Development Of A TaqMan Based Real-time PCR Assay For Detection Of Clonorchis Sinensis DNA In Bile And Gallbladder Stones

Objective:To assess the feasibility of detecting Clonorchis sinensis DNA in the gallbladder bile and gallbladder stones from cholecystolithiasis patients using real-time PCR based on the Taqman probe.Methods:30cases of gallbladder bile and gallbladder stones were randomly selected from the patients underwent removal stones without gallbladder excision during August-September,2011. These specimens were first conducted microscopic examination, and then extracted DNA. Meanwhile, the stools of the patients were collected and performed microscopic detection. According to the cytochrome C oxidase subunit1gene of C. sinensis, to design and synthesis of specific primers, TaqMan probe, and the real-time fluorescence PCR for C. sinensis detection was established. Subsequently, the sensitivity and specificity of the method and the feasibility of detecting C. sinensis DNA in bile and stones were assessed.Results:1. The detection limit of this assay was0.1pg of adult C. sinensis genomic DNA, and this assay produced negative amplification reaction with Schistosoma japonicum, Ascaris lumbricoides, Guangzhou Angiostrongylus and Toxoplasma.2. Fourteen cases of gallbladder bile were found C. sinensis eggs (positive rate of46.7%), the result was coincident with the result from real-time fluorescent PCR (the positive coincidence rate was100%);16cases of gallbladder stones were found C. sinensis eggs (positive rate of53.3%), while19cases of gallbladder stones DNA were real-time PCR-positive results (positive rate63.3%);9of30 stools (30%) were found C. sinensis eggs.Conclusions:1. Cytochrome C oxidase subunit1gene (COX-1) of C. sinensis can be used as target gene for diagnosis of C. sinensis adult worms and eggs in the bile or stones specimens.2. The COX-1gene detection of gallbladder stones through real-time PCR maybe the most effective means for the judgement of C. sinensis infection. This method is fast, simple, high accuracy, and has an important guiding significance for the cholecystolithiasis patients with removal stones without gallbladder remove in postoperative treatment, relapse prevention and the improvement of the diagnosis of cholecystolithiasis complicated with C. sinensis infection.