CN103667493A - Preprocessing method for detecting salmonella live bacteria DNA detection in meat and meat products - Google Patents
Preprocessing method for detecting salmonella live bacteria DNA detection in meat and meat products Download PDFInfo
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Abstract
The invention discloses a preprocessing method for detecting salmonella live bacteria DNA in meat and meat products. The method comprises the following steps: firstly processing meat and meat products, adding PMA into a processed sample, controlling the final concentration of the PMA to be 15 micrograms/mL, sufficiently and uniformly mixing the mixture, and incubating for 3min in a dark place; illuminating for 3min at the position of 20cm below a 500w halogen tungsten lamp; extracting the genome DNA of the processed sample; and performing real-time fluorescence PCR quantitative detection on salmonella live bacteria in the sample by taking the extracted genome DNA as a template. The method can rapidly detect pathogenic live bacteria in meat and meat product matrix, so that the interference of food matrix turbidity and bacterial pollution background values is avoided, and the detection effectiveness is improved.
Description
Technical field
The present invention relates to the detection method of a kind of Salmonellas, particularly relate to the detection method of Salmonella viable bacteria in a kind of meat and meat product.
Background technology
Gene test is one of method for quick of pathogenic bacterium, but at present None-identified viable bacteria and dead bacterium, has reduced the validity of pathogenic microbes detect.Because only have the germ of " work " could retain the virulence of former bacterium and pathogenic, just food safety formed to potential threat.And lost former bacterium power and pathogenic through " extremely " germ of the various approach deactivations such as high temperature, ultraviolet ray, can't threaten food safety.Therefore detection of pathogens key is to distinguish the bacterium of " work " bacterium and " extremely ".It is effectively to detect key link and the technology of pathogenic bacterium with the detection method of dead bacterium that viable bacteria is distinguished in research.
Due to the disunity of " active bacterium " definition, the method for bacterial detection activity is also more, and the relative merits of its detection see the following form.
Summary of the invention
The present invention is after having compared the relative merits of various viable bacterias detections, particular problem for the examined food substrate impact of PMA-qPCR viable bacteria detection technique, the pre-treating process of Salmonellas viable bacteria DNA detection in a kind of meat and meat product is provided, to reach better viable bacteria, detects effect.
The DNA that present method is utilized dead bacterium in smelling of nitrine the third ingot (propidium monoazide, PMA) covalent attachment sample, by DNA extraction, removes the DNA of dead bacterium, reaches the object that viable bacteria detects.Object of the present invention is achieved through the following technical solutions:
1, a pre-treating process for Salmonellas viable bacteria DNA detection in meat and meat product, comprises the steps:
(1) sample preparation: after sample is pulverized, pat 5min with the physiological saline homogeneous of 10 times, 3000r/m is centrifugal, gets supernatant liquor 500 μ L standby in 1.5 mL centrifuge tubes.
(2) PMA processes: in above-mentioned 500 μ L supernatant liquors, add PMA working fluid, hatch dark place, is placed under halogen tungsten lamp and irradiates.In During Illumination, centrifuge tube is placed on trash ice (avoids temperature to rise).
(3) sample DNA extracts: PMA treatment solution is operated according to general DNA extraction method.
(4) take the sample genomic dna that step (3) extracts is template, carries out the Salmonella viable bacteria in real-time fluorescence PCR detection by quantitative sample.
2, the pre-treating process of Salmonellas viable bacteria DNA detection in a kind of meat as above and meat product, the final concentration that in step (2), PMA processes is 10-15 μ g/mL.
3, the pre-treating process of Salmonellas viable bacteria DNA detection in a kind of meat as above and meat product, the time that in step (2), hatch dark place is 3min, and halogen tungsten lamp radiation treatment method is: PMA treatment solution is placed in illumination 3min in 20cm place under 500 w halogen tungsten lamps.
4, the pre-treating process of Salmonellas viable bacteria DNA detection in a kind of meat as above and meat product, step (3) sample DNA extracting method comprises phenol/chloroform method or boiling method.
