Leão Sylvia Cardoso, Bernardelli Amelia, Cataldi Angel, Zumarraga Martin, Robledo Jaime, Realpe Teresa, Mejía Gloria Isabel, da Silva Telles Maria Alice, Chimara Erica, Velazco Maritza, Fernandez Jorge, Rodrigues Pamela Araya, Guerrero Martha Inirida, León Clara Ines, Porras Tania Bibiana, Rastogi Nalin, Goh Khye Seng, Suffys Philip, da Silva Rocha Adalgisa, dos Santos Netto Diogo, Ritacco Viviana, López Beatriz, Barrera Lucia, Palomino Juan Carlos, Martin Anandi, Portaels Françoise
Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo-Escola Paulista de Medicina, Rua Botucatu, 862 3 degrees andar, São Paulo 04023-062, Brazil.
J Microbiol Methods. 2005 May;61(2):193-9. doi: 10.1016/j.mimet.2004.11.015. Epub 2004 Dec 25.
The identification of mycobacterial species in clinical isolates is essential for making patient care decisions. Polymerase chain reaction (PCR) restriction enzyme analysis (PRA) is a simple and rapid identification method, based on amplification of 441 bp of the hsp65 gene and restriction with BstEII and HaeIII. As a contribution to the validation of PRA, a multicenter study was performed in eight laboratories located in Argentina, Brazil, Colombia, Chile, and Guadeloupe. Each laboratory received 18 coded isolates from the collection of the Institute of Tropical Medicine (Antwerp, Belgium), representing duplicates of nine laboratory strains: Mycobacterium terrae CIPT 140320001, Mycobacterium scrofulaceum CIPT 140220031, Mycobacterium flavescens ATCC 14474, Mycobacterium triviale ATCC 23292, Mycobacterium nonchromogenicum ATCC 19530, Mycobacterium chitae ATCC 19627, Mycobacterium abscessus ATCC 19977, Mycobacterium kansasii ATCC 12478, and Mycobacterium peregrinum ATCC 14467. A detailed protocol including amplification, enzymatic digestion, and gel preparation was provided to each laboratory. Two laboratories identified correctly all 18 (100%) isolates, one identified correctly 17 (94.5%), two identified 14 (77.7%), one identified 11 (61%), and two identified 8 (44.4%) isolates. Errors detected in laboratories with more than 77% accuracy were associated with electrophoresis running conditions and an unspecific amplicon produced by a single strain. Lower accuracy was mainly related to inappropriate use of DNA markers and insufficient training in interpretation of patterns. In conclusion, the PRA method was readily implemented in some Latin American and Caribbean laboratories of mycobacteria, but improvements in critical points, as gel running conditions and training in interpretiation of patterns, are needed in order to improve accuracy. In others, improvement in critical points is still necessary.
临床分离株中分枝杆菌菌种的鉴定对于做出患者护理决策至关重要。聚合酶链反应（PCR）限制性酶切分析（PRA）是一种简单快速的鉴定方法，基于对hsp65基因441 bp片段的扩增以及用BstEII和HaeIII进行酶切。作为对PRA验证工作的一项贡献，在阿根廷、巴西、哥伦比亚、智利和瓜德罗普岛的八个实验室开展了一项多中心研究。每个实验室从热带医学研究所（比利时安特卫普）的菌株库中收到18株编码菌株，这些菌株代表了9种实验室菌株的重复样本：地分枝杆菌CIPT 140320001、瘰疬分枝杆菌CIPT 140220031、微黄分枝杆菌ATCC 14474、平凡分枝杆菌ATCC 23292、非产色分枝杆菌ATCC 19530、契塔分枝杆菌ATCC 19627、脓肿分枝杆菌ATCC 19977、堪萨斯分枝杆菌ATCC 12478和偶然分枝杆菌ATCC 14467。向每个实验室提供了一份详细的方案，包括扩增、酶切消化和凝胶制备。两个实验室正确鉴定了所有18株（100%）菌株，一个实验室正确鉴定了17株（94.5%），两个实验室鉴定了14株（77.7%），一个实验室鉴定了11株（61%），两个实验室鉴定了8株（44.4%）菌株。在准确率超过77%的实验室中检测到的错误与电泳运行条件以及单个菌株产生的非特异性扩增子有关。较低的准确率主要与DNA标记物使用不当以及模式解读方面的培训不足有关。总之，PRA方法在一些拉丁美洲和加勒比地区的分枝杆菌实验室中易于实施，但为了提高准确性，需要在关键点上进行改进，如凝胶运行条件和模式解读培训。在其他方面，关键点的改进仍然是必要的。
