Memon Atta Muhammad, Chen Fangzhou, Khan Sher Bahadar, Guo Xiaozhen, Khan Rajwali, Khan Farhan Anwar, Zhu Yinxing, He Qigai
State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.
Department of Livestock Management, Breeding and Genetics, The University of Agriculture Peshawar, Peshawar, Pakistan.
Anim Biotechnol. 2023 Nov;34(5):1807-1814. doi: 10.1080/10495398.2022.2052304. Epub 2022 May 20.
Rotaviruses are rising as zoonotic viruses worldwide, causing the lethal dehydrating diarrhea in children, piglets, and other livestock of economic importance. A simple, swift, cost-effective, highly specific, and sensitive antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) was developed for detection of porcine rotavirus-A (PoRVA) by employing rabbit (capture antibody) and murine polyclonal antibodies (detector antibody) produced against VP6 of PoRVA (RVA/Pig-tc/CHN/TM-a/2009/G9P23). Reactivity of the both polyclonal antibodies was confirmed by using an indirect ELISA, western-blot analysis and indirect fluorescence assay against rVP6 protein and PoRVA. The detection limit of AC-ELISA was found 50 ng/ml of PoRVA protein. The relative sensitivity and specificity of this in-house AC-ELISA were evaluated for detection of PoRVA from porcine diarrhea samples, and results were compared with that of RT-PCR and TaqMan RT-qPCR. The relative sensitivity and specificity of AC-ELISA compared with those of TaqMan RT-qPCR were found as and respectively, with the strong agreement (κ ) between these two techniques. Furthermore, AC-ELISA could not detect any cross-reactivity with porcine epidemic diarrhea virus, transmissible gastro-enteritis virus, pseudo rabies virus and porcine circovirus-2. This in-house AC-ELISA efficiently detected PoRVA from clinical samples, which suggests that this technique can be used for large-scale surveillance and timely detection of rotavirus infection in the porcine farms.
轮状病毒在全球范围内作为人畜共患病毒不断增加，可导致儿童、仔猪和其他具有经济重要性的家畜发生致命性脱水腹泻。通过使用针对猪轮状病毒A（PoRVA，RVA/Pig-tc/CHN/TM-a/2009/G9P23）的VP6产生的兔（捕获抗体）和鼠多克隆抗体（检测抗体），开发了一种简单、快速、经济高效、高度特异且灵敏的抗原捕获酶联免疫吸附测定（AC-ELISA）用于检测PoRVA。通过使用针对rVP6蛋白和PoRVA的间接ELISA、western印迹分析和间接荧光测定法，证实了两种多克隆抗体的反应性。发现AC-ELISA对PoRVA蛋白的检测限为50 ng/ml。对该自制AC-ELISA从猪腹泻样本中检测PoRVA的相对敏感性和特异性进行了评估，并将结果与RT-PCR和TaqMan RT-qPCR的结果进行了比较。与TaqMan RT-qPCR相比，AC-ELISA的相对敏感性和特异性分别为[具体数值未给出]和[具体数值未给出]，这两种技术之间具有很强的一致性（κ值）。此外，AC-ELISA未检测到与猪流行性腹泻病毒、传染性胃肠炎病毒、伪狂犬病病毒和猪圆环病毒2的任何交叉反应。这种自制AC-ELISA能够有效地从临床样本中检测到PoRVA，这表明该技术可用于猪场轮状病毒感染的大规模监测和及时检测。
