Mining of novel species-specific primers for PCR detection of Listeria monocytogenes based on genomic approach

Abstract

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 × 10(3), 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.

Genomics-based Sensitive and Specific Novel Primers for Simultaneous Detection of Burkholderia glumae and Burkholderia gladioli in Rice Seeds

“…Therefore, research has aimed at improving the specificity and sensitivity of detection of plant pathogens. With the recent availability of genome sequences of several strains of bacterial pathogens, the comparative genomics approach has proven useful for screening and identifying novel specific molecular targets to facilitate the accurate and highly specific detection of several bacterial species ( Baek et al, 2018 ; Papaiakovou et al, 2017 ; Tao et al, 2015 ). In this study, novel primer pairs targeting unique and conserved loci generated by comparative genomics sensitively and specifically detected two major bacterial pathogens of rice, B. glumae and B. gladioli .…”

“…The availability of the whole genome sequences of several strains of bacterial pathogens paved the way for improvements of the molecular applications including more accurate molecular detection ( Tao et al, 2015 ). Previous studies have shown that gene probing from comparative genomic analysis is an efficient method for providing target-specific molecular markers by selection of the most appropriate species-specific sequences ( Baek et al, 2018 ; Papaiakovou et al, 2017 ).…”

Phenotypic and Genotypic Characteristics of Non-Hemolytic L. monocytogenes Isolated from Food and Processing Environments

“…The most similar aLM isolates were in groups C and D. The presence of the LMOf2365_2721 gene responsible for encoding glycosyl hydrolase, which is a key enzyme for carbohydrate metabolism, was detected in all model LM and aLMs, while only in group B, represented by isolates from soil (273, 288, 352, and 140) and beetroot (B54), was the LMOf 2365_0970 gene (encoding a conserved hypothetical proteinase) confirmed. These genes can be used to detect LM in food and are more specific than hly or prfA genes [24].…”

Chronic intramammary infection by Listeria monocytogenes in a clinically healthy goat – a case report

“…Molecular identification was performed using 3 sets of species-specific primers on the case isolate as well as on the reference L. monocytogenes strain ATCC 19115 and on a characterized L. ivanovii strain as controls, as described by Tao and coworkers [13] with minor modifications. Amplification conditions were modified to further increase stringency by raising annealing temperature to 61 °C for primer sets Lm13 and Lm20, and to 65 °C for Lm8.…”