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

A technology for staphylococcus aureus and detection methods, applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve the problems of detection methods and detection kits for staphylococcus aureus, etc. Achieve the effect of wide application range, high sensitivity and strong specificity

Inactive Publication Date: 2010-11-03
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
  • Primer, detection method and detection reagent kit for detecting staphylococcus aureus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Amplification of the nuc gene of known strains

[0047] 1) Design of primer set

[0048] The 548---795bp nucleotide sequence of Staphylococcus aureus-specific gene nuc is screened out by consulting the literature and using BLAST software analysis, for six sites of this fragment (these six sites are respectively: 548-568bp, 584-602bp , 624-643bp, 679-703bp, 742-763bp, 777-795bp) LAMP primers were designed and synthesized to obtain the following primers; primer design was completed by LAMP-specific primer design software combined with molecular biology analysis software Advance Vector NTI.

[0049] serial number 1

[0050] Forward outer primer F3-nuc GTCAACCAATGACATTCAGAC

[0051] serial number 2

[0052] Reverse outer primer B3-nuc AACTTTAGCCAAGCCTTGA

[0053] serial number 3

[0054] Forward internal primer FIP-nuc

[0055] ATGCACTTGCTTCAGGACCACACCTGAAACAAAGCATCC

[0056] serial number 4

[0057] reverse inner primer BIP-nuc

[0058] GAAGTCGAGTTTGACAA...

Embodiment 2

[0101] 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:

[0102] The reaction system is: (the total reaction volume is 25ul)

[0103] Element

stock solution concentration

Volume (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

[0104] MgSO 4

Bst DNA Polymerase Buffer

Bst DNA Polymerase

wxya 2 o

100mM

10×

8U / ul

0.5

2.5

0.5

10.0

2.0mM

0.16U / ul

[0105] In addition to the nucleic acid template, the above reaction system can be simplified to amplification reaction solution, enzyme and doubl...

Embodiment 3

[0112] 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:

[0113] The reaction system is: (the total reaction volume is 25ul)

[0114] Element

stock solution concentration

Volume (ul)

Final concentration

nucleic acid template

FIP-nuc

BIP-nuc

50uM

50uM

1.0

1.0

1.0

2.0uM

2.0uM

[0115] F3-nuc

B3-nuc

betaine

dNTP

MgSO 4

Bst DNA Polymerase Buffer

Bst DNA Polymerase

wxya 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

[0116] In addition to the nucleic acid template, the above reaction system can be simplified to amplification reaction solution, enzyme and double distilled wa...

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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 fragmentof 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 rapid detection technology for food-borne pathogens based on a loop-mediated isothermal amplification (LAMP) technology. It particularly relates to a primer set specific to a specific gene fragment of Staphylococcus aureus; and 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] 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. Although the...

Claims

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Application Information

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Patent Type & Authority Patents(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