Lagatie Ole, Merino Michelle, Batsa Debrah Linda, Debrah Alexander Y, Stuyver Lieven J
Janssen Diagnostics, Janssen R&D, Turnhoutseweg 30, 2340, Beerse, Belgium.
Kumasi Centre for Collaborative Research into Tropical medicine, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana.
Parasit Vectors. 2016 Dec 1;9(1):624. doi: 10.1186/s13071-016-1913-7.
Diagnostic procedures for the diagnosis of infection with the nematode parasite Onchocerca volvulus are currently based on the microscopic detection of microfilariae in skin biopsies. Alternative approaches based on amplification of parasitic DNA in these skin biopsies are currently being explored. Mostly this is based on the detection of the O-150 repeat sequence using PCR based techniques.
An isothermal, loop-mediated amplification method has been designed using the mitochondrial O. volvulus cox1 gene as a target.
Analysis of dilution series of synthetic DNA containing the targeted sequence show a non-linear dose-response curve, as is usually the case for isothermal amplification methods. Evaluation of cross-reactivity with the heterologous sequence from the closely related parasites Wuchereria bancrofti, Loa loa and Brugia malayi demonstrated strong specificity, as none of these sequences was amplified. The assay however amplified both O. volvulus and O. ochengi DNA, but with a different melting point that can be used to discriminate between the species. Evaluation of this assay in a set of skin snip biopsies collected in an endemic area in Ghana showed a high correlation with O-150 qPCR and also demonstrated a similar sensitivity. Compared to qPCR, LAMP had a sensitivity of 88.2% and a specificity of 99.2%.
We have developed a sensitive and specific loop-mediated amplification method for detection of O. volvulus DNA in skin biopsies that is capable of providing results within 30 min.
目前，用于诊断线虫寄生虫旋盘尾丝虫感染的诊断程序是基于对皮肤活检组织中微丝蚴的显微镜检测。目前正在探索基于这些皮肤活检组织中寄生虫DNA扩增的替代方法。大多数情况下，这是基于使用基于PCR的技术检测O - 150重复序列。
设计了一种以线粒体旋盘尾丝虫细胞色素c氧化酶亚基1（cox1）基因为靶标的等温环介导扩增方法。
对含有靶向序列的合成DNA稀释系列的分析显示出非线性剂量反应曲线，这是等温扩增方法通常的情况。对与密切相关的寄生虫班氏吴策线虫、罗阿丝虫和马来布鲁线虫的异源序列的交叉反应性评估显示出很强的特异性，因为这些序列均未被扩增。然而，该检测方法扩增了旋盘尾丝虫和欧氏盘尾丝虫的DNA，但熔解温度不同，可用于区分这两个物种。在加纳一个流行地区收集的一组皮肤剪取活检组织中对该检测方法的评估显示，与O - 150定量PCR高度相关，并且灵敏度相似。与定量PCR相比，环介导等温扩增法的灵敏度为88.2%，特异性为99.2%。
我们开发了一种灵敏且特异的环介导扩增方法，用于检测皮肤活检组织中的旋盘尾丝虫DNA，该方法能够在30分钟内得出结果。
