Macdonald Thomas E, Helma Charles H, Ticknor Lawrence O, Jackson Paul J, Okinaka Richard T, Smith Leonard A, Smith Theresa J, Hill Karen K
Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM 87545, USA.
Appl Environ Microbiol. 2008 Feb;74(3):875-82. doi: 10.1128/AEM.01539-07. Epub 2007 Dec 14.
Ten variable-number tandem-repeat (VNTR) regions identified within the complete genomic sequence of Clostridium botulinum strain ATCC 3502 were used to characterize 59 C. botulinum strains of the botulism neurotoxin A1 (BoNT/A1) to BoNT/A4 (BoNT/A1-A4) subtypes to determine their ability to discriminate among the serotype A strains. Two strains representing each of the C. botulinum serotypes B to G, including five bivalent strains, and two strains of the closely related species Clostridium sporogenes were also tested. Amplified fragment length polymorphism analyses revealed the genetic diversity among the serotypes and the high degree of similarity among many of the BoNT/A1 strains. The 10 VNTR markers amplified fragments within all of the serotype A strains but were less successful with strains of other serotypes. The composite multiple-locus VNTR analysis of the 59 BoNT/A1-A4 strains and 3 bivalent B strains identified 38 different genotypes. Thirty genotypes were identified among the 53 BoNT/A1 and BoNT/A1(B) strains, demonstrating discrimination below the subtype level. Contaminating DNA within crude toxin preparations of three BoNT/A subtypes (BoNT/A1 to BoNT/A3) also supported amplification of all of the VNTR regions. These markers provide clinical and forensics laboratories with a rapid, highly discriminatory tool to distinguish among C. botulinum BoNT/A1 strains for investigations of botulism outbreaks.
在肉毒梭菌菌株ATCC 3502的完整基因组序列中鉴定出的10个可变数目串联重复序列(VNTR)区域,用于对59株肉毒中毒神经毒素A1(BoNT/A1)至BoNT/A4(BoNT/A1-A4)亚型的肉毒梭菌菌株进行特征分析,以确定它们区分A型菌株的能力。还测试了代表肉毒梭菌B至G各血清型的两株菌株,包括五株二价菌株,以及两株密切相关的梭状芽孢杆菌。扩增片段长度多态性分析揭示了各血清型之间的遗传多样性以及许多BoNT/A1菌株之间的高度相似性。这10个VNTR标记在所有A型菌株中均扩增出片段,但在其他血清型菌株中的扩增效果较差。对59株BoNT/A1-A4菌株和3株二价B型菌株进行的复合多位点VNTR分析鉴定出38种不同的基因型。在53株BoNT/A1和BoNT/A1(B)菌株中鉴定出30种基因型,表明在亚型水平以下具有区分能力。三种BoNT/A亚型(BoNT/A1至BoNT/A3)粗毒素制剂中的污染DNA也支持所有VNTR区域的扩增。这些标记为临床和法医实验室提供了一种快速、高度有区分能力的工具,用于区分肉毒梭菌BoNT/A1菌株,以调查肉毒中毒疫情。