A New Duplex Reverse Transcription PCR for Simultaneous Detection of Viable Cryptosporidium parvum Oocysts and Giardia duodenalis Cysts

Abstract

To simultaneously detect viable Cryptosporidium parvum oocysts and Giardia duodenalis cysts for the purpose of reducing time and cost spent. A duplex reverse transcription polymerase chain reaction (RT-PCR) method was newly developed. Using duplex RT-PCR method for the hsp70 gene, viable (oo)cyst concentrations of 10(1) and 10(3) (oo)cysts/100 microL could be detected for C. parvum and G duodenalis, respectively. However, after heat-shock stimulation the expression of hsp70 mRNAs was detectable at 10(0) and 10(1) (oo)cysts/100 microL concentrations of C. parvum and G duodenalis, respectively. Thus, the detection sensitivity was significantly increased when the viable (oo)cysts were exposed to heat shock. This study describes a new duplex RT-PCR method for hsp70 gene to detect the viable (oo)cysts of the C. parvum and G duodenalis with less time consumed and at a lower cost. This newly developed duplex RT-PCR method may be used to detect these parasites not only in aquatic environments but also in clinical samples.