State Key Laboratory of Agromicrobiology, Huazhong Agricultural University, Wuhan, Hubei 430070, China; Key Laboratory of Horticultural Crop (Fruit Trees) Biology and Germplasm Creation of the Ministry of Agriculture, Huazhong Agricultural University, Wuhan, Hubei 430070, China.
State Key Laboratory of Agromicrobiology, Huazhong Agricultural University, Wuhan, Hubei 430070, China; The Key Laboratory of Plant Pathology of Hubei Province, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, China.
J Virol Methods. 2019 Jan;263:105-110. doi: 10.1016/j.jviromet.2018.10.021. Epub 2018 Oct 29.
A highly sensitive nested multiplex reverse transcription-polymerase chain reaction (nmRT-PCR) assay was developed for the simultaneous detection of Apple chlorotic leaf spot virus (ACLSV), Apple stem grooving virus (ASGV) and Apple stem pitting virus (ASPV) infecting pear trees. In the assay, a set of three forward primers specific to each of the three viruses and a universal reverse primer was used as external primers in the first-round PCR, which was followed by a second-round PCR developed previously. The nmRT-PCR assay was 10 times more sensitive than conventional mRT-PCR assay in detecting the three viruses in in vitro pear plantlets. This assay was subsequently used to detect these viruses in leaf and bark samples of cultivated and wild pear trees from orchards and demonstrated to be highly sensitive and reliable. This is the first report describing a use of nmRT-PCR for the sensitive and simultaneous detection of the three viruses infecting pear plants. The assay would be useful for the certification of pear planting materials and surveillance of nursery stocks.
一种高度敏感的嵌套多重逆转录-聚合酶链反应（nmRT-PCR）检测方法被开发出来，用于同时检测感染梨树的苹果褪绿叶斑病毒（ACLSV）、苹果茎沟病毒（ASGV）和苹果茎痘病毒（ASPV）。在该检测方法中，第一轮 PCR 使用了一组针对三种病毒的三条特异性正向引物和一条通用反向引物，然后是之前开发的第二轮 PCR。nmRT-PCR 检测方法在检测体外梨苗中三种病毒时比常规 mRT-PCR 检测方法灵敏 10 倍。该检测方法随后被用于检测果园中栽培和野生梨树的叶片和树皮样本中的这些病毒，证明其具有高度的灵敏性和可靠性。这是首次描述 nmRT-PCR 用于敏感和同时检测感染梨树的三种病毒的报道。该检测方法将有助于梨种植材料的认证和苗圃苗木的监测。
