A multiple reverse transcription PCR assay for simultaneous detection of four main viruses in kiwifruit

Kiwifruit ( Actinidia spp.) is an important horticultural crop in the world. Previous studies showed that a number of viruses were found in kiwifruit around the world. To detect the multiple viruses in kiwifruit quickly and effectively, a multiplex reverse transcription polymerase chain reaction (multiplex RT-PCR) method was developed to detect four viruses simultaneously in kiwifruit: Actinidia chlorotic ringspot-associated virus (AcCRaV), Actinidia virus 1 (AcV-1), Actinidia virus A (AcVA), Citrus leaf blotch virus (CLBV), respectively. Four pairs of primers were designed according to the coat protein (CP) genes of the aforementioned viruses and then were confirmed by RT-PCR assay. Actin gene (ACT1) was used as an internal control to improve reliability of the multiplex RT-PCR. Determination of detection limit of the multiplex RT-PCR showed that this method could detect four viruses at the dilution of 10 −4 cDNA. Furthermore, the developed multiplex RT-PCR could detect viruses in kiwifruit field samples successfully.