CN104099407B - The amphenicols Drug-resistant gene Quadruple-PCR detection kit of animal derived bacterium - Google Patents

The amphenicols Drug-resistant gene Quadruple-PCR detection kit of animal derived bacterium Download PDF

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CN104099407B
CN104099407B CN201310127290.1A CN201310127290A CN104099407B CN 104099407 B CN104099407 B CN 104099407B CN 201310127290 A CN201310127290 A CN 201310127290A CN 104099407 B CN104099407 B CN 104099407B
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沈建忠
吴聪明
汪洋
李君�
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China Agricultural University
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Abstract

The invention discloses a kind of amphenicols Drug-resistant gene Quadruple-PCR detection kit of animal derived bacterium.The invention provides the primer sets of bacterial detection to amphenicols drug resistance, be made up of primer pair 1, primer pair 2, primer pair 3 and primer pair 4; Experiment of the present invention proves, the present invention according to the nucleotide sequence design and synthesis 4 of 4 amphenicols Drug-resistant genes (cfr, fexA, fexB, floR) main in bacterium to corresponding Specific PCR primers, utilize Quadruple-PCR to treat amphenicols Drug-resistant gene in bacterial detection to increase, there is the features such as efficient, quick, cost is low, practical.

Description

The amphenicols Drug-resistant gene Quadruple-PCR detection kit of animal derived bacterium
Technical field
The present invention relates to biological technical field, particularly relate to the amphenicols Drug-resistant gene Quadruple-PCR detection kit of animal derived bacterium.
Background technology
Antibiotic medicine serves key effect in control human and animal's infectivity epidemic disease and reduction infectious disease incidence and mortality ratio.But the raising gradually of the intensive degree along with animal rearing, dosage constantly increases, and makes bacterial drug resistance produce and propagates more rapid, thus brings great challenge for the treatment of bacteriosis.Current bacterial resistance sex chromosome mosaicism has become one of the problem of the field such as clinical application in global range, newtype drug research and development focus the most.
Amphenicols medicine comprises paraxin, thiamphenicol and florfenicol, this type of medicine because of its has a broad antifungal spectrum, absorb fast, distribution in vivo on veterinary clinic, extensively play very important effect.Amphenicols mechanism of drug action is mainly by being combined with 50S subunit, and the effect of blocking peptide acyltransferase, disturbs and be combined with 50S subunit with amino acid whose aminoacyl-tRNA terminal, thus new peptide chain formation is obstructed, and arrestin matter is synthesized.
Along with the going deep into of research of the genotoxic potential to paraxin and thiamphenicol, residual and resistance, the many countries comprising China forbid its application on veterinary clinic successively.The nineties in 20th century, the new synthesizing amide alcohols Broad spectrum antibiotics of the first spirit of the U.S.-Schering-Plough's development puts goods on the market, transformation and the modification of structure make florfenicol have obvious advantage than paraxin and thiamphenicol in security and validity, are mainly used in the bacteriosis (colon bacillus, klebsiella, pasteurella haemolytica, pasteurella multocida, Proteus mirabilis, streptococcus aureus, actinobacillus pleuropneumoniae and salmonella typhi etc.) for the treatment of ox, pig, fowl and fish.In April, 1999, florfenicol was in China's approval listing, was widely used in livestock and poultry breeding industry, became one of most widely used veterinary antibiotic gradually.But along with increasing of medication, also constantly occur clinically to the bacterial strain of florfenicol resistance, this certainly will bring serious threat to clinical treatment.
Bacterial drug resistance detects for the clinical accurate medication of guidance, and timely diagnoses and treatment has great importance.Traditional Drug Resistance Detection carrys out the phenotype of bacterial detection resistance, although the method can observe bacterium intuitively to the sensitivity of certain medicine and resistance, but first need to carry out separation and Culture and purifying to bacterium, and complex operation, experience dependency are strong, consuming time longer, far from can meet the needs of clinical quick diagnosis and treatment.Meanwhile, because traditional method detects genotypic expression type under artificial condition in vitro, therefore more to its uncertain factor had an impact, and only can the phenotypic resistance characteristic of bacterial detection, its implicit type resistance can not be detected.
Along with increasing Protocols in Molecular Biology is applied to the detection of resistance, be not only clinical resistance detection and provide responsive fast and more reliable method, also in explanation resistance mechanism, playing keying action simultaneously, providing important scientific basis for carrying out the monitoring of animal source pathogenic bacteria resistance to drugs with epidemiological study and developing of new antibiotic.Originally, conventional round pcr bonding probes qualification or electrophoretic analysis are to the detection of single drug resistant gene having played important effect.But clinically, even the antibiotic resistance of same class also may be due to different mechanism, even same mechanism also may be caused by different drug resistant genes, and due to the transferability of some drug resistant genes, clinical strains demonstrates increasing multidrug resistant, and traditional substance PCR due to Testing index single, far can not meet the requirement of clinical detection, therefore, multiplex PCR arises at the historic moment, and achieves multiobject detection.
