CN104212900B - For the composition that methicillin-resistant and/or resistance to tsiklomitsin streptococcus aureus detect - Google Patents

For the composition that methicillin-resistant and/or resistance to tsiklomitsin streptococcus aureus detect Download PDF

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CN104212900B
CN104212900B CN201410452992.1A CN201410452992A CN104212900B CN 104212900 B CN104212900 B CN 104212900B CN 201410452992 A CN201410452992 A CN 201410452992A CN 104212900 B CN104212900 B CN 104212900B
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郑秋月
于珂
高鹭
马惠蕊
战晓微
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Abstract

The invention provides a kind of composition detected for methicillin-resistant and/or resistance to tsiklomitsin streptococcus aureus, comprise, primer pair SEQ? ID? NO.1/2, and, primer pair SEQ? ID? NO.3/4 and SEQ? ID? at least one pair of primer in NO.5/6.And set up mPCR-DHPLC/ electrophoresis detection test kit and method further on this basis, detect the staphylococcus aureus resistance bacterial strain of pathogenic staphylococcus, MRSA and resistance to tsiklomitsin in food.Use composition of the present invention and method to while carrying out identification of bacteria to streptococcus aureus, MRSA and tetracycline resistance gene can be detected simultaneously, in one-time detection, namely identify the streptococcus aureus in sample, MRSA and resistance to erythromycin streptococcus aureus simultaneously.There is the advantage of fast high-flux, and it is consuming time short to examine side, simple to operation, a large amount of labours and financial resources can be saved, be applicable to the requirement of rapid detection.

Description

For the composition that methicillin-resistant and/or resistance to tsiklomitsin streptococcus aureus detect
Technical field
The invention belongs to technical field of biological, relate to the detection of the drug resistant gene in food and biological product entrained by drug-resistant S. aureus, the mPCR especially for methicillin-resistant and/or tsiklomitsin staphylococcus aureus resistance gene detects.
Technical background
Food borne bacteria resistance causes the attention of people in recent years gradually, and the World Health Organization is using " resistance resistance " theme as the World Hygiene Day in 2011.From 1997, repeatedly hold a meeting discussion Antibiotics usage of the international organizations such as Food and Argriculture OrganizationFAO (FAO), the World Health Organization (WHO) and OIE (OIE) formulated on human medical and sanitarian impact the global criterion controlling food animal antibiotic resistance after food animal.The country such as European Union, the U.S., Japan, Australia establishes the monitoring net of food animal resistant organism and Antibiogics usage amount one after another, has carried out large-scale monitoring.China using food borne bacteria resistance as important food safety and public health problem.
At present, " the antimicrobial agents disk diffusion method of animal derived bacterium and dilution method sensitiveness test operative norm that the clinical and Laboratory Standard council (ClinicalandLaboratoryStandardsInstitute, CLSI) of the domestic method Main Basis to animal derived bacterial drug resistance works out; Recognized Standards---the third edition " and industry standard the mensuration disk diffusion method of bacterial drug resistance " in animal and the goods thereof " (SN/T1944-2007) of inspection and quarantine carry out external microbial culture drug susceptibility test, the result of this drug sensitivity testing in vitro cannot assess the harm of bacterial drug resistance to human body, and the PCR detecting drug resistant gene can judge Resistant strain comparatively accurately, the false resistance avoiding detected bacterial strain to produce because of drug sensitivity testing in vitro or false sensitivity phenomenon.Because bacterial drug resistance is mainly derived from and detection to bacterial infection patients in clinical laboratory medicine, and animal derived bacterial resistance Journal of Sex Research is started late, the attention of people is not enough, thus few to the bacterial drug resistance detection method in animal derived bacterium and food, this just may cause drug-resistant bacteria to be propagated to the mankind by food chain, and causes spreading of drug-resistant bacteria.Staphylococcus aureus in food Resistant strain, when infecting the mankind because its resistance causes patient treatment difficulty, is difficult to cure; Its resistance determining factor also can between bacterial strain Spreading and diffusion, cause Resistant strain incidence constantly to rise, harm is serious.For preventing endurance strain from food, especially propagate to the mankind through food chain in animal food, the microorganism resistance method of inspection set up in advance for food is very important.
