Kit capable of simultaneously detecting 12 diarrhea pathogenic bacteria and application thereof
A technology of pathogenic bacteria and kits, applied in the field of multiplex PCR detection, can solve the problems of less research in the detection of bacterial pathogens, achieve good application prospects, reduce amplification preference, and have the effects of strong specificity
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Embodiment 1
[0054] Example 1 Design of specific chimeric primers
[0055] By consulting OIE, national standard, industry standard and related literature, determine the pathogenic genes or species-specific genes of 12 common diarrheal pathogens, namely bfpA gene and eaeA gene of E. coli EPEC, elt gene and stp gene of E. coli ETEC , Sth gene, stx1 and stx2 genes of E. coli EHEC, aggR gene of E. coli EAEC, virA gene of E. coli EIEC, virA gene of Shigella, ompC gene of Salmonella, hipO gene of Campylobacter jejuni, Campylobacter coli The ceuE gene of Vibrio cholerae, the ompW gene of Vibrio cholerae, the toxR gene of Vibrio parahaemolyticus, the ail gene of Yersinia enterocolitica. Check the literature and various materials, citing existing or self-designed primer sequences for the above genes. When designing primers, download the relevant nucleic acid sequences from the GenBank database, and use software such as DNAStar and PrimerPremier5.0 to select the conservative regions to design primers....
Embodiment 2
[0066] Example 2 Establishment of multiple PCR detection system for simultaneous detection of 12 diarrhea pathogens
[0067] 1. Preparation of multiplex PCR detection template
[0068] (1) Construction of standard plasmid: Amplify the nucleic acid DNA of the corresponding pathogen with the 3'-end part of the specific chimeric primer (that is, the part after the chimeric primer sequence is removed from the universal primer sequence), and the amplified product is connected to the vector plasmid pMD19-TSimple , Using E. ColiJM109 as the host strain, construct a standard plasmid. A total of 15 standard plasmids were constructed for 15 detection targets.
[0069] (2) Extraction and concentration determination of standard plasmids: QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany) was used to extract plasmids from host bacterial cultures carrying standard plasmids; the concentration of nucleic acid solution was determined by NanoDropND-1000 with ultra-micro spectrophotometer.
[0070] (3...
Embodiment 3
[0103] Example 3 Validation of multiplex PCR for simultaneous detection of 12 diarrhea pathogens
[0104] 1. Specificity experiment
[0105] 279 strains were used to verify the specificity of the present invention, including 207 strains belonging to the detection scope of the present invention: 43 strains of Vibrio cholerae, 20 strains of Vibrio parahaemolyticus, 6 strains of Campylobacter jejuni, 6 strains of Campylobacter coli, 24 strains Salmonella, 10 strains of Shigella, 5 strains of EIEC, 19 strains of EAEC, 26 strains of EPEC, 45 strains of ETEC, and 2 strains of EHEC; 72 strains not included in the detection scope of the present invention: 40 strains of non-pathogenic E. coli, 18 strains of Aeromonas, 6 strains of Vibrio mimicus, 6 strains of Vibrio fluvialis, 1 strain of Vibrio vulnificus and 1 strain of Edwards tarda. For Vibrio cholerae, Vibrio parahaemolyticus, Campylobacter jejuni, Campylobacter coli, Salmonella, EIEC&Shigella and EAEC, only one electrophoretic band w...
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