CN103540666B - Multiplex PCR (Polymerase Chain Reaction) detection method of enterobacter cloacae in sewage outlet water body - Google Patents
Multiplex PCR (Polymerase Chain Reaction) detection method of enterobacter cloacae in sewage outlet water body Download PDFInfo
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- C12Q2537/10—Reactions characterised by the reaction format or use of a specific feature the purpose or use of
- C12Q2537/143—Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
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Abstract
The invention discloses a multiplex PCR (Polymerase Chain Reaction) detection method of enterobacter cloacae in a sewage outlet water body. The detection method comprises the following steps: preparing an enterobacter cloacae antibody solution; preparing an immunomagnetic bead solution; enriching enterobacter cloacae in a to-be-detected water sample by the immunomagnetic bead solution to obtain a to-be-detected sample solution with enriched enterobacter cloacae; extracting a DNA (Deoxyribose Nucleic Acid) template by a DNA extraction kit; and performing PCR detection on multiple pairs of primers; and if obtaining four target gene segments 816bp, 1507bp, 502bp and 1216bp, judging that the water sample contains enterobacter cloacae. According to the method, the enterobacter cloacae in the water body has a high enrichment degree, which is beneficial to detection and the method is sensitive in detection; in addition, since multiple pairs of primers are detected simultaneously and the correctness is high.
Description
Technical field
The present invention relates to the PCR detection technique of bacterium, be specifically related to the multi-PCR detection method of enterobacter cloacae in sewage draining exit water body.
Background technology
Enterobacter cloacae (
enterobacter cloacae) be one of member of enterobacter, this bacterium is Grain-negative tubbiness bacillus, is extensively present in occurring in nature, and all can detect in the enteron aisle of humans and animals, ight soil water, earth, plant, enterobacter cloacae has become the more and more important pathogenic bacteria of hospital infection.Therefore the resident that in sewage draining exit water body, the detection of enterobacter cloacae just can react these cities and towns infects the situation of enterobacter cloacae.Multiple PCR technique carries out the process increased for the different zones of multiple DNA profiling or same template, can once increase multiple target gene or gene fragment, makes the accuracy of detection higher.
Summary of the invention
Technical problem to be solved by this invention is to provide the multi-PCR detection method of enterobacter cloacae in a kind of sewage draining exit water body, and the method carries out enrichment to the enterobacter cloacae in sewage draining exit water body, and detects with multipair primer simultaneously, and detection sensitivity is high, and accuracy is high.
The present invention solves the problems of the technologies described above adopted technical scheme: the multi-PCR detection method of enterobacter cloacae in sewage draining exit water body, and its step is as follows:
Prepared by a, enterobacter cloacae antibody-solutions: the single colony inoculation of picking enterobacter cloacae is in by the casein soybean liquid-agar liquid nutrient medium (TSBA) of trypsin digestion, concussion overnight incubation, under 7000 ~ 8000 revs/min centrifugal 5 minutes, precipitation physiological saline washes 1 time, again with physiological saline suspend, add mass percentage concentration be 0.5% ~ 1% formaldehyde spend the night; Under next day 7000 ~ 8000 revs/min centrifugal 5 minutes, precipitation physiological saline washed 3 times, and to be made into concentration respectively with physiological saline be 10
7~ 10
9cfu/ml enterobacter cloacae bacterium liquid and concentration are 10
10concentration is 10 by cfu/ml enterobacter cloacae bacterium liquid
7~ 10
9cfu/ml enterobacter cloacae bacterium liquid 0.2ml abdominal injection first immunisation in mouse, one week, interval is 10 by concentration
7~ 10
9cfu/ml enterobacter cloacae bacterium liquid 0.2ml abdominal injection is second time immunity in mouse, then every one week, is 10 by concentration
