Malandraki Ioanna, Beris Despoina, Isaioglou Ioannis, Olmos Antonio, Varveri Christina, Vassilakos Nikon
Benaki Phytopathological Institute, Department of Phytopathology, Laboratory of Virology, Athens, Greece.
Plant Protection and Biotechnology Centre, Instituto Valenciano de Investigaciones Agrarias (IVIA), Moncada, Valencia, Spain.
PLoS One. 2017 Jul 27;12(7):e0180877. doi: 10.1371/journal.pone.0180877. eCollection 2017.
A one-step multiplex real-time reverse transcription polymerase chain reaction (RT-qPCR) based on TaqMan probes was developed for the simultaneous detection of Apple mosaic virus (ApMV), Apple stem pitting virus (ASPV) and Apple stem grooving virus (ASGV) in total RNA of pome trees extracted with a CTAB method. The sensitivity of the method was established using in vitro synthesized viral transcripts serially diluted in RNA from healthy, virus-tested (negative) pome trees. The three viruses were simultaneously detected up to a 10-4 dilution of total RNA from a naturally triple-infected apple tree prepared in total RNA of healthy apple tissue. The newly developed RT-qPCR assay was at least one hundred times more sensitive than conventional single RT-PCRs. The assay was validated with 36 field samples for which nine triple and 11 double infections were detected. All viruses were detected simultaneously in composite samples at least up to the ratio of 1:150 triple-infected to healthy pear tissue, suggesting the assay has the capacity to examine rapidly a large number of samples in pome tree certification programs and surveys for virus presence.
基于TaqMan探针开发了一种一步多重实时逆转录聚合酶链反应（RT-qPCR），用于同时检测用CTAB法从梨果类果树总RNA中提取的苹果花叶病毒（ApMV）、苹果茎痘病毒（ASPV）和苹果茎沟病毒（ASGV）。该方法的灵敏度通过使用在来自健康、经病毒检测（阴性）的梨果类果树RNA中连续稀释的体外合成病毒转录本进行确定。从天然三重感染苹果树的总RNA制备的健康苹果组织总RNA中，三种病毒在总RNA稀释至10-4时仍能同时被检测到。新开发的RT-qPCR检测方法比传统的单重RT-PCR至少灵敏100倍。该检测方法用36个田间样本进行了验证，检测到9个三重感染和11个双重感染。在复合样本中，至少在三重感染与健康梨组织比例为1:150时，所有病毒仍能同时被检测到，这表明该检测方法有能力在梨果类果树认证计划和病毒存在调查中快速检测大量样本。
