Method of duplex fluorescence PCR - improved molecule beacon for detecting pathogenesis bacterium stemmed from eating source

A technology for improving molecular beacons and food-borne pathogenic bacteria, applied in biochemical equipment and methods, and microbial measurement/inspection, etc., can solve the problem of cumbersome operations and the inability to quickly detect multiple food-borne pathogens at the same time , time-consuming and other problems, to achieve the effect of simple operation, intuitive and clear result observation, and high sensitivity

Inactive Publication Date: 2009-05-06
SHENZHEN CENT FOR DISEASE CONTROL & PREVENTION +1
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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 the detection of food-borne pathogens by dual fluorescent PCR-improved molecular beacons is provided

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  • Method of duplex fluorescence PCR - improved molecule beacon for detecting pathogenesis bacterium stemmed from eating source
  • Method of duplex fluorescence PCR - improved molecule beacon for detecting pathogenesis bacterium stemmed from eating source
  • Method of duplex fluorescence PCR - improved molecule beacon for detecting pathogenesis bacterium stemmed from eating source

Examples

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Effect test

example 1

[0084] In this example, the detection of Vibrio cholerae and Vibrio parahaemolyticus by dual fluorescent PCR-modified molecular beacon is taken as an example. According to the sequence of Vibrio cholerae enterotoxin gene (ctxA) and Vibrio parahaemolyticus thermostable direct hemolysin gene (TDH) sequence published by GenBank and comparative analysis, a pair of primers and probes were designed respectively.

[0085] Primer 1 and probe 1 of ctxA gene

[0086] ctxA-F: 5′-TCCGGAGCATAGAGCTTGGA-3′,

[0087] ctxA-R: 5′-TCGATGATCTTGGAGCATTCC-3′

[0088] ctxA-Probe:

[0089] HEX-5′- CCGTGGATTCATCATGCACCGCC ACGG-3'Dabcyl,

[0090] Primer 2 and probe 2 of TDH gene

[0091] TDH-F: 5'-AAACATCTGCTTTTGAGCTTCCA-3',

[0092] TDH-L: 5′-CTCGAACAACAAACAATATCTCATCATCAG-3′,

[0093] TDH-Prcbe:

[0094] FAM5′-CCGGGG TGTCCCTTTTTCCTGCCCCCGG -Dabcyl,

[0095] Test strains: 104 strains of 12 kinds of bacteria. Including 1 strain of Salmonella (Salmonella), 1 strain of Shigella (Shigella), 5 ...

example 2

[0124] A pair of primers and probes were designed according to the sequences of Salmonella invasion gene (ivnA) and Shigella invasion plasmid antigen H gene (ipaH) published by GenBank and comparative analysis.

[0125] Primer 1 for invA gene:

[0126] invA-F: 5'-GCGGAATATC(I)ATGACGCAGCT-3'

[0127] invA-L: 5'-CGCTACGTTTTGCTTCACGG-3'

[0128] Probe 1 for the invA gene:

[0129] 5'-FAM-CCG CCATTTGTATTGGTTGTTACGGC GG-DABCYL-3'

[0130] Primer 2 for ipaH gene:

[0131] ipaH-F: 5'-TGAAGGAAATGCGTTTCTATG-3'

[0132] ipaH-L: 5'-AGGGAGAACCAGTCCGTAAA-3'

[0133] Probe 2 of ipaH gene:

[0134] ROX5'-CACG GCCGAAGCTATGGTCAGAAGCCGTG -3'-DABCYL

[0135] Reagent components: 10×PCR buffer, MgCl 2 , Taq enzymes, and dNTPs were purchased from Dalian Bao Biological Company, China.

[0136] 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 ...

example 3

[0159] A pair of primers and probes were designed according to the sequences of the thermostable nuclease gene (NUC) of Staphylococcus aureus and the hemolysin gene (hly) of Listeria monocytogenes published by GenBank and comparative analysis.

[0160] A pair of primers for NUC gene 1:

[0161] NUC-F: 5'-GGCAATACGCAAAGAGGTT-3'

[0162] NUC-L: 5'-CTTCTTCTATTTACGCCGTTATC-3'

[0163] Probe 1 for NUC gene:

[0164] ROX-5'-CGATGCA GTCTAAGTAGCTCAGCAAATGCATC G-3’-DABCYL

[0165] A pair of primers for the hly gene 2:

[0166] hly-F: 5'-TGCAAGTCCTAAGACGCCA-3'

[0167] hly-L: 5'—CACTGCATCTCCGTGGTATACTAA—3'

[0168] Probe 2 of hly gene:

[0169] FAM-5'-CGCG CTTGTATATACTTATCGATTTCATCCGCGCG -3'-DABCYL

[0170] Reagent components: 10×PCR buffer, MgCl 2 , Taq enzyme, and dNTP were purchased from Dalian Bao Biological Company.

[0171] Test strains: 85 strains of 13 kinds of bacteria. Including 1 strain of Salmonella (Salmonella), 1 strain of Shigella (Shigella), 1 strain of Vib...

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Abstract

This invention relates to a method of detection of food-borne pathogenic bacteria using double fluorescent PCR and modified molecular beacons. Corresponding pair of primers 1 and a modified molecular beacon probe 1 and another pair of primers 2 and another modified molecular beacon probe are designed according to the specific gene sequence of the two 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 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 by fluorescent PCR-improved 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 serologic...

Claims

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Application Information

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