10cfu/ml enterobacter cloacae bacterium liquid 0.2ml is injected in booster immunization in mouse, after booster immunization the 4th day, extracts eyeball of mouse and gets blood, incubation 0.5h at blood is placed in 37 DEG C, at 4 DEG C, 3000 revs/min of centrifugal 15min, obtain supernatant, and this supernatant is enterobacter cloacae antibody-solutions;
B, prepared by immunomagnetic beads solution: get 100 ~ 200 μ l carboxyl magnetic beads and be placed in centrifuge tube, add 500 μ L activation buffer, vortex oscillator mixes, centrifuge tube is positioned on Magneto separate frame, treat that nanometer magnetic bead is adsorbed on centrifugal tube wall completely, shift out liquid in pipe, nanometer magnetic bead is washed once with activation buffer, in centrifuge tube, activation buffer 500 μ l is added after washing, carbodiimide 0.5mg and N-hydroxy-succinamide 1mg, vortex oscillator mixes, 20 min are activated under room temperature, shift out liquid in pipe, obtain activated magnetic beads, with coupling buffer washing activated magnetic beads secondary, in centrifuge tube, the above-mentioned enterobacter cloacae antibody-solutions 15 μ l that concentration is 6 mg/ml is added after washing, 3 h are reacted under room temperature, take out supernatant liquor, obtain immunomagnetic beads, with coupling buffer washing immunomagnetic beads secondary, then the coupling buffer 500 μ l added containing mass percentage concentration 1% bovine serum albumin (BSA) closes immunomagnetic beads 30min, obtain immunomagnetic beads solution, described activation buffer is pH is 6.0 NaH containing tween 20 (Tween-20)
2pO
4solution, described coupling buffer is pH is 7.4 NaH containing tween 20
2pO
4solution, in described activation buffer and described coupling buffer, NaH
2pO
4concentration is 0.01M, and the mass percentage concentration of tween 20 is 0.05%,
C, testing sample enrichment: get sewage draining exit water sample 2ml in test tube, add the above-mentioned immunomagnetic beads solution of 0.2ml, the light and slow rotational oscillation 30min of room temperature, shift out liquid in test tube, wash immunomagnetic beads 3 ~ 5 times with phosphoric acid buffer, add 0.5ml sterilized water after washing, test tube is placed in magnetic resolution 3min on Magnetic cell sorting frame, take out immunomagnetic beads, obtain the measuring samples solution of enterobacter cloacae enrichment;
Prepared by d, DNA profiling: measuring samples solution DNA extraction kit extracts DNA, obtains DNA profiling, and concrete extraction is carried out according to DNA extraction kit specification sheets;
E, PCR detect: the PCR reaction system of 30 μ l is: 10 × PCR damping fluid 2.5 μ l, the MgCl of concentration 25mM
2liquid 1.5 μ l, the dNTP liquid 1.5 μ l of concentration 10mM, the T aq DNA polysaccharase 0.2 μ l of concentration 5U/ μ l, concentration is all each 1.0 μ l of primer irp2-F, irp2-R, FhuA-F, FhuA-R, SodB-F, SodB-R, Slt-F, Slt-R of 10 μMs, above-mentioned DNA profiling 2 μ l, surplus is distilled water, PCR reaction conditions is 94 ° of C sex change 5 min, 94 ° of C sex change 45s, 55 ° of C annealing 45s, and 72 ° of C extend 1min, 35 circulations, and 72 ° of C extend 10 min, the PCR reaction product agarose gel electrophoresis testing goal gene fragment of mass percentage concentration 1.4%, if obtain 816bp, 1507bp, 502bp and 1216bp tetra-goal gene fragments, then contain enterobacter cloacae in sewage draining exit water body, the nucleotides sequence of described primer irp2-F is classified as: AAGGATTCGC TGTTACCGGA C, the nucleotides sequence of described primer irp2-R is classified as: AACTCCTGAT ACAGGTGGC, the nucleotides sequence of described primers F huA-F is classified as: CAGCAGAACA ACCGAAGCAA GA, the nucleotides sequence of described primers F huA-R is classified as: TTGAGGTCGT GGCGTAATCG T, the nucleotides sequence of described primer SodB-F is classified as: ACCACCAGGC GTATGTCACT AA, the nucleotides sequence of described primer SodB-R is classified as: TGCCGAAAGA TTTGGCGATT, the nucleotides sequence of described primer Slt-F is classified as: CGCAGCCGCT ACGCACAAAT, the nucleotides sequence of described primer Slt-R is classified as: TCGGTTACAT CGCTACCCAT CA.
Above-mentioned primer is the genome sequence according to enterobacter cloacae, and application primer Express 3.0 software design also screening obtains, and primer is synthesized by the raw work in Shanghai.
