STANDARDISATION OF PAPER BASED PCR FOR DETECTION OF BACTERIA USING 16S rRNA GENE

Abstract

Reliable procedures are necessary to preserve, transport and test clinical, water or food samples, frequently requiring a challenging and costly cold chain to be in place. Effective disease surveillance or detection is thus severely hampered in resource-limited settings, such as those currently present in rural India, where procedures for appropriate laboratory based detection are suboptimal. Hence the recent scientific research is been focused to develop laboratory techniques/devices that can be applied in point of care settings. Point-of care (POC) techniques are getting increasingly popular, due to the advantages provided by them like ease of use, variety of applications, cost effectiveness and ease of disposal. This study deals with the standardization of method for microbial detection from sample collected on filter paper by direct amplification of a gene using PCR. Bacterial presence in a sample is confirmed by amplification of 16S rRNA gene as a proof of concept. 16S rRNA gene detection directly from sample spotted on Whatman filter paper no.3 without any pre-treatment to extract DNA is demonstrated, which is different from the currently practiced methods that include an additional step of DNA extraction from the paper. Robustness of the method was tested using Gram positive and Gram negative bacterial cultures and the sensitivity of detection on Whatman filter paper no. 3 was found to be 40-50 cells.