Use of silica as a carrier to recover and prepare waterborne enteric viruses for detection by RT-PCR.
A rapid, efficient and inexpensive method was developed to concentrate poliovirus type 1 (PV1), rotavirus (RV) and hepatitis A virus (HAV) from artificially spiked samples of tap and surface water. The method consists of adsorbing the viruses to silicon dioxide (SiO2) in the presence of 0.5 mM AlCl3 and adjustment of the pH to 3.5. The silica-adsorbed virus was collected by low speed centrifugation. Viral RNA was then extracted with guanidium thiocyanate (GT), and environmental nucleases and inhibitors of reverse transcriptase and Taq polymerase were further eliminated from concentrates by sequential treatment with GT, ethanol and acetone. Subsequent RT-PCR allowed the detection of as few as 1 to 10 TCID50 of PV1, RV, and HAV in seeded 1 liter samples of tap water. The same protocol was then used with effluents from two local sewage treatment plants. These samples, found to be free of HAV, were most commonly contaminated with enteroviruses and rotaviruses. Addition of 1000 TCID50 of HAV, PV1 or RV to a second 1 liter sample, taken at the same time from the corresponding surface waters allowed detection of the input virus without discernible inhibition by amplification inhibitors. The newly established method seems amenable to scaling up and promising for virus monitoring in different water types. The method is rapid and results can be obtained within 24 to 36 hours.
Leisinger
M
M
Institute of Veterinary Bacteriology, Veterinary Medical Faculty, University of Zürich, Switzerland.
Metzler
A
A
D005243:Feces / Q000821:virology; D006507:Hepatovirus / Q000302:isolation & purification*; D006801:Humans; D017955:Poliovirus / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000302:isolation & purification; D012401:Rotavirus / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D012822:Silicon Dioxide; D014871:Water Microbiology; D014881:Water Supply
D012367:RNA, Viral; D012822:Silicon Dioxide