Compared with prior art, the invention has the advantages that the multi-PCR detection method of enterobacter cloacae in sewage draining exit water body, by the preparation of enterobacter cloacae antibody-solutions and the preparation of immunomagnetic beads solution, the enrichment of immunomagnetic beads solution will be used in water sample to be measured, obtain the measuring samples solution of enterobacter cloacae enrichment, then DNA profiling is extracted by DNA extraction kit, detected by the PCR of multipair primer again, if obtain 816bp, 1507bp, 502bp and 1216bp tetra-goal gene fragments, then contain enterobacter cloacae in water sample; In the method water sample, enterobacter cloacae enrichment degree is high, is beneficial to detection, detects sensitive, and multipair primer detects simultaneously, and accuracy is high.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail.
Embodiment 1
Prepared by enterobacter cloacae antibody-solutions: picking enterobacter cloacae (ATCC 13047) single colony inoculation is concussion overnight incubation in TSBA liquid nutrient medium (being purchased from the raw work in Shanghai), under 7000 ~ 8000 revs/min centrifugal 5 minutes, precipitation physiological saline washes 1 time, again with physiological saline suspend, add mass percentage concentration be 0.5% ~ 1% formaldehyde spend the night; Under next day 7000 ~ 8000 revs/min centrifugal 5 minutes, precipitation physiological saline washed 3 times, and to be made into concentration respectively with physiological saline be 10
7~ 10
9cfu/ml enterobacter cloacae bacterium liquid and concentration are 10
7~ 10
9concentration is 10 by cfu/ml enterobacter cloacae bacterium liquid
7~ 10
9cfu/ml enterobacter cloacae bacterium liquid 0.2ml abdominal injection first immunisation in mouse, one week, interval is 10 by concentration
7~ 10
9cfu/ml enterobacter cloacae bacterium liquid 0.2ml abdominal injection is second time immunity in mouse, then every one week, is 10 by concentration
10cfu/ml enterobacter cloacae bacterium liquid 0.2ml is injected in booster immunization in mouse, after booster immunization the 4th day, extracts eyeball of mouse and gets blood, incubation 0.5h at blood is placed in 37 DEG C, at 4 DEG C, 3000 revs/min of centrifugal 15min, obtain supernatant, and this supernatant is that enterobacter cloacae antibody-solutions is stand-by;
Prepared by immunomagnetic beads solution: getting concentration is that 30mg/ml nanometer magnetic bead liquid 100 ~ 200 μ l is placed in centrifuge tube, add activation buffer 500 μ l again, vortex oscillator mixes, centrifuge tube is positioned on Magneto separate frame, treat that nanometer magnetic bead is adsorbed on centrifugal tube wall completely, shift out liquid in pipe, nanometer magnetic bead is washed once with activation buffer, in centrifuge tube, activation buffer 500 μ l is added after washing, carbodiimide 0.5mg and N-hydroxy-succinamide 1mg, vortex oscillator mixes, 20 min are activated under room temperature, shift out liquid in pipe, obtain activated magnetic beads, with coupling buffer washing activated magnetic beads secondary, in centrifuge tube, the above-mentioned enterobacter cloacae antibody-solutions 15 μ l that coupling buffer 485 μ l and concentration are 6 mg/ml is added after washing, 3 h are reacted under room temperature, take out supernatant liquor, obtain immunomagnetic beads, with coupling buffer washing immunomagnetic beads secondary, then the coupling buffer 500 μ l added containing mass percentage concentration 1%BSA closes magnetic bead 30min, obtain immunomagnetic beads solution for later use, above-mentioned activation buffer is pH 6.0, is the Tween-20 of 0.05%, the NaH of concentration 0.01M containing mass percentage concentration
2pO
4solution, above-mentioned coupling buffer is pH 7.4, be 0.05%Tween-20 containing mass percentage concentration, concentration is the NaH of 0.01M
2pO
4solution,
