RESUMO
The extraction of DNA from manure and the subsequent polymerase chain reaction (PCR) amplification of virulence genes to detect pathogens require an effective method of purification. Four different methods were assessed for their effectiveness in extracting and purifying Escherichia coli O157:H7 DNA from cattle manure: phenol/chloroform purification, phenol/chloroform/Sepharose B4 spin columns, phenol/chloroform/polyvinylpolypyrrolidone (PVPP) spun columns, and Mo Bio UltraClean kit. A PCR assay targeting the shiga-like toxin I gene (sltI) was carried out to determine the effectiveness of the four methods in removing PCR inhibitors from the manure samples. All methods were used to extract a manure slurry and the cleanliness of the samples was tested by the PCR with varying concentrations of spiked E. coli O157:H7 target DNA. The PVPP spun columns and the UltraClean kit had the best detection limit, detecting 20 pg of E. coli DNA (about 2x10(3) cells) per 100 mg of manure. The UltraClean kit and the PVPP spun columns also had the best and similar detection limits of 3x10(4) CFU/100 mg manure when E. coli O157:H7 cells were spiked into the manure sample and purified by all four methods. The enrichment of cells after inoculation into manure was performed using tryptic soy broth at 37 degrees C for 5 h. Both the PVPP spun columns and the UltraClean kit methods were used to purify the enriched samples and were able to detect initial inocula of 6 CFU/100 mg manure, indicating that the two methods were highly efficient in purifying DNA from manure samples.
