Detection of enteric viruses in municipal sewage sludge by a combination of the enzymatic virus elution method and RT-PCR.
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 RT-PCR 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 US EPA 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.
Sano
Daisuke
D
Department of Civil Engineering, Graduate School of Engineering, Tohoku University, Aoba06, 980-8579 Sendai, Japan. sano@water.civil.tohoku.ac.jp
Fukushi
Kensuke
K
Yoshida
Yasuko
Y
Omura
Tatsuo
T
D001681:Biological Assay; D002918:Chymotrypsin / Q000494:pharmacology; D004279:DNA, Viral / Q000032:analysis*; D004770:Enterovirus / Q000235:genetics* / Q000302:isolation & purification / Q000472:pathogenicity*; D004784:Environmental Monitoring; D009113:Muramidase / Q000494:pharmacology; D010206:Papain / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012722:Sewage / Q000821:virology*; D014865:Waste Disposal, Fluid
D004279:DNA, Viral; D012722:Sewage; D009113:Muramidase; D002918:Chymotrypsin; D010206:Papain