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

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Biomedical and Environmental Sciences 2010年第2期23卷 146-150页

作者： SEHEE NAM gyucheol lee Water Analysis and Research Center K-water Daejeon 306-711 Republic of Korea

Objective To simultaneously detect viable Cryptosporidium parvum oocysts and Giardia duodenalis cysts for the purpose of reducing time and cost spent. Methods A duplex reverse transcription polymerase chain reaction （RT-PCR） method was newly developed. Results Using duplex RT-PCR method for the hsp70 gene, viable （oo）cyst concentrations of 10^1 and l0^3 （oo）cysts/100 *** 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 μL 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. Conclusion 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.

关键词： Cryptosporidiumparvum Giardia duodenalis Heat-shock RT-PCR

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Detection and Molecular Characterization of Enteroviruses in Korean Surface Water by Using Integrated Cell Culture Multiplex RT-PCR

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Biomedical and Environmental Sciences 2008年第5期21卷 425-431页

作者： gyucheol lee CHANHEE lee CHANSEUNG PARK SANGGI JEONG Water Analysis and Research Center Kwater Division of Life Sciences Chungbuk National University

Objective To identify waterborne enteric viruses in Korean surface water. Methods Integrated cell culture（ICC）multiplex reverse transcription-polymerase chain reaction （RT-PCR） was simultaneously designed to detect coxsackieviruses （CV）, polioviruses （PV）, and reoviruses （RV）. ICC-multiplex RT-PCR and phylogenetic analysis were conducted using 21 total culturable virus assay （TCVA）-positive sample-inoculated cell cultures. Results CV and RV were detected in 9 samples each, and 3 samples were positive for both CV and RV. PV was not detected in any sample. Molecular phylogenetic analysis of the VP1 gene sequences revealed that CV types B2 and B4 predominated in Korean surface water, and the nucleotide sequences of CV type B2 were clustered with those of CVs isolated from China and Japan. The results suggested that the evolution of these viruses occurred in a region-specific manner. Conclusion CV and RV are detectable in Korean surface water, with a predominance of CV type B2, and the evolution of CV type B2 occur in a region-specific manner.

关键词： ICC-multiplex RT-PCR Coxsackieviruses Molecular phylogeny

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