Multiplex RT-PCR Method for the Simultaneous Detection of Nine Grapevine Viruses

Abstract

Viral diseases are a serious pathological problem for grapevines, and in recent years the need for increasingly specific and rapid diagnostic methods for the selection of propagation materials has grown. Arabis mosaic virus, Grapevine fanleaf virus, Grapevine virus A, Grapevine virus B, Grapevine rupestris stem pitting-associated virus, Grapevine fleck virus, and Grapevine leafroll-associated viruses 1, 2, and 3 are nine of the most widespread viruses that naturally infect grapevines. A multiplex RT-PCR was developed for simultaneous detection of these nine grapevine viruses, in combination with a plant RNA internal control used as an indicator of the effectiveness of the reaction. One to ten fragments specific for the viruses and an internal control were simultaneously amplified from infected samples and identified by their specific molecular sizes in agarose gel. The protocol reported is an update of previously published protocols for RNA extraction and multiplex diagnosis of viruses. After several years of use and hundreds of samples tested, and following validation in several laboratories, this multiplex RT-PCR provides a reliable and rapid method for detecting grapevine viruses from a large number of samples.

Plastome-wide comparison reveals new SNV resources for the authentication of Dendrobium huoshanense and its corresponding medicinal slice (Huoshan Fengdou)

“…In line with this observation, the 6 pairs of ARMS-primer developed in this study also showed high specificity for D. huoshanense . Moreover, combined with real-time PCR technology, PCR products can be detected quantitatively and directly through the intensity of fluorescent signals 58 , 59 . Our results show that all the 6 pairs of ARMS-primer amplified in D. huoshanense have significantly lower C t values than those of other Dendrobium species.…”

“…The amplification refractory mutation system (ARMS), which has been developed for detecting SNVs, is well suited to authenticate the TCM products of Dendrobium , i.e ., D. fimbriatum 20 and D. loddigesii 21 . Recently, real-time fluorescent quantitative polymerase chain reaction (real-time PCR), a highly sensitive method of detecting DNA, has been widely adopted to identify food species, such as wheat, soybean and grapevine 22 , 23 , 24 . Therefore, the RT-ARMS method that combines real-time PCR with ARMS was established for the authentication of D. officinale and its medicinal slice (Tiepi Fengdou) 5 .…”

miRVIT: A Novel miRNA Database and Its Application to Uncover Vitis Responses to Flavescence dorée Infection

“…The biological study was performed on plants of the red grape cultivar Barbera ( V. vinifera ) grown in a vineyard located in Cocconato (Piemonte), northwest Italy, and monitored since 2008 for phytoplasma infection ( Maggi et al, 2017 ). FDp and BNp were detected following a phytoplasma enrichment protocol ( Marzachì et al, 1999 ), and the viruses that commonly affect grapevine in Italy were detected by multiplex RT-PCR ( Gambino, 2015 ). Only the plants without both BNp and the most dangerous viruses infecting grapevine were considered for the experimental trial and further divided into three groups: FDp-infected (FD), recovered (R, i.e., plants found positive to FDp infection in the past, but then resulted FDp-negative and symptomless for the last 2 years), and healthy (H).…”

Grapevine Phyllosphere Community Analysis in Response to Elicitor Application against Powdery Mildew

“…Raw reads resulting from the NovaSeq sequencing were trimmed from adaptors and quality filtered using Trimmomatic [59], then mapped against the custom database using BWA and SAM tools [60,61]. Virus and viroid infections were further validated using previously described PCR end-point protocols [62][63][64].…”