Identification of parasite DNA in common bile duct stones by PCR and DNA sequencing

Abstract

We attempted to identify parasite DNA in the biliary stones of humans via PCR and DNA sequencing. Genomic DNA was isolated from each of 15 common bile duct (CBD) stones and 5 gallbladder (GB) stones. The patients who had the CBD stones suffered from cholangitis, and the patients with GB stones showed acute cholecystitis, respectively. The 28S and 18S rDNA genes were amplified successfully from 3 and/or 1 common bile duct stone samples, and then cloned and sequenced. The 28S and 18S rDNA sequences were highly conserved among isolates. Identity of the obtained 28S D1 rDNA with that of Clonorchis sinensis was higher than 97.6%, and identity of the 18S rDNA with that of other Ascarididae was 97.9%. Almost no intra-specific variations were detected in the 28S and 18S rDNA with the exception of a few nucleotide variations, i.e., substitution and deletion. These findings suggest that C. sinensis and Ascaris lumbricoides may be related with the biliary stone formation and development.

Fig. 1

PCR amplification of 28S D1 (A) and 18S rDNA (B) gene fragments from DNA extracted from 15 human common bile stones. The samples were separated via electrophoresis on 2% agarose gel. Lane M: size marker (0.4 µg, 1 kbp plus DNA ladder, Invitrogen). In panel A; Lane 1, 7 and 8: 28S D1 rDNA gene for C. sinensis positive patients (1: a 90-year-old female, 7: a 69-year-old male, 8: a 51-year-old male), In panel B; Lane 7: 18S rDNA gene for A. lumbricoides positive patients (7: a 69-year-old male).

Fig. 2

Nucleotide sequences of a region of the 28S D1 rDNA gene of Clonorchis sinensis from the common bile duct stone extracts compared to modern sequences among C. sinensis and O. viverrini. A dot (.) denotes a same identical nucleotide position. A non-identical nucleotide denotes nucleotide addition in same position. Each of primer sequences on both sides is indicated by an underline. Alignment gaps are indicated by a hyphen. Sequences for each species have been deposited in the GenBank databases (GenBank accession No.: C. sinensis; EF654661 (stone), AF188121 (K; Korea isolate), AF217096 (C; China isolate), O. viverrini (AF408149).

Fig. 3

Nucleotide sequences of a region of the 18S D1 rDNA gene of Ascaris lumbricoides from the common bile duct stone extracts as compared to modern sequences among the family Ascarididae. A dot (.) denotes an identical nucleotide position. A non-identical nucleotide denotes nucleotide addition at the same position. Each of the primer sequences are indicated by an underline. The sequence is from GenBank (EF654660; A. lumbricoides from stone, U94366; A. lumbricoides from human, AF036587; A. suum from pig, and U94383; Toxascaris leonina from dog).