Multiple PCR technique in reaction system, adds multipair primer carry out PCR reaction, the different DNA sequence dna regions of simultaneously increasing in a sample, whether primary first-order equation can the multiple goal gene fragment of enrichment, and exist according to different lengths fragment in electrophoresis result and determine whether containing goal gene.Multiplex PCR, because of the advantage of its high specific, high-level efficiency, low cost, has been applied to the every field of life science.
Summary of the invention
An object of the present invention is to provide the primer sets of drug resistant gene in a kind of detection or auxiliary detection bacterium.
In detection provided by the invention or auxiliary detection bacterium, the primer sets of drug resistant gene, is made up of primer pair 1, primer pair 2, primer pair 3 and primer pair 4;
Described primer pair 1 is made up of the single strand dna shown in sequence 2 in the single strand dna shown in sequence in sequence table 1 and sequence table;
Described primer pair 2 is made up of the single strand dna shown in sequence 4 in the single strand dna shown in sequence in sequence table 3 and sequence table;
Described primer pair 3 is made up of the single strand dna shown in sequence 6 in the single strand dna shown in sequence in sequence table 5 and sequence table;
Described primer pair 4 is made up of the single strand dna shown in sequence 8 in the single strand dna shown in sequence in sequence table 7 and sequence table.
In above-mentioned primer sets, in described primer sets, each bar primer is equimolar ratio;
Described resistance is the medicine of resistance to amphenicols; Described amphenicols medicine is specially florfenicol or paraxin;
Described drug resistant gene is at least one in floR gene, fexB gene, cfr gene and fexA gene.
Another object of the present invention is to provide the PCR reagent of drug resistant gene in a kind of detection or auxiliary detection bacterium.
PCR reagent provided by the invention, is made up of above-mentioned primer sets, pcr amplification damping fluid and water;
The final concentration of each bar primer in described PCR reagent in 4 kinds of primer pairs in described primer sets is 0.2 μm of ol/L;
Described resistance is the medicine of resistance to amphenicols; Described amphenicols medicine is specially florfenicol or paraxin;
Described drug resistant gene is specially at least one in floR gene, fexB gene, cfr gene and fexA gene.
Above-mentioned pcr amplification damping fluid is PremixExTaq solution, and purchased from Takara company Code:D335A, the dNTP that its each component and concentration are the Taq polysaccharase of 1.25U/25 μ l, concentration is 0.4mmol/L, concentration are the Mg of 4mmol/L 2+.
3rd object of the present invention is to provide the PCR kit of drug resistant gene in a kind of detection or auxiliary detection bacterium.
Test kit provided by the invention, comprises above-mentioned primer sets or above-mentioned PCR reagent; Described resistance is the medicine of resistance to amphenicols; Described amphenicols medicine is specially florfenicol or paraxin;
Described drug resistant gene is specially at least one in floR gene, fexB gene, cfr gene and fexA gene.
Above-mentioned primer sets or above-mentioned PCR reagent or mentioned reagent box detect and/or auxiliary detection tested bacteria whether containing the application in drug resistant gene;
Or above-mentioned primer sets or above-mentioned PCR reagent preparation detect and/or auxiliary detection tested bacteria whether containing the application in drug resistant gene product;
Or above-mentioned primer sets or above-mentioned PCR reagent detect and/or the application of auxiliary detection tested bacteria whether in the medicament production of resistance to amphenicols in preparation;
Described resistance is the medicine of resistance to amphenicols; Described amphenicols medicine is specially florfenicol or paraxin; The said products is test kit;
Described drug resistant gene is specially at least one in floR gene, fexB gene, cfr gene and fexA gene.
4th object of the present invention is to provide a kind of detection and/or the method for auxiliary detection tested bacteria whether containing drug resistant gene.
