title: 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 time-consuming 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 8·0), 1·5 mmol l(-1) ethylenediaminetetraacetic acid (EDTA) (pH 8·0), 1·4 mol l(-1) NaCl and 200 μg/mL Proteinase K. Further, 3% PVP (w/v) and β-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.
  
  
  CONCLUSIONS
  Identification of begomoviruses by serology is not suitable due to difficulty in preparing high titre and specific antisera. Begomoviruses are routinely detected by PCR-based techniques using universal or specific primers. However, it is a prerequisite to isolate pure DNA from the samples before PCR. DNA extraction from some plants such as jute, mesta is very difficult due to the presence of mucilage and other impurities. Therefore, we have developed a method of direct PCR without DNA extraction for detection of begomoviruses from these crops. It is the first report of a direct PCR method in jute and mesta.
authors.affiliation: Crop Protection Division, Central Research Institute for Jute and Allied Fibres (CRIJAF), Barrackpore, Kolkata, West Bengal, India.
authors.forename: C
authors.initials: C
authors.lastname: Biswas
authors.forename: P
authors.initials: P
authors.lastname: Dey
authors.forename: S
authors.initials: S
authors.lastname: Satpathy
mesh_terms: D053839:Begomovirus / Q000235:genetics / Q000302:isolation & purification*; D032381:Corchorus / Q000821:virology*; D004279:DNA, Viral / Q000302:isolation & purification; D031584:Hibiscus / Q000821:virology*; D018515:Plant Leaves / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D004279:DNA, Viral
keywords: direct PCR; jute; lysis buffer; mesta