Detection of Citrus psorosis virus Using an Improved One-Step RT-PCR

Abstract

We report an improvement of a one-step reverse transcription polymerase chain reaction (RT-PCR) assay for the detection of Citrus psorosis virus (CPsV; genus Ophiovirus) in citrus trees. Two different sample preparation procedures were compared. The data showed that when using total RNA extracted by the Qiagen procedure more virus isolates could be detected compared with the Trizol procedure. Three pairs of primers reported in literature and selected within the coat protein (CP) region of the virus were used for a reliable detection of CPsV. Only one primer pair has been able to detect all CPsV isolates collected from different citrus regions of Morocco. In addition, this report showed that one-step RT-PCR was more sensitive and more consistent than ELISA test. Notably, 15 out of 22 samples tested positive by one-step RT-PCR could not be detected by Mab Ps29, thus providing a degree of differentiation among isolates. The detection protocol described in this study could be used in citrus certification programs and to test trees in nurseries and commercial orchards for CPsV infection.