Zebardast Nozhat, Yeganeh Farshid, Gharavi Mohammad Javad, Abadi Alireza, Seyyed Tabaei Seyyed Javad, Haghighi Ali
Cellular and Molecular Research Center, Faculty of Medicine, Guilan University of Medical Sciences, Rasht, Iran.
Department of Immunology, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Acta Trop. 2016 Oct;162:233-238. doi: 10.1016/j.actatropica.2016.07.004. Epub 2016 Jul 14.
Entamoeba histolytica, Giardia lamblia and Cryptosporidium spp. are common causes of diarrheal and intestinal diseases all over the world. Microscopic methods are useful in the diagnosis of intestinal parasites (IPs), but their sensitivity was assessed approximately 60 percent. Recently, molecular techniques have been used increasingly for the identification and characterization of the parasites. Among those, in this study we have used multiplex PCR and Real-time PCR with melting curve analysis (qPCR-MCA) for simultaneous detection and differentiation of E. histolytica, E. dispar, E. moshkovskii, G. lamblia and Cryptosporidium spp. in human fecal samples. Twenty DNA samples from 12 E. histolytica and 8 E. dispar samples and twenty stool samples confirmed positive for G. lamblia and Cryptosporidium spp. were analyzed. After DNA extraction from the samples, multiplex PCR was done for detection and differentiation of above mentioned parasites. QPCR-MCA was also performed for the detection and differentiation of 11 isolates of above mentioned parasite in a cycle with a time and temperature. Multiplex PCR was able to simultaneous detect and differentiate of above mentioned parasite in a single reaction. QPCR-MCA was able to differentiate genus and species those five protozoa using melting temperature simultaneously at the same time and temperature programs. In total, qPCR-MCA diagnosed 7/11 isolation of E. histolytica, 6/8 isolation of E. dispar, 1/1 E. moshkovskii Laredo, 10/11 G. Lamblia and 6/11 Cryptosporidium spp. Application of multiplex PCR for detection of more than one species in a test in developing countries, at least in reference laboratories has accurate diagnosis and plays a critical role in differentiation of protozoan species. Multiplex PCR assay with a template and multi template had different results and it seems that using a set of primers with one template has higher diagnostic capability in compare with multi template. The results of this study showed that the use of the qPCR-MCA can be an effective method to simultaneous distinguish of the above mentioned parasites.
溶组织内阿米巴、蓝氏贾第鞭毛虫和隐孢子虫属是全球腹泻和肠道疾病的常见病因。显微镜检查方法对肠道寄生虫（IPs）的诊断很有用，但它们的敏感性约为60%。近年来，分子技术越来越多地用于寄生虫的鉴定和特征分析。其中，在本研究中，我们使用多重PCR和带有熔解曲线分析的实时PCR（qPCR-MCA）同时检测和区分人类粪便样本中的溶组织内阿米巴、迪斯帕内阿米巴、莫斯科维茨内阿米巴、蓝氏贾第鞭毛虫和隐孢子虫属。分析了来自12份溶组织内阿米巴和8份迪斯帕内阿米巴样本的20份DNA样本以及20份经证实蓝氏贾第鞭毛虫和隐孢子虫属呈阳性的粪便样本。从样本中提取DNA后，进行多重PCR以检测和区分上述寄生虫。还进行了qPCR-MCA，以便在一个循环中在特定时间和温度下检测和区分上述11种寄生虫分离株。多重PCR能够在单一反应中同时检测和区分上述寄生虫。qPCR-MCA能够在相同的时间和温度程序下，利用熔解温度同时区分这五种原生动物的属和种。总体而言，qPCR-MCA诊断出7/11的溶组织内阿米巴分离株、6/8的迪斯帕内阿米巴分离株、1/1的拉雷多莫斯科维茨内阿米巴、10/11的蓝氏贾第鞭毛虫和6/11的隐孢子虫属。在发展中国家，至少在参考实验室中，应用多重PCR在一次检测中检测多种物种具有准确的诊断能力，并且在原生动物物种的区分中起着关键作用。单模板和多模板的多重PCR检测结果不同，似乎与多模板相比，使用一组引物和单模板具有更高的诊断能力。本研究结果表明，使用qPCR-MCA可以成为同时区分上述寄生虫的有效方法。