5, the pre-treating process of Salmonellas viable bacteria DNA detection in a kind of meat as above and meat product, quantitative fluorescent PCR the primer and probe sequence in step (4):
Upper primer Pf: as shown in sequence table Seq No.1,
Lower primer Pr: as shown in sequence table Seq No.2,
Probe Pb: as shown in sequence table Seq No.3.
Pathogenic bacterium viable bacteria detection method is more at present, the present invention is after having compared the relative merits of various viable bacterias detections, particular problem for the examined food substrate impact of PMA-qPCR viable bacteria detection technique, set up a kind of DNA detection pre-treating process based on Salmonellas viable bacteria in meat and meat product, to reach better viable bacteria, detected effect.The present invention is directed to actual food product sample substrate PMA is processed and has impact, studied the pre-treating process of meat and meat product food substrate (thering is certain turbidity and bacterial contamination background) Salmonellas viable bacteria DNA detection.Make PMA have more practicality in conjunction with quantitative PCR detection technique, avoided the interference of food substrate turbidity and bacterial contamination background value, improved pathogenic microbes detect speed and validity.
Accompanying drawing explanation
Fig. 1 is the detected result of 2 parts of DNA samples,
Wherein, 1 part is PMA processing sample, and 1 part is the control sample of processing without PMA.
Embodiment
For example the present invention is further explained below:
embodiment 1
A kind of method that detects Salmonella viable bacteria in simulation cold fresh pork sample based on quantitative PCR method
1, material and reagent
Experimental strain: Salmonellas (CMCC (B) 50115).
Food: cold fresh pork, is purchased from market.
Main agents:
DNA extraction liquid formula: 1M Tris-HCl 100 mL; 5M NaCl 20 mL; 0.5M EDTA 100 mL; 20 % SDS 100 Ml; Regulate pH to 8.0; DdH2O constant volume is to 4 L.
PMA: 1mgPMA is dissolved in to the DMSO solution of 200 μ L 20%, obtains the storing solution of 5 μ g/ μ L, keep in Dark Place in-20 ℃.During use, by 10 times of PMA storing solution dilutions, be PMA working fluid afterwards.
2, the preparation of microbial culture and hot inactivated bacteria
(1) preparation of microbial culture-viable bacteria
By the activation of Salmonellas bacterial strain, on HE nutrient agar, rule, in 37 ℃ of constant incubators, cultivate 24h.Picking list colony inoculation is in 50mL nutrient broth, and 37 ℃, 180r/m shaking table is cultured to logarithmic phase.
Draw the inoculum of 1mL in logarithmic phase in 1.5mL centrifuge tube, be placed in immediately cooled on ice process 50s in boiling water bath after.By this, process, cell walls and cytolemma are had damage in various degree and reach the impaired object of germ.
(2) preparation of hot inactivated bacteria
After cold fresh pork is pulverized, add the physiological saline homogeneous of 10 times to pat 5min, 3000r/m is centrifugal, gets supernatant liquor 400 μ L in 1.5 mL centrifuge tubes, sterilizing.By the different ratios of viable bacteria (μ L)/inactivated bacteria (μ L): 0/100,1/99,5/95,10/90,20/80,50/50,70/30,100/0 adds respectively above-mentioned viable bacteria and inactivated bacteria, form the different weight percentage that viable bacteria accounts for total count, this is analog sample, prepares 2 parts.
(3) PMA processes
In 1 part of 500 μ L analog sample of above-mentioned preparation, add PMA working fluid, making PMA final concentration is 15 μ g/mL, fully mixes rear dark place and hatches 3min.Centrifuge tube lid is opened, be placed in the rayed 3min of 20cm place under 500 w halogen tungsten lamps.In During Illumination, centrifuge tube is placed on trash ice (avoids temperature to rise), and this is PMA processing sample, and another part processed without PMA, is control sample.
