A simple, rapid and solvent free nucleic acid extraction protocol for detection of banana bunchy top virus by polymerase chain reaction and loop-mediated isothermal amplification.

Abstract

Banana is known to have high content of polyphenols, polysaccharides and tannins which inhibits polymerase chain reaction (PCR) while detecting the viral pathogens. Multiple-step nucleic acid (NA) extraction protocols are common, but more expensive, time consuming, laborious, and may lead to cross contamination. A simple extraction protocol (SEP) for preparing viral NA from leaves and aphids for detection of BBTV by PCR was developed. Inclusion of sodium sulphite and polyvinylpyrrolidone in the extraction buffer minimized the interferences due to polyphenols and polysaccharides, and an homogenization step has increased the percent detection from plants exhibiting various types of symptoms over virus release protocols reported previously. The detection efficacy for the template obtained in this SEP was comparable with that of templates obtained with a CTAB method and a commercial DNA extraction kit for NA extract preparation. The sensitivity test in PCR showed that this assay could detect 0.1 pg/μl plasmid DNA which is equivalent to 1 × 104 copies. Virus could be detected using SEP from freeze dried as well as CaCl2 dried samples of BBTV infected banana leaves. This methodology provides quality PCR product for direct sequencing suitable for identification and characterization of BBTV. The NA extract prepared by SEP is suitable for BBTV detection in quantitative PCR using SYBR Green chemistry and loop-mediated isothermal amplification assay. This protocol is sensitive, rapid, less prone to contamination, economical, and has potential for large-scale application in surveys, surveillance, quarantine, and certification programmes.