Key Laboratory of Molecular Biophysics of Ministry of Education, Department of Biomedical Engineering, College of Life Science and Technology, Center for Human Genome Research, Huazhong University of Science and Technology, Wuhan, China.
Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening, Jiangsu Key Laboratory of Marine Biological Resources and Environment, Co-Innovation Center of Jiangsu Marine Bio-industry Technology, School of Pharmacy, Jiangsu Ocean University, Lianyungang, China.
J Fish Dis. 2021 Nov;44(11):1753-1763. doi: 10.1111/jfd.13492. Epub 2021 Jul 8.
Shrimp is a globally popular seafood. Shrimp farming has been challenged by various infectious diseases that lead to significant economic losses. The prevention of two important shrimp infectious diseases, the acute hepatopancreatic necrosis disease (AHPND) and the Enterocytozoon hepatopenaei (EHP) infection, is highly dependent on early and accurate diagnostic. On-site monitoring of the two diseases in shrimp farming facilities demands point-of-care-testing (POCT) type of diagnostic assays. This study established a duplex recombinase polymerase amplification (RPA) and lateral flow dipstick (LFD) combined assay that could simultaneously diagnose the two diseases. The optimized RPA-LFD assay could finish the diagnostic in 35 min with good specificity, and the sensitivity reached 10 and 10 gene copies per reaction for EHP and AHPND, respectively, which were at the same level as the currently available molecular diagnostic assays. Test results of clinical samples showed 100% agreement of this assay with the industrial standard nested polymerase chain reaction (PCR) assays, and samples with both diseases were simultaneously identified. Because of the isothermal 37℃ amplification and the visual reading of the signal on dipsticks, the dependence on equipment is minimal. This duplex RPA-LFD assay is well suited for simultaneous POCT diagnostic of the two important shrimp infectious diseases. Moreover, the principle can be applied to multiplex POCT diagnostic of other infectious diseases in aquaculture.
虾是一种全球流行的海鲜。各种传染性疾病给虾类养殖带来了挑战，导致了重大的经济损失。预防两种重要的虾类传染病，急性肝胰腺坏死病（AHPND）和对虾杆状病毒（EHP）感染，高度依赖于早期和准确的诊断。在虾类养殖场设施中对这两种疾病进行现场监测需要即时检测（POCT）类型的诊断检测。本研究建立了一种双重重组酶聚合酶扩增（RPA）和侧流纸条（LFD）联合检测方法，可以同时诊断这两种疾病。优化后的 RPA-LFD 检测法在 35 分钟内完成诊断，具有良好的特异性，EHP 和 AHPND 的灵敏度分别达到 10 和 10 个基因拷贝/反应，与目前可用的分子诊断检测法水平相当。临床样本的检测结果表明，该检测法与工业标准嵌套聚合酶链反应（PCR）检测法完全一致，并且可以同时鉴定出同时患有两种疾病的样本。由于在 37℃的等温条件下进行扩增，并且可以在纸条上直观地读取信号，因此对设备的依赖性很小。这种双重 RPA-LFD 检测法非常适合同时进行两种重要的虾类传染病的即时 POCT 诊断。此外，该原理可应用于水产养殖中其他传染病的多重 POCT 诊断。