Testing sample enrichment: get Xiangshan County of Ningbo City Xiangshan Bay sewage draining exit water sample 2ml in test tube, add the above-mentioned immunomagnetic beads solution of 0.2ml, the light and slow rotational oscillation 30min of room temperature, shift out liquid in test tube, wash immunomagnetic beads 3 ~ 5 times with phosphoric acid buffer, add 0.5ml sterilized water after washing, test tube is placed in magnetic resolution 3min on Magnetic cell sorting frame, take out immunomagnetic beads, obtain the measuring samples solution of enterobacter cloacae enrichment;
Prepared by DNA profiling: measuring samples solution DNA extraction kit (being purchased from the raw work in Shanghai) extracts DNA, and obtain DNA profiling, concrete extraction is carried out according to DNA extraction kit specification sheets;
PCR detects: the PCR reaction system of 30 μ l is: 10 × PCR damping fluid 2.5 μ l, the MgCl of concentration 25mM
2liquid 1.5 μ l, the dNTP liquid 1.5 μ l of concentration 10mM, the T aq DNA polysaccharase 0.2 μ l of concentration 5U/ μ l, concentration is all each 1.0 μ l of primer irp2-F, irp2-R, FhuA-F, FhuA-R, SodB-F, SodB-R, Slt-F, Slt-R of 10 μMs, above-mentioned DNA profiling 2 μ l, surplus is distilled water, PCR reaction conditions is 94 ° of C sex change 5 min, 94 ° of C sex change 45s, 55 ° of C annealing 45s, and 72 ° of C extend 1min, 35 circulations, and 72 ° of C extend 10 min, the PCR reaction product agarose gel electrophoresis testing goal gene fragment of mass percentage concentration 1.4%, obtain 816bp, 1507bp, 502bp and 1216bp tetra-gene fragments, containing enterobacter cloacae in sewage draining exit water body, the nucleotides sequence of primer irp2-F is classified as: AAGGATTCGC TGTTACCGGA C, the nucleotides sequence of primer irp2-R is classified as: AACTCCTGAT ACAGGTGGC, the nucleotides sequence of primers F huA-F is classified as: CAGCAGAACA ACCGAAGCAA GA, the nucleotides sequence of primers F huA-R is classified as: TTGAGGTCGT GGCGTAATCG T, the nucleotides sequence of primer SodB-F is classified as: ACCACCAGGC GTATGTCACT AA, the nucleotides sequence of primer SodB-R is classified as: TGCCGAAAGA TTTGGCGATT, the nucleotides sequence of primer Slt-F is classified as: CGCAGCCGCT ACGCACAAAT, the nucleotides sequence of primer Slt-R is classified as: TCGGTTACAT CGCTACCCAT CA.
Embodiment 2
Substantially the same manner as Example 1, difference is that sewage draining exit water sample is taken from the east of a river, river, Fenghua, Ningbo City sewage draining exit, and PCR obtains 816bp gene fragment after detecting, not containing enterobacter cloacae in sewage draining exit water body.
Embodiment 3
Substantially the same manner as Example 1, difference is that sewage draining exit water sample is taken from Fenghua, Ningbo City Jiang Haishu sewage draining exit, and PCR obtains 1216bp gene fragment after detecting, not containing enterobacter cloacae in sewage draining exit water body.
Embodiment 4
Substantially the same manner as Example 1, difference is that sewage draining exit water sample is taken from the Yong Jiang east of a river, Ningbo City sewage draining exit, obtains 816bp, 502bp bis-gene fragments, not containing enterobacter cloacae in sewage draining exit water body.
Embodiment 5
Substantially the same manner as Example 1, difference is that sewage draining exit water sample is taken from the Yong Jiang north of the Changjiang River, standing wave city sewage draining exit, obtains 816bp, 1507bp, 502bp and 1216bp tetra-gene fragments, containing enterobacter cloacae in sewage draining exit water body.
Embodiment 6: specificity and susceptibility
With the DNA profiling of embodiment 1 prepare detect with PCR substantially identical, difference is just directly with enterobacter cloacae (ATCC 13047), the Pseudomonas fluorescens (ATCC 13525) close with enterobacter cloacae, Wdwardsiella tarda (ATCC15947), intestinal bacteria (ATCC25922) carry out DNA profiling preparation and PCR detects, just enterobacter cloacae obtains 816bp, 1507bp, 502bp and 1216bp tetra-goal gene fragments, other 3 kinds of other bacteriums still have to 1-2 gene fragment, illustrate that specificity is high.Enterobacter cloacae DNA profiling concentration gradient 1-100ng, detects with PCR respectively, detects and be limited to≤100cfu/ml, illustrate that susceptibility is strong.