Streptococcus aureus (Staphylococcusaureus) is common food-borne pathogens, because this bacterium can produce enterotoxin, can food poisoning be caused, also can cause the animal suffering from disease such as poultry class, especially one of the main pathogenic fungi causing mammitis of cow.Along with the antibiotic abuse of veterinary drug, drug-resistant S. aureus spreads the whole world, wherein be called as the methicillin-resistant staphylococcus aureus (methicillinresistantStaphylococcusaureus of " superbacteria ", MRSA) almost resistance in various degree is all had to other microbiotic to except glycopeptide antibiotics, difficulty is treated after infection, and the danger to all antibiotic bacterium resistances may be caused under the selection pressure of medicine, about 50% is reached in MRSA in the national Bacterial resistance surveillance report of Mohnarin2011 year, along with its spread in china of drug tolerant bacteria and the transfer MRSA of resistance determining factor are in animal (especially in the cultivation of poultry class), animal derived food (meat, breast class etc.) and other varieties of food items in all detect, and incidence is also raising year by year, report, in the commercially available chicken of China some areas all there is resistance in streptococcus aureus, and MRSA recall rate reaches 13%, the recall rate of cow's milk source MRSA can reach about 22%, therefore, particularly important to the prevention and control of this bacterioid.Tsiklomitsin is as broad-spectrum antibacterial class microbiotic, for a long time, unreasonable, abuse can not only cause superinfection, accelerate the generation of resistant organism, domestic have report food source property streptococcus aureus to the resistant rate of tsiklomitsin close to 50%, and in animal cultivation, Fourth Ring is usually admixed in feed and is used, and causes the resistant rate of animal derived bacterium to tsiklomitsin higher.The phenomenon that this large amount of Resistant strain occurs often ignore by people, but its harm is extremely serious, can cause the failure of Animal diseases prevention and control on the one hand, and animal source resistant organisms a large amount of is on the other hand by the have a big risk large increase of food chain transport to human body.If pathogenic bacteria then directly causes the failure of human disease treatment, if avirulence resistant organism, then drug resistant gene may be transferred to other pathogenic bacterias in human intestine diseases induced.And domestic MRSA recall rate of carrying tetracycline resistance gene is apparently higher than abroad, but with external genotype unlike, the domestic streptococcus aureus to tetracycline resistant of China mainly carries tetM gene.
Therefore, expect the method setting up more accurately sensitive aimed detection drug resistant gene, in order to judge methicillin-resistant and/or the staphylococcus aureus strains of resistance to tsiklomitsin more accurately, and the false resistance effectively avoiding detected bacterial strain to produce because of drug sensitivity testing in vitro or false sensitivity phenomenon.
Summary of the invention
An object of the present invention, is the composition being provided for methicillin-resistant and/or the detection of resistance to tsiklomitsin streptococcus aureus, comprises,
Primer pair SEQIDNO.1/2, and,
At least one pair of primer in primer pair SEQIDNO.3/4 and SEQIDNO.5/6.
Table is described below about these primer pairs:
As preferred technical scheme, the described composition detected for methicillin-resistant and/or resistance to tsiklomitsin streptococcus aureus, comprise described whole 3 group-specific primerses pair, that is: primer pair SEQIDNO.1/2, primer pair SEQIDNO.3/4 and primer pair SEQIDNO.5/6 simultaneously.
Described composition is applied to PCR reaction, the goal gene fragment that may contain in specific aim amplification testing sample, these goal gene fragments comprise: the Pseudomonas specific gene nuc Gene of pathogenic staphylococcus, methicillin-resistant staphylococcus aureus resistance factor of determination mecA gene and tetracycline resistant factor of determination tetM gene.
