Liu Lu-Yuan, Ye He-Yi, Chen Tsang-Hai, Chen Tsung-Chi
Department of Plant Industry, National Pingtung University of Science and Technology, Pingtung, 91201, Taiwan.
Department of Biotechnology, Asia University, Wufeng, Taichung, 41354, Taiwan.
Virol J. 2017 Jan 10;14(1):1. doi: 10.1186/s12985-016-0669-1.
Tospoviruses, the plant-infecting genus in the family Bunyaviridae, are thrips borne and cause severe agricultural losses worldwide. Based on the serological relationships of the structural nucleocapsid protein (NP), the current tospoviruses are divided into six serogroups. The use of NP-antisera is convenient for virus detection, but it is insufficient to identify virus species grouped in a serogroup due to the serological cross-reaction. Alternatively, virus species can be identified by the N gene amplification using specific primers. Tomato spotted wilt virus (TSWV) is the type species of the genus Tospovirus and one of the most destructive plant viruses. Eight known tospoviruses, Alstroemeria necrotic streak virus (ANSV), Chrysanthemum stem necrosis virus (CSNV), Groundnut ringspot virus (GRSV), Impatiens necrotic spot virus (INSV), Melon severe mosaic virus (MeSMV), Pepper necrotic spot virus (PNSV), Tomato chlorotic spot virus (TCSV) and Zucchini lethal chlorosis virus (ZLCV), sharing serological relatedness with TSWV in NP, are grouped in the TSWV serogroup. Most of the TSWV-serogroup viruses prevail in Europe and America. An efficient diagnostic method is necessary for inspecting these tospoviruses in Asia, including Taiwan.
A microarray platform was developed for simultaneous detection and identification of TSWV-serogroup tospoviruses. Total RNAs extracted from Chenopodium quinoa leaves separately inoculated with ANSV, CSNV, GRSV, INSV, TCSV and TSWV were used for testing purposes. The 5'-biotinylated degenerate forward and reverse primers were designed from the consensus sequences of N genes of TSWV-serogroup tospoviruses for reverse transcription-polymerase chain reaction (RT-PCR) amplification. Virus-specific oligonucleotide probes were spotted on the surface of polyvinyl chloride (PVC) chips to hybridize with PCR products. The hybridization signals were visualized by hydrolysis of NBT/BCIP with streptavidine-conjugated alkaline phosphatase. The microarray was further applied to diagnose virus infection in field crop samples.
Amplicons of approximately 0.46 kb were amplified from all tested TSWV-serogroup tospoviruses by RT-PCR using the degenerate primer pair Pr-dTS-f/Pr-dTS-r. Virus species were identified on chips by hybridization of PCR products with respective virus-specific probes. The microarray was successfully used to diagnose TSWV infection in field pepper samples.
In this study, a rapid, sensitive and precise microarray method has been developed to simultaneously detect and identify six TSWV-serogroup tospoviruses. The microarray platform provides a great potential to explore tospoviruses that can help researchers and quarantine staff to prevent invasions of tospoviruses.
番茄斑萎病毒属是布尼亚病毒科中感染植物的一个属，由蓟马传播，在全球范围内造成严重的农业损失。根据结构核衣壳蛋白（NP）的血清学关系，目前的番茄斑萎病毒属病毒分为六个血清群。使用NP抗血清便于病毒检测，但由于血清学交叉反应，不足以鉴定血清群中的病毒种类。另外，可以使用特异性引物通过N基因扩增来鉴定病毒种类。番茄斑萎病毒（TSWV）是番茄斑萎病毒属的模式种，也是最具破坏性的植物病毒之一。八种已知的番茄斑萎病毒属病毒，即六出花坏死条纹病毒（ANSV）、菊花茎坏死病毒（CSNV）、花生环斑病毒（GRSV）、凤仙花坏死斑病毒（INSV）、甜瓜严重花叶病毒（MeSMV）、辣椒坏死斑病毒（PNSV）、番茄褪绿斑病毒（TCSV）和西葫芦致死褪绿病毒（ZLCV），在NP上与TSWV具有血清学相关性，被归为TSWV血清群。大多数TSWV血清群病毒在欧洲和美洲流行。在亚洲，包括台湾，需要一种有效的诊断方法来检测这些番茄斑萎病毒属病毒。
开发了一种微阵列平台，用于同时检测和鉴定TSWV血清群的番茄斑萎病毒属病毒。从分别接种了ANSV、CSNV、GRSV、INSV、TCSV和TSWV的藜麦叶片中提取的总RNA用于测试。根据TSWV血清群番茄斑萎病毒属病毒N基因的共有序列设计5'-生物素化简并正向和反向引物，用于逆转录-聚合酶链反应（RT-PCR）扩增。将病毒特异性寡核苷酸探针点样在聚氯乙烯（PVC）芯片表面，与PCR产物杂交。用链霉亲和素偶联的碱性磷酸酶水解NBT/BCIP使杂交信号可视化。该微阵列进一步应用于诊断田间作物样本中的病毒感染。
使用简并引物对Pr-dTS-f/Pr-dTS-r通过RT-PCR从所有测试的TSWV血清群番茄斑萎病毒属病毒中扩增出约0.46 kb的扩增子。通过PCR产物与各自病毒特异性探针的杂交在芯片上鉴定病毒种类。该微阵列成功用于诊断田间辣椒样本中的TSWV感染。
在本研究中，开发了一种快速、灵敏且精确的微阵列方法，用于同时检测和鉴定六种TSWV血清群番茄斑萎病毒属病毒。该微阵列平台为探索番茄斑萎病毒属病毒提供了巨大潜力，可帮助研究人员和检疫人员预防番茄斑萎病毒属病毒的入侵。
