Quadruple fluorescent PCR primer set, probe set, kit and method for detecting four pathogenic bacteria in drinking water

A technology for pathogenic bacteria and drinking water, which is applied in the field of quadruple fluorescent PCR primers for detecting four pathogenic bacteria in drinking water, can solve the problems of complicated operation steps, large differences, and various reagents, and achieve environmental friendliness, The effect of high efficiency and low detection limit

Active Publication Date: 2020-05-26
贵州省产品质量检验检测院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These patent applications differ greatly from the drinking water standard requirements in terms of test samples and test objects, and there are problems such as a wide variety of reagents and complicated operation steps.

Method used

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  • Quadruple fluorescent PCR primer set, probe set, kit and method for detecting four pathogenic bacteria in drinking water
  • Quadruple fluorescent PCR primer set, probe set, kit and method for detecting four pathogenic bacteria in drinking water
  • Quadruple fluorescent PCR primer set, probe set, kit and method for detecting four pathogenic bacteria in drinking water

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Construction and verification of a quadruple fluorescent PCR kit for detecting four pathogenic bacteria in drinking water

[0048] ① Primer set and probe set: As shown in Table 1 and Table 2, synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd., dilute the dry powder of primers and probes to 100 μmol / L as a stock solution, and prepare 10 μmol according to Table 3 / L as the working fluid.

[0049] Table 3 Preparation of 100μmol / L stock solution into 10μmol / L use solution

[0050]

[0051] ②Fluorescent PCR reagents: common commercially available fluorescent PCR reagents, selected in this example Path-ID TM qPCR Master Mix.

[0052] ③ Positive control: Take all the primer stock solution and dilute to 10 μmol / L respectively. Inoculate Coliformbacteria, E.faecalis, P.aeruginosa, C.perfringens to BGLB liquid medium, KF streptococcus liquid medium, CN liquid medium, SPS liquid medium, etc. to enrich bacteria respectively, and extract D...

Embodiment 2

[0067] Example 2 Provincial environmental monitoring risk samples - detection of surface water samples

[0068] A surface water sample was donated by the Guizhou Academy of Environmental Sciences. The naked eye can see that it is relatively turbid with a little precipitation. Take 1L samples and use an outsourced DNA extraction kit (Mobio DNA Isolation Kit and its 0.22μm filter membrane) to extract DNA, the DNA purity is OD 260 / OD 280 =1.75, the concentration is 5.13ng / μL, and stored at -20°C. Afterwards, the DNA was concentrated to 50 ng / μL with a column film concentration tube (Millipore Microcon DNA fast flow (PCR grade)) for detection, and the primer set, probe set, positive control, negative control, blank control, etc. Kit components, fluorescent reagent selection ABITaqMan TM Environmental Master Mix 2.0, on-machine detection (ABI fluorescent PCR instrument, model 7500Fast).

[0069] The test results are shown in Table 8: ①Quality control standard: FAM, VIC, NE...

Embodiment 3

[0072] Example 3 Provincial Food Supervision Sampling Samples——Detection of Bottled Drinking Water and Bottled Drinking Natural Mineral Water Samples

[0073] Take a bottled drinking water sample (set as #1 sample) and a bottled drinking natural mineral water sample (set as #2 sample) in the provincial food supervision and sampling, take 1L of each sample to pass through a 0.22μm filter membrane, filter the membrane The slices were cut into 4 parts, and inoculated into BGLB liquid medium, KF streptococcus liquid medium, CN liquid medium, and SPS liquid medium respectively, and the bacteria were enriched according to the temperature and time required by the medium instructions. Take 250 μL of bacterial liquid and mix it into 1 mL sample, and use the purchased DNA extraction kit (QiagenMagAttract Hmw DNA kit) to extract sample DNA on a small magnetic stand (Qiagen MagAttract Magnetic Rack). The DNA purity of #1 sample and #2 sample are both OD 260 / OD 280 =1.79, the concentrati...

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Abstract

The invention discloses a quadruple fluorescent PCR primer set for detecting four pathogenic bacteria in drinking water. The quadruple fluorescent PCR primer set comprises primers for coli groups, streptococcus faecalis, pseudomonas aeruginosa and clostridium perfringens, and is specifically shown as Seq. ID No. 1 to Seq. ID No. 8. The invention belongs to the technical field of gene engineering detection, mutual interference cannot be caused for each primer and each probe, amplification can be carried out on specific objective sequences only, a fluorescence signal is stimulated, non-objectivesequences are free of amplification or fluorescence signal, accurate detection for the four pathogenic bacteria in drinking water can be realized, and the quadruple fluorescent PCR primer set has theadvantages of high specificity, small standard error, short detection time, reagent cost saving and the like and is applicable for detecting the indexes of the pathogenic bacteria in the standard ofdrinking water.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering detection, and in particular relates to a quadruple fluorescent PCR primer set, a probe set, a kit and a method for detecting four pathogenic bacteria in drinking water. Background technique [0002] Food is the most important thing for the people, food safety is the basic pillar of social harmony, and the quality and safety of drinking water is the top priority. In recent years, with the in-depth study of disease control and prevention, national standards, industry standards, and detailed rules for supervision and sampling at all levels have clarified the detection indicators of pathogenic bacteria in drinking water. Currently, for ordinary drinking water, such indicators mainly include Coliformbacteria, Pseudomonas aeruginosa, and for drinking mineral water, such indicators mainly include Coliform bacteria, Enterococcus faecalis, Pseudomonas aeruginosa P. aeruginosa and Clostridium ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/10C12Q1/04C12N15/11
CPCC12Q1/686C12Q1/689C12Q2600/16C12Q2600/166C12Q2537/143C12Q2563/107C12Q2545/113
Inventor 孙端方罗绍楠左泽彦董睿李春宇田志强张谦黄家瑞
Owner 贵州省产品质量检验检测院
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