|LI, JILIAN - Chinese Academy Of Agricultural Sciences|
|CORNMAN, SCOTT - Us Geological Survey (USGS)|
|Murphy, Charles - Charlie|
|PENG, WENJUN - Chinese Academy Of Agricultural Sciences|
|WU, JIE - Chinese Academy Of Agricultural Sciences|
|BONCRISTIANI, HUMBERTO - University Of North Carolina|
|ZHOU, LIANG - Emory University, School Of Medicine|
|Chen, Yanping - Judy|
Submitted to: mBio
Publication Type: Other
Publication Acceptance Date: 6/1/2014
Publication Date: 8/20/2014
Citation: Li, J., Cornman, S.R., Evans, J.D., Pettis, J.S., Zhao, Y., Murphy, C.A., Peng, W., Wu, J., Boncristiani, H., Zhou, L., Hammond, J., Chen, Y. 2014. Reply to conclusive evidence of replication of a plant virus in honeybees is lacking. mBio. 5(3):e01250-14.
Technical Abstract: This reply provides clarifications regarding comment “Conclusive Evidence of Replication of a Plant Virus in Honeybees Is Lacking” about our paper “Systemic Spread and Propagation of a Plant-Pathogenic Virus in European Honeybees, Apis mellifera” [mBio 2014, 5(1):e00898-13]. The strand-specific real time RT-PCR method have been utilized for detecting and measuring specific strands of viral RNA and demonstrating virus replication in infected hosts. However, there have been concerns regarding specificity of the method due to false priming events. We identified that the major problem associated with amplification of non-targeted viral RNA strand was due to carryover of primer from Reverse Transcription (RT) to the subsequent PCR amplification. We therefore improved the method by employing double cDNA purification to collect cDNA and to remove short fragment of oligonucleotides and enzymatic reagents. Our results of validation proved that the strand specificity could be ensured by the standard strand-specific tagged RT-PCR method coupled with double steps of cDNA purification. This explanation should help to allay concerns about the plausibility of non-target amplification of strand-specific real time RT-PCR method employed in our study.