Method of multiplex fluorescence PCR ¿C improved molecule beacon for detecting pathogenesis bacterium stemmed from eating source

A technology for improving molecular beacons and food-borne pathogens, which is applied in biochemical equipment and methods, microbial measurement/inspection, etc. Pathogenic bacteria and other problems, to achieve the effect of simple operation, high sensitivity, and intuitive and clear result observation

Active Publication Date: 2006-07-05
SHENZHEN CENT FOR DISEASE CONTROL & PREVENTION +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to overcome the shortcomings of the existing technology such as cumbersome operation and long time consumption, as well as the inability to quickly detect multiple food-borne pathogens at the same time, a method for detecting food-borne pathogens by multiple fluorescent PCR-improved molecular beacons is provided

Method used

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  • Method of multiplex fluorescence PCR ¿C improved molecule beacon for detecting pathogenesis bacterium stemmed from eating source
  • Method of multiplex fluorescence PCR ¿C improved molecule beacon for detecting pathogenesis bacterium stemmed from eating source
  • Method of multiplex fluorescence PCR ¿C improved molecule beacon for detecting pathogenesis bacterium stemmed from eating source

Examples

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Effect test

example 1

[0125] In this example, the detection of Salmonella and Shigella by dual fluorescent PCR-improved molecular beacons is taken as an example to analyze the method for the detection of foodborne pathogens by multiplex fluorescent PCR-improved molecular beacons. According to the sequences of Salmonella invasion gene (ivnA) and Shigella invasion plasmid antigen H gene (ipaH) published by GenBank and comparative analysis, a pair of primers and probes were designed respectively as described above.

[0126] Test strains: 132 strains of 14 kinds of bacteria. Including 1 strain of enterotoxigenic E. coli (ETEC), 1 strain of invasive E. coli (EIEC), 1 strain of O157: H7 E. coli (O157: H7), 1 strain of pathogenic E. coli Enteropathogenic E.coli (EPEC), 1 strain of Citrobacter, 1 strain of Escherichia coli, 1 strain of Vibrio parahaemolyticus (V.parahaemolyticus), 1 strain of cholera (V.cholera ) Vibrio, 1 strain of Staphylococcus aureus (S.aureus), 1 strain of Listeria monocytogenes (Lis...

example 2

[0152] In this example, the detection of Shigella, O157:H7 Escherichia coli and Listeria monocytogenes by triple fluorescent PCR-improved molecular beacon is taken as an example. According to GenBank, the sequence of the coding invasion plasmid antigen H gene (ipaH) of Shigella, the sequence of the hemolysin gene rfbE of Escherichia coli O157:H7, and the sequence of the hemolysin gene (hly) of Listeria monocytogenes were published, respectively The designed pair of primers and probes and their amplified fragments are the same as those described above.

[0153] Test strains: O157: H7 Escherichia coli (O157: H7), Listeria monocytogenes, Shigella (various serotypes). All strains were isolated and identified in accordance with the National Inspection Standard of "Food Microbiology" promulgated by the People's Republic of China.

[0154] The method for simultaneously detecting Shigella, O157:H7 Escherichia coli, and Listeria monocytogenes by utilizing the above-mentioned fluoresce...

example 3

[0174] This example illustrates a method for the simultaneous detection of Shigella, Vibrio cholerae, Staphylococcus aureus, and Escherichia coli O157:H7 by quadruple fluorescent PCR-modified molecular beacons. According to GenBank, the sequence of the coding invasion plasmid antigen H gene (ipaH) of Shigella, the sequence of the enterotoxin gene ctxA of Vibrio cholerae, the sequence of the thermostable nuclease gene NUC of Staphylococcus aureus, and the sequence of Escherichia coli O157:H7 were released according to GenBank. The rfbE sequence of the hemolysin gene, a pair of primers and probes and amplified fragments designed respectively are the same as those described above.

[0175] Test strains: Shigella, Vibrio cholerae, Staphylococcus aureus, Escherichia coli O157:H7. All strains were isolated and identified in accordance with the National Inspection Standard of "Food Microbiology" promulgated by the People's Republic of China.

[0176] The method for simultaneously de...

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Abstract

This invention relates to a method of detection of food-borne pathogenic bacteria using multiple fluorescent PCR and modified molecular beacons. Several pairs of primers and modified molecular beacon probes are designed according to the specific gene sequence of the pathogenic bacteria to be tested, which is amplified using fluorescent PCR and modified molecular beacon. The amplified product is crossbred with molecular beacon, and the fluorescent intensity of the reaction system is tested with the necessary circle times for intended threshold as a judgment for results, that is, negative for Ct of 0 or 40 and positive for Ct less than 38. This invention, which is advanced in high sensibility, significant specificity, simple operation and intuitionistic observation, is applicable in simultaneous detection of any two kinds, three kinds and four kinds of randomly combined bacteria and food-borne pathogenic bacteria as well as specimen in large amounts.

Description

technical field [0001] The invention relates to a method for detecting food-borne pathogenic bacteria with a fluorescent PCR molecular beacon. Background technique [0002] There is a high incidence of foodborne diseases such as Vibrio cholerae causing cholera, Salmonella typhi and Shigella causing typhoid and dysentery respectively. Cholera, typhoid, and dysentery are key infectious diseases in my country. The incidence of food poisoning caused by Vibrio parahaemolyticus, Salmonella, Staphylococcus aureus, Bacillus cereus, Proteus, etc. accounts for a relatively high proportion of the incidence of foodborne diseases in my country. At present, these food-borne pathogenic bacteria are still detected by the traditional methods of bacterial isolation, culture and identification. This traditional detection method is cumbersome to operate, time-consuming and labor-intensive. It usually takes 5 days, and the longest time is one month. Moreover, the biochemical and serological te...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
Inventor 扈庆华李庆阁郑琳琳石晓路郑薇薇王冰庄志雄刘小立张顺祥
Owner SHENZHEN CENT FOR DISEASE CONTROL & PREVENTION
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