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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Bee Research Laboratory » Research » Publications at this Location » Publication #305476
Title: Reply to conclusive evidence of replication of a plant virus in honeybees is lacking

Author
item LI, JILIAN - Chinese Academy Of Agricultural Sciences
item CORNMAN, SCOTT - Us Geological Survey (USGS)
item Evans, Jay
item Pettis, Jeffery
item Zhao, Yan
item Murphy, Charles
item PENG, WENJUN - Chinese Academy Of Agricultural Sciences
item WU, JIE - Chinese Academy Of Agricultural Sciences
item BONCRISTIANI, HUMBERTO - University Of North Carolina
item ZHOU, LIANG - Emory University, School Of Medicine
item Hammond, John
item 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.

Interpretive Summary:

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.