Mining of novel species-specific primers for PCR detection of Listeria monocytogenes based on genomic approach
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Listeria monocytogenes in contaminated food is considered as a serious health threat for consumers due to its high mortality rate. The objective of this study was to obtain novel species-specific target-genes and primers for the molecular detection of L. monocytogenes using a comparative genomic approach. By comparative analysis of L. monocytogenes and non-L. monocytogenes genome sequences in the GenBank database with BLAST program, 26 specific target sequences were used as candidates and the primers were designed for L. monocytogenes species-specificity verification by using PCR assay. Finally, the three genes LMOf2365_0970, LMOf2365_2721 and mpl were identified to have L. monocytogenes species-specificity and be unique as detection targets for diagnostic application. The species-specific primer Lm8 of gene LMOf2365_0970, Lm13 of gene LMOf2365_2721 and Lm20 of gene mpl showed better specificity and sensitivity than the primers described previously. The PCR detection limits of the three specific primer sets were 430, 43, 4.3 fg/μL for genomic DNA, and 5 × 103, 50, 5 cfu/mL for pure culture of L. monocytogenes. There was no interference in specificity of detecting L. monocytogenes by co-culture with other foodborne pathogens in high concentration. Moreover, after 6–8 h of enrichment, L. monocytogenes in the artificially contaminated milk samples at an inoculum dose of 38 cfu/10 mL milk could be detected successfully with the studied three primers. Therefore, the three specific genes and primers can be applied to establish a novel rapid and accurate method for detecting L. monocytogenes in food materials.
KeywordsListeria monocytogenes Species-specific primer Specificity Sensitivity Comparative genome approach
The authors would like to thank social development program of Jiangsu Province and independent innovation program of Jiangsu Province for its financial support (Grant Nos. BE2012746 and CX (12)3087).
This study was funded by Social development program of Jiangsu Province (Grant Number BE2012746) and Independent innovation program of Jiangsu Province (Grant Number CX(12)3087).
Compliance with ethical standards
Conflict of interest
Author Tingting TAO, Qiming Chen, Xiaomei Bie, Fengxia Lu and Zhaoxin Lu declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
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