(4) DNA extraction
In PMA treatment solution, add 750 μ L DNA extracting solutions, vibration mixes rear boiling water bath 5min; Add phenol/chloroform (V/V=1:1) 700 μ L, vibration mixes, the centrifugal 5min of 13000r/m; Draw supernatant to the new aseptic centrifuge tube of correspondence, add equal-volume chloroform, mix the centrifugal 5min of rear 13000r/m; Draw supernatant to the new aseptic centrifuge tube of correspondence, add the Virahol of 0.6 times of volume precooling, mix the centrifugal 5min of 13000r/m; Remove gently supernatant, be inverted on thieving paper, be stained with dry liquids; Add 700 μ L 75% ethanol, put upside down washing; The centrifugal 5min of 13000r/m; Remove gently supernatant, be inverted on thieving paper, be stained with dry liquids as far as possible; The centrifugal 10s of 4000r/m blots residual liquid drying at room temperature 1-5min as far as possible with micro sample adding appliance; Abandon supernatant, precipitation is dissolved in 20 μ L nucleic acid lysates.Gained DNA solution is standby below being kept at-18 ℃.
embodiment 2
Fluorescence quantitative PCR detection
1, the design of primer and probe
The sample genomic dna that extracts of take carries out fluorescent PCR, the object bacteria in qualitative detection sample as template.Design quantitative fluorescent PCR primer sequence used as follows:
Upstream primer Pf is: 5 '-TCGTCATTCCATTACCTAC-3 '
Downstream primer Pr is: 5 '-AAAC GTTGAAAAACTGAGGAC-3 '
Probe Pb is: 5 '-FAM – TCTGGTTG ATTTCCTGATCGCA-BHQ1.
2, fluorescence quantitative PCR detection
Utilize ABI 7500 fluorescent PCR instrument to detect 2 duplicate samples, by mixed solution at 95 ℃ of denaturation 2min; Then 95 ℃ of 10 s; 60 ℃ of 40 s (collection fluorescent signal), totally 40 circulations; Finally in 40 ℃ of insulation 10min.Detected result as shown in Figure 1.
Described mixed solution is composed as follows:
| 10×(NH4) 2SO 4 buffer | 2.5μL |
| 25 mM MgCl 2 | 3.5μL |
| Primer probe mixture | 1μL |
| 25 mM dNTPs | 1μL |
| Taq archaeal dna polymerase (5U) | 0.5μL |
| ddH2O | 14.5μL |
| DNA extraction liquid | 2μL |
Be illustrated in figure 1 the detected result of 2 duplicate samples: 1 part is PMA processing sample, 1 part is the control sample of processing without PMA.PMA sample is along with the raising of viable bacteria ratio, and Ct value reduces gradually, and what the rear detection of PMA processing was described is viable bacteria.When viable bacteria per-cent is less than 1%, the sample Ct value that PMA processes is 0, and control sample Ct value is constant, illustrates that PMA combines DNA in hot inactivated bacteria, has effectively stoped PCR to detect hot inactivated bacteria, reduces false positive results and occurs; When in sample, viable bacteria per-cent is 100%, PMA treatment group is identical with control group Ct, illustrates that PMA does not detect and disturbs viable bacteria PCR.
< 110 Entry-Exit Inspection and Quarantine Bureau's inspection and quarantine technique centers, > Heilungkiang
The pre-treating process of Salmonellas viable bacteria DNA detection in < 120 > meat and meat product
＜160＞3
＜210＞1
＜211＞19
＜212＞DNA
< 213 > primer Pf artificial sequences
＜400＞1
＜210＞2
＜211＞21
＜212＞DNA
< 213 > primer Pr artificial sequences
＜400＞2
＜210＞3
＜211＞22
＜212＞DNA
< 213 > probe Pb artificial sequences
＜400＞3
TCTGGTTGAT TTCCTGATCG CA 22
Claims (4)
1. a pre-treating process for Salmonellas viable bacteria DNA detection in meat and meat product, is characterized in that, comprises the steps:
(1) sample preparation: after sample is pulverized, pat 5min with the physiological saline homogeneous of 10 times, 3000r/m is centrifugal, gets supernatant liquor 500 μ L standby in 1.5mL centrifuge tube;
(2) PMA processes: in above-mentioned 500 μ L supernatant liquors, add PMA working fluid, hatch dark place, is placed under halogen tungsten lamp and irradiates;
(3) sample DNA extracts: PMA treatment solution is operated according to general DNA extraction method;
(4) take the sample genomic dna that step (3) extracts is template, carries out the Salmonella viable bacteria in real-time fluorescence PCR detection by quantitative sample.
