Mwendwa Fridah, Mbae Cecilia K, Kinyua Johnson, Mulinge Erastus, Mburugu Gitonga Nkanata, Njiru Zablon K
Institute of Tropical Medicine and Infectious Diseases, Jomo Kenyatta University of Agriculture and Technology, P.O Box 62000-0200, Nairobi, Kenya.
Centre for Microbiology Research, Kenya Medical Research Institute, P.O Box 19464-00202, Nairobi, Kenya.
BMC Res Notes. 2017 Mar 31;10(1):142. doi: 10.1186/s13104-017-2466-3.
Entamoeba histolytica, the causative agent for amoebiasis is a considerable burden to population in the developing countries where it accounts for over 50 million infections. The tools for detection of amoebiasis are inadequate and diagnosis relies on microscopy which means a significant percent of cases remain undiagnosed. Moreover, tests formats that can be rapidly applied in rural endemic areas are not available.
In this study, a loop-mediated isothermal test (LAMP) based on 18S small subunit ribosomal RNA gene was designed with extra reaction accelerating primers (stem primers) and compared with the published LAMP and PCR tests in detection of E. histolytica DNA in clinical samples.
The stem LAMP test indicated shorter time to results by an average 11 min and analytical sensitivity of 10 (30 pg/ml) compared to the standard LAMP and PCR which showed sensitivities levels of 10 (3 ng/ml) and 10 (~30 ng/ml) respectively using tenfold serial dilution of DNA. In the analysis of clinical specimens positive for Entamoeba spp. trophozoites and cysts using microscopy, the stem LAMP test detected E. histolytica DNA in 36/126, standard LAMP test 20/126 and PCR 17/126 cases respectively. There was 100% agreement in detection of the stem LAMP test product using fluorescence of SYTO-9 dye in real time machine, through addition of 1/10 dilution of SYBR Green I and electrophoresis in 2% agarose gel stained with ethidium bromide.
The stem LAMP test developed in this study indicates potential towards detection of E. histolytica.
溶组织内阿米巴是阿米巴病的病原体，在发展中国家给人群带来了相当大的负担，这些国家有超过5000万例感染。阿米巴病的检测工具不足，诊断依赖显微镜检查，这意味着很大比例的病例仍未被诊断出来。此外，没有可在农村流行地区快速应用的检测形式。
在本研究中，基于18S小亚基核糖体RNA基因设计了一种带有额外反应加速引物（茎环引物）的环介导等温扩增试验（LAMP），并与已发表的LAMP和PCR试验在检测临床样本中溶组织内阿米巴DNA方面进行比较。
与标准LAMP和PCR相比，茎环LAMP试验显示出平均缩短11分钟的出结果时间，分析灵敏度为10（约30 pg/ml），而标准LAMP和PCR分别使用DNA的十倍系列稀释显示灵敏度水平为10（约3 ng/ml）和10（约30 ng/ml）。在对显微镜检查显示溶组织内阿米巴滋养体和包囊阳性的临床标本进行分析时，茎环LAMP试验分别在36/126例中检测到溶组织内阿米巴DNA，标准LAMP试验在20/126例中检测到，PCR在17/126例中检测到。通过添加1/10稀释的SYBR Green I并在溴化乙锭染色的2%琼脂糖凝胶中进行电泳，使用实时机器中SYTO-9染料的荧光对茎环LAMP试验产物的检测有100%的一致性。
本研究中开发的茎环LAMP试验显示出检测溶组织内阿米巴的潜力。
