Vibrio parahaemolyticus is a bacteria that is a major cause of food poisoning. Thermostable direct hemolysin (TDH) is known as one of the food poisoning factors produced by this pathogen. This kit allows real-time PCR detection of the V. parahaemolyticus thermostable direct hemolysin gene (tdh gene).

This kit includes Takara Ex Taq HS, a hot-start PCR enzyme, which prevents non-specific amplification caused by mispriming or primer-dimer formation during reaction mixture preparation or other pre-cycling steps. The use of Takara Ex Taq HS, therefore, allows high-sensitivity detection. This kit enables amplification products to be detected by cycling-probe technology, which provides highly sensitive detection through the combined use of an RNA-DNA chimeric probe and RNase H. This enables efficient detection of specific sequences of the gene fragment during and after amplification. One end of the probe is labeled with a fluorescent moiety and the other end with a quencher. When intact, this probe does not emit fluorescence due to the action of the quencher. However, when it forms a hybrid with the complementary sequence of an amplification product, RNase H cleaves RNA in the chimeric probe, resulting in strong fluorescent-signal emission. The amount of amplified product can be monitored by measuring the intensity of emitted fluorescence.

This kit incorporates the FAM-labeling probe for detecting the tdh gene target and the ROX-labeling probe for detecting the internal control. By simultaneously monitoring two wavelengths, tdh gene detection and false-negative monitoring through detection of the internal control DNA can be achieved in a single tube. The kit relies on real-time PCR detection, which requires no electrophoresis and yields results quickly.

This kit was developed in cooperation with Dr. Kanji Sugiyama, Shizuoka Institute of Environment and Hygiene.