Two of object of the present invention is to provide a kind of test kit detected for methicillin-resistant and/or resistance to tsiklomitsin streptococcus aureus, comprise the composition detected for methicillin-resistant and/or resistance to tsiklomitsin streptococcus aureus of the invention described above, i.e. primer pair.
Obviously, described test kit can be applicable to pcr amplification, in described test kit, except comprising above-mentioned Auele Specific Primer pair, can also comprise other component for PCR reaction, described component includes but not limited to Taq DNA polymerase, dNTP, 10 × PCR damping fluid and water.
The object of further aspect of the present invention is to provide a kind of method detecting methicillin-resistant and/or resistance to tsiklomitsin streptococcus aureus, comprise the step of PCR reaction, described PCR reaction uses the composition for methicillin-resistant and/or the detection of resistance to tsiklomitsin streptococcus aureus of the present invention mentioned above to be mPCR amplimer.
In specific embodiment, PCR reaction system cumulative volume 25 μ L in the method for detection methicillin-resistant of the present invention and/or resistance to erythromycin streptococcus aureus, comprise: testing sample DNA solution 1 μ l, the Taq DNA polymerase 0.25 μ L of 5U/ μ L, the water of PCR reaction solution 12 μ L and surplus;
Containing 10mMTrisHCl, 50mMKCl, 25mMMgCl in described PCR reaction solution 2, dNTP each 2.5mM and 0.1 μM primer pair SEQIDNO.1/2 (SEQIDNO.1, each 0.1 μM of SEQIDNO.2), the primer pair SEQIDNO.3/4 (SEQIDNO.3 of 0.1 μM, each 0.1 μM of SEQIDNO.4) and the primer pair SEQIDNO.5/6 (SEQIDNO.5, SEQIDNO.6) of 0.2 μM.
In specific embodiment, in the method for detection methicillin-resistant of the present invention and/or resistance to tsiklomitsin streptococcus aureus, PCR reaction parameter is:
Denaturation: 94 DEG C, 1min;
Enter circulation: 94 DEG C of sex change 30s, 57 DEG C of annealing 90s, 72 DEG C extend 90s, 30 circulations;
Stop extending: 72 DEG C, 10min.
To the product of mPCR, the method for DHPLC or electrophoresis can be adopted to carry out further interpretation of result:
One of specific embodiments, PCR reaction product is carried out to the step of DHPLC analysis, analysis condition is as follows:
Chromatographic column: PS-DVB & C18DNASep chromatographic column, 4.6mm × 50mm, granularity 3 μm;
Column temperature: 50 DEG C;
According to volume ratio, moving phase is:
0min：55.0％A，45.0％B；
0.5min：50.2％A，49.8％B；
3.6min：41.8％A，58.2％B；
6.8min：38.2％A，61.8％B；
9.9min：36.3％A，63.7％B；
13.0min：35.0％A，65.0％B；
A is 50mlTEAA and the mixing of 250 μ l acetonitriles, adds sterilizing ultrapure water and is settled to 1000ml gained solution; B is the mixing of 50mlTEAA and 250ml acetonitrile, adds sterilizing ultrapure water and is settled to 1000ml gained solution;
Flow velocity: 0.9mL/min;
Detector: fluorimetric detector, light source 150WXenon lamp; Excitation spectrum bandwidth 15nm; Emission spectrum bandwidth 15.3nm; Detection sensitivity: at wavelength 350nm integration 2s;
Applied sample amount: PCR primer 10 μ L.
