Detection of enteric viruses in municipal sewage sludge by a combination of the enzymatic virus elution method and RT-PCR
Detection of enteric viruses in municipal sewage sludge by a combination of the enzymatic virus elution method and RT-PCR
复制标题
DOI：
10.1016/s0043-1354(03)00208-2
复制
发表时间：
2003-08-01
期刊：
影响因子：
12.8
通讯作者：
Omura, T
中科院分区：
文献类型：
--
作者：
Sano, D;Fukushi, K;Omura, T
许多研究人员已经报道，致病性肠道病毒可以保留在城市污水污泥中。虽然RT-PCR技术已广泛应用于各种环境样品中的病毒检测，但由于基因扩增的抑制物质，RT-PCR在检测污水污泥中的病毒方面存在困难。然而，酶病毒洗脱（EVE）方法与RTPCR的组合，使其能够有效地检测病毒在污水污泥。EVE法中污泥絮体的酶解增强了接种脊髓灰质炎病毒1型（PV 1）的污水污泥中的病毒洗脱，并通过RT-PCR进行PV 1的检测，没有任何抑制。相反，使用10%牛肉提取物溶液的USEPA方法中的病毒测定中的RT-PCR的应用是不实际的，因为抑制病毒基因扩增。使用溶菌酶（多糖降解酶）、木瓜蛋白酶（蛋白酶）和胰凝乳蛋白酶（蛋白酶）的EVE方法与RT-PCR的组合导致31%的病毒回收效率，但是没有观察到这些酶对病毒回收效率的协同效应。溶菌酶或木瓜蛋白酶的EVE法是一种很有前途的病毒洗脱方法，可用于污水污泥中病毒的RT-PCR检测。(C)2003爱思唯尔科技有限公司版权所有。
Pathogenic enteric viruses can be retained in municipal sewage sludge as has been reported by many researchers. Although the RT-PCR technique has been extensively employed for the virus detection from various environmental samples, the application of RT-PCR to the detection of viruses in sewage sludge has the difficulty because of inhibitory substances to the gene amplification. However, a combination of the enzymatic virus elution (EVE) method with RTPCR made it possible to effectively detect viruses in sewage sludge. The enzymatic breakdown of sludge flocs in the EVE method enhanced the virus elution from poliovirus 1 (PV1)-inoculated sewage sludge, and the detection of PV1 was performed by RT-PCR without any inhibitions. On the contrary, the application of RT-PCR to the viral assay in the USEPA method using the 10% beef extract solution was not practical because of inhibitions to the viral gene amplification. The combination of the EVE method using lysozyme (polysaccharide-degrading enzyme), papain (protease), and chymotrypsin (protease) with RT-PCR resulted in a virus recovery efficiency of 31%, but a synergistic effect of these enzymes on the virus recovery efficiency was not observed. The EVE method using lysozyme or papain could be a promising procedure for the virus elution from sewage sludge in detecting these viruses with RT-PCR. (C) 2003 Elsevier Science Ltd. All rights reserved.