Evaluation of QIAamp DNA mini kit for removing of inhibitors in detection of Cryptosporidium parvum oocysts in water samples by a nested- PCR assay

ABSTRACT

In recent years, there has been a dramatic increase in the occurrence of waterborne disease outbreaks caused by the Cryptosporidium parvum, and presence of this protozoan parasite in drinking water is a significant health problem faced by the water industry. A new strategy for detection of Cryptosporidium oocysts in water samples is PCR' based techniques. In this study a nested' PCR assay was designed for the specific amplification of a 199 bp DNA fragment of the gene encoding the heat shock protein [hsp70] of Cryptosporidium parvum oocysts. In order to prevent the inhibition of PCR amplification by substances contained in water samples, three DNA purification methods including QIAamp DNA mini kit, InstaGene Matrix, MagExtractor ' Genome were compared in concentrates of tap water samples spiked with the oocysts. After it was found that the QIAamp is only efficient purification technique, the efficiency of QIAamp and immunomagnetic separation for nested'PCR assay of various water samples was compared. The results show that QIAamp provide a useful and rapid tool for removing of PCR inhibitors. It seems that QIAamp purification- nested PCR assay is a sensitive, rapid and cost effective method for detection of Cryptosporidium parvum oocysts in clean water samples with turbidity < 2 nephelometric turbidity unit [NTU]