Development and evaluation of one-step RT-qPCR TaqMan multiplex panels applied to six viruses occurring in lily and tulip bulbs

IF 2.2 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods Pub Date : 2024-06-18 DOI:10.1016/j.jviromet.2024.114987

M.P.E. van Gent-Pelzer , A.M. Dullemans , M. Verbeek, P.J.M. Bonants, T.A.J. van der Lee

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Abstract

One-step RT-qPCR TaqMan assays have been developed for six plant viruses with considerable economic impact in the growing of tulip and lily bulbs: lily mottle virus, lily symptomless virus, lily virus X, Plantago asiatica mosaic virus, tulip breaking virus and tulip virus X. To enhance efficacy and cost-efficiency these assays were combined into multiplex panels. Four different multiplex panels were designed, each consisting of three virus assays and an adapted assay for the housekeeping gene nad5 of lilies and tulips, that acts as an internal amplification control. To eliminate false negative results due to variation in the viral genome sequences, for each target virus two assays were developed on distinct conserved genomic regions. Specificity, PCR efficiency and compatibility of primers and probes were tested using gBlock constructions. Diagnostic samples were used to evaluate the strategy. High Throughput Sequencing of a set of the diagnostic samples, further verified the presence or absence of the viruses in the RNA samples and sequence variations in the target genes. This interchangeable multiplex panel strategy may be a valuable tool for the detection of viruses in certification, surveys and virus diagnostics.

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开发和评估适用于百合和郁金香球茎中六种病毒的一步式 RT-qPCR TaqMan 多重检测板。

针对对郁金香和百合球茎种植有重大经济影响的六种植物病毒（百合斑驳病毒、百合无症状病毒、百合病毒 X、车前草花叶病毒、郁金香破损病毒和郁金香病毒 X）开发了一步式 RT-qPCR TaqMan 检测方法。设计了四种不同的多重检测板，每种检测板由三种病毒检测和一种百合和郁金香看家基因 nad5（作为内部扩增对照）的改良检测组成。为了消除因病毒基因组序列变异而导致的假阴性结果，针对每种目标病毒都开发了针对不同保守基因组区域的两种检测方法。利用 gBlock 结构测试了引物和探针的特异性、PCR 效率和兼容性。诊断样本用于评估该策略。一组诊断样本的高通量测序进一步验证了 RNA 样本中病毒的存在与否以及目标基因的序列变异。这种可互换的多重面板策略可能是在认证、调查和病毒诊断中检测病毒的重要工具。

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来源期刊

Journal of virological methods 医学-病毒学

CiteScore

5.80

自引率

0.00%

发文量

209

审稿时长

41 days

期刊介绍： The Journal of Virological Methods focuses on original, high quality research papers that describe novel and comprehensively tested methods which enhance human, animal, plant, bacterial or environmental virology and prions research and discovery. The methods may include, but not limited to, the study of: Viral components and morphology- Virus isolation, propagation and development of viral vectors- Viral pathogenesis, oncogenesis, vaccines and antivirals- Virus replication, host-pathogen interactions and responses- Virus transmission, prevention, control and treatment- Viral metagenomics and virome- Virus ecology, adaption and evolution- Applied virology such as nanotechnology- Viral diagnosis with novelty and comprehensive evaluation. We seek articles, systematic reviews, meta-analyses and laboratory protocols that include comprehensive technical details with statistical confirmations that provide validations against current best practice, international standards or quality assurance programs and which advance knowledge in virology leading to improved medical, veterinary or agricultural practices and management.