23.8363.37 and 23.0863.97 (Mean 6SD) respectively. Of all the patients, 49.7% (91/183), 14.2% (26/183), 30.1% (55/183) and 6.0% (11/183) were with moderate weight, underweight, over- weight and obesity. During the operation, no indications such as bile leakage and common bile duct injury occurred. The number of stones from each patient ranged from one to hundreds; single stones and multiple stones was found in 21.9% (40/183) and 78.1% (143/ 183) of patients respectively. All stones from one patient were counted as one case; all the 183 cases of stones were analyzed in the present study. Microscopic Examination of Gallbladder Stones (Chinese National Inventive Patent Number: ZL201010123552.3) Gallbladder stones were washed twice with distilled water, and then dried at 60uC for 12 hours. Subsequently, stones were split, and about 10 mg of each layer was weighed if the hierarchical structure of the profile was clear. Otherwise, about 10 mg was weighed directly, put in a mortar and 300 ml of 0.9% NaCl was added. The stones were ground thoroughly and then filtered with a 260 mesh nylon yarn. Each filtrate was then smeared onto 3–4 labeled slides and scored for the presence or absence of C. sinensis eggs under an Olympus System Microscope (BX51, Japan). Detection of C. sinensis DNA in the Gallbladder Stones by Real-time Fluorescent PCR Gallbladder stones that were scored positive or negative for eggs (thirty each) based on morphology were chosen randomly from the pool of stone samples, for confirmation of the presence of C. sinensis DNA using real-time fluorescent PCR. DNA from a C. sinensis adult was used as a positive control. Extraction of DNA. Adult of C. sinensis: The adult worm of C. sinensis obtained from clinical patients was first washed twice with 0.9% NaCl, then dried on the filter paper and ground thoroughly in a mortar by adding liquid nitrogen constantly. Only a single specimen was used and DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen) according to manufacturer’s instructions. Briefly, the grinding powder was suspended into 180 ml of ATL buffer, and then 20 ml proteinase-K was added and incubated at 58uC for 4 h with brief vortexing every 30 min. Thereafter, 200 ml AL buffer and 200 ml absolute alcohol was added successively and mixed by vortexing for 15–20 s. Finally, the genomic DNA was collected using DNeasy Mini spin column and eluted in 100 ml elution buffer (AE) and stored at 220uC until use. Gallbladder stones: For mechanical breaking, 20 mg of the stones were weighed and frozen in liquid nitrogen gas for 10 min, and then ground thoroughly in a mortar. After adding 1 ml 0.9% NaCl, the suspension was transferred to a new 2.0 Ep tube and centrifuged at 12000 rpm for 10 min. The pellet was treated and used for DNA extraction with a DNeasy Blood Tissue Kit (Cat.No. 69504, Qiagen, Germany) in accordance with the manufacturer’s instructions. Fluorescent PCR. The primers and Taqman probe were designed to detect the cytochrome C oxidase subunit 1 gene of C. sinensis (GenBank: FJ965388.1) using the Beacon Designer-v 7.51 software. It was then submitted to the BLAST program of NCBI for specific analysis. These primers Cs-F (59-GGTTTGGTAT- GATTAGTCACATTTG-39) and Cs-R. (59-ACCACCCTACCCAGACAAAC-39) amplify a 121 bp fragment of the COI sequence. The minor-groove-binding TaqMan-probe Cs-P. (59-JOE-AGCAAACATAGCCAACACCAAGCCC-BHQ1-39) was used to detect the C. sinensis specific product. The real-time Cholecystolithiasis Is Associated with Clonorchis PCR assay was run in a total reaction volume of 50 ml. The final concentration of the reaction solution included 0.2 mM of both forward and reverse primers, 0.2 mM of the TaqMan probe, 25 ml of Premix Ex TaqTM (Takara), 1 ml ROX buffer and 2 ml templates. The real-time PCR cycling parameters were set as follows: initial 95uC for 30 s, then 45 cycles of 15 s at 95uC and 31 s at 60uC. PCR amplification, detection, and data analysis were performed with an ABI 7300 fluorescence quantitative PCR instrument (Applied Biosystems, Foster City, CA, USA). Composition Analysis of Gallbladder Stones by Fourier Transform Infrared (FTIR) Spectroscopy Gallbladder stones samples were analyzed with a Bruker (TENSOR27, Germany) FTIR spectrometer in the frequency range 400–4000 cm-1 at 4 cm-1 resolution. For each measure- ment, 2 mg of finely powdered stone sample mixed with 300 mg KBr was used to make discs. Control spectrums were acquired from standard substances, which were of high quality (99% pure) and purchased from Sigma Chemical Company (St. Louis, MO, USA). Through comparisons of the spectra between gallbladder stones and standard controls, gallbladder stones were grouped as cholesterol stones, pigment stones or mixed stones [25,26]. Observation of Gallbladder Stones with Scanning Electron Microscopy (SEM) Thirty egg-positive gallbladder stones were chosen for SEM analysis. Stones were washed thoroughly with distilled water and dried at 60uC. Then, they were split and one piece was taken from each layer if the hierarchical structure of the profile was clear; otherwise, one piece from the surface and one piece from the core were taken and then fixed on the sample table and dried at 60uC overnight. Then, the stones were sputter-coated with gold (ETD- 2000, China) and observed under a ZEISS (EVO LS10, Germany) SEM and photographed. The EHT was 20 kv. Statistical Analysis Age was analyzed as mean 6SE, the detection rate was analyzed using a chi square test and the partitions of the chi square method were used for multiple comparisons using SPSS v.11.5 software. P,0.05 was regarded as statistically significant. Results C. sinensis Eggs were Detected in the Gallbladder Stones using Light Microscopy and Egg-positive Gallbladder Stones were Mainly Pigment Stones C. sinensis eggs are yellow-brown in color, resemble sesame seeds, have a thick shell and a small operculum in the front end, shouldering in the junction of the shell and operculum, with occasional visible small protrusions in the obtuse posterior end. Through microscopic examination, C. sinensis eggs were found in 122 of 183 gallbladder stones, providing a detection rate of 66.7%. Meanwhile, large portions of the eggs were adhered to or wrapped by bilirubinate granules and/or mucoid matter. Based on these observations, eggs in the gallbladder stones were preliminarily judged as C. sinensis eggs (as shown in Figure 1). To determine the main composition of the stones, we analyzed the stones using FTIR spectroscopy. Results showed that the 183 gallbladder stones were composed of 115 pigment stones, 45 mixed stones and 23 cholesterol stones. The characteristic spectrum of the three types of stones is shown in Figure 2 and the spectrum of the standard controls is shown in Figure 3. The FTIR spectroscopy analysis revealed that 122 egg-positive PLoS ONE | www.plosone.org 3 August 2012 | Volume 7 | Issue 8 | e42471