Development of a real-time PCR assay for the detection of Clonorchis sinensis DNA in gallbladder bile and stone samples from patients with cholecystolithiasis.
High prevalence of cholecystolithiasis in parts of East Asia has been postulated to be associated with Clonorchis sinensis infection. This study describes the development of a TaqMan-based real-time PCR assay for the detection of C. sinensis DNA in gallbladder bile and stone samples from patients with cholecystolithiasis. Primers and probe targeting the cytochrome c oxidase subunit 1 gene of mitochondrial DNA proved to be highly specific for C. sinensis and did not amplify other related heterogeneous DNA samples. The detection limit of this assay was 0.1 pg of adult C. sinensis genomic DNA. All of the egg-positive samples determined by microscopy yielded positive results by real-time PCR assay and that genetic testing of gallbladder stones using real-time PCR was considered as the most effective means for assessing C. sinensis infection status. This assay not only contributes to a greater understanding of stone pathogenesis but also benefits patients with cholecystolithiasis by facilitating effective diagnosis, treatment, and relapse prevention.
Institute of Gallbladder Disease of Panyu, Guangzhou 511470, China. fqj1958@163.com
Tie
T
Qiao
Pei-Ming
PM
Zheng
Rui-Hong
RH
Ma
Xiao-Bing
XB
Luo
Zhen-Liang
ZL
Luo
D000328:Adult; D000818:Animals; D001208:Asia; D001646:Bile / Q000469:parasitology*; D041761:Cholecystolithiasis / Q000150:complications*; D003003:Clonorchiasis / Q000175:diagnosis* / Q000469:parasitology; D003004:Clonorchis sinensis / Q000235:genetics / Q000302:isolation & purification*; D017931:DNA Primers / Q000235:genetics; D018509:DNA, Helminth / Q000235:genetics / Q000302:isolation & purification*; D003576:Electron Transport Complex IV / Q000235:genetics; D005260:Female; D005704:Gallbladder / Q000469:parasitology*; D006801:Humans; D008297:Male; D008875:Middle Aged; D025202:Molecular Diagnostic Techniques / Q000379:methods; D015345:Oligonucleotide Probes / Q000235:genetics; D010274:Parasitology / Q000379:methods; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D017931:DNA Primers; D018509:DNA, Helminth; D015345:Oligonucleotide Probes; D003576:Electron Transport Complex IV