Method for detecting living streptococcus agalactiae in milk by SDS-PMA-qPCR (sodium dodecyl sulfate-propidium monoazide-quantitative polymerase chain reaction) method

A technology of streptococcus lactis and live bacteria, which is applied in the field of detection of pathogenic bacteria in milk samples, can solve the problems of false positive and false negative results, achieve high specificity, improve sensitivity, and promote the effect of penetration

Inactive Publication Date: 2019-02-12
新疆农业科学院农业质量标准与检测技术研究所
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Problems solved by technology

[0004] The present invention provides a method for detecting viable Streptococcus agalactiae in milk by SDS-PMA-qPCR, which overcomes the deficiencies of the prior art and can effectively solve the problem of qPCR for detecting Streptococcus agalactiae in milk in the prior art The detection method and the PMA-qPCR detection method are likely to cause false positive or false negative results

Method used

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  • Method for detecting living streptococcus agalactiae in milk by SDS-PMA-qPCR (sodium dodecyl sulfate-propidium monoazide-quantitative polymerase chain reaction) method
  • Method for detecting living streptococcus agalactiae in milk by SDS-PMA-qPCR (sodium dodecyl sulfate-propidium monoazide-quantitative polymerase chain reaction) method
  • Method for detecting living streptococcus agalactiae in milk by SDS-PMA-qPCR (sodium dodecyl sulfate-propidium monoazide-quantitative polymerase chain reaction) method

Examples

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Effect test

Embodiment 1

[0022] Example 1, the SDS-PMA-qPCR method for detecting viable Streptococcus agalactiae in milk was carried out according to the following steps: the first step, processing the milk sample to be tested: shake the milk sample to be tested, and take 2 mL of the milk sample in EP tube, then add SDS stock solution 10 to it 4 μg / mL 4 μL [the concentration of the SDS stock solution should be added to it is 20 μg / mL, but because the volume of SDS with a final concentration of 20 μg / mL added to the 2 mL milk sample is too small (only 0.008 μL), in practice, 0.008 μL It is very difficult to measure, it is easy to cause errors, and it is easy to cause distortion of the final test results. Therefore, using this concentration of SDS mother solution is not only easy to measure, but also the actual final addition amount is consistent with the addition amount of 20μg / mL SDS], adding the final concentration Mixture A was obtained for 2 μL of 10 mg / mL PMA, and then the mixture A was incubated ...

Embodiment 2

[0027] Example 2, as an optimization of the previous example, CTAB included 100 nM Tris-HCL pH 8, 1.4 M NaCl and 20 nM EDTA.

Embodiment 3

[0028] Embodiment 3, as an optimization of the above embodiment, the DNA extraction solution is prepared from phenol, chloroform and isopropanol, wherein the volume ratio of phenol:chloroform:isopropanol=25:24:1.

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Abstract

The invention relates to the technical field of technology for detecting pathogenic bacteria in a milk sample, and in particular to a method for detecting living streptococcus agalactiae in milk by anSDS-PMA-qPCR (sodium dodecyl sulfate-propidium monoazide-quantitative polymerase chain reaction) method. The method is carried out according to the following steps: (Step 1) SDS-PMA treatment of a to-be-detected milk sample; (Step 2) extraction of DNA; (Step 3) qPCR amplification reaction; (Step 4) determination of result. Before DNA is extracted, the method disclosed by the invention can be used for extracting DNA better, and the sensitivity of the detection method is increased; before PMA is used for treating bacteria, SDS can be used for enhancing the permeability of PMA into the bacteria, moreover, an appropriate SDS concentration can effectively promote the permeation effect of PMA into dead bacteria, and the combination of both can accurately differentiate living and dead streptococcus agalactiae cells. To sum up, dead streptococcus agalactiae in the to-be-detected milk sample cannot interfere with the method disclosed by the invention, the method can accurately detect out thespecific content of streptococcus agalactiae in quantified milk, moreover, the specificity is high, and a false positive result can be prevented.

Description

technical field [0001] The invention relates to the technical field of detection technology of pathogenic bacteria in milk samples, and relates to a method for detecting the viable bacteria of Streptococcus agalactiae in milk by SDS-PMA-qPCR method. Background technique [0002] Streptococcus agalactiae is a gram-positive, catalase-negative pathogen that usually causes recessive mastitis in dairy cows, and is the most infectious and fastest-spreading Streptococcus spp. Pathogenic bacteria, also an important infectious pathogen for humans and animals, may cause food poisoning. The presence of Streptococcus agalactiae in raw milk is also a potential source of pathogens in the dairy food chain, followed by food contamination. risk. Conventional biological methods are the gold standard for the detection of Streptococcus agalactiae, but it is labor-intensive, time-consuming and has low specificity. Therefore, the establishment of a rapid, accurate and sensitive method for the de...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/10C12R1/46
CPCC12N15/1003C12Q1/686C12Q1/689C12Q2561/113C12Q2563/107C12Q2545/114
Inventor 赵艳坤陈贺刘慧敏蔡建星李进王成
Owner 新疆农业科学院农业质量标准与检测技术研究所
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