用于同时检测类病毒和植原体的一步多重定量逆转录聚合酶链反应

One-Step Multiplex Quantitative RT-PCR for the Simultaneous Detection of Viroids and Phytoplasmas.

作者信息

Malandraki Ioanna, Varveri Christina, Vassilakos Nikon

机构信息

Department of Phytopathology, Laboratory of Virology, Benaki Phytopathological Institute, Athens, Greece.

出版信息

Methods Mol Biol. 2019;1875:151-157. doi: 10.1007/978-1-4939-8837-2_12.

DOI:10.1007/978-1-4939-8837-2_12

PMID:30362002

Abstract

A one-step multiplex quantitative reverse transcription polymerase chain reaction protocol is described, for the detection in pome trees of Pear blister canker viroid and Apple scar skin viroid, together with universal detection of phytoplasmas. Total nucleic acids extraction is performed according to a modified CTAB protocol and TaqMan MGB probes are used to surpass high genetic variability of viroids. The multiplex real-time assay is at least ten times more sensitive than conventional protocols and its features make it suitable for rapid and massive screening of pome fruit trees phytoplasmas and viroids in certification schemes and surveys.

摘要

本文描述了一种一步多重定量逆转录聚合酶链反应方案，用于检测梨树上的梨疱溃疡类病毒和苹果锈果类病毒，同时对植原体进行通用检测。总核酸提取按照改良的CTAB方案进行，TaqMan MGB探针用于克服类病毒的高遗传变异性。多重实时检测比传统方案至少敏感十倍，其特性使其适用于在认证计划和调查中对梨果类果树的植原体和类病毒进行快速大量筛查。