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Primer, detection method and detection reagent kit for detecting shigella

A Shigella, detection method technology, applied in biochemical equipment and methods, microbe determination/inspection, DNA/RNA fragments, etc., can solve the problem of detection methods and detection kits that have not detected Shigella, etc. problems, to 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 is currently no detection method and detection kit for Shigella using the loop-mediated isothermal amplification method

Method used

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  • Primer, detection method and detection reagent kit for detecting shigella

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Amplification of the ipaH gene of known strains

[0054] A) Design of primer set

[0055] The 1095-1299bp nucleic acid sequence of Shigella-specific gene ipa H was selected by consulting literature and analyzing with BLAST software, targeting the six sites of the fragment (1095-1112bp, 124-1141bp, 1173-1194bp, 1207). -1227bp, 1261-1279bp, 1280-1299bp) designed and synthesized LAMP primers to obtain the following primers; the primer design was completed by LAMP special primer design software combined with molecular biology analysis software Advance Vector NTI.

[0056] Serial number 1

[0057] Forward primer F3-ipa ATACCGTCTCTGCACGCA

[0058] Serial number 2

[0059] Reverse outer primer B3-ipa TCGAAAAGGCCTTCTGATGC

[0060] Serial number 3

[0061] Forward inner primer FIP-ipa GCGAAAGACTGCTGTCGAAGCTTTCCGTGAACAGGTCGCT

[0062] Serial number 4

[0063] Reverse inner primer BIP-ipa TGCCACTGAGAGCTGTGAGGACTGATGGACCAGGAGGGTT

[0064] The sequences of the 6 sites are:

[...

Embodiment 2

[0103] The difference between this embodiment and the first embodiment is that in this embodiment, in the gene amplification stage based on the LAMP method, in order to accelerate the amplification reaction speed, the amplification reaction solution further includes:

[0104] Serial number 5

[0105] Forward loop primer LF-ipa GGCACTGAGTTTTTCCAGCCAT

[0106] Serial number 6

[0107] Reverse loop primer LB-ipa CGCTCACATGGAACAATCTCCGG

[0108] at this time:

[0109] The forward loop primer LF-ipa: amplify the complementary sequence starting from 1144-1165bp (atggctggaaaaactcagtgcc);

[0110]The reverse loop primer LB-ipa: Amplification starts with a sequence of 1236-1258 bp (cgctcacatggaacaatctccgg).

[0111] At this time, the reaction system is: (the total volume of the reaction is 25ul)

[0112] Ingredients

[0113] After adding the forward loop primer LF-ipa and the reverse loop primer LB-ipa, it only needs to be kept in a constant temperature water bath at a temperatur...

Embodiment 3

[0115] The difference between this embodiment and the first embodiment is that, in this embodiment, the reaction system used for gene amplification based on the LAMP method is:

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

[0117] Ingredients

Concentration of storage solution

Quantity (ul)

Final concentration

Nucleic acid template

FIP-ipa

BIP-ipa

F3-ipa

B3-ipa

LF-ipa

LB-ipa

betaine

25uM

25uM

7.5uM

7.5uM

20uM

20uM

4M

1.0

1.0

1.0

0.5

0.5

0.75

0.75

5.0

1.0uM

1.0uM

0.15uM

0.15uM

0.6uM

0.6uM

0.8M

[0118] dNTP

mgsO 4

Bst DNA Polymerase Buffer

Bst DNA Polymerase

ddH 2 O

10mM

100mM

10×

8U / ul

2.5

0.5

2.5

0.5

8.5

1.0mM

2.0mM

0.16U / ul

[0119] Except for the nucleic acid template, the above reaction system can be simplified as an amplification reaction...

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PUM

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Abstract

The invention relates to a technique for fast detecting food-borne pathogens based on a loop-mediated isothermal amplification, LAMP technique. A primer for detection of shigella can augment the specific base sequence of a target gene which is the ipaH-GenBank (accession no. M32063) of the shigella, and the primer is complementary to a part of or a complementary chain of the nucleic acid sequence on the 1095-1299bp loci on the target gene. The invention provides a primer unit having specificity to a specific gene fragment of the shigella, and through detecting whether or not the detecting specimen in a reagent box of the primer unit contains the specific gene fragment of the shigella, determines whether the shigella exists in the specimen or not.

Description

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

Claims

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Application Information

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