联合免疫磁珠分离-分子信标-逆转录-聚合酶链反应法检测环境样本中的甲型肝炎病毒
Combined immunomagnetic separation-molecular beacon-reverse transcription-PCR assay for detection of hepatitis A virus from environmental samples.
作者信息
Abd El Galil Khaled H, El Sokkary M A, Kheira S M, Salazar Andre M, Yates Marylynn V, Chen Wilfred, Mulchandani Ashok
机构信息
Department of Microbiology, Mansoura University, Egypt.
出版信息
Appl Environ Microbiol. 2004 Jul;70(7):4371-4. doi: 10.1128/AEM.70.7.4371-4374.2004.
Abstract
In this study, a molecular-beacon-based real-time reverse transcription (RT)-PCR assay was developed to detect the presence of hepatitis A virus (HAV) in environmental samples. A 125-bp, highly conserved 5' noncoding region of HAV was targeted. The sensitivity of the real-time RT-PCR assay was tested with 10-fold dilutions of viral RNA, and a detection limit of 1 PFU was obtained. The specificity of the assay was demonstrated by testing with other environmental pathogens and indicator microorganisms, and only HAV was positively identified. When combined with immunomagnetic separation, the real-time RT-PCR assay successfully detected as few as 20 PFU in seeded groundwater samples. Because of its simplicity and specificity, this assay has broad applications for the rapid detection of HAV in contaminated foods or water.
摘要
在本研究中，开发了一种基于分子信标的实时逆转录（RT）-PCR检测方法，用于检测环境样本中甲型肝炎病毒（HAV）的存在。靶向HAV高度保守的5'非编码区的125bp片段。通过对病毒RNA进行10倍稀释来测试实时RT-PCR检测方法的灵敏度，获得了1个噬斑形成单位（PFU）的检测限。通过检测其他环境病原体和指示微生物证明了该检测方法的特异性，且仅能阳性鉴定出HAV。当与免疫磁分离相结合时，实时RT-PCR检测方法成功地在接种的地下水样本中检测到低至20个PFU的HAV。由于其简单性和特异性，该检测方法在快速检测受污染食品或水中的HAV方面具有广泛的应用。
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