The development of duplex real-time PCR for detection of Listeria monocytogenes and Shigella
- VernacularTitle:单核细胞增生李斯特菌与志贺菌双重实时PCR检测技术的建立
- Author:
Wei XU
;
Sufang LI
;
Jun LIU
;
Dian HU
- Publication Type:Journal Article
- Keywords:
Duplex real-time PCR;
Listeria monocytogenes;
Shigella;
Food detection
- From:
Chinese Journal of Microbiology and Immunology
2008;28(10):946-950
- CountryChina
- Language:Chinese
-
Abstract:
Objective To develop a rapid,sensitive,specific and accurate quantitative duplex real-time PCR assay for detection of Listeria monocytogenes and Shigella.Methods Two sets of specific primers and probes were selected according to Listeria monocytogencs hly gene and Shigella ipaH gene.The target hly and iPaH fragments were amplified by PCR,and used to construct recombinant pGEM-T-hly and pGEM-T-ipaH respectively.The two recombinant circular plasmid DNAs were linearized with EcoR I that did not cut within the target DNA fragment.The ten-fold dilutions of plasmid were subjected to the standard quantitation curve in duplex real-time PCR assay.Various genomic DNAs of Listeria innocua,Listeria weshimeri,Salmonella,Staphylococcus aureus,Bacillus subtilis,Escherichia coli and Proteus were used as negative controls to confirm the specificity of duplex real-time PCR assay.The assay was also used to detect Listeria monocytogenes and Shigella in artificially contaminated sterilized skim milk.Results The recombinant plasmids were constructed successfully,hly probe(rAM and TAMRA double labelled)and ipaH probe (HEX and TAMRA double labelled)were used to develop an optimized PCR successfuliv.Conclusion The selected primers and probes showed high specificity for these two target bacteria,the linear range of the assay was good(105-101 copies/μl,R2≥0.998)and sensitivity Was 10 copies/PCR.Following a DNA extraction method which combined EZ Spin Colum Genomic DNA Isolation Kit(BBI)/Phenol-chloroform,the sensitivity of assay Was 102CFU/ml for both Listeria monocytogenes and Shigella in artificially contaminated sterilized skim milk,which equivalents to 10 CFU/PCR.
