Centers for Disease Control and Prevention, Atlanta, Georgia 30333, USA.
Foodborne Pathog Dis. 2010 Feb;7(2):129-36. doi: 10.1089/fpd.2009.0371.
The PulseNet USA subtyping network recently established a standardized protocol for multiple-locus variable-number tandem repeat analysis (MLVA) to characterize Shiga toxin-producing Escherichia coli O157. To enable data comparisons from different laboratories in the same database, reproducibility and high quality of the data must be ensured. The aim of this study was to test the robustness and reproducibility of the proposed standardized protocol by subjecting it to a multilaboratory validation process and to address any discrepancies that may have arisen from the study. A set of 50 strains was tested in 10 PulseNet participating laboratories that used capillary electrophoresis instruments from two manufacturers. Six out of the 10 laboratories were able to generate correct MLVA types for 46 (92%) or more strains. The discrepancies in MLVA type assignment were caused mainly by difficulties in optimizing polymerase chain reactions that were attributed to technical inexperience of the staff and suboptimal quality of reagents and instrumentation. It was concluded that proper training of staff must be an integral part of technology transfer. The interlaboratory reproducibility of fragment sizing was excellent when the same capillary electrophoresis platform was used. However, sizing discrepancies of up to six base pairs for the same fragment were detected between the two platforms. These discrepancies were attributed to different dye and polymer chemistries employed by the manufacturers. A novel software script was developed to assign alleles based on two platform-specific (Beckman Coulter CEQ8000 and Applied Biosystems Genetic Analyzer 3130xl) look-up tables containing fragment size ranges for all alleles. The new allele assignment method was validated at the PulseNet central laboratory using a diverse set of 502 Shiga toxin-producing Escherichia coli O157 isolates. The validation confirmed that the script reliably assigned the same allele for the same fragment regardless of the platform used to size the fragment.
美国脉冲网络最近建立了一个用于对产志贺毒素大肠杆菌 O157 进行多位点可变数目串联重复分析（MLVA）的标准化协议，以对其进行特征分析。为了能够在同一个数据库中比较来自不同实验室的数据，必须确保数据的可重复性和高质量。本研究的目的是通过多实验室验证过程来测试所提出的标准化协议的稳健性和可重复性，并解决研究中可能出现的任何差异。将一套 50 株菌株在 10 个参与脉冲网络的实验室中进行了测试，这些实验室使用了来自两个制造商的毛细管电泳仪器。在 10 个实验室中，有 6 个实验室能够为 46 株（92%）或更多菌株生成正确的 MLVA 类型。MLVA 类型赋值的差异主要是由于工作人员技术经验不足和试剂及仪器质量不理想导致聚合酶链反应难以优化所致。研究得出结论，必须将工作人员的适当培训作为技术转让的一个组成部分。当使用相同的毛细管电泳平台时，片段大小的实验室间可重复性非常好。然而，在两个平台之间检测到同一片段的大小差异高达 6 个碱基对。这些差异归因于制造商采用的不同染料和聚合物化学。开发了一种新的软件脚本，根据包含所有等位基因片段大小范围的两个平台特定（贝克曼库尔特 CEQ8000 和应用生物系统遗传分析仪 3130xl）查找表，对等位基因进行赋值。在脉冲网络中心实验室使用一组 502 株不同的产志贺毒素大肠杆菌 O157 分离株对新的等位基因赋值方法进行了验证。验证结果表明，该脚本可以可靠地为同一片段分配相同的等位基因，而不管用于对片段进行大小分析的平台如何。