Method provided by the invention, comprises the steps: to carry out Quadruple-PCR amplification by above-mentioned primer sets or above-mentioned PCR reagent to described testing sample, detects PCR primer;
If the PCR primer obtained has 200bp, 348bp, 534bp and 683bp size, then tested bacteria contains or candidate contains 4 kinds of drug resistant genes: floR gene, fexB gene, cfr gene and fexA gene; If the PCR primer obtained does not have any one size of 200bp, 348bp, 534bp and 683bp, then tested bacteria not containing or candidate containing in 4 kinds of drug resistant genes any one: floR gene, fexB gene, cfr gene and fexA gene;
If the PCR primer obtained has 200bp size, then tested bacteria contains or candidate contains floR gene; If the PCR primer obtained does not have 200bp size, then tested bacteria not containing or candidate not containing floR gene;
If the PCR primer obtained has 348bp size, then tested bacteria contains or candidate contains fexB gene; If the PCR primer obtained does not have 348bp size, then tested bacteria not containing or candidate not containing fexB gene;
If the PCR primer obtained has 534bp size, then tested bacteria contains or candidate contains cfr gene; If the PCR primer obtained does not have 534bp size, then tested bacteria not containing or candidate not containing cfr gene;
If the PCR primer obtained has 683bp size, then tested bacteria contains or candidate contains fexA gene; If the PCR primer obtained does not have 683bp size, then tested bacteria not containing or candidate not containing fexA gene;
If the PCR primer obtained has 200bp and 348bp size, then tested bacteria contains or candidate contains floR gene and fexB gene; If the PCR primer obtained does not have 200bp and 348bp size, then tested bacteria not containing or candidate not containing floR gene and fexB gene;
If the PCR primer obtained has 200bp and 534bp size, then tested bacteria contains or candidate contains floR gene and cfr gene; If the PCR primer obtained does not have 200bp and 534bp size, then tested bacteria not containing or candidate not containing floR gene and cfr gene;
If the PCR primer obtained has 200bp and 683bp size, then tested bacteria contains or candidate contains floR gene and fexA gene; If the PCR primer obtained does not have 200bp and 683bp size, then tested bacteria not containing or candidate not containing floR gene and fexA gene;
If the PCR primer obtained has 348bp and 534bp size, then tested bacteria contains or candidate contains fexB gene and cfr gene; If the PCR primer obtained does not have 348bp and 534bp size, then tested bacteria not containing or candidate not containing fexB gene and cfr gene;
If the PCR primer obtained has 348bp and 683bp size, then tested bacteria contains or candidate contains fexB gene and fexA gene; If the PCR primer obtained does not have 348bp size, then tested bacteria not containing or candidate not containing fexB gene and fexA gene;
If the PCR primer obtained has 534bp and 683bp size, then tested bacteria contains or candidate contains cfr gene and fexA gene; If the PCR primer obtained does not have 534bp size and 683bp size, then tested bacteria not containing or candidate not containing cfr gene and fexA gene.
In aforesaid method, the annealing temperature of described Quadruple-PCR amplification is 55 DEG C; The method of described detection PCR primer is electrophoresis detection or order-checking.
5th object of the present invention is to provide a kind of primer pair.
Primer pair provided by the invention is any one in following 4 kinds: primer pair 1, primer pair 2, primer pair 3 and primer pair 4;
Described primer pair 1 is made up of the single strand dna shown in sequence 2 in the single strand dna shown in sequence in sequence table 1 and sequence table;
Described primer pair 2 is made up of the single strand dna shown in sequence 4 in the single strand dna shown in sequence in sequence table 3 and sequence table;
Described primer pair 3 is made up of the single strand dna shown in sequence 6 in the single strand dna shown in sequence in sequence table 5 and sequence table;
Described primer pair 4 is made up of the single strand dna shown in sequence 8 in the single strand dna shown in sequence in sequence table 7 and sequence table.
Experiment of the present invention proves, the present invention according to the nucleotide sequence design and synthesis 4 of 4 amphenicols Drug-resistant genes (cfr, fexA, fexB, floR) main in bacterium to corresponding Specific PCR primers, utilize Quadruple-PCR to treat amphenicols Drug-resistant gene in bacterial detection to increase, can disposablely filter out tested bacteria whether containing in 4 amphenicols Drug-resistant genes any one or multiple, also can be used for the qualification tested bacteria whether medicine of resistance to amphenicols; The features such as primer of the present invention and method have efficiently, quick, cost is low, practical.
Accompanying drawing explanation
Fig. 1 is the amplification of Quadruple-PCR
Fig. 2 is the amplification of bacterial strain W9-2
Fig. 3 is the amplification of bacterial strain EF-01
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Part bacterial strain used in following embodiment and the literature reference recording this bacterial strain and the drug resistant gene contained as shown in table 1:
The drug resistant gene that table 1 is the 6 strain medicine of resistance to amphenicols bacterial strain literature references and contains
The above-mentioned bacterial strains public all can obtain from China Agricultural University.
