX-M-1 group, CTX-M-2 group, CTX-M-9 group, OXA-1 group, OXA-2 group, OXA-10 group, PER, GES, VEB, DHA, ACT-1 to carry out drug resistance analysis, corresponding primer sequence is:
P1：AGGAAGAGTATGATTCAACA
P2：CTCGTCGTTTGGTATGGC
P1：TGCGCAAGCTGCTGACCAGC
P2：TTAGCG(T/C)TGCCAGTGCTCGA
P1：ATGCGTT(A/T)T(A/G)TTCGCCTGTG
P2：GGCGCTCAGATGCTGCGC
P1：A(A/G)GCGCTTCCCGGCGACGTG
P2：TTAGCG(T/C)TGCCAGTGCTCGA
P1：ATGGTTAAAAAATCACTGCGC
P2：TCCCGACGGCTTTCCGCCTT
P1：ATGATGACTCAGAGCATTCG
P2：TCCCGACGGCTTTCCGCCTT
P1：CGGCCTGTATTTCGCTGTTG
P2：TCCCGACGGCTTTCCGCCTT
P1：CTGTTGTTTGGGTTTCGCAAG
P2：CTTGGCTTTTATGCTTGATG
P1：CAGGCGC(T/C)GTTCG(T/C)GATGAGTT
P2：GCC(T/C)TCTATCCAGTAATCGCC
P1：GTCTTTC(A/G)AGTACGGCATTA
P2：GATTTTCTTAGCGGCAACTTA
P1：AGTCAGCGGCTTAGATA
P2：CGTATGAAAAGGACAATC
P1：ATGCGCTTCATTCACGCAC
P2：CTATTTGTCCGTGCTCAGG
P1：GCGGTAATTTAACCAGA
P2：GCCTATGAGCCAGTGTT
P1：AACTTTCACAGGTGTGCTGGGT
P2：CCGTACGCATACTGGCTTTGC
P1：TCGGTAAAGCCGATGTTGCGG
P2：CTTCCACTGCGGCTGCCAGTT
Beneficial effect of the present invention is as follows: the present invention selects at present that more common hospital infection strain---Klebsiella Pneumoniae is a research object for use, utilization polymerase chain reaction (PCR) and dna sequencing technology and gene order are than reciprocity modern molecular biology and information technology, system is comprehensively studied the resistance mechanism of Klebsiella Pneumoniae, discovery causes the drug resistant gene of the anti-beta-lactam class of Klebsiella Pneumoniae medicine, use antibacterials to have important value to instructing clinical rational, can reduce hospital's incidence of nosocomial infection, reduce mortality ratio, save social medical resource.Achievement has good society and economic benefit, and popularizing application prospect is extensive.
The invention will be further described below in conjunction with the drawings and specific embodiments.
Description of drawings
Fig. 1 is a blaTEM gene PCR electrophorogram of the present invention; [M: molecular weight marker from top to bottom is respectively 1000,900,800,700,600,500,400,300,250,200,150,100,50bp, P: positive control, N: negative control, S: the sample positive]
Fig. 2 is blaCTX-M-1 group's gene PCR electrophorogram of the present invention; [M: molecular weight marker from top to bottom is respectively 1000,900,800,700,600,500,400,300,250,200,150,100,50bp, P: positive control, N: negative control, S: the sample positive]
Fig. 3 is a blaDHA gene PCR electrophorogram of the present invention; [M: molecular weight marker from top to bottom is respectively 1000,900,800,700,600,500,400,300,250,200,150,100,50bp, P: positive control, N: negative control, S: the sample positive]
Fig. 4 is blaCTX-M-15 type gene sequencing figure of the present invention;
Fig. 5 is blaSHV type gene sequencing figure of the present invention.
Embodiment
1 materials and methods
1.1 bacterium source and evaluation
80 strains be separation from January, 2006～2007 patient's clinical samples between year October, be respectively: 53 parts of sputums, 8 parts of urines, 4 parts of bile, 5 parts of festers, 4 parts of drainage liquid, 3 parts of blood, 3 parts of throat swabs.All strains examined all uses ATB Bacteria Identification instrument to identify that the bacterial classification post-sampling is standby.
