Development of a recombinase polymerase amplification assay for the diagnosis of banana bunchy top virus in different banana cultivars
Recombinase polymerase amplification (RPA) is a rapid, isothermal amplification method with high specificity and sensitivity. In this study, an assay was developed and evaluated for the detection of banana bunchy top virus (BBTV) in infected banana plants. Three oligonucleotide primer pairs were designed from the replicase initiator protein gene sequences of BBTV to function both in RPA as well as in polymerase chain reaction (PCR). A total of 133 symptomatic as well as asymptomatic banana leaf samples from various cultivars were collected from the different regions of India and evaluated for BBTV infection using the RPA assay. BBTV was efficiently detected using crude leaf sap in RPA and the results obtained were consistent with PCR-based detection using purified DNA as template. To our knowledge, this is the first report of reliable diagnosis of BBTV infection by RPA using crude leaf sap as a template.
- 7.Joshi M, Deshpande JD (2011) Polymerase chain reaction: methods, principles and acpplication. Int J Biomed Res 7:81–89Google Scholar
- 9.Mahadev SR, Thamilarasan SK, Kathithachalam A (2013) PCR detection of banana bunchy top virus (BBTV) at tissue culture level for the production of virus-free planting materials. Int Res J Biol Sci 2(6):22–26Google Scholar
- 14.Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, p 2344 (3 volume set) Google Scholar
- 15.Selvarajan R, Balasubramanian V, Kavitha K, Kavitha KS, Sathiamoorthy S, Ahlawat YS (2008) Detection of banana bunchy top virus and banana streak Mysore virus by PCR: Impact of storing virus infected banana samples. Indian J Virol 19:28–33Google Scholar
- 17.Selvarajan R, Balasubramanian V, Dayakar S, Sathiamoorthy S, Ahlawat YS (2010) Evaluation of immunological and molecular techniques for the detection of different isolates of banana bunchy top virus in India. Indian Phytopathol 63(3):333–336Google Scholar
- 18.Selvarajan R, Sheeba MM, Balasubramanian V (2011) Simultaneous detection of episomal banana streak Mysore virus and banana bunchy top virus using multiplex RT-PCR. Curr Sci 100:31–34Google Scholar