Reverse transcriptase real-time PCR for detection and quantification of viable Campylobacter jejuni directly from poultry faecal samples
Reverse transcriptase real-time PCR for detection and quantification of viable Campylobacter jejuni directly from poultry faecal samples
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DOI：
10.1016/j.resmic.2011.10.007
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发表时间：
2012-01-01
影响因子：
2.6
通讯作者：
Bang, Dang Duong
中科院分区：
文献类型：
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作者：
Bui, Xuan Thanh;Wolff, Anders;Bang, Dang Duong
弯曲杆菌属。是全世界人类细菌性腹泻的最常见原因。因此，需要一种快速可靠的检测和定量检测该病原体的方法。在本研究中，我们建立了一种直接从鸡粪样品中检测和定量检测空肠弯曲菌活菌的逆转录实时定量聚合酶链式反应(RT-qPCR)方法。将该方法和基于DNA的实时定量聚合酶链式反应(QPCR)方法与细菌培养方法的结果进行了比较。使用细菌培养和RT-qPCR方法，在添加空肠弯曲菌和自然污染的粪便样本中，都可以在长达5天的时间内检测到活的空肠弯曲菌细胞。我们发现，当培养法不能计数活的空肠弯曲菌细胞时，没有获得RT-qPCR信号。相比之下，使用基于DNA的qPCR方法，可以检测到死亡或不活的弯曲杆菌细胞，即使在储存20天后，所有测试样本都是阳性的。所建立的直接从鸡粪中检测和定量检测空肠弯曲菌活细胞的方法可用于进一步研究弯曲杆菌在环境中的生存情况。(C)2011年巴斯德研究所。爱思唯尔·马森公司出版。版权所有。
Campylobacter spp. is the most common cause of bacterial diarrhoea in humans worldwide. Therefore, rapid and reliable methods for detection and quantification of this pathogen are required. In this study, we have developed a reverse transcription quantitative real-time PCR (RT-qPCR) for detection and quantification of viable Campylobacter jejuni directly from chicken faecal samples. The results of this method and a DNA-based quantitative real-time PCR (qPCR) method were compared with those of a bacterial culture method. Using bacterial culture and RT-qPCR methods, viable C. jejuni cells could be detected for up to 5 days in both the C. jejuni spiked and the naturally contaminated faecal samples. We found that no RT-qPCR signals were obtained when viable C. jejuni cells could not be counted by the culture method. In contrast, using a DNA-based qPCR method, dead or non-viable Campylobacter cells were detected, and all tested samples were positive, even after 20 days of storage. The developed method for detection and quantification of viable C. jejuni cells directly from chicken faecal samples can be used for further research on the survival of Campylobacter in the environment. (C) 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.