Ducey Thomas F, Page Brent, Usgaard Thomas, Borucki Monica K, Pupedis Kitty, Ward Todd J
Microbial Genomics and Bioprocessing Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture, 1815 North University Street, Peoria, IL 61604, USA.
Appl Environ Microbiol. 2007 Jan;73(1):133-47. doi: 10.1128/AEM.01453-06. Epub 2006 Nov 3.
Listeria monocytogenes is a facultative intracellular pathogen responsible for food-borne disease with high mortality rates in humans and is the leading microbiological cause of food recalls. Lineage I isolates of L. monocytogenes are a particular public health concern because they are responsible for most sporadic cases of listeriosis and the vast majority of epidemic outbreaks. Rapid, reproducible, and sensitive methods for differentiating pathogens below the species level are required for effective pathogen control programs, and the CDC PulseNet Task Force has called for the development and validation of DNA sequence-based methods for subtyping food-borne pathogens. Therefore, we developed a multilocus genotyping (MLGT) assay for L. monocytogenes lineage I isolates based on nucleotide variation identified by sequencing 23,251 bp of DNA from 22 genes distributed across seven genomic regions in 65 L. monocytogenes isolates. This single-well assay of 60 allele-specific probes captured 100% of the haplotype information contained in approximately 1.5 Mb of comparative DNA sequence and was used to reproducibly type a total of 241 lineage I isolates. The MLGT assay provided high discriminatory power (Simpson's index value, 0.91), uniquely identified isolates from the eight listeriosis outbreaks examined, and differentiated serotypes 1/2b and 4b as well as epidemic clone I (ECI), ECIa, and ECII. In addition, the assay included probes for a previously characterized truncation mutation in inlA, providing for the identification of a specific virulence-attenuated subtype. These results demonstrate that MLGT represents a significant new tool for use in pathogen surveillance, outbreak detection, risk assessment, population analyses, and epidemiological investigations. DNA sequences were deposited in the GenBank database under accession numbers DQ 812146 to DQ 812517, DQ 843664 to DQ 844598, and AY 512391 to AY 512502.
单核细胞增生李斯特菌是一种兼性胞内病原体，可引发食源性疾病，在人类中死亡率很高，是食品召回的主要微生物原因。单核细胞增生李斯特菌的I系分离株是一个特别的公共卫生问题，因为它们导致了大多数散发性李斯特菌病病例以及绝大多数疫情爆发。有效的病原体控制计划需要快速、可重复且灵敏的方法来区分种以下水平的病原体，美国疾病控制与预防中心（CDC）脉冲网络工作组呼吁开发和验证基于DNA序列的食源性病原体亚型分型方法。因此，我们基于对65株单核细胞增生李斯特菌分离株中分布于七个基因组区域的22个基因的23,251 bp DNA进行测序所鉴定出的核苷酸变异，开发了一种针对单核细胞增生李斯特菌I系分离株的多位点基因分型（MLGT）检测方法。这种包含60个等位基因特异性探针的单孔检测方法捕获了约1.5 Mb比较DNA序列中所含单倍型信息的100%，并用于对总共241株I系分离株进行可重复分型。MLGT检测方法具有高鉴别力（辛普森指数值为0.91），能唯一鉴定出所检测的八起李斯特菌病疫情中的分离株，还能区分血清型1/2b和4b以及流行克隆I（ECI）、ECIa和ECII。此外，该检测方法包含针对先前已鉴定的inlA基因截断突变的探针，可用于鉴定一种特定的毒力减弱亚型。这些结果表明，MLGT是一种用于病原体监测、疫情检测、风险评估、群体分析和流行病学调查的重要新工具。DNA序列已存入GenBank数据库，登录号为DQ 812146至DQ 812517、DQ 843664至DQ 844598以及AY 512391至AY 512502。
