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

A technology for Vibrio hemolyticus and detection method, which is applied in the directions of biochemical equipment and methods, determination/inspection of microorganisms, DNA/RNA fragments, etc., and can solve the problem of no detection kit for Vibrio parahaemolyticus, etc., Achieve a wide range of applications, high sensitivity and specificity

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 detection kits using the loop-mediated isothermal amplification method to detect Vibrio parahaemolyticus, as well as reports on primers and primer sets specific to specific gene fragments of Vibrio parahaemolyticus tih

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Amplification of the tih gene of known bacterial strains

[0048] 1) Design of primer set

[0049] The 946---1140bp nucleic acid sequence of vibrio parahaemolyticus specific gene tih 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: 946-965bp, 968- 987bp, 1010-1031bp, 1043-1062bp, 105-1122bp, 1123-1140bp) 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.

[0050] serial number 1

[0051] Forward outer primer F3-tih GGCACAAGCGATGTACTACA

[0052] serial number 2

[0053] Reverse outer primer B3-tih GACGCTGCACACTCAGAG

[0054] serial number 3

[0055] Forward inner primer FIP-tih

[0056] GCGCAGAAGTTAGCGTCTCGAAGCGCAAGGTTACAACATCAC

[0057] serial number 4

[0058] Reverse inner primer BIP-ti...

Embodiment 2

[0097] 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:

[0098] The reaction system is: (the total reaction volume is 25ul)

[0099] Element

stock solution concentration

Volume (ul)

Final concentration

nucleic acid template

FIP-tih

BIP-tih

F3-tih

B3-tih

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

[0100] In addition to the nucleic acid template, the above reaction system can be simplified to amplification reaction solution, e...

Embodiment 3

[0107] 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:

[0108] The reaction system is: (the total reaction volume is 25ul)

[0109] Element

stock solution concentration

Volume (ul)

Final concentration

nucleic acid template

FIP-tih

BIP-tih

F3-tih

B3-tih

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

[0110] In addition to the nucleic acid template, the above reaction system can be simplified to amplification reaction solution, enz...

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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 vibrio parahemolyticus can augment the specific base sequence of a target gene which is the tih-GenBank (accession no. AY578148) of the vibrio parahemolyticus, and the primer is complementary to a part of or a complementary chain fragment of the nucleic acid sequence on the 946-1140 loci on the target gene. The invention provides a primer having specificity to a specific gene fragment of the vibrio parahemolyticus, and through detecting whether or not the detecting specimen in a reagent box of the primer unit contains the specific gene fragment of the vibrio parahemolyticus, determines whether the vibrio parahemolyticus 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 for a specific gene fragment of Vibrio parahaemolyticus; it also relates to a detection method and a detection kit for detecting Vibrio parahaemolyticus with a loop-mediated isothermal amplification method using the primers and primer set . 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 vari...

Claims

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Application Information

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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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