CN101392287A - Aminoglycosides drug resistance gene quadruple-PCR detection technology by animal origin bacteria - Google Patents

Abstract

The invention relates to a fourfold PCR detecting technology for detecting drug resistance genes of zoonotic bacteria to aminoglycoside drugs. The technology is designed according to the nucleic acid sequence of four major drug resistance genes (aphA3, aacC4, aadA and aacC2) of aminoglycoside drugs and four corresponding specific PCR primers are synthesized; and the fourfold PCR technology is used for amplifying the four drug resistance genes. By the utilization of the technology, the appearance of an agarose gel electrophoresi specific band can be adopted for determining the result and four aminoglycoside drug resistance genes in clinical isolates can be detected.

Description

A kind of animal derived bacterium is to aminoglycoside medicaments drug resistant gene quadruple PCR detection technique

[invention field]

The present invention relates to animal derived bacterium to aminoglycoside medicaments drug resistant gene quadruple PCR detection technique, belonging to the Medical Molecular Biology field, specifically is to aminoglycoside medicaments drug resistant gene quadruple PCR detection technique and the application in the animal derived bacterium resistance detects thereof about animal derived bacterium.

[background technology]

One, the significance of bacterial drug resistance and rapid detection thereof:

China has become one of country that bacterial drug resistance is the most serious in the world.People's health has been arrived in drug-resistance of bacteria serious harm.Drug-resistance of bacteria also constantly increases the weight of in the world wide, on people doctor and the veterinary clinic even " the super resistant organism " that occurred pasting medical help, i.e. and drug-resistant bacteria (pan-resistance) entirely.Therefore countries in the world and international organization show great attention to animal and use the influence of antibacterials to food safety and human health.A large amount of research data both domestic and external shows that bacterium recall rate and resistant rate are all in swift and violent increase all over the world, and the resistance problem becomes more and more serious.The problem of bacterial drug resistance is extremely urgent, so carry out the chemical sproof investigation of China's animal derived bacterium comprehensively, foundation simply, the bacterial drug resistance detection method is the matter of utmost importance of pendulum in ours in front so that further scientific understanding and grasp bacterial drug resistance produce rule, mechanism of transmission and the mechanism of action fast and accurately.

Along with the problem of bacterial drug resistance is constantly aggravated, drug sensitive test detection method and means become more and more important.At present, drug sensitive test detection method commonly used mainly contains two big classes: a big class is that direct bacterial detection is to different antibiotic susceptibility, and drug-resistant phenotype detection method, this is that present laboratory is carried out resistance and detected direct and the most the most frequently used method, but its recall rate is subjected to influence of various factors such as the pH of measuring method, incubation temperature, time, substratum and concentration bigger.Therefore, must learn its measuring method and carry out strictness control, otherwise be difficult to obtain correct result.Another shortcoming of drug-resistant phenotype detection method is exactly must be through loaded down with trivial details bacterium purification procedures, and sense cycle is long, is unfavorable for instructing clinical medication fast and accurately.Drug-resistant phenotype detection method commonly used has: (1) disk diffusion method, judge sensitivity to measure bacteriostatic diameter, and intermediary or resistance, the influence of the beastly many factors of this method is as inoculation bacterium amount, preincubate time, antibiotic content and diffusive force, plate thickness etc.(2) dilution method can be measured certain medicine to detecting the minimal inhibitory concentration (MIC) of bacterium.Its shortcoming is consuming time, effort.(3) E test is to be released by Sweden AB Biodisk company in 1988, and this method can directly be measured the MIC of medicine to bacterium to be measured in conjunction with dilution method and diffusion ratio juris and characteristics.Its shortcoming is to cost an arm and a leg, and the result is subjected to multiple factor affecting as disk diffusion method.(4) instrumentation and automatic assay are as Vitek system, MicroScan system etc.By detecting turbidity or the fluorescence intensity of fluorescent indicator or the hydrolysis reaction sentence read result of fluorogenic substrate, the automatization assessing instrument also has certain limitation.At first be that automatic equipment is costly, secondly plant and instrument operation, maintenance requirement height are for some slow growing bacteriums and postpone to express the bacterium of resistance inducible enzyme, are difficult in the specific time to detect, often or cause omission.Simultaneously the drug-resistant phenotype detection method is the phenotype resistant characterization on the tentative check of external use medicine bacterium, latent type resistance that can not bacterial detection.

