用于检测大麦幼苗中大麦条纹花叶病毒株系的一步免疫捕获实时逆转录聚合酶链反应检测方法的开发。

Development of a one-step immunocapture real-time RT-PCR assay for the detection of barley stripe mosaic virus strains in barley seedlings.

作者信息

Zarzyńska A, Jeżewska M, Trzmiel K, Hasiów-Jaroszewska B

出版信息

Acta Virol. 2014;58(1):81-5. doi: 10.4149/av_2014_01_81.

DOI:10.4149/av_2014_01_81

Abstract

A one-step immunocapture real-time RT-PCR (IC-real-time RT-PCR) was developed for efficient detection of barley stripe mosaic virus (BSMV) in barley seedlings. The novel detection system was designed using a primer set targeting the conserved region in the triple gene block 2 (TGB2) to expand its capacity to detect all BSMV strains. This assay was evaluated for its efficiency in detecting BSMV in barley seedlings. Using the immunocapture sample preparation, real-time RT-PCR was able to detect BSMV in samples, which were indicated as negative by ELISA. The sensitivity of detection in the real-time RT-PCR was as low as 50 fg/µl of total viral RNA under optimal reaction conditions. This level of sensitivity indicated that the one-step IC-real-time RT-PCR developed in the present study could be used for routine plant and seed health assays.

摘要

开发了一种一步免疫捕获实时逆转录聚合酶链反应（IC-实时RT-PCR）方法，用于高效检测大麦幼苗中的大麦条纹花叶病毒（BSMV）。使用靶向三基因块2（TGB2）保守区域的引物对设计了这种新型检测系统，以扩大其检测所有BSMV毒株的能力。对该检测方法在检测大麦幼苗中BSMV的效率进行了评估。使用免疫捕获样品制备方法，实时RT-PCR能够检测到酶联免疫吸附测定（ELISA）显示为阴性的样品中的BSMV。在最佳反应条件下，实时RT-PCR的检测灵敏度低至50 fg/µl总病毒RNA。这种灵敏度水平表明，本研究中开发的一步IC-实时RT-PCR可用于常规植物和种子健康检测。