Embodiment 1, for detecting the acquisition of the primer of the amphenicols Drug-resistant gene of animal derived bacterium
Be classified as sequence 9 according to 4 its nucleotides sequences of amphenicols Drug-resistant gene cfr(main in bacterium), its nucleotides sequence of fexA(is classified as sequence 10), its nucleotides sequence of fexB(is classified as sequence 11), its nucleotides sequence of floR(is classified as sequence 12) nucleotide sequence design and synthesis 4 to corresponding Specific PCR primers, specific as follows:
The Specific PCR primers pair of drug resistant gene cfr:
For:5 '-CTCTAGCCAACCGTCAAG-3 ' (sequence 1),
Rev:5 '-AAGCAGCGTCAATATCAATC-3 ' (sequence 2);
The Specific PCR primers pair of drug resistant gene fexA:
For:5 '-ATTCTGGCAAGTGGTAGTC-3 ' (sequence 3),
Rev:5 '-CCTGGCAACAATATCATTCC-3 ' (sequence 4);
The Specific PCR primers pair of drug resistant gene fexB:
For:5 '-CCAACGCCAATACAACCA-3 ' (sequence 5),
Rev:5 '-TCCTGTTCTCGGTGTGAT-3 ' (sequence 6);
The Specific PCR primers pair of drug resistant gene floR:
For:5 '-GGATGGCAGGCGATATTC-3 ' (sequence 7);
Rev:5 '-GAGAAGAAGACGAAGAAGGT-3 ' (sequence 8);
Synthesize the special primer of above-mentioned drug resistant gene cfr, fexA, fexB, floR according to prior art, with ultrapure water dilution, make the concentration of each bar primer be 10 μm of ol/L.
Embodiment 2, for detecting the application of the primer of the amphenicols Drug-resistant gene of animal derived bacterium
One, the application of primer in the amphenicols Drug-resistant gene or resistance of qualification bacterium
1, the extraction of animal derived bacterium Resistance detection and genomic dna
Above-mentioned bacterium EF-01, W9-2, LYP-C-BCTb11 are all carried out resistance to amphenicols drug testing: at brain heart infusion (BHI) plate streaking of the florfenicol containing 10mg/L, 37 ° of C incubated overnight, what can grow is resistance to florfenicol bacterium, and result bacterium EF-01, W9-2, LYP-C-BCTb11 are resistance to florfenicol bacterium.
Adopt and boil the genomic dna that bacterium method extracts bacterium EF-01, W9-2, the LYP-C-BCTb11 of the medicine of resistance to amphenicols respectively, obtain EF-01 genomic dna, W9-2 genomic dna, LYP-C-BCTb11 genomic dna (280 μ g/mL).
The three strain strain gene groups extracted respectively are got 10 μ l equal-volume mixing as positive control sample.
2, Quadruple-PCR amplification
PremixExTaq solution (Takara company Code:D335A), the dNTP that its each component and concentration are the Taq polysaccharase of 1.25U/25 μ l, concentration is 0.4mmol/L, concentration are the Mg of 4mmol/L 2+.
20 μ lPCR amplification systems are: 10 μ lPremixExTaq solution, 0.4 μ l concentration are the specificity upstream and downstream primer pair (final concentration of each bar primer in amplification system is 0.2 μm of ol/L) of 10 μm of ol/L drug resistant genes cfr, fexA, fexB, floR and ultrapure water and load an aseptic PCR and react in thin-walled tube, and adding water to cumulative volume is 19.5 μ l; Add 0.5 μ l bacterial genomes DNA more wherein as template, can PCR amplification system be formed.
Each component of above-mentioned PCR amplification system and final concentration following (concentration of 20 μ l systems) thereof: the Mg of dNTP, 2mmol/L of 0.2mmol/L 2+, the Taq enzyme of 0.5U, the specificity upstream and downstream primer final concentration of drug resistant gene cfr, fexA, fexB, floR be 0.2 μm of ol/L and water.
20 μ lPCR amplification systems are increased according to following pcr amplification condition, wherein template is respectively above-mentioned 1 positive control sample obtained, test strains (in the resistance to florfenicol bacterium in pig source that 2010 gather on pig farm, Shandong Province, the nose swab obtained is rule on brain heart infusion (BHI) agar plate of the florfenicol containing 10mg/L, 37 ° of C incubated overnight, the bacterial strain of resistance to florfenicol of acquisition) genomic dna, negative control (water):
Pcr amplification loop parameter is: 95 DEG C of denaturation 5min, afterwards 32 circulations, and each circulation is 95 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 40s, and after loop ends, 72 DEG C extend 8min, 4 DEG C of preservations.
Obtain PCR primer.
Get the PCR primer of 7 μ l, point sample is in 2% agarose gel electrophoresis plate hole, and 150V voltage, electrophoresis 25min, obtains picture by gel imaging system.
Electrophoretic band is reclaimed order-checking,
Result as shown in Figure 1, swimming lane 1-4 is respectively: No. 1 swimming lane is DL2000DNAMarker, No. 2 swimming lanes are EF-01, W9-2, LYP-C-BCTb11 tri-strain bacterium mixutre genome, No. 3 swimming lanes are negative control (water), No. 4 swimming lanes are that (test strains is the resistance to florfenicol bacterium in pig source gathered on pig farm, Shandong Province in 2010 to test strains, the nose swab obtained is rule on brain heart infusion (BHI) agar plate of the florfenicol containing 10mg/L, 37 ° of C incubated overnight, the bacterial strain obtained), can find out, swimming lane 2 is EF-01, W9-2, LYP-C-BCTb11 tri-strain bacterium mixutre genome has 200bp, 348bp, 534bp, the band of 683bp, illustrate that the drug resistant gene that three kinds of bacterial strains contain can increase in primary first-order equation system simultaneously, and it is unaffected, swimming lane 4 only has the band of 200bp, test strains is described only containing floR gene.
