Evaluation of DNA Melting Curve Analysis Real-Time PCR for Detection and Differentiation of Cryptosporidium Species

Cryptosporidium spp. are protozoan parasites that infect various vertebrate hosts. Although 10 species are recognized, at least five types have been found to infect human(1). Thus, the identification of Cryptosporidium spp. and genotypes is important for the assessment of public health issues and for tracking infection or contamination sources(2). Presently, the identification of Cryptosporidium spp. and genotypes is made mostly by PCR-RFLP or sequencing(3,4). Amplification of Cryptosporidium DNA fragments by PCR proved to constitute a sensitive and specific method to genotype Cryptosporidium. The PCR prot°Cols reported, however, require further pr°Cessing of the amplicon, which is time consuming and prone to false-positive results due to cross contamination. Recently developed closedtube, real time PCR methods can circumvent these problems(5). The light cycler system (R°Che Molecular Bi°Chemicals, Lewes, United Kingdom) is a combined thermal cycler and fluorimeter that allows PCR product detection and identification with a variety of fluorescence chemistries. The light-cycler system can provide a rapid PCR result because of very fast temperature transition rates and online analysis of data. In addition, there is a reduced risk of contamination because product detection is accomplished in a closed system(6). PCR amplicon detection is possible using the dye SYBR Green 1, which fluoresces only when bound to doublestranded DNA. On completion of PCR, amplicons are identified by measurement of their melting temperatures (Tms). It is possible to interpret the results of multiplex PCR using this system if the Tms of the specific amplicons are sufficiently different and constant to allow differentiation(7). Evaluation of DNA Melting Curve Analysis Real-Time PCR for Detection and Differentiation of Cryptosporidium Species Rasha H. Soliman1 and Ahmad A. Othman2 Departments of Medical Parasitology, Faculty of Medicine, Suez Canal University, Ismailia1 and Tanta University, Tanta2, Egypt