Total viral DNA and RNA were extracted

Total viral DNA and RNA were extracted see more from fecal specimens prepared in phosphate-buffered saline at 10%(wt/vol) using the QIAamp MinElute Virus Spin Kit (Qiagen, Hilden, Germany) according to the manufacturer’s recommendations. HuCV, enteric Adv and HAstV were detected by PCR as described previously [8–10]. G. lamblia and Ent. histolytica were detected using direct microscopy with a saline

preparation of the specimen. The clinical history and physiological findings of each patient were documented on standardized case report forms. Fecal samples from five healthy and five hospitalized children at the same location but with no apparent diarrhea were analyzed as controls. Libraries of the 16S rRNA gene were constructed

for each fecal sample, with a minimum size of 100 analyzable sequences [11]. Analyzing dominant fecal bacterial species by 16S rRNA gene Momelotinib sequence technology All fecal samples were collected in triplicate; one for timely isolation and detection of the enteric pathogens; one stored at −20°C for 16S rRNA sequence analysis; and one stored in 20% glycerol at −80°C for isolation of the putative pathogens suggested by the 16S rRNA gene analysis. Go6983 The DNA was extracted from a 200-mg fecal sample, which was measured and adjusted to 100 ng/μl of each sample for PCR. The universal eubacterial primers 27 F-519R (5’-agagtttgatcmtggctcag-3’ and 5’-gwattaccgcggckgctg-3’) were used to Tobramycin amplify a 500-bp region of the 16S rRNA gene. LaTaq polymerase (TaKaRa, Dalian, China) was used for PCR under the following conditions: 95°C for 5 min, followed by 20 cycles of: 95°C for 30 s, 52°C for 30 s, and 72°C for 1 min; and a final elongation step at 72°C for 10 min. The PCR products were extracted from sliced gels and cloned into the pGEMR-T Easy Vector System (Promega, Madison, WI,

USA). They were then transformed into competent E. coli JM109. A total of 130 white clones for each fecal sample were randomly selected for enrichment. The purified plasmid DNA was used for sequence analysis. To verify the repeatability, we repeated the 16S rRNA gene analysis of feces at admission for nine children with diarrhea of unknown etiology. The 16S rRNA gene sequences were analyzed for chimeric constructs using the Chimera Check program within the Ribosomal Database Project. Species-level identification was performed using a 16S rRNA gene sequence similarity of ≥99% compared with the prototype strain sequence in the GenBank. Identification at the genus level was defined as a 16S rRNA gene sequence similarity of ≥97% with that of the prototype strain sequence in the GenBank, and the sequences were listed by genus. The sequences matched attributable to either E. coli or Shigella sp. were listed as E. coli/Shigella sp.

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