Another big class is to detect its drug resistant gene type by methods such as molecular biology, and the drug resistant gene detection method.This class detection method mainly contains the sequencing of bacterial plasmid collection of illustrative plates, plasmid fingerprinting collection of illustrative plates, plasmid elimination test, PCR and PCR-restriction fragment length polymorphism (PCR-RFLP), PCR-single strand conformation polymorphism (PCR-SSCP), Southern blot, Dot blot, nucleic acid probe detection, drug resistant gene chip detection, DNA etc.Wherein, PCR (comprising other method that PCR-based derives) and the method that is derived by hybridization are used at most, also have most prospect.

PCR method has sensitivity, advantage such as special, quick, the goal gene that can detect denier detects, and do not need can provide strong foundation for clinical application fast and accurately through the time-consuming bacterium separation and purification stage, be widely used in the bacterial resistance Journal of Sex Research.Simultaneously, when the MIC value of drug-resistant phenotype detection was in threshold value, PCR equimolecular biology techniques also can be judged its resistance exactly.But conventional PCR method can not satisfy the needs of drug sensitive test, because it once can only detect a drug resistant gene.The practicality of conventional PCR method on drug sensitive test detects is not high.

Multiplex PCR is to add many primer to be increased simultaneously to a plurality of goal gene in same reaction system.It can set up internal contrast in detecting the gene process; Can indicate the quantity of template; Several goal gene simultaneously can increase; The time and the reagent that consume are few, reduce setup time and raise the efficiency; Can carry out the detection of sample and goal gene in large quantities.Since Chamberlain (1988) first Application multiplex PCR increases a plurality of human dystrophin genes, multiplex PCR has developed into a kind of current techique, is widely used in pathogenic agent discriminating, sex screening, linkage analysis, legal medical expert's research, template quantitatively and the genetic diseases diagnosis.In recent years, in the multiple PCR technique detection that is applied to bacterial drug resistance more and more widely.Song Wei etc. (2001) [Song Wei etc. produce plasmid-mediated AmpC β-Nei phthalein amine enzyme gram-negative bacteria genotype and resistance research, Zhongshan University's doctorate paper].(2003) [[Birgit Strommenger such as Birgit Strommenger, Christiane Kettlitz, Guido WernerMultiplex PCR Assay for Simultaneous Detection of Nine Clinically Relevant AntibioticResistance Genes in Staphylococcus aureus Clin Microbiol.2003 September; 41 (9): 4089-4094] designed the distribution situation of nine kinds of clinical common antibiotics drug resistant genes in staphylococcus; (2003) such as Philip E.Coudron, Nancy D.Hanson, and Michael W.Climo[Philip E.Coudron Nancy D.Hanson, and Michael W.Climo Occurrence of Extended-Spectrum and AmpC Beta-Lactamases in Bloodstream Isolatesof Klebsiella pneumoniae:Isolates Harbor Plasmid-Mediated FOX-5and ACT-1AmpCBeta-LactamasesJ Clin Microbiol.2003 February; 41 (2): 772-777] detected the situation of in 190 strain klebsiellas, producing plasmid-mediated type AmpC β-Nei Xiananmei drug resistant gene.

[Manisha Mehrotra such as Manisha Mehrotra, Gehua Wang, and Wendy M.JohnsonMultiplex PCR for Detection of Genes for Staphylococcus aureus Enterotoxins, Exfoliative Toxins, Toxic Shock Syndrome Toxin1, and Methicillin Resistance.J Clin Microbiol.2000 March; 38 (3): 1032-1035.] (1999) successful Application multiple PCR technique detects product enterotoxin entA-entE gene and the methicillin-resistant gene mecA in the streptococcus aureus.

Up to the present, yet there are no the relevant reported literature of using quadruple PCR bacterial detection aminoglycoside drug resistant gene at home.

