[Development of efficient DNA isolation procedures for Cryptosporidium and Trichinella PCR detection in fecal...

PCR detection of genetic material of the parasites present in faeces may be an alternative for microscopic and serological tests routinely used for diagnosing parasitic enteral infections. However, small amount of target DNA combined with low efficiency of total DNA extraction, and presence of PCR inhibitors in the samples to be amplified, may cause false negative detection results. The aim of this work was to evaluate the impact of DNA isolation procedure used on the amplification of DNA fragments from the genomes of protozoan Cryptosporidium parvum and the nematode Trichinella spiralis. Two methods based on different principles of biological material lysis were evaluated; NucliSENS miniMAG employing simultaneously applied chemical lysis and mechanical disruption or mechanical disruption followed by enzymatic lysis in case of QIAamp DNA Stool Mini Kit. Both of the analyzed systems for nucleic acids purification allowed isolation of DNA from purified Cryptosporidium parvum oocysts and Trichinella spiralis muscle larva mixed with mouse or pig faeces. However, sensitivity of PCR detection of DNA obtained by enzymatic lysis based method was lower compared to the one achieved with DNA isolated by chemical lysis. When QIAamp DNA Stool Mini Kit procedure was used detection limit was 5 and 10 fold higher for Cryptosporidium parvum and Trichinella spiralis, respectively. Only NucliSENS miniMAG procedure combined with mechanical disruption of the faecal material allowed detection of Cryptosporidium sp. in human fecal samples collected from children with diahorrea, and detection of Trichinella spiralis in stool samples obtained, on days 8 to 12 post infection, from mice experimentally infected with 500 muscle larvae per mouse. Thorough mechanical disruption with simultaneous chemical lysis allows efficient isolation ofDNA of the investigated intestinal parasites present in stool and the subsequent PCR detection.