Wu Y D, Xu M J, Wang Q Q, Zhou C X, Wang M, Zhu X Q, Zhou D H
State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No. 1, Xujiaping, Chengguan District, Lanzhou 730046, Gansu Province, China.
College of Animal Science, South China Agricultural University, Guangzhou 510642, Guangdong Province, China.
Vet Parasitol. 2017 Aug 30;243:199-203. doi: 10.1016/j.vetpar.2017.06.026. Epub 2017 Jun 27.
Toxoplasma gondii infects all warm-blooded vertebrates, resulting in a great threat to human health and significant economic loss to the livestock industry. Ingestion of infectious oocysts of T. gondii from the environment is the major source of transmission. Detection of T. gondii oocysts by existing methods is laborious, time-consuming and expensive. The objective of the present study was to develop a recombinase polymerase amplification (RPA) method combined with a lateral flow (LF) strip for detection of T. gondii oocysts in the soil and water. The DNA of T. gondii oocysts was amplified by a pair of specific primers based on the T. gondii B1 gene over 15min at a constant temperature ranging from 30°C to 45°C using RPA. The amplification product was visualized by the lateral flow (LF) strip within 5min using the specific probe added to the RPA reaction system. The sensitivity of the established assay was 10 times higher than that of nested PCR with a lower detection limit of 0.1 oocyst per reaction, and there was no cross-reactivity with other closely related protozoan species. Fifty environmental samples were further assessed for the detection validity of the LF-RPA assay (B1-LF-RPA) and compared with nested PCR based on the B1 gene sequence. The B1-LF-RPA and nested PCR both showed that 5 out of the 50 environmental samples were positive. The B1-LF-RPA method was also proven to be sufficiently tolerant of existing inhibitors in the environment. In addition, the advantages of simple operation, speediness and cost-effectiveness make B1-LF-RPA a promising molecular detection tool for T. gondii.
刚地弓形虫可感染所有温血脊椎动物，对人类健康构成巨大威胁，并给畜牧业造成重大经济损失。从环境中摄入刚地弓形虫的感染性卵囊是主要传播途径。用现有方法检测刚地弓形虫卵囊既费力、耗时又昂贵。本研究的目的是开发一种结合侧流（LF）试纸条的重组酶聚合酶扩增（RPA）方法，用于检测土壤和水中的刚地弓形虫卵囊。使用RPA，在30°C至45°C的恒定温度下，一对基于刚地弓形虫B1基因的特异性引物在15分钟内扩增刚地弓形虫卵囊的DNA。使用添加到RPA反应体系中的特异性探针，在5分钟内通过侧流（LF）试纸条使扩增产物可视化。所建立检测方法的灵敏度比巢式PCR高10倍，检测下限为每个反应0.1个卵囊，并且与其他密切相关的原生动物物种无交叉反应。进一步评估了50份环境样品用于LF-RPA检测（B1-LF-RPA）的有效性，并与基于B1基因序列的巢式PCR进行比较。B1-LF-RPA和巢式PCR均显示50份环境样品中有5份呈阳性。B1-LF-RPA方法也被证明对环境中现有的抑制剂具有足够耐受性。此外，操作简单、快速和成本效益高的优点使B1-LF-RPA成为一种有前景的刚地弓形虫分子检测工具。