<110> University Of Ningbo
The multi-PCR detection method of enterobacter cloacae in <120> sewage draining exit water body
<160> 8
<170> PatentIn version 3.5
<210> 1
<211> 21
<212> DNA
<213> artificial sequence
<400> 1
AAGGATTCGC TGTTACCGGA C 21
<210> 2
<211> 19
<212> DNA
<213> artificial sequence
<400> 2
AACTCCTGAT ACAGGTGGC 19
<210>3
<211> 22
<212> DNA
<213> artificial sequence
<400> 3
CAGCAGAACA ACCGAAGCAA GA 22
<210> 4
<211> 21
<212> DNA
<213> artificial sequence
<400> 4
TTGAGGTCGT GGCGTAATCG T 21
<210> 5
<211> 22
<212> DNA
<213> artificial sequence
<400> 5
ACCACCAGGC GTATGTCACT AA 22
<210> 6
<211> 20
<212> DNA
<213> artificial sequence
<400> 6
TGCCGAAAGA TTTGGCGATT 20
<210> 7
<211> 20
<212> DNA
<213> artificial sequence
<400> 7
CGCAGCCGCT ACGCACAAAT 20
<210> 8
<211> 22
<212> DNA
<213> artificial sequence
<400> 8
TCGGTTACAT CGCTACCCAT CA 22
Claims (1)
1. the multi-PCR detection method of enterobacter cloacae in sewage draining exit water body, is characterized in that step is as follows:
Prepared by a, enterobacter cloacae antibody-solutions: the single colony inoculation of picking enterobacter cloacae is in by the casein soybean liquid-agar liquid nutrient medium of trypsin digestion, concussion overnight incubation, under 7000 ~ 8000 revs/min centrifugal 5 minutes, precipitation physiological saline washes 1 time, again with physiological saline suspend, add mass percentage concentration be 0.5% ~ 1% formaldehyde spend the night; Under next day 7000 ~ 8000 revs/min centrifugal 5 minutes, precipitation physiological saline washed 3 times, and to be made into concentration respectively with physiological saline be 10
7~ 10
9cfu/ml enterobacter cloacae bacterium liquid and concentration are 10
10concentration is 10 by cfu/ml enterobacter cloacae bacterium liquid
7~ 10
9cfu/ml enterobacter cloacae bacterium liquid 0.2ml abdominal injection first immunisation in mouse, one week, interval is 10 by concentration
7~ 10
9cfu/ml enterobacter cloacae bacterium liquid 0.2ml abdominal injection is second time immunity in mouse, then every one week, is 10 by concentration
10cfu/ml enterobacter cloacae bacterium liquid 0.2ml is injected in booster immunization in mouse, after booster immunization the 4th day, extracts eyeball of mouse and gets blood, incubation 0.5h at blood is placed in 37 DEG C, at 4 DEG C, 3000 revs/min of centrifugal 15min, obtain supernatant, and this supernatant is enterobacter cloacae antibody-solutions;
B, prepared by immunomagnetic beads solution: get 100 ~ 200 μ l carboxyl magnetic beads and be placed in centrifuge tube, add 500 μ L activation buffer, vortex oscillator mixes, centrifuge tube is positioned on Magneto separate frame, treat that nanometer magnetic bead is adsorbed on centrifugal tube wall completely, shift out liquid in pipe, nanometer magnetic bead is washed once with activation buffer, in centrifuge tube, activation buffer 500 μ l is added after washing, carbodiimide 0.5mg and N-hydroxy-succinamide 1mg, vortex oscillator mixes, 20 min are activated under room temperature, shift out liquid in pipe, obtain activated magnetic beads, with coupling buffer washing activated magnetic beads secondary, in centrifuge tube, the above-mentioned enterobacter cloacae antibody-solutions 15 μ l that concentration is 6 mg/ml is added after washing, 3 h are reacted under room temperature, take out supernatant liquor, obtain immunomagnetic beads, with coupling buffer washing immunomagnetic beads secondary, then the coupling buffer 500 μ l added containing mass percentage concentration 1% bovine serum albumin closes immunomagnetic beads 30min, obtain immunomagnetic beads solution, described activation buffer is pH is 6.0 NaH containing tween 20
2pO
4solution, described coupling buffer is pH is 7.4 NaH containing tween 20
2pO
4solution, in described activation buffer and described coupling buffer, NaH
