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Dual fluorescent quantitative PCR (polymerase chain reaction) detection method and kit for two pathogenic bacteria in prepackaged drinking water

A technology for fluorescence quantification and detection reagents, which is applied in the field of fluorescence quantitative PCR detection, can solve the problems of long detection time and the detection limit cannot meet the limit requirements of pathogenic bacteria, and achieves low reagent cost, high practical value and good stability. Effect

Pending Publication Date: 2022-04-12
北京市食品安全监控和风险评估中心(北京市食品检验所)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The sampling volume does not meet the standard requirements for prepackaged drinking water (250mL); the detection limit cannot meet the limit requirements for pathogenic bacteria in the standard (pathogenic bacteria cannot be detected); the detection time is long (2-3 days)

Method used

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  • Dual fluorescent quantitative PCR (polymerase chain reaction) detection method and kit for two pathogenic bacteria in prepackaged drinking water
  • Dual fluorescent quantitative PCR (polymerase chain reaction) detection method and kit for two pathogenic bacteria in prepackaged drinking water
  • Dual fluorescent quantitative PCR (polymerase chain reaction) detection method and kit for two pathogenic bacteria in prepackaged drinking water

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1 detects the construction and verification of the double fluorescent PCR kit of two kinds of pathogenic bacteria (pseudomonas aeruginosa and faecal streptococcus) in drinking water

[0064] 1. Design of primers and probes

[0065] According to the Pseudomonas aeruginosa-specific gyrB gene coding region sequence published by GenBank, specific primers and probes for Pseudomonas aeruginosa were designed, and the sequences are as follows (SEQ ID NO:1-3):

[0066] Upstream primer PA-F: 5'-GGCGTGGGTGTGGAAGTC-3'

[0067] Downstream primer PA-R: 5'-AGAACCTGCTCTGCTTCACCA-3'

[0068] Fluorescent probe PA-P: 5'-FAM-TGCAGTGGAACGACA-MGB-3'

[0069] According to the sequence of the 23S rDNA gene coding region of Streptococcus faecalis retrieved from GenBank, specific primers and probes for Streptococcus faecalis were designed. The sequences are as follows (SEQ ID NO: 4-6):

[0070] Upstream primer EF-F: 5'-GAGGACCGAACCCACGTA-3'

[0071] Downstream primer EF-R: 5'-CAGT...

Embodiment 2

[0081] Embodiment 2 Specificity and Sensitivity Investigation

[0082] 1. Specificity test

[0083] Using the primer set and probe set of Example 1, Pseudomonas aeruginosa and Streptococcus faecalis DNA are used as positive controls, and Salmonella, Bacillus cereus, Listeria, Proteus, Micrococcus luteus, Staphylococcus aureus The DNA of cocci, Clostridium perfringens, Citrobacter, and Escherichia coli were used as negative controls, and were detected with PCR kits to evaluate the specificity of the established fluorescent PCR method.

[0084] Experimental results such as figure 1 As shown, except Pseudomonas aeruginosa and Streptococcus faecalis with amplification curves and Ct value <30, other animal-derived templates had no amplification. It can be seen that the primers and probes provided by the present invention have good specificity and no cross-reaction with other microbial species.

[0085] 2. Sensitivity test

[0086] ①Pick the colonies of Pseudomonas aeruginosa an...

Embodiment 3

[0090] Example 3 Evaluation of the DNA Precipitation Effect of the DNA Precipitation Agent

[0091] Taking Pseudomonas aeruginosa as an example, take 10 4 Dilute bacterial suspension 1.0mL, add to 250mL sterilized water. After filtration, the bacterial DNA on the filter membrane was extracted. Two filter membranes were used to extract DNA and detected according to the aforementioned method, and the other two filter membranes were used to extract DNA and detected according to the aforementioned method (without adding RNA carrier). The result is as figure 2 shown. The Ct value of the sample with RNA carrier filter membrane was 16.6, and the Ct value of the sample without RNA carrier filter membrane was 27.1. Therefore, the yield of cell DNA can be improved by adding RNA carrier.

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Abstract

The invention discloses a dual fluorescent quantitative PCR (Polymerase Chain Reaction) detection method and kit for two pathogenic bacteria in pre-packaged drinking water. The kit comprises primers and probes (SEQ ID NO: 1-6) for detecting the pseudomonas aeruginosa and the streptococcus faecalis. The kit is good in specificity, the detection method is rapid and simple, the accuracy is high, the sensitivity is good, and a new method is provided for detecting the two pathogenic bacteria of the pseudomonas aeruginosa and the streptococcus faecalis in the pre-packaged drinking water. The dual fluorescent quantitative PCR method provided by the invention can accurately and rapidly realize detection of pseudomonas aeruginosa and streptococcus faecalis in drinking water, can complete detection within one day, has the advantages of rapidness, specificity, sensitivity, high flux, good stability, reagent cost saving and the like, and is suitable for two pathogenic bacteria indexes of pseudomonas aeruginosa and streptococcus faecalis in drinking water standards.

Description

technical field [0001] The invention relates to a fluorescent quantitative PCR detection technology, in particular to a double fluorescent quantitative PCR detection method and a kit for two pathogenic bacteria (pseudomonas aeruginosa and fecal streptococcus) in prepackaged drinking water. Background technique [0002] Pseudomonas aeruginosa (Pseudomonas aeruginosa) is a common water-borne and food-borne pathogen, which can produce a variety of harmful substances such as extracellular enzymes, endotoxins, enterotoxins, and hemolysin after entering the body and proliferating in large quantities , causing pathological changes or even necrosis of the tissues and organs of infected human animals, and then causing acute gastroenteritis, sepsis, meningitis and other diseases, which seriously threaten life and health. In people's daily production and life, Pseudomonas aeruginosa exists in all types of water, and it has strong resistance to physical and chemical methods such as disi...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6851C12N15/11C12R1/46C12R1/385
Inventor 王立平王丹李赫婧薛晨玉姜洁刘艳琴吴燕涛王凯毅李树垚王青龙何瑞云何湘漪
Owner 北京市食品安全监控和风险评估中心(北京市食品检验所)
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