Extract the genomic dna of test strains, order-checking, can find out, the genomic dna of test strains is really only containing the gene of resistance to amphenicols floR;
Extract EF-01, W9-2, LYP-C-BCTb11 tri-genomic dna of strain bacterium respectively, order-checking, can find out, EF-01 contains fexB(348bp)+cfr (534bp), W9-2 contains fexA (683bp)+cfr (534bp), and LYP-C-BCTb11 contains cfr (534bp)+floR (200bp).
Therefore, the primer sets that the specificity upstream and downstream primer pair of above-mentioned drug resistant gene cfr, fexA, fexB, floR can be adopted to form or PCR reagent detect as follows to tested bacteria:
Whether detect tested bacteria containing resistance to amphenicols drug gene: floR gene, fexB gene, cfr gene and/or fexA gene:
If the PCR primer obtained has 200bp, 348bp, 534bp and 683bp size, then tested bacteria contains or candidate contains 4 kinds of drug resistant genes: floR gene, fexB gene, cfr gene and fexA gene; If the PCR primer obtained does not have any one size of 200bp, 348bp, 534bp and 683bp, then tested bacteria not containing or candidate containing in 4 kinds of drug resistant genes any one: floR gene, fexB gene, cfr gene and fexA gene;
If the PCR primer obtained has 200bp size, then tested bacteria contains or candidate contains floR gene; If the PCR primer obtained does not have 200bp size, then tested bacteria not containing or candidate not containing floR gene;
If the PCR primer obtained has 348bp size, then tested bacteria contains or candidate contains fexB gene; If the PCR primer obtained does not have 348bp size, then tested bacteria not containing or candidate not containing fexB gene;
If the PCR primer obtained has 534bp size, then tested bacteria contains or candidate contains cfr gene; If the PCR primer obtained does not have 534bp size, then tested bacteria not containing or candidate not containing cfr gene;
If the PCR primer obtained has 683bp size, then tested bacteria contains or candidate contains fexA gene; If the PCR primer obtained does not have 683bp size, then tested bacteria not containing or candidate not containing fexA gene;
If the PCR primer obtained has 200bp and 348bp size, then tested bacteria contains or candidate contains floR gene and fexB gene; If the PCR primer obtained does not have 200bp and 348bp size, then tested bacteria not containing or candidate not containing floR gene and fexB gene;
If the PCR primer obtained has 200bp and 534bp size, then tested bacteria contains or candidate contains floR gene and cfr gene; If the PCR primer obtained does not have 200bp and 534bp size, then tested bacteria not containing or candidate not containing floR gene and cfr gene;
If the PCR primer obtained has 200bp and 683bp size, then tested bacteria contains or candidate contains floR gene and fexA gene; If the PCR primer obtained does not have 200bp and 683bp size, then tested bacteria not containing or candidate not containing floR gene and fexA gene;
If the PCR primer obtained has 348bp and 534bp size, then tested bacteria contains or candidate contains fexB gene and cfr gene; If the PCR primer obtained does not have 348bp and 534bp size, then tested bacteria not containing or candidate not containing fexB gene and cfr gene;
If the PCR primer obtained has 348bp and 683bp size, then tested bacteria contains or candidate contains fexB gene and fexA gene; If the PCR primer obtained does not have 348bp size, then tested bacteria not containing or candidate not containing fexB gene and fexA gene;
If the PCR primer obtained has 534bp and 683bp size, then tested bacteria contains or candidate contains cfr gene and fexA gene; If the PCR primer obtained does not have 534bp size and 683bp size, then tested bacteria not containing or candidate not containing cfr gene and fexA gene.
Detect the tested bacteria whether medicine of resistance to amphenicols (being specially resistance to florfenicol):
If the PCR primer obtained has at least one in 200bp, 348bp, 534bp, 683bp, then the tested bacteria medicine of resistance to amphenicols or candidate's medicine of resistance to amphenicols;
If the PCR primer obtained does not have any one in 200bp, 348bp, 534bp, 683bp, then the tested bacteria not medicine of resistance to amphenicols or the candidate not medicine of resistance to amphenicols.