To described DHPLC detected result, judge according to following standard:
Detect sample to occur without amplification absorption peak, can judge that sample result is as feminine gender, direct report does not detect corresponding pathogenic bacterium;
Detect sample nuc gene and occur typical PCR primer absorption peak, and when absorption peak is greater than 3mV, can judge that this sample is as streptococcus aureus, and mecA, tetM gene is without amplification absorption peak, can judge that this bacterium is as MSSA, and without tetracycline resistance gene tetM;
Detect sample nuc gene and occur typical PCR primer absorption peak, and absorption peak is when being greater than 3mV, can judge that this sample is as streptococcus aureus, there is typical PCR primer absorption peak in mecA gene simultaneously, and tetM gene is without amplification absorption peak, can judge that this sample is as MRSA, and without tetracycline resistance gene tetM;
Detect sample nuc gene and occur typical PCR primer absorption peak, and absorption peak is when being greater than 3mV, can judge that this sample is as streptococcus aureus, there is typical PCR primer absorption peak in mecA gene simultaneously, and there is typical PCR primer absorption peak in tetM gene, can judge that this sample is as MRSA, and to tetracycline resistant;
Detect sample and occur typical PCR primer absorption peak, but when absorption peak is less than 3mV, suggestion sample detects after Zengjing Granule again again; Results peaks of reforming absorption value is still less than 3mV then for negative, otherwise is probable positive.
To the analysis of described mPCR amplification, specific embodiments two is steps of electrophoretic analysis: 10 μ LPCR products, in 3% agarose gel electrophoresis, and voltage 200V, electric current 190mA, time 20min, observations on ultraviolet transmission analyser.
To described electrophoresis detection result, judge according to following standard:
Detect the amplified band that corresponding size appears in sample nuc gene, can judge that this sample is as streptococcus aureus, and mecA, tetM gene is without the amplified band of corresponding size, can judge that this bacterium is as MSSA, and without tetracycline resistance gene tetM gene;
Detect the amplified band that corresponding size appears in sample nuc gene, can judge that this sample is as streptococcus aureus, there is the amplified band of corresponding size in mecA gene simultaneously, and tetM gene is without the amplified band of corresponding size, can judge that this sample is as MRSA, and without tetracycline resistance gene tetM gene;
Detect the amplified band that corresponding size appears in sample nuc gene, can judge that this sample is as streptococcus aureus, mecA, tetM gene occurs that the amplified band of corresponding size can judge that this sample is as MRSA simultaneously, and to tetracycline resistant.
The present invention is directed to factor of determination tetM gene design specificity multiplex PCR probe and the test kit of the detection nuc Gene of Staphylococcus aureus in food, MRSA resistance factor of determination mecA gene and resistance to tsiklomitsin, and set up mPCR-DHPLC/ electrophoretic detection further, detect the staphylococcus aureus resistance bacterial strain of pathogenic staphylococcus, MRSA and resistance to tsiklomitsin in food.Use composition of the present invention and method to while carrying out identification of bacteria to streptococcus aureus, MRSA and tetracycline resistance gene can be detected simultaneously, there is the advantages such as fast high-flux, meet microorganism in food and detect rapidly demand accurately.Use the mPCR-DHPLC/ electrophoresis method set up of the present invention in one-time detection, namely can identify streptococcus aureus in sample, MRSA and resistance to erythromycin streptococcus aureus simultaneously.And it is consuming time short to examine side, simple to operation, a large amount of labours and financial resources can be saved, be applicable to the requirement of rapid detection.
