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Fluorescent detection kit and method for Streptococcus suis 2 type nucleic acid containing 89K pathogenicity island gene

A technology of Streptococcus suis and virulence island, which is applied in the directions of fluorescence/phosphorescence, microbial determination/inspection, biochemical equipment and methods, etc., and can solve the problems of simultaneous detection of Streptococcus suis and 89K virulence island genes, etc.

Inactive Publication Date: 2009-05-27
ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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Problems solved by technology

[0003] At present, there are common PCR, multiplex PCR and fluorescent quantitative PCR methods for establishing rapid diagnosis methods for SS2 nucleic acid, but there is no detection method for simultaneous detection of Streptococcus suis and 89K virulence island gene

Method used

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  • Fluorescent detection kit and method for Streptococcus suis 2 type nucleic acid containing 89K pathogenicity island gene
  • Fluorescent detection kit and method for Streptococcus suis 2 type nucleic acid containing 89K pathogenicity island gene
  • Fluorescent detection kit and method for Streptococcus suis 2 type nucleic acid containing 89K pathogenicity island gene

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Embodiment 1

[0089] Utilize the TaqMan-MGB fluorescent quantitative PCR method of the present invention to detect 1 suspected SS2 patient emergency sample in Zhejiang Province, the result is positive, see image 3 .

[0090] (1) Determination of standard curve:

[0091] ①Obtain the standard solution:

[0092] Using the specific 16S rRNA of Streptococcus suis and the DNA of Streptococcus suis containing 89K virulence island gene as templates, the target fragments of 16S rDNA and 89K virulence island gene were respectively amplified, and the amplified products were cloned into pMD18-T vector (Takara company), transformed into DH5α Escherichia coli, picked positive clones and proliferated, extracted plasmid DNA for sequence determination to identify the inserted sequence, and determined the concentration of plasmid DNA to convert the copy number.

[0093] Take the aforementioned cloning plasmid standard 10 10 copies / ml, according to 10-fold dilution to make 10 9 ~10 1 copies / ml gradient ...

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Abstract

The invention provides a rapid kit and a method for detecting genes containing 89k pathogenicity island Streptococcus type II through a Taqman-MGB double fluorescent probe PCR. The specific primer and the specific probe of the kit include a 16S upstream primer of 5'- AGCGCAGGCGGTTTGA-3', a 16S downstream primer of 5'-ACAGTTTCCAA AGCGTACTATGGTTA-3', a 16S specific probe of 5'-FAM-AGTCTG AAGTAAAAGGC-MGB-3', a 89K upstream primer of 5'-ATAAAAATACCGCT GTTGGATGGA-3', a 89K downstream primer of : 5'-CGTCTACACCGAATT GTT TTGCA-3', and a 89K specific probe of 5'-HEX- CATGGGAGATATAAAG A AC -MGB-3', wherein FAM and HEX are reporting fluorescent groups, and HGB is a decorative group. The method provided by the invention has easy and convenient sampling, rapid evaluation, high sensitivity and accurate results, and can be applied to early diagnosis of SS2 onset.

Description

(1) Technical field [0001] The invention relates to a Taqman-MGB dual fluorescent probe PCR detection kit and a detection method containing 89K virulence island gene Streptococcus suis type 2 nucleic acid. (2) Background technology [0002] Streptococcus suis is an important zoonotic pathogen. From the first report in 1968 to 2007, more than 409 cases of human infection with Streptococcus suis type 2 (SS2) have occurred worldwide. As early as the autumn of 1990 to the spring of 1991, streptococcal diseases had occurred in some areas of the Yangtze River Delta in my country, and the symptoms were streptococcal toxic shock syndrome (STSS). From 1998 to 2005, SS2 outbreaks occurred in Jiangsu and Sichuan. There were many deaths, and most of the patients showed STSS, but what pathogenic factor caused the disease and its pathogenic mechanism have not yet been identified. In 2007, it was reported in the literature that the 1998 Jiangsu isolate and the 2005 Sichuan isolate from cli...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/14G01N21/64
Inventor 王志刚朱水荣
Owner ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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