摘要
以往甲型肝炎病毒(HAV)的检测主要依赖细胞培养法,但此法所需时间长、敏感性低、特异性不强。虽然反转录PCR等技术已被用于粪便或食品中HAV的检测,但常规反转录PCR操作繁琐、污染环节多,极易造成污染,致使实验结果不稳定或出现假阳性结果。为克服PCR检测HAV的缺点和不足,我们在实验基础上建立了一步单管反转录PCR技术检测水中的HAV,采用热裂解法提取样品中HAV病毒的总RNA,反转录与PCR反应一步进行,整个反应在一个反应管中进行,操作方法较简单,中间可能污染的环节较少,因此,最大限度的减少了由环境造成的可能污染。我们设计合成的引物(H1—H2)在HAV病毒核酸的VP1区,只特异地扩增HAV核酸片断,具有较强的特异性。改进后的反转录PCR的敏感性与常规反转录PCR比有所提高,可检测到细胞培养悬液或水中10个TCID_(50)的HAV。
A simple, sensitive and specific PCR was developed for the detection of hepatitis A virus (HAV)RNA in samples from water. The strategy included a direct extraction of HAV RNA from samples by thermal cracking, one step PCR amplification in combination with the step of reverse transcription, and only one tube was used in the whole reaction which minimized the contamination risks. The one-step and a single tube PCR method was used to detect the presence of HAV RNA in water or cell culturesand the minimum concentration of HAVs detectable was 10 TCID50.
出处
《中国卫生检验杂志》
1996年第2期63-66,共4页
Chinese Journal of Health Laboratory Technology