Two, bacterium is to the resistance mechanism and the drug resistant gene thereof of aminoglycoside antibiotics

The microbiotic of, wide spectrum efficient as a class, aminoglycoside medicaments (Aminogtycosides AGs) plays important effect clinically, especially treatment by Gram-negative bacteria effect brilliance on the caused severe infections.Since nineteen forty-four, Streptomycin sulphate was found, a series of aminoglycoside medicaments were cured and veterinary clinic in the people by rapid Application and Development, and wherein Streptomycin sulphate, kantlex, gentamicin become the central model of such medicine.But along with the extensive and a large amount of use of aminoglycoside medicaments, also more and more to the Resistant strain of this class antibiotics resistance, the resistance spectrum is more and more wider, and this has greatly limited the development of aminoglycoside antibiotics.Bacterium is a lot of to the mechanism that aminoglycoside antibiotics produces resistance, at present generally acceptedly mainly contains three kinds: 1, produce resistance by reducing the picked-up of aminoglycoside antibiotics or reducing medicine accumulation in vivo.2, change ribosome bind site and produce resistance.3, produce resistance [Llano-Setelo B by expressing aminoglycoside antibiotics modifying enzyme (AMEs), Azucena E F Jr, Kotra L P, et al.Aminoglyeosides modified by resistanceenzymes display diminished binding to the bactefial ribosomal aminoacyl-tRNA site.Chem Biol, 2002,9 (4): 455-463] bacterium produces resistance mainly by this mechanism realization to aminoglycoside antibiotics.Specifically, the aminoglycoside antibiotics modifying enzyme can be divided into three major types again, i.e. aminoglycoside phosphotransferase (APHs), aminoglycoside acetyltransferase (AAC), aminoglycoside adenosyl transferase (ANT).There are many subclass can modify hydroxyl or amino on the different positions in each fermentoid again, cause different drug-resistant phenotypes.Therefore, the detection of aminoglycoside medicaments drug resistant gene just can reflect that bacterium is to such antibiotic resistance situation from molecular level.

This test kit filters out aphA3, aacC4, four AMEs genes of aadA, aacC2 goal gene as the quadruple pcr amplification according to documents and materials and clinical detection result.

[summary of the invention]

One of purpose of the present invention provides the quadruple PCR detection technique method of a kind of rapid detection animal derived bacterium to the aminoglycoside medicaments drug resistant gene

The objective of the invention is to reach by the following technical programs:

A kind of animal derived bacterium is to aminoglycoside medicaments drug resistant gene quadruple PCR detection technique, contain pcr template and prepare reagent and bacterial resistance gene quadruple pcr amplification reagent, it is characterized in that: it is by washings that described pcr template prepares reagent, sample preparation liquid is formed, described bacterial resistance gene quadruple pcr amplification reagent comprises that 10 times of PCR damping fluid components are 50mMKCl, the 10mM Tris.HCl of pH8.3 and 0.01% gelatin, concentration is the dGTP of 2.5mmol/L, dCTP, dATP and dTTP, concentration is 25mM/L MgCl2, four big class drug resistant genes are the aphA3 gene, the aacC4 gene, the aadA gene, the specificity upstream and downstream primer concentration of aacC2 gene is 25mmol/L, and concentration is 2.5U/ μ lTaq archaeal dna polymerase and ultrapure water.Described animal derived bacterium is as follows to the final concentration of each component of aminoglycoside medicaments drug resistant gene quadruple pcr amplification reagent: 1 times of PCR damping fluid, the final concentration of dGTP, dCTP, dATP and dTTP is 0.25mmol/L, the final concentration of MgCl2 is 2.5mM/L, the final concentration of 4 class medicine drug resistant gene Auele Specific Primer aphA3, aacC4, aadA and aacC2 is respectively 0.125mmol/L, 0.2mmol/L, 0.45mmol/L and 0.225mmol/L, and Taq archaeal dna polymerase final concentration is 1U/50 μ l.

A kind of optimal technical scheme is characterized in that: after only needing centrifugal collection thalline in the described sample pre-treatments, handling through respective handling liquid can be standby.

