Development of Real‐time RT‐PCR Assays for Detection and Quantification of Pineapple Mealybug Wilt‐associated Viruses‐1

Abstract

AbstractPineapple mealybug wilt‐associated virus‐1 (PMWaV‐1) is a major causative agent for pineapple disease. A real‐time, one‐step reverse transcription (RT)–polymerase chain reaction (PCR) assay using TaqMan probes was developed for the detection of PMWaV‐1. Real‐time RT‐PCR, targeting the amplification of heat‐shock protein 70 homolog (hsp70 h) gene fragment, was highly specific and capable of distinguishing PMWaV‐1 from PMWaV‐2 and PMWaV‐3, which are two other common virus species in pineapple plants. Sensitivity assay revealed that real‐time RT‐PCR was approximately 100‐ to 1000‐fold more sensitive than standard one‐step RT‐PCR, when total RNA of infected plant and PMWaV‐1 hsp70 h transcript were used, respectively. This real‐time RT‐PCR assay is rapid, specific and sensitive, which can be used for PMWaV‐1 diagnosis as well as surveillance.