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Primer, detection method and detection reagent kit for detecting cholera vibrio

A Vibrio cholerae detection method technology, applied in biochemical equipment and methods, microbial measurement/inspection, DNA/RNA fragments, etc., can solve the problems of detection methods and detection kits for Vibrio cholerae, etc. Achieve the effect of wide application range, strong specificity and high sensitivity

Inactive Publication Date: 2008-04-02
ZHUHAI DISEASE PREVENTION & CONTROL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are currently no reports on the detection method and detection kit for Vibrio cholerae using the loop-mediated isothermal amplification method, and the LAMP primer set specific to the specific gene fragment of Vibrio cholerae ompW

Method used

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  • Primer, detection method and detection reagent kit for detecting cholera vibrio

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Amplification of the ompW gene of known bacterial strains

[0055] 1) Design of primer set

[0056] Go out the 14---220bp nucleotide sequence of vibrio cholerae specificity gene ompW by consulting literature and analyze and screen with BLAST software, aim at six sites of this fragment (these six sites are respectively:

[0057] 14-34bp, 35-55bp, 88-112bp, 118-142bp, 179-198bp, 199-220bp) LAMP primers were designed and synthesized to obtain the following primers; primers were designed through LAMP special primer design software combined with molecular biology analysis software Advance Vector NTI completed.

[0058] serial number 1

[0059] Forward outer primer F3-omp CGGGTACTAATAATGCATTGA

[0060] serial number 2

[0061] Reverse outer primer B3-omp CGATCATAAATACCCAAGGATT

[0062] serial number 3

[0063] Forward internal primer FIP-omp

[0064] GGAACCAGCTATCATTGAGCATATAGTGATTTAAAACTGGACGACT

[0065] serial number 4

[0066] reverse inner primer BIP-om...

Embodiment 2

[0108] The difference between this embodiment and Embodiment 1 is that in this embodiment, in the gene amplification stage based on the LAMP method, in order to speed up the amplification reaction speed, the amplification reaction solution also includes:

[0109] serial number 5

[0110] Forward loop primer LF-omp GTTAGCAGCAAGTCCCCATG

[0111] serial number 6

[0112]Reverse loop primer LB-omp TACAAAGCAGGTGCAGATGC

[0113] at this time:

[0114] The forward loop primer LF-omp: amplifies the complementary sequence starting from 56-75bp (catggggacttgctgctaac);

[0115] The reverse loop primer LB-omp: amplification starts at 157-176bp (tacaaagcaggtgcagatgc)

[0116] Now the reaction system is: (total reaction volume is 25ul)

[0117] Element

[0118] After adding the above-mentioned forward loop primer LF-omp and reverse loop primer LB-omp, it is only necessary to keep the temperature in a constant temperature water bath at 65°C for about 40 minutes, and then inac...

Embodiment 3

[0120] The difference between this example and Example 2 is that in this example, the reaction system used for gene amplification based on the LAMP method is: (the total reaction volume is 25ul)

[0121] Element

stock solution concentration

Quantity (ul)

Final concentration

nucleic acid template

FIP-omp

BIP-omp

F3-omp

B3-omp

LF-omp

LB-omp

betaine

dNTP

MgSO 4

Bst DNA Polymerase Buffer

25uM

25uM

7.5uM

7.5uM

20uM

20uM

4M

10mM

100mM

10×

1.0

1.0

1.0

0.5

0.5

0.75

0.75

5.0

2.5

0.5

2.5

1.0uM

1.0uM

0.15uM

0.15uM

0.6uM

0.6uM

0.8M

1.0mM

2.0mM

[0122] Bst DNA Polymerase

wxya 2 o

8U / ul

0.5

8.5

0.16U / ul

[0123] In addition to the nucleic acid template, the abov...

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PUM

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Abstract

The invention relates a technique for fast detecting food-borne pathogens based on a loop-mediated isothermal amplification, LAMP technique. A primer for diction of comma bacillus can augment the specific base sequence of a target gene which is the ompW-GenBank (accession no. AF001009) of the comma bacillus, and the primer is complementary to a part of or a complementary chain fragment of the nucleic acid sequence on the 14-220 loci on the target gene. The invention provides a primer having specificity to a specific gene fragment of the comma bacillus, and through detecting whether or not the detecting specimen in a reagent box of the primer unit contains the specific gene fragment of the comma bacillus, determines whether the comma bacillus exists in the specimen or not.

Description

technical field [0001] The invention relates to a rapid detection technology for food-borne pathogens based on a loop-mediated isothermal amplification (LAMP) technology. It particularly relates to a primer and a primer set specific to a specific gene fragment of Vibrio cholerae; it also relates to a detection method and a detection kit for detecting Vibrio cholerae by using the primer and the primer set with a loop-mediated isothermal amplification method. Background technique [0002] High incidence of foodborne illness, caused by Salmonella, Shigella, Staphylococcus aureus, Proteus, Vibrio cholerae, Vibrio parahaemolyticus and E.coli O157:H7, rotavirus and norovirus Food poisoning, whose incidence rate accounts for a very high proportion in the incidence of foodborne diseases in my country, is a serious public health problem. [0003] At present, the detection of foodborne pathogens mainly relies on pathogen isolation methods, immunological methods and various PCR methods...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCY02A50/30
Inventor 魏泉德张彩虹谭爱军张丽荣
Owner ZHUHAI DISEASE PREVENTION & CONTROL CENT
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