Establishment of multiplex real-time RT-PCR and one-step multiplex RT-PCR in detecting of CEVd and HSVd. (A) Standard curves of CEVd and HSVd for absolute quantification obtained by plotting Ct values versus actual pTOPO-CEVd and pTOPO-HSVd copy number. The Ct values for each dilution are the means of three replicates. (B) One-step multiplexe RT-PCR detected CEVd and HSVd transcripts alone and in combination. Samples A and B were collected from the field. Lane 1, viroids co-infection citrus sample; 2,3, unknown samples in the field; 4, Mixed 100 ng/μL RNA transcripts of plasmids of pTOPO-CEVd and pTOPO-HSVd; 5, 100 ng/μL RNA transcript of plasmid pTOPO-CEVd; 6, 100 ng/μL RNA transcript of plasmid pTOPO-HSVd; 7, healthy control; 8, water control; M, 100-bp molecular marker.