Accompanying drawing explanation
Accompanying drawing 8 width of the present invention, respectively:
Fig. 1: the MRSA of resistance to tsiklomitsin bacterial strain mPCR-electrophoresis detection result;
Fig. 2: the MRSA of resistance to tsiklomitsin bacterial strain mPCR-DHPLC detected result;
The method specificity test result of Fig. 3: mPCR-electrophoresis detection methicillin-resistant and/or resistance to tsiklomitsin streptococcus aureus,
The method specificity test result of Fig. 4: mPCR-DHPL detection methicillin-resistant and/or resistance to tsiklomitsin streptococcus aureus,
In Fig. 3 and 4,1. Listeria Monocytogenes; 2. Salmonella typhimurium; 3. enterococcus faecalis; 4. intestinal bacteria; 5. Vibrio parahemolyticus; 6. Pseudomonas aeruginosa; 7. streptococcus aureus;
Fig. 5: mPCR-electrophoresis detection methicillin-resistant and/or resistance to tsiklomitsin streptococcus aureus method sensitivity test result,
Fig. 6: mPCR-DHPLC detects methicillin-resistant and/or resistance to tsiklomitsin streptococcus aureus method sensitivity test result,
In Fig. 5 and 6,1:5.4 × 10 6cfu/mL; 2:5.4 × 10 5cfu/mL; 3:5.4 × 10 4cfu/mL; 4:5.4 × 10 3cfu/mL; 5:5.4 × 10 2cfu/mL;
Fig. 7: actual bacterial strain sample mPCR-DHPLC detected result,
Fig. 8: actual bacterial strain sample mPCR-electrophoresis detection result,
In Fig. 7 and 8,1. carry the MRSA bacterial strain of texM drug resistant gene; 2.MRSA bacterial strain; 3.MSSA bacterial strain.
In above-mentioned accompanying drawing, X-coordinate is retention time (unit: minute min), and ordinate zou represents absorption peak strength of signal (unit: millivolt mV).
Embodiment
Following non-limiting example, it is further described the foundation of present method and application thereof, can make the present invention of those of ordinary skill in the art's comprehend, but not limit the present invention in any way.
If without specified otherwise, the main agents that this part uses, instrument and merchandise resources thereof are: the reagent such as bacterial genomes DNA small scale purification test kit (TakaRaMiniBESTBacterialGenomicDNAExtractionkit), Taq enzyme and PCR damping fluid are all purchased from precious biotechnology (Dalian) company limited; Triethylamine acetyl salt (TEAA, chromatographically pure) is purchased from Transgenomic company; Acetonitrile (chromatographically pure) is purchased from Fisher company; Regular-PCR instrument PE24000 (PerkinElmer company, the U.S.); Denaturing high-performance chromatography instrument NAV-99-4500 (Transgenomic company, the U.S.); Supercentrifuge centrifuge5804 (Eppendorf company, Germany).
The equal purchased from American Culture Collection (ATCC) of the present invention's reference culture used and Chinese medicine Microbiological Culture Collection administrative center (CMCC), in table 1.Each bacterial strain uses strain preservative tube-80 DEG C preservation, and activation culture etc. are all carried out according to relevant national standard (GB), inspection and quarantine industry standard (SN) or internal authority standard method.
Table 1 test strain
Embodiment 1
(1) the design of primer, synthesis and the assembling of test kit:
Factor of determination tetM for the detection nuc Gene of streptococcus aureus, MRSA resistance factor of determination mecA gene and resistance to tsiklomitsin designs primer.The present embodiment determine detect primer sequence and expanding fragment length as shown in table 2:
Table 2
Be designed for the test kit of detection on this basis.This test kit comprises the Taq DNA polymerase and PCR reaction solution that concentration is 5U/ μ L; Containing 10mMTrisHCl, 50mMKCl, 25mMMgCl in PCR reaction solution wherein 2, dNTP (dATP, dGTP, dCTP and dTTP) each 2.5mM and above-mentioned 3 pairs of streptococcus aureuses primer pair (concentration is as shown in table 2).
⑵mPCR：
The multi-primers using above-mentioned steps (1) designed and correspondingly test kit, carry out pcr amplification, comprise the steps:
1. the preparation of measuring samples: adopt isolation kit method to prepare testing sample DNA genome:
A, food samples:
Product 25g (position of clip sample is with reference to national standard method) is taken food with aseptic technique, add in the aseptic triangular flask or slap type homogenizing bag that 225mLBPW is housed after pulverizing, rock 3 ~ 5min or bounce 1min, cultivating 18 ~ 24h by after triangular flask or homogenizing bag sealing at 36 DEG C.
Use bacterial genomes to extract test kit and extract its genomic dna, produce pcr template.Mark, directly as pcr template or-20 DEG C of preservations.
