Journal of Pure and Applied MicrobiologyVol. 3 No. 2

One-Step Gram PCR Assay for Rapid and Accurate Bacterial Differentiation

K. Krishnamurthy*, Deepti Rao, Poonam Agarwal and Gururaja Ramavana

Camson Bio Technologies Ltd, Bangaluru, Karnataka, India.

Received on 17 June 2009 and accepted on 25 July 2009

 

ABSTRACT

Gram differentiation through conventional Gram staining is not a reliable method due to high incidence of false positive results. Molecular techniques such as PCR provide a better alternative. A simple, rapid and accurate method has been developed that enables exact differentiation of Gram positive and negative bacteria using a one step PCR assay. The one-step PCR using Gram specific primers resulted in a single 1025bp amplicon in Gram negative bacteria and a 355bp amplicon in addition to 1025bp amplicon in Gram positive bacteria. All tested bacteria were identified correctly through a single PCR reaction and showed complete concordance with biochemical and Gram staining results. This protocol proves to be a rapid, powerful and sensitive tool in determining the Gram status of bacteria. This difference in banding patterns between Gram positive and negative bacteria can be exploited for their clear-cut differentiation.

Keywords : Multiplex PCR, Nested PCR, Ribotyping, Gram staining.