A kind of optimal technical scheme is characterized in that: the composition that described pcr template prepares reagent wash liquid is: 5% glycerine.

A kind of optimal technical scheme is characterized in that: the composition that described pcr template prepares the reagent sample treatment solution is: 60% glycerine.

A kind of optimal technical scheme is characterized in that: described animal derived bacterium is the four big class aminoglycoside drug resistant gene upstream and downstream primers that concentration proportioning is the suitable amplifications that mix to the quadruple pcr amplification of aminoglycoside medicaments drug resistant gene.

A kind of optimal technical scheme is characterized in that: described four kinds of drug resistant genes are that specificity upstream and downstream primer sequence is as follows: aphA3:(For:5 '-TGACTGGGCACAACAGACAA-3 ' Rev:5 '-CGGCGATACCGTAAAGCAC-3 ') aacC4:(For:5 '-ATGACCTTGCGATGCTCTATGA-3 ' Rev:5 '-CGAATGCCTGGCGTGTTT-3 ') aadA:(For:5 '-ATCTGGCTATCTTGCTGACA-3 ' Rev:5 '-TATGACGGGCTGATACTGG-3 ') aacC2:(For:5 '-ACCCTACGAGGAGACTCTGAATG-3 ' Rev:5 '-CCAAGCATCGGCATCTCATA-3 ')

A kind of animal derived bacterium may further comprise the steps aminoglycoside medicaments drug resistant gene quadruple PCR detection technique:

1.PCR the preparation method of template is as follows:

(1) the LB culture media shaking vase is cultivated and is increased bacterium, comprises animal excreta sample, tissue juice, blood or other body fluid;

(2) get 1ml and cultivate the bacterium liquid of 4-6h in aseptic 1.5mlEP pipe through the LB culture media shaking vase, the centrifugal 3min of 8000-10000rin, abandon supernatant, add the 1ml pcr template again and prepare reagent wash liquid, repeated centrifugation once, abandon supernatant, add the 50-200ul pcr template at last and prepare the reagent sample treatment solution, standby behind the concussion mixing.

2. animal derived bacterium is as follows to the collocation method of aminoglycoside medicaments drug resistant gene quadruple pcr amplification reagent:

(1) according to prior art synthetic above-mentioned drug resistant gene Auele Specific Primer aphA3, aacC4, aadA and aacC2, with the dilution of 1mmolTris.Hcl-0.1mmolEDTA damping fluid, making its concentration is 25mmol/L;

(2) get 10 times PCR damping fluid, concentration respectively is dGTP, dCTP, dATP and the dTTP mixture of 2.5mmol/L, and concentration is 25mM/LMgCl ₂, the specificity upstream and downstream primer that it is aphA3 gene, aacC4 gene, aadA gene, aacC2 gene that concentration is 25mmol/L four big class drug resistant genes, concentration is that 2.5U/ μ l Taq archaeal dna polymerase and ultrapure water are packed in the aseptic PCR reaction thin-walled tube.

3. animal derived bacterium is to the composition of reagent in the aminoglycoside medicaments drug resistant gene quadruple PCR detection technique:

(1) gets 10 times PCR damping fluid 5 μ l, concentration respectively is dGTP, dCTP, dATP and the dTTP mixture 4 μ l of 2.5mmol/L, concentration is 25mM/L MgCl25 μ l, concentration is 2.5U/ μ l Taq archaeal dna polymerase 0.4 μ l, and the specificity upstream and downstream primer that it is aphA3 gene, aacC4 gene, aadA gene, aacC2 gene that concentration is 25mmol/L four big class drug resistant genes 0.25 μ l, 0.4 μ l, 0.9 μ l, 0.45 μ l respectively reacts in the thin-walled tube in an aseptic PCR.

(2) add the template 5 μ l prepared, moisturizing adds 20 μ l mineral oil to the cumulative volume 50 μ l and binds and get final product.

