Purpose: This study aims to refine MLVA primers and validate the MLVA assay using an inter-laboratory approach. The aim is to further expand a 2500 strain database containing MLVA information with an additional 2000 strains in order to allow for their implementation in routine analysis.
Methods: L. monocytogenes DNA was amplified in two multiplex PCR reactions using eight primer combinations targeting seven specific VNTR loci. Fluorescent PCR fragments were separated using an ABI 3730 Genetic Analyzer. The fragment data were analyzed using GeneMapper and BioNumerics software to determine isolate relationships. Method comparison between MLVA and PFGE was conducted using Comparative Partitions analysis.
Results: The MLVA protocol provided high discriminatory power, enhanced amplification efficiency and data quality. Inter-laboratory validation of the protocol using 60 strains of different subtypes/sources indicated that the MLVA protocol was reproducible and repeatable among three participating laboratories. Comparative Partitions analysis involving 378 strains with 215 PFGE types yielded Simpson’s Diversity index values of 0.998 and 0.991 for MLVA and PFGE, respectively. The adjusted Wallace coefficients were 0.292 when MLVA was used as a primary typing method and 0.075 when PFGE was a primary subtyping method, indicating that the MLVA protocol was more discriminatory in some cases. Data analysis of ~4000 strains revealed predominant/persistent L. monocytogenes genotypes and their potential epidemiological links to clinically important strains.
Significance: The inter-laboratory validated MLVA protocol, along with the updated L. monocytogenes MLVA database, allows for implementation of MLVA for real-time subtyping of this pathogen to minimize listeriosis.