2pO
4concentration is 0.01M, and the mass percentage concentration of tween 20 is 0.05%,
C, testing sample enrichment: get sewage draining exit water sample 2ml in test tube, add the above-mentioned immunomagnetic beads solution of 0.2ml, the light and slow rotational oscillation 30min of room temperature, shift out liquid in test tube, wash immunomagnetic beads 3 ~ 5 times with phosphoric acid buffer, add 0.5ml sterilized water after washing, test tube is placed in magnetic resolution 3min on Magnetic cell sorting frame, take out immunomagnetic beads, obtain the measuring samples solution of enterobacter cloacae enrichment;
Prepared by d, DNA profiling: measuring samples solution DNA extraction kit extracts DNA, obtains DNA profiling, and concrete extraction is carried out according to DNA extraction kit specification sheets;
E, PCR detect: the PCR reaction system of 30 μ l is: 10 × PCR damping fluid 2.5 μ l, the MgCl of concentration 25mM
2liquid 1.5 μ l, the dNTP liquid 1.5 μ l of concentration 10mM, the T aq DNA polysaccharase 0.2 μ l of concentration 5U/ μ l, concentration is all each 1.0 μ l of primer irp2-F, irp2-R, FhuA-F, FhuA-R, SodB-F, SodB-R, Slt-F, Slt-R of 10 μMs, above-mentioned DNA profiling 2 μ l, surplus is distilled water, PCR reaction conditions is 94 ° of C sex change 5 min, 94 ° of C sex change 45s, 55 ° of C annealing 45s, and 72 ° of C extend 1min, 35 circulations, and 72 ° of C extend 10 min, the PCR reaction product agarose gel electrophoresis testing goal gene fragment of mass percentage concentration 1.4%, if obtain 816bp, 1507bp, 502bp and 1216bp tetra-goal gene fragments, then contain enterobacter cloacae in sewage draining exit water body, the nucleotides sequence of described primer irp2-F is classified as: AAGGATTCGC TGTTACCGGA C, the nucleotides sequence of described primer irp2-R is classified as: AACTCCTGAT ACAGGTGGC, the nucleotides sequence of described primers F huA-F is classified as: CAGCAGAACA ACCGAAGCAA GA, the nucleotides sequence of described primers F huA-R is classified as: TTGAGGTCGT GGCGTAATCG T, the nucleotides sequence of described primer SodB-F is classified as: ACCACCAGGC GTATGTCACT AA, the nucleotides sequence of described primer SodB-R is classified as: TGCCGAAAGA TTTGGCGATT, the nucleotides sequence of described primer Slt-F is classified as: CGCAGCCGCT ACGCACAAAT, the nucleotides sequence of described primer Slt-R is classified as: TCGGTTACAT CGCTACCCAT CA.
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| CN106609303B (en) * | 2015-10-27 | 2020-02-28 | 天津市第三中心医院 | Primer and method for detecting enterobacter cloacae O20 type |
| CN106609305B (en) * | 2015-10-27 | 2020-02-28 | 天津市第三中心医院 | Primer and method for detecting enterobacter cloacae O21 type |
| CN106609304B (en) * | 2015-10-27 | 2020-02-28 | 天津市第三中心医院 | Primer and method for detecting enterobacter cloacae O12 type |
| CN107132350A (en) * | 2015-12-02 | 2017-09-05 | 南昌大学 | A kind of nano antibody affine in immunity sorbing material of specific bond aflatoxin |
| CN106284247B (en) * | 2016-08-29 | 2018-10-30 | 山东胜伟盐碱地科技有限公司 | A kind of irrigation canals and ditches monitoring salt concentration and water level in real time |
| CN107012216B (en) * | 2017-04-06 | 2020-09-15 | 南方医科大学南方医院 | LAMP primer group, kit and rapid detection method for detecting enterobacter cloacae |
| CN110295240B (en) * | 2019-07-11 | 2023-01-24 | 南开大学 | Specific primers for multiple serotypes of enterobacter cloacae and multiplex PCR detection method |
| CN114686600B (en) * | 2022-02-24 | 2023-12-12 | 宁波大学 | Primer group and method for meat detection based on seven-fold PCR technology |
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| CN102382888A (en) * | 2011-11-08 | 2012-03-21 | 宁波大学 | Immunomagnetic bead -FQ-PCR detection method for Shewanella putrefaciens |
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