Filardo Fiona, Vukovic Peter, Sharman Murray, Gambley Cherie, Campbell Paul
Queensland Department of Agriculture and Fisheries, Ecosciences Precinct, GPO Box 267, Brisbane, QLD 4001, Australia.
Plants (Basel). 2022 Sep 5;11(17):2325. doi: 10.3390/plants11172325.
Assays for the high throughput screening of crops for virus monitoring need to be quick, easy, and low cost. One method involves using tissue blot immunoassays (TBIA), where plant stems are blotted onto nitrocellulose membrane and screened with available antibodies against a range of viruses. TBIAs are inexpensive but limited by antibody availability and specificity. To circumvent the antibody limitations, we developed the tissue blot hybridization chain reaction (TB-HCR). As with TBIA, plant stems are blotted onto a nitrocellulose membrane, however, TB-HCR involves using nucleic acid probes instead of antibodies. We demonstrated for the first time that TB-HCR can be used for plant viruses by designing and testing probes against species from several virus genera including , , , , and . We also explored different hairpin reporter methods such as biotin/streptavidin-AP and the Alexa Fluor-488 Fluorophore. TB-HCR has applications for low-cost diagnostics for large sample numbers, rapid diagnostic deployment for new viruses, and can be performed as a preliminary triage assay prior to downstream applications.
用于作物高通量病毒监测筛选的检测方法需要快速、简便且成本低廉。一种方法是使用组织印迹免疫分析（TBIA），即将植物茎部印迹到硝酸纤维素膜上，并用针对多种病毒的现有抗体进行筛选。TBIA成本低廉，但受抗体可用性和特异性的限制。为了规避抗体的局限性，我们开发了组织印迹杂交链式反应（TB-HCR）。与TBIA一样，将植物茎部印迹到硝酸纤维素膜上，然而，TB-HCR使用核酸探针而非抗体。我们首次通过设计和测试针对包括……等几个病毒属物种的探针，证明了TB-HCR可用于植物病毒检测。我们还探索了不同的发夹报告方法，如生物素/链霉亲和素-碱性磷酸酶和Alexa Fluor-488荧光团。TB-HCR可用于大量样本的低成本诊断、新病毒的快速诊断部署，并且可在下游应用之前作为初步筛选检测。