Two, specific detection
1, resistance to amphenicols drug testing
Resistance to florfenicol detects: get the 6 strain medicine of resistance to amphenicols bacterial strain EF-01, W9-2,3-38, LYP-C-BCTb11, W3, EH-1 and 6 strain amphenicols medicaments insensitive bacterial strain S1-S6(all gathered slaughterhouse, Ningxia health pig anus swab in 2012 years), carry out resistance to florfenicol detection, specific as follows: bacterial strain common not containing the BHI(brain heart infusion agar of medicine) dull and stereotyped on can grow, and can not grow on brain heart infusion (BHI) agar plate of the florfenicol containing 10mg/L, then florfenicol is responsive; Bacterial strain common not containing the BHI(brain heart infusion agar of medicine) dull and stereotyped on can grow, and to grow on brain heart infusion (BHI) agar plate of the florfenicol containing 10mg/L, then resistance to florfenicol;
Resistance to paraxin detects: get the 6 strain medicine of resistance to amphenicols bacterial strain EF-01, W9-2,3-38, LYP-C-BCTb11, W3, EH-1 and 6 strain amphenicols medicaments insensitive bacterial strain S1-S6, carry out resistance to paraxin detection, specific as follows: bacterial strain common not containing the BHI(brain heart infusion agar of medicine) dull and stereotyped on can grow, and can not grow on brain heart infusion (BHI) agar plate of the paraxin containing 32mg/L, then Chloramphenicol-sensitive; Bacterial strain common not containing the BHI(brain heart infusion agar of medicine) dull and stereotyped on can grow, and to grow on brain heart infusion (BHI) agar plate of the paraxin containing 32mg/L, then resistance to paraxin;
Through above-mentioned detection, bacterial strain EF-01, W9-2,3-38, LYP-C-BCTb11, W3, EH-1 resistance to fluorobenzene Buddhist nun also resistance to paraxin;
Bacterial strain S1-S6 is also to Chloramphenicol-sensitive to florfenicol sensitivity.
The literature reference of record of the 6 strain medicine of resistance to amphenicols bacterial strains and the result of the drug resistant gene contained after gene order-checking as shown in table 1 below.
2, PCR detects
Extract the genomic dna of the 6 strain medicine of resistance to amphenicols bacterial strain EF-01, W9-2,3-38, LYP-C-BCTb11, W3, EH-1 and 6 strain amphenicols medicaments insensitive bacterial strain S1-S6 respectively as template, with above-mentioned one 2 PCR amplification system and pcr amplification condition increase, obtain PCR primer.
Get the PCR primer of 7 μ l, point sample is in 2% agarose gel electrophoresis plate hole, and 150V voltage, electrophoresis 25min, obtains picture by gel imaging system.
Electrophoretic band is reclaimed order-checking,
Whether detect tested bacteria containing resistance to amphenicols drug gene: floR gene, fexB gene, cfr gene and/or fexA gene:
If the PCR primer obtained has 200bp, 348bp, 534bp and 683bp size, then tested bacteria contains or candidate contains 4 kinds of drug resistant genes: floR gene, fexB gene, cfr gene and fexA gene; If the PCR primer obtained does not have any one size of 200bp, 348bp, 534bp and 683bp, then tested bacteria not containing or candidate containing in 4 kinds of drug resistant genes any one: floR gene, fexB gene, cfr gene and fexA gene;
If the PCR primer obtained has 200bp size, then tested bacteria contains or candidate contains floR gene; If the PCR primer obtained does not have 200bp size, then tested bacteria not containing or candidate not containing floR gene;
If the PCR primer obtained has 348bp size, then tested bacteria contains or candidate contains fexB gene; If the PCR primer obtained does not have 348bp size, then tested bacteria not containing or candidate not containing fexB gene;
If the PCR primer obtained has 534bp size, then tested bacteria contains or candidate contains cfr gene; If the PCR primer obtained does not have 534bp size, then tested bacteria not containing or candidate not containing cfr gene;
If the PCR primer obtained has 683bp size, then tested bacteria contains or candidate contains fexA gene; If the PCR primer obtained does not have 683bp size, then tested bacteria not containing or candidate not containing fexA gene;
If the PCR primer obtained has 200bp and 348bp size, then tested bacteria contains or candidate contains floR gene and fexB gene; If the PCR primer obtained does not have 200bp and 348bp size, then tested bacteria not containing or candidate not containing floR gene and fexB gene;
If the PCR primer obtained has 200bp and 534bp size, then tested bacteria contains or candidate contains floR gene and cfr gene; If the PCR primer obtained does not have 200bp and 534bp size, then tested bacteria not containing or candidate not containing floR gene and cfr gene;
If the PCR primer obtained has 200bp and 683bp size, then tested bacteria contains or candidate contains floR gene and fexA gene; If the PCR primer obtained does not have 200bp and 683bp size, then tested bacteria not containing or candidate not containing floR gene and fexA gene;
If the PCR primer obtained has 348bp and 534bp size, then tested bacteria contains or candidate contains fexB gene and cfr gene; If the PCR primer obtained does not have 348bp and 534bp size, then tested bacteria not containing or candidate not containing fexB gene and cfr gene;
If the PCR primer obtained has 348bp and 683bp size, then tested bacteria contains or candidate contains fexB gene and fexA gene; If the PCR primer obtained does not have 348bp size, then tested bacteria not containing or candidate not containing fexB gene and fexA gene;
If the PCR primer obtained has 534bp and 683bp size, then tested bacteria contains or candidate contains cfr gene and fexA gene; If the PCR primer obtained does not have 534bp size and 683bp size, then tested bacteria not containing or candidate not containing cfr gene and fexA gene.
