Detection and identification of viable Listeria monocytogenes by real-time PCR
- VernacularTitle:实时荧光RT-PCR检测活性单核细胞增生李斯特菌方法的建立
- Author:
Dazhi JIN
;
Jijuan CAO
;
Zheng ZHANG
;
Mingjie XIE
;
Shuirong ZHU
- Publication Type:Journal Article
- Keywords:
Real-time PCR;
Listeria monocytogenes
- From:
Chinese Journal of Microbiology and Immunology
2008;28(10):941-945
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish a rapid,sensitive and specific assay based on real-time PCR combined with reverse transcription for detecting and identifying viable Listeria monocytogenes.Methods The hlyA gene of Listeria monocytogenes was chosen as target,and then the primers and TaqMan probe were designed.Both ends of probe were modified with two different fluorescence groups.The PCR reaction was optimized systematically.The mRNA of Listeria monocytogenes was extracted,and then reverse transcription was performed through random primer.The cDNA Was detected by real-time PCR.Then the specificity,sensitivity and reproducibility of real-time PCR were estimated.In final,real-time PCR was applied to detect 20 mocked double-blind samplea.Results Viable Listeria monocytogenes were detected by real-time PCR accurately and quickly,and meanwhile,none of other bacteria and non-viable Listeria monocytogenes could be identified.The sensitivity was 10 CFU/ml in pure culture and 103CFU/ml for mocked samples respectively.The coefficient of variation of intra-assay and inter-assay Was less than 5%.When this assay was applied directly to identify 20 mocked double-blind samples,10 of these were positive to viable Listeria monocytogenes,5 were negative to non-viable Listeria monocytogenes,and 5 were negative to other pathogens.Conclusion It is demonstrated that real-time PCR is a reliable,accurate and feasible assay for viable Listeria monocytogenes.The establishment of this assay provided complete data for analysis and diagnosis in the field of food safety and epidemiologic survey.