1.2 antibacterials sensitivity test
Drug sensitive test is by U.S. CLSI version requirement in 2006.Quality Control bacterial strain: escherichia coli ATCC25922, Klebsiella Pneumoniae ATCC700603 Pseudomonas aeruginosa ATCC27853, available from Ministry of Health Clinical Laboratory center.
1.3 drug sensitive test
Antibacterials sensitization test trace broth dilution method.
1.4 beta lactamase gene test
1.4.1 template preparation
Choose the pure culture bacterium colony and insert in the 0.5ml centrifuge tube (in preset 200ng/ml Proteinase K solution 200 μ l), 56 ℃ of water-baths 2 hours change 95 ℃ of water-baths 10 minutes, add Chelex-10040 μ l, centrifugal (15000rpm) 30 seconds.Supernatant liquor is the template liquid of gene test, and-20 ℃ of refrigerators are preserved standby.
1.4.2 the detection of gene
Adopt polymerase chain reaction (PCR) method, wherein primer sequence sees Table 1.Various target gene pcr amplification systems are: every reaction system P
1, P
2Each 0.5 μ M of primer, each mM of dNTPs200, KCl10mM, (NH
4)
2SO
48mM, MgCl
22mM, Tris-HCl (pH9.0) 10mM, NP
400.5%, BSA 0.02% (wt/vol), Taq DNA pol 1U.Total reaction volume 20 μ l (wherein template liquid 5 μ l).Pcr amplification product〉the 500bp thermal circulation parameters is: 93 ℃ of pre-sex change 2min, 93 ℃ of 60s → 55 ℃ 60s → 72 ℃ of 60s circulated for 35 cycles then, and last 72 ℃ extend to 5min.Pcr amplification product＜500bp thermal circulation parameters is: 93 ℃ of pre-sex change 2min, and 93 ℃ of 30s → 55 ℃ 30s → 72 ℃ of 60s circulated for 35 cycles then, and last 72 ℃ extend to 5min.Product the purpose band suitable with the positive control molecule occur and is judged to the positive through 1% agarose gel electrophoresis.
Table 1:PCR primer sequence
| The gene title | Primer sequence (5 ' → 3 ') | Product length |
| blaTEM | P1：AGGAAGAGTATGATTCAACA | 535bp |
| P2：CTCGTCGTTTGGTATGGC | ||
| blaSHV | P1：TGCGCAAGCTGCTGACCAGC | 305bp |
| P2：TTAGCG(T/C)TGCCAGTGCTCGA | ||
| blaLEN | P1：ATGCGTT(A/T)T(A/G)TTCGCCTGTG | 591bp |
| P2：GGCGCTCAGATGCCTGCGC | ||
| blaOKP | P1：A(A/G)GCGCTTCCCGGCGACGTG | 362bp |
| P2：TTAGCG(T/C)TGCCAGTGCTCGA | ||
| BlaCTX-M-1 group | P1：ATGGTTAAAAAATCACTGCGC | 833bp |
| P2：TCCCGACGGCTTTCCGCCTT | ||
| BlaCTX-M-2 group | P1：ATGATGACTCAGAGCATTCG | 833bp |
| P2：TCCCGACGGCTTTCCGCCTT | ||
| BlaCTX-M-9 group | P1：CGGCCTGTATTTCGCTGTTG | 793bp |
| P2：TCCCGACGGCTTTCCGCCTT | ||
| BlaOXA-1 group | P1：CTGTTGTTTGGGTTTCGCAAG | 440bp |
| P2：CTTGGCTTTTATGCTTGATG | ||
| BlaOXA-2 group | P1：CAGGCGC(T/C)GTTCG(T/C)GATGAGTT | 233bp |
| P2：GCC(T/C)TCTATCCAGTAATCGCC | ||
| BlaOXA-10 group | P1：GTCTTTC(A/G)AGTACGGCATTA | 822bp |
| P2：GATTTTCTTAGCGGCAACTTA | ||
| blaPER | P1：AGTCAGCGGCTTAGATA | 978bp |
| P2：CGTATGAAAAGGACAATC | ||
| blaGES | P1：ATGCGCTTCATTCACGCAC | 846bp |
| P2：CTATTTGTCCGTGCTCAGG | ||
| blaVEB | P1：GCGGTAATTTAACCAGA | 961bp |
| P2：GCCTATGAGCCAGTGTT | ||
| blaDHA | P1：AACTTTCACAGGTGTGCTGGGT | 405bp |
| P2：CCGTACGCATACTGGCTTTGC | ||
| blaACT-1 | P1：TCGGTAAAGCCGATGTTGCGG | 303bp |
| P2：CTTCCACTGCGGCTGCCAGTT |
2 results
2.1 the susceptibility of antibacterials
80 strains separation sees Table 2 from the susceptibility that elderly patients produce the KPN bacterium antibacterials of β-Nei Xiananmei.
