Establishment and application of TaqMan probe-based quantitative real-time PCR for rapid detection and quantification...

Ichthyophthirius multifiliis, a pathogenic ciliate, is a crucial pathogen of freshwater fish and can result in severe economic loss in the aquaculture industry worldwide. It is necessary to develop a sensitive and accurate method for detecting I. multifiliis in farming environments and fish skin and gills to protect fishes from infection of the parasite due to a lack of both safe and effective treatment drugs. The present study established a new TaqMan probe-based quantitative PCR (qPCR) detection method targeting the coding region of the cathepsin L cysteine protease (ICP2) gene of I. multifiliis. The sensitivity, specificity, reproducibility and application for detection and diagnosis of the TaqMan probe-based qPCR method were evaluated. In addition, the linear model between the cycle threshold (Ct) and the logarithmic starting quantity (SQ) of the number of theronts per 1 L of sterile water was developed as Ct = -3.312lg(SQ)+ 34.47 with an R² of 0.9636 and a minimum detection limit of 4 theronts per 1 L of water and could be employed to determine the theront number based on Ct value. The results of the detection of trial infection samples with the TaqMan probe-based qPCR method showed that the tissues of fish individuals infected with I. multifiliis and the tank water samples were positive detection signals. In contrast, the tissues and water samples from uninfected fish individuals and tanks containing healthy fish showed no signals. The detection results demonstrated the reliability of this detection method. Overall, the novel TaqMan probe-based qPCR method with high sensitivity and specificity as well as repeatability for detection of I. multifiliis was a valuable tool in detecting the parasite in farming water, pond sediments, and fish tissues and could provide early warning for prevention of the disease caused by I. multifiliis.