Melting curves were obtained from 55°C to 90°C, with fluorescence measurements taken at every 1°C increase in temperature. All reactions were carried out in triplicate along with a non-template control. Ct values were calculated CBL0137 price under default settings for the absolute quantification using the software provided with the instrument. The equation drawn from the graph was used to calculate the precise number of target molecule (plasmid copy no. or number of bacteria) tested in same reaction plate as standard as well as in sample. Statistical analysis Graph of respective bacterial population is plotted as mean value
with standard error. Each sample was analyzed in triplicate for calculation of significant differences in bacterial population by the Man-Whitney test. P values of 0.05 or below considered as significant. Paired samples collected from healthy volunteers before and after satronidazole treatment were analyzed by
Wilcoxon matched-pairs signed rank test (two tailed). Analysis was done using GraphPad Prism-5 software. Results Screening of E. histolytica positive samples DNA from concentrated cyst was subjected to Dot-blot hybridization. Dot blot analysis of 550 samples yielded TH-302 supplier 39 samples (7%) that were positive for Entamoeba (Figure 1B). The DNA from Entamoeba positive samples were subjected to PCR using species specific primers of E. histolytica and E. dispar (Figure 2C & D). Out of 39 samples, 17 samples (43%) were positive for E. histolytica. None of the samples only in our study population were found positive for both the species of the parasite. Quantification of predominant flora High quality DNA isolated from E. histolytica positive stool sample was subjected to Real Time analysis
to assess the predominant gut flora that included Bacteroides, Bifidobacterium, Eubacterium, selleck screening library Clostridium leptum subgroup, Clostridium coccoides subgroup, Lactobacillus and Ruminococcus. Two subdominant genera Methanobrevibacter smithii and Sulphur reducing bacteria (SRB) were also quantified. Validation of primers designed by us for the above genera have already been reported . In addition to the above primers, here we report a Real time analysis of nim gene copy number for which a standard curve and amplification curve have been drawn that shows specific and efficient quantification with slope = −3.6 and R2 =0.998 (Figure 3A & B). Figure 3 Real-time analysis for quantification of different bacterial genera in Healthy vs E. histolytica positive (Eh + ve) samples. (A) Bacteroides (B) Clostridium coccoides subgroup (C) Clostridium leptum subgroup (D) Lactobacillus (E) Campylobacter (F) Eubacterium. P value = .05 or below was considered significant. CI stands for confidence interval. Our analysis reveals that during healthy conditions, the members of Bacteroides were the most abundant in number among the predominant targeted genera. However, a significant decrease was observed in population of Bacteroides (p = .