INTRODUCTION: Intestinal pathogens are usually diagnosed by isolation and identification of the organisms from fecal cultures. Culture methods are precise, but relatively time-consuming, labor-intensive and difficult for some fastidious intestinal pathogens. Polymerase chain reaction (PCR) is efficient, specific and a sensitive technique for the detection of organisms and are increasingly applied to the diagnosis of infectious diseases. We aimed to evaluate five different methods to extract DNA from stool samples to be used as a PCR template.
MATERIAL AND METHODS: Fecal samples collected from children living in Fortaleza, Ceara, Brazil, were previously defined as positive for Cryptosporidium sp. and enteroaggregative Escherichia coli (EAEC). These stool samples were pretreated with 1) two cycles of freeze-thawing plus alkaline treatment with potassium hydroxide (KOH) and dithiotreitol (DTT), 2) alkaline treatment with KOH and DTT, 3) one cycle of freeze-thawing using liquid nitrogen, 4) three cycles of freeze-thawing using liquid nitrogen, and 5) no pretreatment. All aliquots were submitted to the QIAamp DNA Stool Mini Kit (Qiagen, Valencia, CA). PCR products for specific genes of Cryptosporidium sp. and EAEC were visualized after 1.2% agarose gel electrophoresis and ethidium bromide staining.
RESULTS: Nested PCR amplification of the 18S rRNA gene of Cryptosporidium sp. and a single PCR of the aggR gene of EAEC showed differences among the types of pre-treatment tested. Analysis of gel bands intensity revealed variations among the methods, although all of them had originated bands clearly visible. For Cryptosporidium sp., the techniques 1 (physical + chemical pretreatment), 2 (chemical pretreatment only), and 5 (no pretreatment) resulted bands with similar intensity. For EAEC, the option 5 yielded the best level of detection.
CONCLUSION: Our comparative study indicated that the absence of pretreatment was similar to other methods for Cryptosporidium sp., and was superior for EAEC concerning to the isolation of PCR-amplified DNA from fecal samples. The use of untreated fecal samples directly as target for pathogen DNA extraction reduces time and labor work.
FINANCIAL SUPPORT: FIC/NIH (USA) and CNPq (Brazil)