The susceptibility of table 2 antibacterials
| Antibacterials | The persister number | Resistant rate % | Antibacterials | The persister number | Resistant rate % |
| The amoxycilline Trihydrate bp | 79 | 98.8 | Cefepime | 80 | 100 |
| Amoxycilline Trihydrate bp/clavulanic acid | 70 | 87.5 | Cefoxitin | 30 | 37.5 |
| Piperacillin | 76 | 95.0 | Imipenum | 0 | 0 |
| Piperacillin/his azoles crust | 32 | 40.0 | Meropenem | 0 | 0 |
| Ticarcillin | 80 | 100 | Gentamicin | 59 | 73.8 |
| Ticarcillin/clavulanic acid | 75 | 93.8 | Tobramycin | 41 | 51.2 |
| Cefoxitin | 80 | 100 | Netilmicin | 43 | 53.8 |
| Cephalofruxin | 80 | 100 | Amikacin | 40 | 50.0 |
| Ceftazime | 80 | 100 | Ciprofloxacin | 80 | 100 |
| Cefotaxime | 80 | 100 | Trimethoprim-sulfamethoxazole | 72 | 90.0 |
2.2 beta lactamase gene test result
80 strains separation detects 6 kinds of β-Nei Xiananmei genes such as blaTEM, blaSHV, blaCTX-M-1 group, blaCTX-M-9 group, blaOXA-1 group, blaDHA from the KPN bacterium that elderly patients produce β-Nei Xiananmei, and positive rate is respectively 95% (76/80), 30% (24/80), 51.3% (41/80), 5% (4/80), 5% (4/80), 13.8% (11/80).Have 76 strains to detect a kind of β-Nei Xiananmei gene at least in the 80 strain bacterium, 58 strains detect β-Nei Xiananmei gene more than 2 kinds simultaneously, detect 4 kinds of β-Nei Xiananmei genes at most simultaneously, have only 4 strains not detect the β-Nei Xiananmei gene.BlaTEM gene PCR electrophorogram is seen Fig. 1, blaCTX-M-1 group's gene PCR electrophorogram is seen Fig. 2, blaDHA gene PCR electrophorogram is seen Fig. 3, No. 2 strain blaCTX-M-1 group PCR positive products is blaCTX-M-15 type (in full accord with listed blaCTX-M-15 type sequence) through dna sequencing and online (www.ncbi.nlm.nih.gov/BLASTn) comparison, and Fig. 4 is a CTX-M-15 type sequencer map; No. 2 strain blaSHV PCR positive products are through dna sequencing and online comparison and blaSHV-28 type, the same a group of blaSHV-43 type, and Fig. 5 is the blaSHV sequencer map, No. 2 strain blaSHV sequences and the blaSHV sequence molecular evolution figure that has logined at U.S. NCBI.
Conclusion
80 strain KPN bacterium beta-lactam class antibacterials resistances are closely related with the product β-Nei Xiananmei.In the 80 strain Klebsiella Pneumoniaes, have 76 strains to detect a kind of beta lactamase gene at least, 58 strains detect beta lactamase gene more than 2 kinds simultaneously, detect 4 kinds of beta lactamase genes at most simultaneously.The positive rate that detects the TEM gene is 95%, the SHV gene is 30%, CTX-M-1 group's gene be 51.3% and OXA-1 group's gene be 5% etc., the TEM gene is the modal β-Nei Xiananmei gene of Klebsiella Pneumoniae, mediation is to the resistance of Ampicillin Trihydrate, penicillin, ceftazidime and cefoxitin etc., and this resistance is transmitted by bacterial plasmid.The CTX-M β-Nei Xiananmei is antibacterials such as hydrolysis cefepime, cephamycin highly, detect the representative of CTX-M type gene to cefepime, cephamycin-type medicine resistance.Klebsiella Pneumoniae SHV gene can mediate the Ampicillin Trihydrate resistance, and this result of study drug-resistant phenotype conforms to beta lactamase gene test situation.OXA type beta lactamase hydrolyzable Oxazacillin detects this fermentoid gene and shows that bacterium is to Oxazacillin, Ampicillin Trihydrate resistance.
