Bari Tanjina, Al Mamun Md Abdullah, Toet Hayley, Rathinasamy Vignesh, Larkins Jo-Ann, Beddoe Travis, Spithill Terry W, Piedrafita David, Greenhill Andrew R
Institute of Innovation, Science and Sustainability (IISS), Federation University, Australia.
Faculty of Medicine, Nursing and Health Sciences, Monash University, VIC, Australia; Department of Parasitology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh.
Vet Parasitol. 2024 Apr;327:110132. doi: 10.1016/j.vetpar.2024.110132. Epub 2024 Jan 19.
Fasciola hepatica causes liver fluke disease in production animals and humans worldwide. Faecal egg counts (FEC) are the most common diagnostic tool for the diagnosis of liver fluke disease. However, FEC has low sensitivity and is often unreliable for the detection of patent infection. In this study, loop-mediated isothermal amplification (LAMP) was optimised and evaluated for the detection of Fasciola hepatica infection, with the aim of increased sensitivity and making it suitable for on-farm application. LAMP was initially conducted under laboratory conditions, optimised to enable visual detection using calcein dye. DNA extraction based on bead-beating was developed to enable on-farm application. LAMP results were compared to FEC and polymerase chain reaction (PCR). Under laboratory conditions, LAMP was conducted using two incubation methods: a conventional PCR thermocycler and a field-deployable LAMP instrument. When compared to a 'rigorous' FEC protocol consisting of multiple counts using a comparatively large volume of faeces and with infection confirmed post-mortem, LAMP was highly sensitive and specific (using silica membrane DNA extraction sensitivity 88 %, specificity 100 %; using sieving and beat-beating DNA extraction sensitivity 98.9 %, specificity 100 %). When applied on-farm, LAMP was compared to conventional FEC, which suggested high sensitivity but low specificity (sensitivity 97 %, specificity 37.5 %). However, further analysis, comparing field LAMP results to laboratory PCR, suggested that the low specificity was likely the outcome of the inability of conventional FEC to detect all true F. hepatica positive samples. Based on the high sensitivity and specificity of LAMP compared to a 'rigorous' FEC protocol and its ability to be used in field settings, the study demonstrates the potential of LAMP for diagnosing F. hepatica infection in agriculture.
肝片吸虫在全球范围内的生产动物和人类中引发肝吸虫病。粪便虫卵计数（FEC）是诊断肝吸虫病最常用的诊断工具。然而，FEC的灵敏度较低，对于检测显性感染往往不可靠。在本研究中，对环介导等温扩增（LAMP）技术进行了优化和评估，以检测肝片吸虫感染，目的是提高灵敏度并使其适用于农场应用。LAMP最初在实验室条件下进行，优化后可使用钙黄绿素染料进行可视化检测。开发了基于珠磨法的DNA提取方法，以实现农场应用。将LAMP结果与FEC和聚合酶链反应（PCR）进行比较。在实验室条件下，LAMP采用两种孵育方法进行：传统的PCR热循环仪和可现场部署的LAMP仪器。与一种“严格的”FEC方案（使用相对大量粪便进行多次计数，并在死后确认感染）相比，LAMP具有高度的敏感性和特异性（使用硅胶膜DNA提取时灵敏度为88%，特异性为100%；使用筛分和珠磨法DNA提取时灵敏度为98.9%，特异性为100%）。在农场应用时，将LAMP与传统FEC进行比较，结果显示其灵敏度高但特异性低（灵敏度97%，特异性37.5%）。然而，进一步分析将现场LAMP结果与实验室PCR结果进行比较，表明低特异性可能是由于传统FEC无法检测到所有真正的肝片吸虫阳性样本所致。基于与“严格的”FEC方案相比LAMP具有高灵敏度和特异性以及其在现场环境中使用的能力，该研究证明了LAMP在农业中诊断肝片吸虫感染的潜力。
