Comparative study of two extraction methods for enteric virus recovery from sewage sludge by molecular methods.
ABSTRACT The aim of this study was to compare two nucleic acid extraction methods for the recovery of enteric viruses from activated sludge. Test samples were inoculated with human adenovirus (AdV), hepatitis A virus (HAV), poliovirus (PV) and rotavirus (RV) and were then processed by an adsorption-elution-precipitation method. Two extraction methods were used: an organic solvent-based method and a silica method. The organic-based method was able to recoup 20% of the AdV, 90% of the RV and 100% of both the PV and HAV from seeded samples. The silica method was able to recoup 1.8% of the AdV and 90% of the RV. These results indicate that the organic-based method is more suitable for detecting viruses in sewage sludge.
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ABSTRACT: Sewage sludge and treated wastewater when contaminated with enteric virus and discharged into the environment, could pose a human health risk. The aim of study was to verify the presence and viability of enteric viruses in sewage sludge and treated wastewater at a local sewage plant in Florianopolis city, Brazil. Sewage sludge was concentrated by organic flocculation and polyethylene glycol precipitation and wastewater by electronegative membrane filtration and ultrafiltration by Centriprep Concentrator. Adenovirus (AdV), hepatitis A virus (HAV), and Rotavirus (RV) were examined for all samples for 12 months and Poliovirus (PV) was also tested for in sewage sludge samples. AdV was the most prevalent in both kind of samples, followed by RV, PV (in sludge) and HAV. Viral viability by cell culture (ICC-PCR) was: AdV: 100%, HAV: 16.7%, PV: 91.7%, RV: 25% in sludge and AdV: 66.6%, HAV: 66.6% and RV: 0% in wastewater. IFA for AdV in sludge ranged from 70 to 300 FFU/ml. QPCR for AdV ranged from 4.6 x 10(4) to 1.2 x 10(6) and from 50 to 1.3 x 10(4) gc/ml in sludge and wastewater, respectively. HAV quantification in sludge ranged from 3.1 x 10(2) to 5.4 x 10(2) gc/ml. In conclusion, it was possible to correlate presence and viability of enteric viruses in the environmental samples analyzed.Water Science & Technology 01/2010; 61(2):537-44. · 1.21 Impact Factor
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ABSTRACT: The polymerase chain reaction (PCR) has become an essential method for the detection of viruses in tissue specimens. However, it is well known that the presence of PCR inhibitors in tissue samples may cause false-negative results. Hence the identification of PCR inhibitors and evaluation and optimization of nucleic acid extraction and preservation methods is of prime concern in virus discovery programs dealing with animal tissues. Accordingly, to monitor and remove inhibitors we have performed comparative analyses of two commonly used tissue storage methods and 5 RNA purification techniques using a variety of animal tissues, containing quantified levels of added MS2 bacteriophages as the indicator of inhibition. The results showed (i) no significant difference between the two methods of sample preservation, viz direct storage at -80C or 4C in RNAlater, (ii) lung rodent tissues contained lower levels of inhibitor than liver, kidney and spleen, (iii) RNA extraction using the EZ1+PK RNA kit was the most effective procedure for removal of RT-PCR inhibitors.Journal of virological methods 03/2013; · 2.13 Impact Factor