Development of sensitive and reliable detection of banana bunchy top virus in abaca (Musa texttilis Nee) by Polymerase Chain Reaction

Issue Date

8-2018

Abstract

In the current abaca rehabilitation and virus resistance breeding programs, it is crucial that sensitive and reliable diagnostics is established for virus indexing of abaca planting materials and for virus resistance screening. This study was conducted to develop a sensitive and reliable detection of Banana bunchy top virus (BBTV) by polymerase chain reaction (PCR) in abaca plant. The conditions for virus detection by PCR were optimized in template DNA extracted by Dellaporta, Sarkosyl and CTAB methods and in varying amounts (undiluted, 1000, 100, 10, 1, 0.1, 0.01 and 0.001 ng) using different primers. Detection of BBTV was most sensitive using template DNA extracted by Dellaporta compared with Sarkosyl and CTAB methods. The detectable at various concentrations up to 0.001 ng. Dellaporta extracted DNA but can be efficiently (100%) detected from 0.1 to 100 ng. The virus was also detectable at 0.001 ng but efficiency was lower with Sarkosyl (0.1 to 10 ng) and CTAB (1 to 100 ng) extracted DNA. The presence of inhibitors prevented the amplification of BBTV in the highly concentrated (undiluted and 1000 ng) Sarkosyl and CTAB but not Dellaporta extracted DNA. Dilution of the template DNA increased the sensitivity of detection. The Musa sequence was amplified in more concentrated DNA (undiluted and 1000 ng) extracted by Dellaporta but not Sarkosyl and CTAB methods, suggesting that Dellaporta extracted contained lesser amount of inhibitors. Detection of BBTV was most consistent using the BBT1/BBT2 primer pair which amplifies the DNA-R component (replicase) of the viral genome. Amplification of an internal control DNA using the Musa tagged microsatellite primers AGMI025 and AGMI026 confirmed the reliability of BBTV detection by PCR.

Source or Periodical Title

Philippine Journal of Crop Science

ISSN

0115-463x

Volume

43

Issue

2

Page

12-19

Document Type

Article

Frequency

tri-quarterly

Physical Description

graphs, dendrograms

Language

English

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