4.PCR the amplification cycles parameter is:

(1) 94 ℃ of pre-sex change 5min;

(2) enter circulation then: 94 ℃ of sex change 50s, 54 ℃ of annealing 45s, 72 ℃ of extension 50s, totally 30 circulations behind last 72 ℃ of extension 5min, are taken out in 4 ℃ of preservations.

5. the drug resistant gene detected result is judged

(1) gets 5 μ lPCR products, behind 1 μ l Loading buffer mixing, point sample is in 2% agarose gel electrophoresis plate hole, 80V voltage, electrophoresis 40min, the band of 284bp, 384bp, 486bp, 677bp appears in the judgement of taking pictures under Ultraluminescence Cheng Xiangyi among the result, then indicate aadA gene, aacC4 gene, aacC2 gene, aphA3 gene respectively.

(2) identification method that directly checks order: each 20 μ l of upstream and downstream primer of purified PCR product 50 μ l and four kinds of genes are served the sea together give birth to the biological company limited of worker and carry out dna sequencing.

The present invention will be further described below by the drawings and specific embodiments, but and do not mean that limiting the scope of the invention.

Description of drawings

Fig. 1 judges the electrophoresis instance graph for a kind of animal derived bacterium to aminoglycoside medicaments drug resistant gene quadruple PCR detection technique result.Wherein No. 1 swimming lane is 100bp DNA Marker, No. 2 positive contrasts of swimming lane, and No. 3 negative contrasts of swimming lane, No. 4 swimming lanes are for detecting positive (containing the aphA3 gene).

Claims (7)

1, a kind of animal derived bacterium is to aminoglycoside medicaments drug resistant gene quadruple PCR detection technique, contain pcr template and prepare reagent and bacterial resistance gene quadruple pcr amplification reagent, it is characterized in that: described pcr template prepares reagent and is made up of washings, sample preparation liquid, described bacterial resistance gene quadruple pcr amplification reagent comprises 10mM Tris.HCl and 0.01% gelatin that 10 times of PCR damping fluid components are 50mMKCl, pH8.3, concentration is dGTP, dCTP, dATP and the dTTP of 2.5mmol/L, and concentration is 25mM/L MgCl ₂, the specificity upstream and downstream primer concentration that four big class drug resistant genes are aphA3 gene, aacC4 gene, aadA gene, aacC2 gene is 25mmol/L, and concentration is 2.5U/ μ l Taq archaeal dna polymerase and ultrapure water.Described animal derived bacterium is as follows to the final concentration of each component of aminoglycoside medicaments drug resistant gene quadruple pcr amplification reagent: 1 times of PCR damping fluid, the final concentration of dGTP, dCTP, dATP and dTTP is 0.25mmol/L, MgCl ₂Final concentration be 2.5mM/L, the final concentration of 4 class medicine drug resistant gene Auele Specific Primer aphA3, aacC4, aadA and aacC2 is respectively 0.125mmol/L, 0.2mmol/L, 0.45mmol/L and 0.225mmol/L, and Taq archaeal dna polymerase final concentration is 1U/50 μ l.

2, drug resistant gene quadruple PCR detection technique according to claim 1 is characterized in that: after only needing centrifugal collection thalline in the described sample pre-treatments, handling through respective handling liquid can be standby.

3, drug resistant gene quadruple PCR detection technique according to claim 1, it is characterized in that: the composition that described pcr template prepares reagent wash liquid is: 5% glycerine.

4, drug resistant gene quadruple PCR detection technique according to claim 1, it is characterized in that: the composition that described pcr template prepares the reagent sample treatment solution is: 60% glycerine.

5, drug resistant gene quadruple PCR detection technique according to claim 1 is characterized in that: described animal derived bacterium is the four big class aminoglycoside drug resistant gene upstream and downstream primers that concentration proportioning is the suitable amplifications that mix to the quadruple pcr amplification of aminoglycoside medicaments drug resistant gene.

