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Primer, detection method and detection reagent kit for detecting bacillus coli O157:H7

A technology for O157 and Escherichia coli, which is applied in biochemical equipment and methods, microbe determination/testing, DNA/RNA fragments, etc., to achieve strong specificity, wide application range 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 E. coli O157:H7 using the loop-mediated isothermal amplification method, and there is no report on the LAMP primer set specific to the specific rfbE gene fragment of E. coli O157:H7

Method used

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  • Primer, detection method and detection reagent kit for detecting bacillus coli O157:H7

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Amplification of the rfb gene of known bacterial strains

[0046] 1) Design of primer set

[0047] The 7462---7671bp nucleic acid sequence of Escherichia coli O157:H7 specific gene rfbE is screened out by consulting the literature and using BLAST software analysis, and is aimed at six sites of this fragment (these six sites are respectively: 7462-7479bp, 7480- 7498bp, 7520-7544bp, 7550-7572bp, 7613-7632bp, 7652-7671bp) LAMP primers were designed and synthesized to obtain the following primers; primer design was completed by LAMP special primer design software combined with molecular biology analysis software Advance Vector NTI.

[0048] serial number 1

[0049] Forward outer primer F3-rfb GTACATTGGCATCGTGTG

[0050] serial number 2

[0051] Reverse outer primer B3-rfb CGAAAACGTGAAATTGCTGA

[0052] serial number 3

[0053] Forward inner primer FIP-rfb

[0054] GAGAGGAATTAAGGAATCACCTTGCGACAGGGTAAAAAACTGGC

[0055] serial number 4

[0056] reverse internal ...

Embodiment 2

[0096] The difference between this example and Example 1 is that in this example, the reaction system used for gene amplification based on the LAMP method is:

[0097] The reaction system is: (the total reaction volume is 25ul)

[0098] Element

stock solution concentration

Quantity (ul)

Final concentration

nucleic acid template

FIP-rfb

BIP-rfb

F3-rfb

B3-rfb

betaine

dNTP

MgSO 4

Bst DNA Polymerase Buffer

Bst DNA Polymerase

wxya 2 o

25uM

25uM

7.5uM

7.5uM

4M

10mM

100mM

10×

8U / ul

1.0

1.0

1.0

0.5

0.5

5.0

2.5

0.5

2.5

0.5

10.0

1.0uM

1.0uM

0.15uM

0.15uM

0.8M

1.0mM

2.0mM

0.16U / ul

[0099] In addition to the nucleic acid template, the above reaction system can be simplified to amplification reaction solution,...

Embodiment 3

[0106] The difference between this example and Example 1 is that in this example, the reaction system used for gene amplification based on the LAMP method is:

[0107] The reaction system is: (the total reaction volume is 25ul)

[0108] Element

stock solution concentration

Quantity (ul)

Final concentration

nucleic acid template

FIP-rfb

BIP-rfb

F3-rfb

B3-rfb

betaine

dNTP

MgSO 4

Bst DNA Polymerase Buffer

Bst DNA Polymerase

wxya 2 o

50uM

50uM

15uM

15uM

7.5M

10mM

150mM

10×

16U / ul

1.0

1.0

1.0

0.5

0.5

5.0

4.0

1.0

2.5

1.0

7.5

2.0uM

2.0uM

0.3uM

0.3uM

1.5M

1.6mM

6.0mM

0.64U / ul

[0109] In addition to the nucleic acid template, the above reaction system can be simplified to amplification reaction solution, e...

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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 colon bacillus O157:H7can augment the specific base sequence of a target gene which is the rfbE-GenBank (accession no. AE005429) of the colon bacillus O157:H7, and the primer is complementary to a part of or a complementary chain fragment of the nucleic acid sequence on the 7462-7471 loci on the target gene. The invention provides a primer having specificity to a specific gene fragment of the colon bacillus O157:H7, and through detecting whether or not the detecting specimen in a reagent box of the primer unit contains the specific gene fragment of the colon bacillus O157:H7, determines whether the colon bacillus O157:H7 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. In particular, it relates to a primer and a primer set specific to a specific gene fragment of Escherichia coli O157:H7; it also relates to a detection method and a detection kit for detecting Escherichia coli O157:H7 by a loop-mediated isothermal amplification method using the primers and the primer set . Background technique [0002] High incidence of foodborne illness, caused by Salmonella, Shigella, Staphylococcus aureus, Proteus, Vibrio cholerae, Vibrio parahaemolyticus and Escherichia 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 met...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/10C12N15/11
Inventor 魏泉德张彩虹谭爱军张丽荣
Owner ZHUHAI DISEASE PREVENTION & CONTROL CENT
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