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Primer, detection method and detection reagent kit for detecting salmonella

A Salmonella, detection method technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc. strong effect

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 Salmonella using the loop-mediated isothermal amplification method

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Amplification of the invA gene of known bacterial strains

[0044] 1) Design of primer set

[0045]The 382---580bp nucleic acid sequence of the Salmonella specific gene inV is screened out by consulting the literature and using BLAST software analysis, and six sites (these six sites are respectively: 382-399bp; 471-490bp; 414- 431bp; 563-580bp; 493-512bp; 534-552bp.) 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.

[0046] serial number 1

[0047] Forward outer primer F3-inv GAACGTGTCGCGGAAGTC

[0048] serial number 2

[0049] Reverse outer primer B3-inv CGGCAATAGCGTCACCTT

[0050] serial number 3

[0051] Forward inner primer FIP-inv GCGCGGCATCCGCATCAATATCTGGATGGTATGCCCGG

[0052] serial number 4

[0053] Reverse inner primer BIP-inv GAACGGCGAAGCGTACTGGACATCGCACCGTCAAAGGAA

[0054] The seq...

Embodiment 2

[0091] 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:

[0092] The reaction system is: (the total reaction volume is 25ul)

[0093] Element

stock solution concentration

Quantity (ul)

Final concentration

nucleic acid template

FIP-inv

BIP-inv

F3-inv

B3-inv

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

[0094] In addition to the nucleic acid template, the above reaction system can be simplified to amplification reaction solution,...

Embodiment 3

[0100] 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:

[0101] The reaction system is: (the total reaction volume is 25ul)

[0102] Element

stock solution concentration

Quantity (ul)

Final concentration

nucleic acid template

FIP-inv

BIP-inv

F3-inv

B3-inv

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

[0103]In addition to the nucleic acid template, the above reaction system can be simplified to amplification reaction solution, enzyme and d...

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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 salmonella can augment the specific base sequence of a target gene which is the invA-GenBank (accession no. DQ644633) of the salmonella, and the primer is complementary to a part of or a complementary chain of the nucleic acid sequence on the 382-580bp loci on the target gene. The invention provides a primer unit having specificity to a specific gene fragment of the salmonella, and through detecting whether or not the detecting specimen in a reagent box of the primer unit contains the specific gene fragment of the salmonella, determines whether the salmonella 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 Salmonella; it also relates to a detection method and a detection kit for detecting Salmonella 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. Althoug...

Claims

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Application Information

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Patent Type & Authority Applications(China)
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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