Nested reverse transcriptase-polymerase chain reaction for the detection of group A rotaviruses
Nested reverse transcriptase-polymerase chain reaction for the detection of group A rotaviruses
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DOI：
10.1046/j.1439-0450.2002.00510.x
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发表时间：
2002-03-01
期刊：
影响因子：
--
通讯作者：
Sachse, K
中科院分区：
文献类型：
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作者：
Elschner, M;Prudlo, J;Sachse, K
轮状病毒是与几乎所有哺乳动物物种的腹泻病相关的重要病原体。在本研究中，开发了一种用于检测A组轮状病毒的巢式逆转录聚合酶链式反应（RT-PCR），该反应基于基因片段6中的目标区域。人、牛、猪的轮状病毒株。对犬科动物、猫科动物、马科动物和绵羊的起源进行了检查。此外，该研究还包括一些粪便样本，其中已经使用 PCR 以外的其他方法检测到了轮状病毒。所有测试的菌株和粪便样本均形成巢式 RT-PCR 产物。 RT-PCR 检测含病毒细胞培养上清液的检测限为 3 x 10(-2) [50%组织培养感染剂量 (TCID50)]，巢式扩增检测限为 3 x 10(-3) TCID50。为了检查样品基质对灵敏度的影响，在轮状病毒阴性粪便样本中掺入了猪轮状病毒 OSU 的含病毒细胞培养悬浮液。目前 PCR 程序的检测限约为每克粪便 1.6 x 10(2) TCID50，并且使用巢式 PCR 可以将其提高一个数量级。本发明用于检测和鉴定A组轮状病毒的方法代表了一种强大的诊断工具，并且被证明适用于不同来源的轮状病毒，包括人类来源的轮状病毒。
Rotaviruses are important pathogens associated with diarrhoeal diseases in almost all species of mammals. In the present study, a nested reverse transcriptase-polymerase chain reaction (RT-PCR) for the detection of group A rotaviruses was developed, which is based on a target region in gene segment 6. Rotavirus strains of human, bovine, porcine. canine, feline, equine, and ovine origin were examined. Furthermore several faecal specimens, in which rotavirus had already been detected using other methods than PCR, were included in the study. A nested RT-PCR product was formed with all strains and faecal samples tested. The detection limit for virus-containing cell culture supernatant was 3 x 10(-2) [50% tissue culture infective dose (TCID50)] by RT-PCR and 3 x 10(-3) TCID50 by nested amplification. In order to examine the influence of the sample matrix on sensitivity, a rotavirus-negative faecal specimen was spiked with virus-containing cell culture suspension of the porcine rotavirus OSU. The detection limit of the present PCR procedure was approximately 1.6 x 10(2) TCID50 per g faeces and could be increased by one order of magnitude using nested PCR. The present method for detection and identification of group A rotaviruses represents a powerful diagnostic tool and was shown to be applicable to rotaviruses of different origin, including human sources.