Giardia duodenalis: improved detection of viable cysts by reverse transcription-PCR of heat shock-inducible hsp70 gene.
Giardia duodenalis is a waterborne protozoan parasite that causes the diarrhoeal disease, giardiasis. Its durable and thick cell wall allows the parasite to exhibit resistance to environmental stresses. Because G. duodenalis exists in a water system at low levels, it is necessary to develop a sensitive method to detect its viability in aquatic environments. In the present study, specific primers for the heat shock protein (hsp) 70 gene were designed on the basis of G. duodenalis genome sequence and bioinformatic analysis. Viable G. duodenalis cysts were successfully distinguished by reverse transcription-PCR (RT-PCR) analysis using these primers. The amplicon of hsp70 was obtained from one cyst of G. duodenalis/100 microl, and this detection sensitivity significantly increased by 10(3)-fold when the cysts were given heat shock treatment. These findings prove that viable G. duodenalis cysts were successfully detected with a high degree of sensitivity by RT-PCR analysis targeting the hsp70 gene of G. duodenalis, thereby suggesting its practical potential for detecting viable G. duodenalis in environmental samples.
Lee
Gyu-Cheol
GC
Water Analysis and Research Center, K-Water, Daejeon 306-711, Republic of Korea. gclee@kwater.or.kr
Nam
Se-Hee
SH
Chae
Jong-Chan
JC
Lee
Chan-Hee
CH
D000818:Animals; D005872:Giardia / Q000145:classification / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics*; D010802:Phylogeny; D012333:RNA, Messenger / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014867:Water / Q000469:parasitology*
D018840:HSP70 Heat-Shock Proteins; D012333:RNA, Messenger; D014867:Water