| Article ID | Journal | Published Year | Pages | File Type |
|---|---|---|---|---|
| 4567045 | Scientia Horticulturae | 2013 | 6 Pages |
•Standardization of Duplex PCR for simultaneous detection of ACLSV and ApMV along with internal control.•Validation of duplex RT-PCR by screening six rootstocks, 16 crab apples and 13 cultivars for presence of both viruses.•Among rootstocks only 2 showed presence of expected size band (432 bp) for ACLSV where as none showed amplification for ApMV.•All crab apples tested were free from infection by both the viruses as no band was visible, whereas among 13 cultivars 11 were found positive for ACLAV and 9 for ApMV.
Apple Chlorotic Leaf Spot Virus (ACLSV; family Betaflexiviridae, genus Trichovirus) and Apple mosaic virus (ApMV; family Bromoviridae, genus Ilarvirus) are economically important viruses of apple (Malus × domestica Borkh.). A duplex reverse transcription polymerase chain reaction (RT-PCR) assay was developed for simultaneous detection of both ACLSV and ApMV along with an internal control (NADH dehydrogenase subunit 5 gene). The internal control was used to minimize the risk of getting false negative results. Specific primers were designed against coat protein gene of ApMV while for ACLSV & internal control previously reported primer sets were used. Firstly the uniplex RT-PCR assay was standardized for the detection of each virus independently. Then duplex RT-PCR protocol for simultaneous detection of ACLSV and ApMV was standardized by using primer pairs ACLSV-CP1F & ACLSV-CP1R and ApMV-CP1F & ApMV-CP5R, respectively, along with primer set for internal control. Robustness of the technique was further validated wherein; duplex RT-PCR was carried out to detect both viruses in crab apples, rootstocks and different popular cultivars of apple. Results reveal that the duplex RT-PCR assay has detection sensitivity as that of uniplex RT-PCR assay for respective viruses. This optimized duplex RT-PCR provides a simple, rapid, sensitive and convenient way for simultaneous detection of ACLSV & ApMV by reducing the time and cost of the supplies.