B, reference culture: the single bacterium colony of picking cultivates 18 ~ 24h in nutrient broth, use bacterial genomes to extract test kit and extract its genomic dna, produce pcr template.Mark, directly as pcr template or-20 DEG C of preservations.
2. pcr amplification:
Get 1 μ l testing sample DNA solution, add the PCR reaction solution in 12 μ l test kits, 0.25 μ LTaqDNA polysaccharase and sterilizing ultrapure water to cumulative volume 25 μ L; The centrifugal 10s of 5000r/min, then carries out pcr amplification by following parameters:
Denaturation: 94 DEG C, 1min;
Enter circulation: 94 DEG C of sex change 30s, 57 DEG C of annealing 90s, 72 DEG C extend 90s, 30 circulations;
Stop extending: 72 DEG C, 10min;
PCR primer 4 DEG C preservation is to be measured;
(3) mPCR product analysis:
1. get 10 μ L steps (2) mPCR products therefrom after 3% agarose gel electrophoresis (200V) detects 20min, observations on ultraviolet transmission analyser, as shown in Figure 1.Visible, amplified fragments size is the band of 147bp is tetracycline resistance gene tetM; The mecA drug resistant gene of amplified fragments size to be the band of 320bp be Renicillin binding protein 2a (PBP2a); Amplified fragments size is the band of 415bp is heat stable nuclease nuc staphylococcus aureus specific Species estimation gene.
2. PCR primer is carried out DHPLC analysis, DHPLC analysis condition is as follows:
Chromatographic column: PS-DVB & C18DNASep chromatographic column, 4.6mm × 50mm, granularity 3 μm;
Column temperature: 50 DEG C;
According to volume ratio, moving phase is:
0min：55.0％A，45.0％B；
0.5min：50.2％A，49.8％B；
3.6min：41.8％A，58.2％B；
6.8min：38.2％A，61.8％B；
9.9min：36.3％A，63.7％B；
13.0min：35.0％A，65.0％B；
Wherein, buffered soln A is 50mlTEAA and the mixing of 250 μ l acetonitriles, adds sterilizing ultrapure water and is settled to 1000ml gained solution; Buffered soln B is the mixing of 50mlTEAA and 250ml acetonitrile, adds sterilizing ultrapure water and is settled to 1000ml gained solution;
Flow velocity: 0.9mL/min;
Detector: fluorimetric detector, light source: 150WXenon lamp; Excitation spectrum bandwidth: 15nm; Emission spectrum bandwidth: 15.3nm; Detection sensitivity: at wavelength 350nm integration 2s;
Applied sample amount: PCR primer 10 μ L.
As shown in Figure 2, the pcr amplification product of three kinds of primers all can produce positive absorption peak to mPCR-DHPLC detected result, and absorption peak and amplified fragments size meet.
Embodiment 2. specific test
In table 1, all reference strain extracts genomic dna, then with these genomic dnas for template, the method set up according to embodiment 1 carries out detections analysis.As shown in Figure 3, in test strain, only streptococcus aureus detects respective strap to mPCR-electrophoresis result, and all the other bacterial strains all show negative result.The mPCR-electrophoretic method detected result specificity that embodiment 1 is set up is high, does not have false positive and false negative result.
As shown in Figure 4, in test strain, only streptococcus aureus detects respective absorption peak to mPCR-DHPLC result, and all the other bacterial strains all show negative result.The PCR-DHPLC detection method that embodiment 1 is set up has very high specificity, does not have false positive and false negative result.
Embodiment 3 detection sensitivity is tested
Streptococcus aureus ATCC51153 is inoculated in TSB substratum, cultivates 24h for 36 ± 1 DEG C, and be 5.4 × 10 by the bacterial number that gradient dilution method or counting process measure enrichment liquid 6cfu/ml, and respectively 10 are carried out to enrichment liquid -2, 10 -3, 10 -4gradient dilution, obtaining bacterial number is 5.4 × 10 5cfu/ml, 5.4 × 10 4cfu/ml, 5.4 × 10 3cfu/ml, 5.4 × 10 2cfu/ml bacterium liquid carries out DNA extraction respectively and analyzes, and as depicted in figures 5 and 6, display nuc gene is minimum detects 5.4 × 10 to mPCR-electrophoresis/DHPLC result 3cfu/ml.
