Abdalhamed Abeer Mostafa, Naser Soad Mohammed, Mohamed Ayman Hamady, Zeedan Gamil Sayed Gamil
Department of Parasitology and Animals Diseases (Infectious Diseases), National Research Centre, Dokki, Giza, Egypt.
Clinical Pathology Research Unit, Department of Parasitology and Animals Diseases, National Research Centre, Dokki, Giza, Egypt.
Iran J Microbiol. 2022 Aug;14(4):574-586. doi: 10.18502/ijm.v14i4.10245.
Rapid diagnosis is a cornerstone for controlling and preventing viral disease outbreaks. The present study is aimed to develop a rapid field diagnostic test based on gold nanoparticles for the detection of lumpy skin diseases (LSD), and foot and mouth diseases (FMD) in animals with high sensitivity and specificity.
FMD and LSD vaccines were used as a source of viruses' antigens for preparing monoclonal antibodies and conjugated with gold nanoparticles that characterized using various techniques such as UV-visible spectrometry, and transmission electron microscopy (TEM). Monoclonal antibodies (mAbs) for each serotype produced in experimental rats and used to capture antibodies for FMDV and/or LSDV. ELISA was used to screen 469 milk samples and 1165 serum samples from naturally infected cattle, buffaloes, sheep, and goats for validation of the lateral flow test (LFT). LSDV DNA was extracted from 117 blood and skin biopsy samples collected from naturally infected cattle during the 2019 outbreak.
The specificity and sensitivity of GNP-LFT were evaluated and compared to Ag-ELISA, Western blot tests (WB), and PCR. A total of 95 FMDV positives out of 469 (20.25%) milk samples and 268 FMDV positives out of 1165 (23.3%) serum samples from natural infected cattle, buffaloes, sheep, and goats examined by ELISA to valid GNPS-LFT Viral LSDV DNA was detected in 60/117 (51.5%) and 31/60 (52.9%). While the GNPS-LFT assay results were 49/117 (41.9%) and 29/60 (48.3%) blood and skin biopsy samples, respectively. The diagnostic sensitivity and specificity of the GNP-LFT test were 72% and 82%, respectively. All vesicular fluid and epithelium samples collected from infected animals were identified as positive by the GNP-LFT and Ag-ELISA. Ag-ELISA, on the other hand, was 90% and 100%. While the developed GNP-LFT used LSDV polyclonal antibodies were similar to ELISA and IgG-WB with a sensitivity of 72.8% and a specificity of 88.8%, respectively.
The GNPS-LFT is a novel immunoassay based on mono or polyclonal antibodies conjugated with gold nanoparticles that provides an accurate, rapid, specific, and sensitive tool for field rapid diagnosis of FMDV and LSDV.
快速诊断是控制和预防病毒性疾病爆发的基石。本研究旨在开发一种基于金纳米颗粒的快速现场诊断测试，用于高灵敏度和特异性地检测动物的结节性皮肤病（LSD）和口蹄疫（FMD）。
口蹄疫和结节性皮肤病疫苗用作病毒抗原来源，用于制备单克隆抗体，并与金纳米颗粒偶联，通过紫外可见光谱和透射电子显微镜（TEM）等各种技术对其进行表征。在实验大鼠中制备每种血清型的单克隆抗体（mAb），并用于捕获口蹄疫病毒（FMDV）和/或结节性皮肤病病毒（LSDV）的抗体。采用酶联免疫吸附测定（ELISA）对469份牛奶样本和1165份来自自然感染牛、水牛、绵羊和山羊的血清样本进行筛查，以验证侧向流动试验（LFT）。从2019年疫情期间自然感染牛采集的117份血液和皮肤活检样本中提取LSDV DNA。
对金纳米颗粒侧向流动试验（GNP-LFT）的特异性和灵敏度进行了评估，并与抗原ELISA、蛋白质免疫印迹试验（WB）和聚合酶链反应（PCR）进行比较。通过ELISA检测的来自自然感染牛、水牛、绵羊和山羊的469份牛奶样本中有95份（20.25%）口蹄疫病毒阳性，1165份血清样本中有268份（23.3%）口蹄疫病毒阳性，以验证金纳米颗粒侧向流动试验。在60/117（51.5%）和31/60（52.9%）的样本中检测到病毒LSDV DNA。而金纳米颗粒侧向流动试验的检测结果分别为血液和皮肤活检样本中的49/117（41.9%）和29/60（48.3%）。GNP-LFT测试的诊断灵敏度和特异性分别为72%和82%。从感染动物采集的所有水疱液和上皮样本通过GNP-LFT和抗原ELISA鉴定为阳性。另一方面，抗原ELISA的灵敏度和特异性分别为90%和100%。而使用LSDV多克隆抗体开发的GNP-LFT与ELISA和IgG-WB相似，灵敏度分别为72.8%，特异性为88.8%。
金纳米颗粒侧向流动试验是一种基于与金纳米颗粒偶联的单克隆或多克隆抗体的新型免疫测定方法，为口蹄疫病毒和结节性皮肤病病毒的现场快速诊断提供了一种准确、快速、特异且灵敏的工具。