Detect the tested bacteria whether medicine of resistance to amphenicols (being specially resistance to florfenicol or resistance to paraxin):
If the PCR primer obtained has at least one in 200bp, 348bp, 534bp, 683bp, then the tested bacteria medicine of resistance to amphenicols or candidate's medicine of resistance to amphenicols;
If the PCR primer obtained does not have any one in 200bp, 348bp, 534bp, 683bp, then the tested bacteria not medicine of resistance to amphenicols or the candidate not medicine of resistance to amphenicols.
Result is as follows:
The PCR primer that bacterial strain EF-01 obtains has 348bp and 534bp size, then this bacterial strain contains fexB gene and cfr gene (the resistance to florfenicol of this bacterial strain or paraxin); The genomic dna extracting this bacterial strain sends to order-checking, containing fexB gene (sequence 11) and cfr gene (sequence 9);
The PCR primer of bacterial strain W9-2 has 683bp and 534bp size, then this bacterial strain contains fexA gene and cfr gene (the resistance to florfenicol of this bacterial strain or paraxin); Extract this strain gene group DNA and send to order-checking, containing fexA gene (sequence 10) and cfr gene (sequence 9);
The PCR primer of bacterial strain 3-38 has 683bp and 534bp size, then this bacterial strain contains fexA gene and cfr gene (the resistance to florfenicol of this bacterial strain or paraxin); Extract this strain gene group DNA and send to order-checking, containing fexA gene (sequence 10) and cfr gene (sequence 9);
The PCR primer that bacterial strain LYP-C-BCTb11 obtains has 534bp and 200bp size, then this bacterial strain contains cfr gene and floR gene (the resistance to florfenicol of this bacterial strain or paraxin); Extract this strain gene group DNA and send to order-checking, containing cfr gene (sequence 9) and floR(sequence 12);
The PCR primer of bacterial strain W3 has 348bp and 534bp size, then this bacterial strain contains fexB gene and cfr gene (the resistance to florfenicol of this bacterial strain or paraxin); Extract this strain gene group DNA and send to order-checking, containing fexB gene (sequence 11) and cfr gene (sequence 9);
The PCR primer that bacterial strain EH-1 obtains has 348bp size, then this bacterial strain contains fexB gene (the resistance to florfenicol of this bacterial strain or paraxin); Extract this strain gene group DNA and send to order-checking, containing fexB gene (sequence 11);
The PCR primer that S1-S6 bacterial strain obtains all does not have any one in 200bp, 348bp, 534bp, 683bp, and therefore S1-S6 bacterial strain is not all containing any one (to florfenicol and paraxin all sensitivities) in cfr, fexA, fexB, floR gene; The genomic dna extracting S1-S6 bacterial strain respectively sends to order-checking, and the genomic dna of each bacterial strain is not all containing cfr(sequence 9), fexA(sequence 10), fexB(sequence 11) and floR gene (sequence 12) in any one.
Therefore, sequencing result is consistent with method of the present invention, illustrates that method of the present invention is correct.
Three, sensitivity technique
The bacterial strain W9-2(of above-mentioned resistance to florfenicol or resistance to paraxin is contained fexA gene and cfr gene) and EF-01(contain fexB gene and cfr gene) the concentration of Genomic DNA solution be all adjusted to 280 μ g/mL, 10 times of gradient dilutions are done respectively with sterilized water, dilute 8 gradients altogether, each gradient gets 0.5 μ l respectively as template, with above-mentioned one 2 PCR amplification system and pcr amplification condition increase, obtain PCR primer.
Get the PCR primer of 7 μ l, point sample is in 2% agarose gel electrophoresis plate hole, and 150V voltage, electrophoresis 25min, obtains picture by gel imaging system.
Result is as shown in Figures 2 and 3:
Fig. 2 is the amplification of bacterial strain W9-2, and wherein, swimming lane 1 is DL2000DNAMarker; Swimming lane 2 ~ 9:DNA template concentrations is respectively 14.0ng, 1.4ng, 140.0pg, 14.0pg, 1.4pg, 140.0fg, 14.0fg, 1.4fg/PCR; Swimming lane 10:ddH 2o.As shown in Figure 2, can see band comparatively clearly at the 6th swimming lane, its drug resistant gene detected is cfr gene, fexA gene, and corresponding DNA concentration is 1.4pg/PCR, and can't see amplified band clearly after the 7th swimming lane.