Embodiment 4. actual sample detects
Get 3 strains isolated streptococcus aureus from actual sample, carry out DNA extraction, and carry out mPCR according to the method that embodiment 1 is set up, and carry out electrophoresis and DHPLC analysis with the condition that embodiment 1 is set up.DHPLC detected result is as accompanying drawing 7, visible:
No. 1 bacterial strain detects 147bp, 320bp, 415bp tri-positive absorption peaks, and this strain bacterium is the MRSA carrying texM drug resistant gene; No. 2 bacterial strains detect 320bp, 415bp two positive absorption peaks, and this strain bacterium is the MRSA not carrying texM drug resistant gene; No. 3 bacterial strains detect 415bp positive absorption peak, and this strain bacterium is the MSSA not carrying erythromycin-resistant gene.
Electrophoresis detection result is as accompanying drawing 8, visible, and its detected result is consistent with DHPLC detected result.By disk diffusion method and automatic bacterial identification and susceptibility instrument, this 3 strain streptococcus aureus is verified that its resistance is respectively: No. 1 bacterial strain is the MRSA of resistance to tsiklomitsin; No. 2 bacterial strains are the MRSA to sensitive tetracycline; No. 3 bacterial strains are the MSSA to sensitive tetracycline.The method detected result set up with this test conforms to, and illustrates that mPCR primer pair designed by the present invention and corresponding method of detection have good effect for Testing and appraisal streptococcus aureus, MRSA and tetracycline resistant bacterial strain.
In the present embodiment, use the Resistant strain that traditional disk diffusion method is identified, after bacteria genus qualification, then need could confirm its resistance through bacterium 24h incubated overnight, and this method and Bacteria Identification susceptibility instrument all need to measure bacterium reduced turbidity, be subject to the impact of testing staff's operation; And disk diffusion method qualification consuming timely needs 3-5 days, workload is large, and qualification result influence factor is many.And the method using the present invention to set up can while qualification streptococcus aureus Pseudomonas, detect its drug resistant gene, average always (the comprising sample preparation) 6h consuming time of method, detects (not comprising sample preparation) consuming time 2.5h, fast easy and simple to handle.

Claims (9)

1., for the composition that methicillin-resistant and/or resistance to tsiklomitsin streptococcus aureus detect, comprise following primer pair:
Primer pair SEQIDNO.1/2, primer pair SEQIDNO.3/4 and primer pair SEQIDNO.5/6.
2., for the test kit that methicillin-resistant and/or resistance to tsiklomitsin streptococcus aureus detect, comprise composition according to claim 1.
3. detect the method for methicillin-resistant and/or resistance to tsiklomitsin streptococcus aureus in food, comprise the step of PCR reaction, it is characterized in that, described PCR reaction uses the composition described in claim 1 to be amplimer.
4. method according to claim 3, is characterized in that, described PCR reaction system cumulative volume 25 μ L, comprising: the Taq DNA polymerase 0.25 μ L of testing sample DNA solution 1 μ l, 5U/ μ L, the water of PCR reaction solution 12 μ L and surplus;
Containing 10mMTrisHCl, 50mMKCl, 25mMMgCl in described PCR reaction solution 2, the primer pair SEQIDNO.1/2 of dNTP each 2.5mM and 0.1 μM, the primer pair SEQIDNO.5/6 of primer pair SEQIDNO.3/4 and 0.2 μM of 0.1 μM.
5. method according to claim 3, is characterized in that, described PCR reaction parameter is:
Denaturation: 94 DEG C, 1min;
Enter circulation: 94 DEG C of sex change 30s, 57 DEG C of annealing 90s, 72 DEG C extend 90s, 30 circulations;
Stop extending: 72 DEG C, 10min.
