A method of direct PCR without DNA extraction for rapid detection of begomoviruses infecting jute and mesta

Abstract

We have developed a simple method of direct PCR (dPCR) without timeconsuming procedures of DNA extraction by directly using the leaf bits for rapid detection of begomoviruses in jute and mesta. The leaf bits were treated with a lysis buffer for 35 min, and the lysate was used as PCR template. Different components and their concentration in lysis buffer systems were optimized and the optimal buffer system composed of 20 mmol l 1 tris (hydroxymethyl aminomethane (Tris)-Cl (pH 80)), 15 mmol l 1 ethylenediaminetetraacetic acid (EDTA) (pH 80), 14 mol l 1 NaCl and 200 lg/mL Proteinase K. Further, 3% PVP (w/v) and b-marcaptoethanol (1% v/v) were additionally added into the buffer in case of jute. Under optimized PCR conditions, both viral DNA as well as plant (jute and mesta) genomic DNA were amplified from the lysate. dPCR required fewer reagents and less incubation time reducing both time and cost of detection.