一种用于诊断日本血吸虫病的新型荧光免疫层析检测条。

A novel fluorescence immunochromatographic assay strip for the diagnosis of schistosomiasis japonica.

机构信息

Key Laboratory of Animal Parasitology, Ministry of Agriculture of China, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China.

University of Reading, Whiteknights, Reading, Berkshire, RG26UA, England.

出版信息

Parasit Vectors. 2021 Jan 6;14(1):8. doi: 10.1186/s13071-020-04511-6.

DOI:10.1186/s13071-020-04511-6

PMID:33407752

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7788720/

Abstract

BACKGROUND

Schistosomiasis japonica is a severe zoonosis. Domestic animals are the primary source of infection and play an important role in disease transmission. Surveillance and diagnosis play key roles in schistosomiasis control; however, current techniques for the surveillance and diagnosis of the disease have limitations. In this study, we developed a novel fluorescence immunochromatographic assay (FICA) strip to detect anti-Schistosoma japonicum antibodies in host serum.

METHODS

A FICA strip was developed for the diagnosis of Schistosoma japonicum in domestic animals. Streptococcus protein G (SPG) and soluble egg antigen (SEA) were transferred onto a nitrocellulose (NC) membrane to form the control line (C) and the test line (T), respectively. With fluorescence activity as well as binding activity to multispecies IgG, the recombinant protein rSPG-RFP was expressed and employed as an antibody indicator in the FICA strips.

RESULTS

The dual gene fusion plasmid was verified by PCR and restriction enzyme digestion. The expressed recombinant protein was 39.72 kDa in size, which was consistent with the predicted molecular weight. The western blot results showed binding activity between rSPG-RFP and IgGs from different hosts. Fluorescence microscopy also showed the fluorescence activity of the protein present. The affinity constant (Ka) values of rSPG-RFP with rabbit, donkey, mouse and goat IgG were 1.9 × 10, 4.1 × 10, 1.7 × 10 and 5.4 × 10, respectively. Moreover, based on the recombinant protein, the test strip for detecting S. japonicum in buffaloes could distinguish positive from negative serum. The lower limit of detection of the FICA strip was 1:10,000. Compared with ELISA, the FICA strips exhibited similar results in the diagnosis of infection in clinical bovine serum samples, with a kappa value of 0.9660 and P < 0.01. The cross-reactivities of the FICA strips with Haemonchus contortus and Schistosoma turkestanicum (30.15% and 91.66%, respectively) were higher than those of ELISA (26.98% and 87.5%, respectively).

CONCLUSIONS

Based on the rSPG-RFP protein that we developed, strip detection can be completed within 15 min. Heightened sensitivity allows the strip to accurately identify schistosome antibodies in serum. In conclusion, this method is convenient, feasible, rapid and effective for detecting S. japonicum.

摘要

背景

日本血吸虫病是一种严重的人畜共患病。家畜是主要的感染源，在疾病传播中起着重要作用。监测和诊断在血吸虫病控制中起着关键作用；然而，目前该疾病的监测和诊断技术存在局限性。本研究开发了一种新的荧光免疫层析检测试剂盒（FICA）条，用于检测宿主血清中的抗日本血吸虫抗体。

方法

为了诊断家畜中的日本血吸虫病，我们开发了一种 FICA 条。链球菌蛋白 G（SPG）和可溶性虫卵抗原（SEA）被转移到硝酸纤维素（NC）膜上，分别形成质控线（C）和检测线（T）。重组蛋白 rSPG-RFP 具有荧光活性和与多物种 IgG 的结合活性，被用作 FICA 条中的抗体指示剂。

结果

双基因融合质粒通过 PCR 和酶切进行验证。表达的重组蛋白大小为 39.72 kDa，与预测的分子量一致。Western blot 结果显示 rSPG-RFP 与来自不同宿主的 IgGs 之间存在结合活性。荧光显微镜也显示了该蛋白的荧光活性。rSPG-RFP 与兔、驴、鼠和山羊 IgG 的亲和常数（Ka）值分别为 1.9×10、4.1×10、1.7×10 和 5.4×10。此外，基于该重组蛋白，我们可以检测水牛中的日本血吸虫病，能够区分阳性和阴性血清。FICA 条的检测下限为 1:10,000。与 ELISA 相比，FICA 条在诊断临床牛血清样本中的感染时具有相似的结果，kappa 值为 0.9660，P<0.01。FICA 条与捻转血矛线虫和土耳其斯坦东毕吸虫的交叉反应性（分别为 30.15%和 91.66%）高于 ELISA（分别为 26.98%和 87.5%）。