Claims (2)
1, a kind of Klebsiella Pneumoniae may further comprise the steps β-Nei Xiananmei drug resistant gene detection method:
(1), select inpatient's Klebsiella Pneumoniae bacterial strain, all strains examined identifies that through the Bacteria Identification instrument bacterial classification post-sampling is standby,
(2), adopt micro-dilution method to measure the susceptibility of antibacterials, carry out antibiotics sensitivity and judge,
(3), adopt the PCR method to detect the β-Nei Xiananmei drug resistant gene, and choose the positive strain of DNA and carry out sequencing analysis, described β-Nei Xiananmei drug resistant gene and corresponding primer sequence are:
Gene title primer sequence
blaTEM P1：AGGAAGAGTATGATTCAACA
P2：CTCGTCGTTTGGTATGGC
blaSHV P1：TGCGCAAGCTGCTGACCAGC
P2：TTAGCG(T/C)TGCCAGTGCTCGA
blaLEN P1：ATGCGTT(A/T)T(A/G)TTCGCCTGTG
P2：GGCGCTCAGATGCTGCGC
blaOKP P1：A(A/G)GCGCTTCCCGGCGACGTG
P2：TTAGCG(T/C)TGCCAGTGCTCGA
BlaCTX-M-1 group P1:ATGGTTAAAAAATCACTGCGC
P2：TCCCGACGGCTTTCCGCCTT
BlaCTX-M-2 group P1:ATGATGACTCAGAGCATTCG
P2：TCCCGACGGCTTTCCGCCTT
BlaCTX-M-9 group P1:CGGCCTGTATTTCGCTGTTG
P2：TCCCGACGGCTTTCCGCCTT
BlaOXA-1 group P1:CTGTTGTTTGGGTTTCGCAAG
P2：CTTGGCTTTTATGCTTGATG
BlaOXA-2 group P1:CAGGCGC (T/C) GTTCG (T/C) GATGAGTT
P2：GCC(T/C)TCTATCCAGTAATCGCC
BlaOXA-10 group P1:GTCTTTC (A/G) AGTACGGCATTA
P2：GATTTTCTTAGCGGCAACTTA
blaPER P1：AGTCAGCGGCTTAGATA
P2：CGTATGAAAAGGACAATC
blaGES P1：ATGCGCTTCATTCACGCAC
P2：CTATTTGTCCGTGCTCAGG
blaVEB P1：GCGGTAATTTAACCAGA
P2：GCCTATGAGCCAGTGTT
blaDHA P1：AACTTTCACAGGTGTGCTGGGT
P2：CCGTACGCATACTGGCTTTGC
blaACT-1 P1：TCGGTAAAGCCGATGTTGCGG
P2：CTTCCACTGCGGCTGCCAGTT。
2, a kind of Klebsiella Pneumoniae as claimed in claim 1 is characterized in that β-Nei Xiananmei drug resistant gene detection method: in the detection of described gene, each target gene pcr amplification system is: every reaction system P
1, P
2Each 0.5 μ M of primer, each mM of dNTPs200, KCl 10mM, (NH
4)
2SO
48mM, MgCl
22mM, Tris-HCl pH9.0 10mM, NP
400.5%, BSA 0.02% wt/vol, Taq DNA pol 1U, total reaction volume 20 μ l, pcr amplification product〉the 500bp thermal circulation parameters is: 93 ℃ of pre-sex change 2min, 93 ℃ of 60s → 55 ℃ 60s → 72 ℃ of 60s then, circulated for 35 cycles, last 72 ℃ extend to 5min, and pcr amplification product＜500bp thermal circulation parameters is: 93 ℃ of pre-sex change 2min, 93 ℃ of 30s → 55 ℃ 30s → 72 ℃ of 60s then, circulated for 35 cycles, last 72 ℃ extend to 5min, and product the purpose band suitable with the positive control molecule occur and is judged to the positive through 1% agarose gel electrophoresis.
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| CN115798575B (en) * | 2023-02-06 | 2023-06-02 | 中国医学科学院北京协和医院 | System and method for predicting sensitivity of klebsiella to ceftazidime |
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