Fig. 3 is the amplification of bacterial strain EF-01, and wherein, swimming lane 1 is DL2000DNAMarker; Swimming lane 2 ~ 9:DNA template concentrations is respectively 14.0ng, 1.4ng, 140.0pg, 14.0pg, 1.4pg, 140.0fg, 14.0fg, 1.4fg/PCR; Swimming lane 10:ddH 2o.As shown in Figure 3, can see band comparatively clearly at the 6th swimming lane, its drug resistant gene detected is cfr gene, fexB gene, and corresponding DNA concentration is 1.4pg/PCR, and can't see amplified band clearly after the 7th swimming lane.
Therefore, comprehensive twice detected result judges that PCR detection sensitivity is as 1.4pg/PCR, has higher sensitivity.

Claims (12)

1. detect or the primer sets of drug resistant gene in auxiliary detection bacterium, be made up of primer pair 1, primer pair 2, primer pair 3 and primer pair 4;
Described primer pair 1 is made up of the single strand dna shown in sequence 2 in the single strand dna shown in sequence in sequence table 1 and sequence table;
Described primer pair 2 is made up of the single strand dna shown in sequence 4 in the single strand dna shown in sequence in sequence table 3 and sequence table;
Described primer pair 3 is made up of the single strand dna shown in sequence 6 in the single strand dna shown in sequence in sequence table 5 and sequence table;
Described primer pair 4 is made up of the single strand dna shown in sequence 8 in the single strand dna shown in sequence in sequence table 7 and sequence table;
In described primer sets, each bar primer is equimolar ratio.
2. primer sets according to claim 1, is characterized in that:
Described resistance is the medicine of resistance to amphenicols;
Described drug resistant gene is at least one in floR gene, fexB gene, cfr gene and fexA gene.
3. detect or the PCR reagent of drug resistant gene in auxiliary detection bacterium, be made up of the primer sets described in claim 1 or 2, pcr amplification damping fluid and water;
The final concentration of each bar primer in described PCR reagent in 4 kinds of primer pairs in described primer sets is 0.2 μm of ol/L;
Described resistance is the medicine of resistance to amphenicols.
4. reagent according to claim 3, is characterized in that: described drug resistant gene is at least one in floR gene, fexB gene, cfr gene and fexA gene.
5. detect or the PCR kit of drug resistant gene in auxiliary detection bacterium, comprise PCR reagent described in primer sets described in claim 1 or 2 or claim 3; Described resistance is the medicine of resistance to amphenicols.
6. test kit according to claim 5, is characterized in that: described drug resistant gene is at least one in floR gene, fexB gene, cfr gene and fexA gene.
7. PCR reagent described in the primer sets described in claim 1 or 2 or claim 3 or reagent according to claim 4 detect and/or auxiliary detection tested bacteria whether containing the application in drug resistant gene; Described be applied as non-diseases diagnoses and treatment application.
8. PCR reagent described in the primer sets described in claim 1 or 2 or claim 3 preparation detect and/or auxiliary detection tested bacteria whether containing the application in the product of drug resistant gene.
9. PCR reagent described in the primer sets described in claim 1 or 2 or claim 3 preparation detect and/or the auxiliary detection tested bacteria whether medicine of resistance to amphenicols product in application.
10. the application according to any one of claim 7-9, is characterized in that: described resistance is the medicine of resistance to amphenicols;
Described drug resistant gene is at least one in floR gene, fexB gene, cfr gene and fexA gene.
Whether 11. 1 kinds of detections and/or auxiliary detection tested bacteria contain the non-diseases diagnoses and treatment method of drug resistant gene, comprise the steps: to carry out Quadruple-PCR amplification by PCR reagent described in the primer sets described in claim 1 or 2 or claim 3 to described testing sample, detect PCR primer;
If the PCR primer obtained has 200bp size, then tested bacteria contains or candidate contains floR gene; If the PCR primer obtained does not have 200bp size, then tested bacteria not containing or candidate not containing floR gene;
If the PCR primer obtained has 348bp size, then tested bacteria contains or candidate contains fexB gene; If the PCR primer obtained does not have 348bp size, then tested bacteria not containing or candidate not containing fexB gene;
If the PCR primer obtained has 534bp size, then tested bacteria contains or candidate contains cfr gene; If the PCR primer obtained does not have 534bp size, then tested bacteria not containing or candidate not containing cfr gene;
If the PCR primer obtained has 683bp size, then tested bacteria contains or candidate contains fexA gene; If the PCR primer obtained does not have 683bp size, then tested bacteria not containing or candidate not containing fexA gene.
12., according to method described in claim 11, is characterized in that:
The annealing temperature of described Quadruple-PCR amplification is 55 DEG C;
The method of described detection PCR primer is electrophoresis detection or order-checking.
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