histolytica genome. NIM-F&R primers amplified 458 bp fragment of nim gene from stool
sample DNA. This amplified fragment of 458 bp was cloned in pGEMT-easy vector and sequenced to ensure the amplification of correct gene. The clone was subsequently used as a standard for quantification of nim gene by Real Time-PCR. PCR-RFLP of nim gene Primers NIM-F and NIM-R were used to amplify all the members of nim gene family from stool sample DNA. Members of nim gene family were differentiated by digesting the PCR product with restriction enzymes HpaII and TaqI. HpaII digests nimA, nimC, nimD at selleck chemicals llc different loci but not nimB and nimE where as TaqI digests nimA, nimB, nimE at different loci but not nimC and nimD[19]. Reference strains Genus specific primers ALK inhibitor were used to amplify respected genera from DNA of stool sample of healthy individual. The amplified product was cloned and sequenced and sequences were deposited in EMBL database to obtain the accession numbers (Table 2).These 16S rRNA gene fragment containing plasmids were used as reference strains. Table 2 Accession number of reference strain used in the study Bacteria Source Accession no. Bacteroides Stool of healthy individual AM117604 Methanobrevibacter Stool of healthy individual FN813615
Eubacterium Stool of healthy individual FN813614 Lactobacillus Stool of healthy individual AM042701 Bifidobacterium Stool of healthy individual AM042698 Clostridium Stool of healthy individual AM042697 Campylobacter Stool of healthy individual AM042699 Ruminococcus Stool of healthy individual FN823053 Sulfate-reducing bacteria Stool of healthy individual FN995351 Real time PCR analysis of bacterial population Quantification was done using ABI-7500 machine and power syber green PCR master mix kit from Applied Biosystems, USA. Standard curve was the method
of choice for absolute quantification of bacteria. Standard curve was made using serial dilutions of selleck chemicals plasmid (containing 16S rRNA gene fragment) of known concentrations on tenfold basis. With the molecular weight of the plasmid and insert known, it is possible to calculate the copy number as follows: Step 1: Determining molecular weight (mw) Weight in Daltons (g/mol) = (bp size of double stranded product)(330 Da x 2nt/bp) Step 2: Molecular L-gulonolactone oxidase weight to copy number X g/mol/Avogadro’s number (6.023 × 1023 molecules/mole) = X g/molecule Where X = the weight of one molecule or copy Where bp = base pairs, nt = nucleotides [20] Real time PCR runs were performed in 96 well optical plates (each containing 1x PCR master mix, 4 pm/μl forward and reverse primer(optimized concentration) and 1μl plasmid DNA of tenfold dilutions or 1μl DNA from samples in 20μl reaction) for 40 cycles using an ABI 7500 sequence detector (Applied biosystems). Default 7500 cycle conditions were used with only change in the annealing temperature.