6. method according to claim 3, is characterized in that, also comprise the step of PCR reaction product being carried out to DHPLC analysis, analysis condition is as follows:
Chromatographic column: PS-DVB & C18DNASep chromatographic column, 4.6mm × 50mm, granularity 3 μm;
Column temperature: 50 DEG C;
According to volume ratio, moving phase is:
0min：55.0％A，45.0％B；
0.5min：50.2％A，49.8％B；
3.6min：41.8％A，58.2％B；
6.8min：38.2％A，61.8％B；
9.9min：36.3％A，63.7％B；
13.0min：35.0％A，65.0％B；
A is 50mlTEAA and the mixing of 250 μ l acetonitriles, adds sterilizing ultrapure water and is settled to 1000ml gained solution; B is the mixing of 50mlTEAA and 250ml acetonitrile, adds sterilizing ultrapure water and is settled to 1000ml gained solution;
Flow velocity: 0.9mL/min;
Detector: fluorimetric detector, light source 150WXenon lamp; Excitation spectrum bandwidth 15nm; Emission spectrum bandwidth 15.3nm; Detection sensitivity: at wavelength 350nm integration 2s;
Applied sample amount: PCR primer 10 μ L.
7. method according to claim 6, is characterized in that, judges described DHPLC detected result according to following standard:
Detect sample to occur without amplification absorption peak, can judge that sample result is as feminine gender, direct report does not detect corresponding pathogenic bacterium;
Detect sample nuc gene and occur typical PCR primer absorption peak, and when absorption peak is greater than 3mV, can judge that this sample is as streptococcus aureus, and mecA, tetM gene is without amplification absorption peak, can judge that this bacterium is as MSSA, and without tetracycline resistance gene tetM;
Detect sample nuc gene and occur typical PCR primer absorption peak, and absorption peak is when being greater than 3mV, can judge that this sample is as streptococcus aureus, there is typical PCR primer absorption peak in mecA gene simultaneously, and tetM gene is without amplification absorption peak, can judge that this sample is as MRSA, and without tetracycline resistance gene tetM;
Detect sample nuc gene and occur typical PCR primer absorption peak, and absorption peak is when being greater than 3mV, can judge that this sample is as streptococcus aureus, there is typical PCR primer absorption peak in mecA gene simultaneously, and there is typical PCR primer absorption peak in tetM gene, can judge that this sample is as MRSA, and to tetracycline resistant;
Detect sample and occur typical PCR primer absorption peak, but when absorption peak is less than 3mV, suggestion sample is reformed after Zengjing Granule again; Results peaks of reforming absorption value is still less than 3mV then for negative, otherwise is probable positive.
8. method according to claim 3, is characterized in that, also comprises the step of PCR reaction product being carried out to electrophoretic analysis: 10 μ LPCR products, in 3% agarose gel electrophoresis, voltage 200V, electric current 190mA, electrophoresis time is 20min, observations on ultraviolet transmission analyser.
9. method according to claim 8, is characterized in that, judges described electrophoresis result according to following standard:
Detect the amplified band that corresponding size appears in sample nuc gene, can judge that this sample is as streptococcus aureus, and mecA, tetM gene is without the amplified band of corresponding size, can judge that this bacterium is as MSSA, and without tetracycline resistance gene tetM;
Detect the amplified band that corresponding size appears in sample nuc gene, can judge that this sample is as streptococcus aureus, there is the amplified band of corresponding size in mecA gene simultaneously, and tetM gene is without the amplified band of corresponding size, can judge that this sample is as MRSA, and without tetracycline resistance gene tetM;
Detect the amplified band that corresponding size appears in sample nuc gene, can judge that this sample is as streptococcus aureus, mecA, tetM gene occurs that the amplified band of corresponding size can judge that this sample is as MRSA simultaneously, and to tetracycline resistant.
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