2. the pre-treating process of Salmonellas viable bacteria DNA detection in a kind of meat according to claim 1 and meat product, is characterized in that: the final concentration that in step (2), PMA processes is 10-15 μ g/mL.
3. the pre-treating process of Salmonellas viable bacteria DNA detection in a kind of meat according to claim 1 and meat product, it is characterized in that: the time that in step (2), hatch dark place is 3min, and halogen tungsten lamp radiation treatment method is: PMA treatment solution is placed in illumination 3min in 20cm place under 500 w halogen tungsten lamps.
4. the pre-treating process of Salmonellas viable bacteria DNA detection in a kind of meat according to claim 1 and meat product, is characterized in that: step (3) sample DNA extracting method comprises phenol/chloroform method or boiling method.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104830835A (en) * | 2015-05-13 | 2015-08-12 | 黑龙江省乳品工业技术开发中心 | Method for inhibiting pollution of nucleic acid amplification product and application |
| CN105784798A (en) * | 2016-03-31 | 2016-07-20 | 山东五洲检测有限公司 | Detection method for salmonella content in meat |
| CN106399489A (en) * | 2016-08-31 | 2017-02-15 | 北京卓诚惠生生物科技股份有限公司 | Fluorescent PCR primer and probe group and kit for detecting living bacteria in clinical sample |
| CN109371112A (en) * | 2018-11-27 | 2019-02-22 | 中国计量大学 | A kind of livestock and poultry salmonella viable bacteria rapid detection method |
| CN109762807A (en) * | 2019-03-01 | 2019-05-17 | 华中农业大学 | The kit and application method that DNA of bacteria extracts in a kind of meat |
| CN113046451A (en) * | 2021-03-19 | 2021-06-29 | 厦门承葛医学检验实验室有限公司 | Live bacterium quantification method of flora and application thereof |
-
2013
- 2013-12-17 CN CN201310690144.XA patent/CN103667493B/en not_active Expired - Fee Related
Non-Patent Citations (2)
| Title |
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| 李聪聪: "PMA-qPCR 方法用于监测超声波灭菌效率的适用性及其应用", 《现代食品科技》 * |
| 王宏勋 等: "不同脂肪比例的冷鲜猪肉馅中菌相组成比较研究", 《中国酿造》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104830835A (en) * | 2015-05-13 | 2015-08-12 | 黑龙江省乳品工业技术开发中心 | Method for inhibiting pollution of nucleic acid amplification product and application |
| CN105784798A (en) * | 2016-03-31 | 2016-07-20 | 山东五洲检测有限公司 | Detection method for salmonella content in meat |
| CN106399489A (en) * | 2016-08-31 | 2017-02-15 | 北京卓诚惠生生物科技股份有限公司 | Fluorescent PCR primer and probe group and kit for detecting living bacteria in clinical sample |
| CN109371112A (en) * | 2018-11-27 | 2019-02-22 | 中国计量大学 | A kind of livestock and poultry salmonella viable bacteria rapid detection method |
| CN109762807A (en) * | 2019-03-01 | 2019-05-17 | 华中农业大学 | The kit and application method that DNA of bacteria extracts in a kind of meat |
| CN113046451A (en) * | 2021-03-19 | 2021-06-29 | 厦门承葛医学检验实验室有限公司 | Live bacterium quantification method of flora and application thereof |
| CN113046451B (en) * | 2021-03-19 | 2022-08-23 | 厦门承葛医学检验实验室有限公司 | Live bacterium quantification method of flora and application thereof |
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