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A method of direct PCR without DNA extraction for rapid detection of begomoviruses infecting jute and mesta
Author(s) -
Biswas C.,
Dey P.,
Satpathy S.
Publication year - 2014
Publication title -
letters in applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.698
H-Index - 110
eISSN - 1472-765X
pISSN - 0266-8254
DOI - 10.1111/lam.12196
Subject(s) - lysis buffer , dna extraction , lysis , chromatography , tris , proteinase k , dna , extraction (chemistry) , biology , ethylenediaminetetraacetic acid , polymerase chain reaction , microbiology and biotechnology , virology , chemistry , biochemistry , gene , organic chemistry , chelation
We have developed a simple method of direct PCR (d PCR ) 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 μ g/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. d PCR required fewer reagents and less incubation time reducing both time and cost of detection. Significance and Impact of the Study 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.