Ko Gwangpyo, Jothikumar Narayanan, Hill Vincent R, Sobsey Mark D
University of Texas Health Science Center, School of Public Health, 1200 Herman Pressler Dr. RAS W-634, Houston, TX 77225, USA.
J Virol Methods. 2005 Aug;127(2):148-53. doi: 10.1016/j.jviromet.2005.02.017. Epub 2005 Apr 18.
Adenoviruses are among the most persistent and ubiquitous viruses in water and wastewater, but some of them are difficult to detect due to non-cytopathogenicity and slow growth in cell cultures. A TaqMan real-time RT-PCR method in conjunction with cell culture infectivity was developed to rapidly detect mRNA produced by infectious adenoviruses in water samples. Only infectious adenoviruses were detected by this method, based on their ability to produce mRNA during replication in cell culture. The mRNA of Ad41 was detected as soon as 3 days after infection at levels as low as 5 infectious units (IU) per cell culture. In order to confirm that our methods detected only infectious viruses, 1-ml volumes of 10(4)IU of Ad41 were exposed to different free chlorine doses. No mRNA was detected in cells inoculated with Ad41 treated with the highest free chlorine dose of 100 mg min/l. However, mRNA of adenovirus was detected in cells inoculated with virus that was untreated or exposed to a lower free chlorine dose of 1 mg min/l. These results suggest that mRNA detection by real-time RT-PCR is a sensitive and specific method to detect low levels of infectious adenoviruses in water and other environmental media within 1-3 days.
腺病毒是水和废水中最持久且分布最广的病毒之一，但其中一些由于无细胞病变效应以及在细胞培养中生长缓慢而难以检测。开发了一种结合细胞培养感染性的TaqMan实时逆转录聚合酶链反应（RT-PCR）方法，以快速检测水样中感染性腺病毒产生的信使核糖核酸（mRNA）。基于感染性腺病毒在细胞培养中复制时产生mRNA的能力，该方法仅能检测到感染性腺病毒。在感染后3天就能检测到Ad41的mRNA，在每个细胞培养物中低至5个感染单位（IU）的水平即可检测到。为了确认我们的方法仅检测感染性病毒，将1毫升含10⁴个IU的Ad41暴露于不同剂量的游离氯中。在用最高游离氯剂量100毫克·分钟/升处理的Ad41接种的细胞中未检测到mRNA。然而，在用未处理或暴露于较低游离氯剂量1毫克·分钟/升的病毒接种的细胞中检测到了腺病毒的mRNA。这些结果表明，通过实时RT-PCR检测mRNA是一种在1至3天内检测水和其他环境介质中低水平感染性腺病毒的灵敏且特异的方法。
