Chalmers G, Martin S W, Prescott J F, Boerlin P
Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada.
Vet Microbiol. 2008 Apr 1;128(1-2):126-35. doi: 10.1016/j.vetmic.2007.09.018. Epub 2007 Oct 2.
Clostridium perfringens is a well-characterized bacterial species which can be both commensal and pathogenic in humans and many animals. Genetic typing of the bacterium is often used for molecular epidemiological purposes, and can be useful for observing population structures as well. Analysis of the variable number of tandem repeats (VNTRs) within the genome, called multiple-locus VNTR analysis (MLVA) provides genetic information useful for molecular typing. A MLVA typing method has been developed recently by Sawires and Songer [Sawires, Y.S., Songer, J.G., 2005. Multiple-locus variable-number tandem repeat analysis for strain typing of Clostridium perfringens. Anaerobe 11, 262-272] for C. perfringens. A novel MLVA protocol is described here, with the aim of investigating the discriminatory potential of the method, and to obtain preliminary data on the population structure of C. perfringens from a wide variety of C. perfringens sources. This protocol uses new loci in noncoding regions of the chromosome, and also makes use of capillary electrophoresis for more precise results and for high-throughput typing. DNA sequencing of amplicons was performed to ensure inclusion of conserved tandem repeats within each locus. Fifty-four epidemiologically unrelated isolates from a local collection obtained from 11 different animal species were typed at 6 loci. Thirty-five unique MLVA types were obtained, resulting in a Simpson's index of diversity of 0.975. Epidemiologically related isolates (n=27) previously typed by pulsed-field gel electrophoresis (PFGE) were also examined with MLVA and the congruency of the two methods was found to be very high. All 81 isolates were successfully typed with MLVA, and polymerase chain reactions (PCR) were automated using robotics and 96-well plates, with PCR product sizes determined using capillary electrophoresis. Reproducibility was also shown to be very high.
产气荚膜梭菌是一种特征明确的细菌物种，在人类和许多动物中既可以是共生菌，也可以是致病菌。该细菌的基因分型常用于分子流行病学目的，也有助于观察种群结构。对基因组内串联重复序列可变数目（VNTR）的分析，即多位点VNTR分析（MLVA），可提供用于分子分型的遗传信息。Sawires和Songer [Sawires, Y.S., Songer, J.G., 2005. Multiple-locus variable-number tandem repeat analysis for strain typing of Clostridium perfringens. Anaerobe 11, 262 - 272] 最近开发了一种针对产气荚膜梭菌的MLVA分型方法。本文描述了一种新的MLVA方案，旨在研究该方法的鉴别潜力，并从多种产气荚膜梭菌来源获得有关产气荚膜梭菌种群结构的初步数据。该方案使用染色体非编码区的新位点，还利用毛细管电泳以获得更精确的结果和高通量分型。对扩增子进行DNA测序，以确保每个位点包含保守的串联重复序列。对从11种不同动物物种的本地样本中获得的54株流行病学无关的分离株进行了6个位点的分型。获得了35种独特的MLVA类型，辛普森多样性指数为0.975。还对先前通过脉冲场凝胶电泳（PFGE）分型的流行病学相关分离株（n = 27）进行了MLVA检测，发现两种方法的一致性非常高。所有81株分离株均成功通过MLVA分型，并使用机器人技术和96孔板实现了聚合酶链反应（PCR）自动化，通过毛细管电泳确定PCR产物大小。结果还表明重复性非常高。
