Multiple-locus variable-number tandem repeat analysis using multiplex polymerase chain reaction and next-generation sequencing - A novel high-throughput method for subtyping Listeria strains

Listeria monocytogenes is significantly associated with listeriosis infection. Several species of Listeria other than L. monocytogenes directly affect the food industry. In the present study we developed a novel subtyping method for tracking the source of L. monocytogenes and L. innocua in large-scale investigations using multiple-locus variable-number tandem repeat analysis (MLVA) coupled with next-generation sequencing (NGS). Forty-eight L. monocytogenes and L. innocua strains isolated from a food processing plant and the environment were used for the amplification of 15 specific variable number tandem repeat (VNTR) loci. The results exhibited 100% concordance for amplicon detection using both the subtyping methods. Coupling of MLVA with capillary electrophoresis (CE) or NGS detected 38 and 89 different alleles, respectively. MLVA coupled with NGS showed significantly higher discriminating power than MLVA coupled with CE. In MLVA coupled with NGS method, locus JLR1 of L. monocytogenes had 54% discrimination while locus TR3 had 100% discrimination solely in L. innocua. In addition, MLVA coupled with NGS method had significantly higher discrimination power (DI = 1.0 with 48 MLVA patterns) than that of MLVA coupled with CE (DI = 0.79 with 15 MLVA patterns). These results indicate the advantage of MLVA coupled with NGS method in detecting not only the length but also sequence polymorphism in a single read run. MLVA coupled with NGS led to significant reduction in labor and cost than CE and the traditional Sanger sequencing. Thus, MLVA coupled with NGS is an effective and feasible technique for the molecular typing of Listeria species in food industries.