A novel ready-to-use dry-reagent polymerase chain reaction for detection of Escherichia coli & Shigella species

Background & objectives: Polymerase chain reaction (PCR) has wide acceptance for rapid identification of pathogens and also for diagnosis of infectious conditions. However, because of economic and expertise constraints, a majority of small or peripheral laboratories do not use PCR. The objective of the present study was to develop a dry-reagent PCR assay as an alternative to conventional PCR to assess its applicability in routine laboratory practice using malB gene for identification of Escherichia coli as a model. Methods: A total of 184 isolates were selected for the study comprising clinical isolates of E. coli and non-E. coli including Shigella sp. and a few other control strains. The DNA was isolated from all the isolates. The isolated DNA as well as the overnight grown bacterial cultures were subjected to both conventional wet PCR and dry-reagent PCR. Results: The genomic DNA isolated from E. coli showed amplification of malB gene in both conventional wet and dry-reagent PCR and the band was observed at 491 bp. In dry-reagent PCR, the overnight grown E. coli cells also showed positive result. The non-E. coli strains other than Shigella sp. showed negative in both conventional wet and dry-reagent PCR. Shigella sp. showed positive in both conventional wet and dry-reagent PCR. Interpretation & conclusions: Considering the elimination of genomic DNA isolation step, and similar results with the conventional wet PCR, dry-reagent PCR may be a good alternative for the conventional wet PCR.

Identification of multiple strains of Porphyromonas gingivalis using heteroduplex polymerase chain reaction in varying severity of chronic periodontitis

Aim: Research has demonstrated that there are multiple strains of Porphyromonas gingivalis with varying potency to cause periodontal disease. The current study aims at using heteroduplex polymerase chain reaction (PCR) to detect the strain diversity of P. gingivalis in periodontitis lesions of varying severity in a sample of the Indian population. Materials and Methods: Subgingival plaque samples were collected from 60 individuals with varying severity of chronic periodontitis and 30 individuals with a clinically healthy periodontium. The samples were subjected to PCR analysis to identify P. gingivalis, followed by heteroduplex analysis to identify the strain diversity in a given sample. Bacterial culture was carried out as a comparative standard. Results: Of the 56 samples that were positive for P. gingivalis by PCR, 54 samples yielded eight different heteroduplex patterns. Analysis of these patterns indicated that two strains of P. gingivalis were present in 41 individuals (45.6%) and three strains were present in 13 individuals (14.4%). Detection of P. gingivalis by PCR was significantly more in the periodontitis group as compared to the healthy group. Conclusions: Species-specific PCR and heteroduplex analysis provide a simple and accurate method to analyse the strain diversity of P. gingivalis. P. gingivalis was detected in both healthy periodontal sites as well as sites with periodontitis. The presence of two or three P. gingivalis strains was seen in 60% of the samples.