猪轮状病毒胶体金检测试纸条的研制及应用

doi:10.16431/j.cnki.1671-7236.2024.03.034

摘要/Abstract

摘要： 【目的】利用2株猪轮状病毒(Porcine rotavirus,PoRV)单克隆抗体,研制胶体金检测试纸条,用于PoRV的临床快速检测。【方法】制备40 nm粒径的胶体金溶液标记单克隆抗体4G5,将单克隆抗体5G4和羊抗鼠IgG分别包被在硝酸纤维素膜上,作为检测线和质控线。通过优化胶体金溶液最适pH、标记抗体和检测抗体的最佳使用量,制备PoRV胶体金检测试纸条;对试纸条的灵敏度、敏感性、特异性、重复性和稳定性进行评价。通过检测50份猪粪便样品,计算试纸条与RT-PCR方法的符合率,最后采用试纸条进行1 249份临床样品的测定。【结果】经优化后试纸条的最佳参数如下:胶体金溶液最佳pH为8.0,标记抗体4G5和检测抗体5G4最佳使用量分别为14.4 μg/mL和1 mg/mL。试纸条能检测出1∶2 000稀释的PoRV OSU株(G5型)病毒液,灵敏度为10^4.5 TCID₅₀/mL,且检测PoRV G9、G3和G4型毒株均为阳性。检测猪传染性胃肠炎病毒(Transmissible gastroenteritis virus,TGEV)、猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)、猪δ冠状病毒(Porcine deltacoronavirus,PDCoV)、猪伪狂犬病病毒(Porcine pseudorabies virus,PRV)和猪圆环病毒2型(Porcine circovirus type 2,PCV2)均为阴性。批内和批间重复性、稳定性良好。检测50份猪粪便样品,与RT-PCR方法的阳性符合率为97.5%(39/40),阴性符合率为100%(10/10),总体符合率为98%(49/50)。检测2022年在河南、山西省猪场收集的临床样品1 249份,阳性检出率为5.2%。【结论】本研究制备的PoRV胶体金检测试纸条具有良好的敏感性、特异性和重复性等优点,与RT-PCR方法的符合率较高,可用于规模化猪场PoRV的快速检测。

关键词: 猪轮状病毒(PoRV); 胶体金检测试纸条; 快速检测

Abstract: 【Objective】 Two strains of monoclonal antibodies of Porcine rotavirus (PoRV) were used to establish PoRV colloidal gold test strip for rapid detection of PoRV.【Method】 The 40 nm colloidal gold particles were used to label the monoclonal antibody 4G5.The monoclonal antibody 5G4 and sheep anti-mouse IgG were coated on the surface of nitrocellulose membrane as the detection line and the control line,respectively.The colloidal gold test strip was established by optimizing the optimal pH of colloidal gold solution,the optimal usage amount of the labeled antibody and the detection antibody.The sensitivity,specificity,repeatability and stability of the strip were assessed.The coincidence rate of the strip and RT-PCR was calculated by detecting 50 pig fecal samples,and then the strip was put to clinical use by detecting 1 249 clinical samples.【Result】 The optimal parameters of the test strip were as follows:The optimal pH of colloidal gold solution was 8.0,the optimal usage amounts of labeled antibody 4G5 and detection antibody 5G4 were 14.4 μg/mL and 1 mg/mL,respectively.The strip could detect a 1∶2 000 dilution of PoRV OSU strain (genotype G5),the sensitivity was 10^4.5 TCID₅₀/mL,and the detection results of PoRV G9,G3 and G4 strains were all positive.And no cross reaction occured with Transmissible gastroenteritis virus (TGEV),Porcine epidemic diarrhea virus (PEDV),Porcine deltacoronavirus (PDCoV),Porcine pseudorabies virus (PRV) and Porcine circovirus type 2 (PCV2).The assay results showed that the strip had good repeatability in and between batches and stability.50 pig fecal samples were detected by the strip and RT-PCR,the results showed that the positive coincidence rate was 97.5% (39/40),the negative coincidence rate was 100% (10/10),and the overall coincidence rate was 98% (49/50).1 249 clinical samples which were collected from pig farms in Henan and Shanxi provinces in 2022 were detected by the test strip,and the positive rate of PoRV was 5.2%.【Conclusion】 The colloidal gold strip developed in this study had good sensitivity,specificity and repeatability,and high coincidence rate with RT-PCR method,and could be used for PoRV rapid detection in industrialized pig farm.

Key words: Porcine rotavirus (PoRV); colloidal gold test strip; rapid detection

中图分类号:

S852.65⁺9.4

李妍花, 周兵强, 康笃利, 郝丽影, 汪志艳, 赵少若, 黄甜, 庞杏豪, 刘俊阳, 楚园园, 张云静, 高晓静, 邓均华, 田克恭. 猪轮状病毒胶体金检测试纸条的研制及应用[J]. 中国畜牧兽医, 2024, 51(3): 1223-1231.

LI Yanhua, ZHOU Bingqiang, KANG Duli, HAO Liying, WANG Zhiyan, ZHAO Shaoruo, HUANG Tian, PANG Xinghao, LIU Junyang, CHU Yuanyuan, ZHANG Yunjing, GAO Xiaojing, DENG Junhua, TIAN Kegong. Development and Application of Colloidal Gold Test Strip Against Porcine Rotavirus[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(3): 1223-1231.

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