HOW GOOD ARE THE MICROBIAL DETECTION TECHNIQUES: CAN WE EFFICIENTLY DETECT PATHOGENS IN BIOSOLIDS?

Biosolids are defined as the solid, semisolid or liquid residue generated during the treatment of domestic sewage in a treatment facility. Biosolids can be recycled and applied as fertilizer to improve and maintain soil productivity. However, 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 within the environment including biosolids and can limit the use of PCR. Some of the inhibitors characterized in environmental samples include bile salts, collagen, fulvic acid, heparin, hemoglobin, humic acids, polysaccharides, urea, and melanin.The main objective of this study was to determine the appropriateness, reliability, and cost-effectiveness of molecular techniques for the detection of pathogens at various stages of biosolids production and management and to compare with the traditional culture-based techniques. Five commercially available DNA/RNA purification kits listed below were compared for the detection of pathogens in biosolids; QIAamp DNA Mini Kit (Qiagen), High Pure RNA Isolation Kit (Roche), Fast DNA Spin Kit (Q-bio gene), Powersoil DNA isolation Kit (Mobio), and Ultra Clean Soil DNA Kit (MoBio). Biosolids samples were collected and pathogens were eluted using elution buffer containing 3% Beef Extract and 3% Citric Acid. The sample eluent was then purified using above-mentioned kits. The purified sample was then analyzed by PCR/RT-PCR for the presence of viruses. Recovery efficiency of the pathogen recovery methods was established by processing biosolids samples spiked with MS2, PRD1, and Poliovirus type 2. The optimized virus recovery method was applied to biosolids samples for the detection of indigenous adenovirus and rotavirus.Results indicate that the Qiagen DNA purification kit and Powersoil kit was able to effectively purify nucleic acid from biosolids for PCR application and was able to accurately amplify Poliovirus genome in biosolids. Ten fold dilutions of the sample concentrate aided in lowering the inhibitor concentrations in the final reaction. Nine out of ten sub samples of land-applied biosolids tested positive for adenovirus by PCR, supporting the reproducibility of developed technique. In addition, sequencing data of the PCR products indicates Human Adenovirus Type 41 isolate. The newly developed technique increases the applicability of PCR amplification for the detection of pathogen in biosolids. Applicability of our molecular method can offer a better classification tool for Biosolids in respect to microbial pathogens (Class A and Class B).