6, drug resistant gene quadruple PCR detection technique according to claim 1, it is characterized in that: described four kinds of drug resistant genes are that specificity upstream and downstream primer sequence is as follows:

aphA3：

For：5’-TGACTGGGCACAACAGACAA-3’Rev：5’-CGGCGATACCGTAAAGCAC-3’aacC4：

For：5’-ATGACCTTGCGATGCTCTATGA-3’Rev：5’-CGAATGCCTGGCGTGTTT-3’aadA：

For：5’-ATCTGGCTATCTTGCTGACA-3’Rev：5’-TATGACGGGCTGATACTGG-3’aacC2：

For：5’-ACCCTACGAGGAGACTCTGAATG-3’Rev：5’-CCAAGCATCGGCATCTCATA-3’。

7, a kind of animal derived bacterium may further comprise the steps aminoglycoside medicaments drug resistant gene quadruple PCR detection technique:

[1] preparation method of pcr template is as follows:

(1) the LB culture media shaking vase is cultivated and is increased bacterium, comprises animal excreta sample, tissue juice, blood or other body fluid;

(2) get 1ml and cultivate the bacterium liquid of 4-6h in aseptic 1.5mlEP pipe through the LB culture media shaking vase, the centrifugal 3min of 8000-10000rin, abandon supernatant, add the 1ml pcr template again and prepare reagent wash liquid, repeated centrifugation once, abandon supernatant, add the 50-200ulPCR template at last and prepare the reagent sample treatment solution, standby behind the concussion mixing.

[2] animal derived bacterium is as follows to the collocation method of aminoglycoside medicaments drug resistant gene quadruple pcr amplification reagent:

(1) according to prior art synthetic above-mentioned drug resistant gene Auele Specific Primer aphA3, aacC4, aadA and aacC2, with the dilution of 1mmolTris.Hcl-0.1mmolEDTA damping fluid, making its concentration is 25mmol/L;

(2) get 10 times PCR damping fluid, concentration respectively is dGTP, dCTP, dATP and the dTTP mixture of 2.5mmol/L, and concentration is 25mM/L MgCl ₂, the specificity upstream and downstream primer that it is aphA3 gene, aacC4 gene, aadA gene, aacC2 gene that concentration is 25mmol/L four big class drug resistant genes, concentration is that 2.5U/ μ l Taq archaeal dna polymerase and ultrapure water are packed in the aseptic PCR reaction thin-walled tube.

[3] animal derived bacterium is to the composition of reagent in the aminoglycoside medicaments drug resistant gene quadruple PCR detection technique:

(1) get 10 times PCR damping fluid 5 μ l, concentration respectively is dGTP, dCTP, dATP and the dTTP mixture 4 μ l of 2.5mmol/L, and concentration is 25mM/L MgCl ₂5 μ l, concentration is 2.5U/ μ l Taq archaeal dna polymerase 0.4 μ l, and the specificity upstream and downstream primer that it is aphA3 gene, aacC4 gene, aadA gene, aacC2 gene that concentration is 25mmol/L four big class drug resistant genes 0.25 μ l, 0.4 μ l, 0.9 μ l, 0.45 μ l respectively reacts in the thin-walled tube in an aseptic PCR.

(2) add the template 5 μ l prepared, moisturizing adds 20 μ l mineral oil to the cumulative volume 50 μ l and binds and get final product.

[4] the pcr amplification loop parameter is:

(1) 94 ℃ of pre-sex change 5min;

(2) enter circulation then: 94 ℃ of sex change 50s, 54 ℃ of annealing 45s, 72 ℃ of extension 50s, totally 30 circulations behind last 72 ℃ of extension 5min, are taken out in 4 ℃ of preservations.

[5] the drug resistant gene detected result is judged

(1) gets 5 μ lPCR products, behind 1 μ l Loading buffer mixing, point sample is in 2% agarose gel electrophoresis plate hole, 80V voltage, electrophoresis 40min, the band of 284bp, 384bp, 486bp, 677bp appears in the judgement of taking pictures under Ultraluminescence Cheng Xiangyi among the result, then indicate aadA gene, aacC4 gene, aacC2 gene, aphA3 gene respectively.

(2) identification method that directly checks order: each 20 μ l of upstream and downstream primer of purified PCR product 50 μ l and four kinds of genes are served the sea together give birth to the biological company limited of worker and carry out dna sequencing.

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