Primer, detection method and detection reagent kit for detecting staphylococcus aureus

A technology of staphylococcus and detection method, which is applied in the directions of biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of detection methods and detection kits that have not detected Staphylococcus aureus, etc., To achieve the effect of wide application, 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 detection methods and kits for the detection of Staphylococcus aureus using the loop-mediated isothermal amplification method, as well as LAMP primers and primer sets specific to specific gene segments of Staphylococcus aureus.

Method used

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  • Primer, detection method and detection reagent kit for detecting staphylococcus aureus
  • Primer, detection method and detection reagent kit for detecting staphylococcus aureus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047]Example 1 Amplification of the nuc gene of known strains

[0048] A) Design of primer set

[0049] The 548---795bp nucleic acid sequence of the Staphylococcus aureus-specific gene nuc was selected by consulting the literature and analyzing with BLAST software, targeting the six sites of the fragment (the six sites were 548-568bp, 584-602bp, respectively) , 624-643bp, 679-703bp, 742-763bp, 777-795bp) designed and synthesized LAMP primers, and obtained the following primers; the 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 primer F3-nuc GTCAACCAATGACATTCAGAC

[0052] Serial number 2

[0053] Reverse outer primer B3-nuc AACTTTAGCCAAGCCTTGA

[0054] Serial number 3

[0055] Forward inward primer FIP-nuc

[0056] ATGCACTTGCTTCAGGACCACACCTGAAACAAAGCATCC

[0057] Serial number 4

[0058] Reverse inner primer BIP-nuc

[0059] GAAGTCGAGTTTGACAAAGGTCAAA...

Embodiment 2

[0102] 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:

[0103] The reaction system is: (the total reaction volume is 25ul)

[0104] Ingredients

Concentration of storage solution

Quantity (ul)

Final concentration

Nucleic acid template

FIP-nuc

BIP-nuc

F3-nuc

B3-nuc

betaine

dNTP

25uM

25uM

7.5uM

7.5uM

4M

10mM

1.0

1.0

1.0

0.5

0.5

5.0

2.5

1.0uM

1.0uM

0.15uM

0.15uM

0.8M

1.0mM

[0105] mgsO 4

Bst DNA Polymerase Buffer

Bst DNA Polymerase

ddH 2 O

100mM

10×

8U / ul

0.5

2.5

0.5

10.0

2.0mM

0.16U / ul

[0106] Except for the nucleic acid template, the above reaction system can be simplified as an amplification reaction solution, enzyme and double distilled water.

[0107] Amplification reaction...

Embodiment 3

[0113] 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:

[0114] The reaction system is: (the total reaction volume is 25ul)

[0115] Ingredients

Concentration of storage solution

Quantity (ul)

Final concentration

Nucleic acid template

FIP-nuc

BIP-nuc

50uM

50uM

1.0

1.0

1.0

2.0uM

2.0uM

[0116] F3-nuc

B3-nuc

betaine

dNTP

mgsO 4

Bst DNA Polymerase Buffer

Bst DNA Polymerase

ddH 2 O

15uM

15uM

7.5M

10mM

150mM

10×

16U / ul

0.5

0.5

5.0

4.0

1.0

2.5

1.0

7.5

0.3uM

0.3uM

1.5M

1.6mM

6.0mM

0.64U / ul

[0117] Except for the nucleic acid template, the above reaction system can be simplified to an amplification reaction solution, enzyme and double distilled water.

[0118] Amplification reaction so...

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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 staphylococcus aureus can augment the specific base sequence of a target gene which is the nuc-GenBank (accession no. V01281) of the staphylococcus aureus, and the primer is complementary to a part of or a complementary chain fragment of the nucleic acid sequence on the 548-795loci on the target gene. The invention provides a primer having specificity to a specific gene fragment of the staphylococcus aureus, and through detecting whether or not the detecting specimen in a reagent box of the primer unit contains the specific gene fragment of the staphylococcus aureus, determines whether the staphylococcus aureusexists 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 set specific to specific gene fragments of Staphylococcus aureus; it also relates to a detection method and a detection kit for detecting Staphylococcus aureus by using the 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, and it 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. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12Q1/14
Inventor 魏泉德张彩虹谭爱军张丽荣
Owner ZHUHAI DISEASE PREVENTION & CONTROL CENT
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