Abstract
An improved reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay which uses 1μL of RNA extract was evaluated. The original RT-LAMP assay using 5μL of RNA extract showed generally high-sensitive detection of Cryptosporidium in water samples; but in some types of water such as pigsty effluents and it’s downstream river water, this assay failed to detect Cryptosporidium. This failure may be caused by substances in those water samples which block gene amplification. To eliminate the effect of gene amplification-blocking substances, we decreased the quantity of the RNA extract used for the RT-LAMP assay from 5μL to 1μL. The detection limit of the improved RT-LAMP assay was as low as 6×10-3 oocysts/LAMP test tube, which theoretically enables us to detect even one Cryptosporidium oocyst in a water sample. This improved RT-LAMP assay succeeded in detecting Cryptosporidium oocysts much more sensitively not only in pigsty effluents but also in other water samples as well.
