New strategies for the recovery and detection of microbial pathogens and indicators in various types of biosolids

Biosolids are defined as the solid, semisolid or liquid residue generated during the treatment of domestic sewage in a treatment facility. These biosolids may contain high levels of pathogens which can pose a threat to public health. Currently, little research has been done on pathogen detection within biosolids using molecular methods such as Polymerase Chain Reaction (PCR). Substances that inhibit enzyme activity are present at elevated levels in the environment including biosolids and can limit the use of PCR. The focus of this project was to develop biosolids sample processing methods for monitoring pathogens using molecular techniques. The approach used in this study was based on the isolation of pathogens from biosolids followed by the application of molecular techniques. In the first phase, nine methods were compared for recovering microorganisms from biosolids. The three top performing (glycine and beef extract based) methods were further optimized and compared with the methods recommended by the USEPA. The recovery/elution method developed in this study proved superior to USEPA methods for recovering bacteria, viruses and helminths from biosolids. Finally, glycine based method was selected because of its consistent performance over a broad range of biosolid types. The sample concentrates by glycine based method contained less amounts of humic acid like materials than the sample concentrates by beef extract based method; therefore, are more compatible with molecular methods for the detection of pathogens.; Five commercially available kits were compared for removing PCR inhibitors from sample concentrates and the Qiagen DNA purification kit was selected for integration into the new sample processing protocol, which included a phenol-chloroform extraction prior to the Qiagen cleaning step. The lower limit of detection for DNA by the new method was established between 10 and 100 ng in a 100mul PCR reaction. The sample concentrates cleaned by the newly developed method were shown to be compatible with PCR, RT-PCR, and Quantitative RT-PCR.; Our newly developed technique increases the applicability of PCR amplification for the detection of pathogens in biosolids. Applicability of our molecular method can offer better classification for Biosolids in respect to microbial pathogens (Class A and Class B).