Diagnosis and identification of Leishmania spp. from Giemsa-stained slides, by real-time PCR and melting curve analysis in south-west of Iran.

S Khademvatan, N Neisi, S Maraghi, J Saki
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引用次数: 39

Abstract

Background: The aim of present study was describing a real-time PCR assay for the diagnosis and direct identification of Leishmania species on Giemsa-stained slides in south-west of Iran.

Materials and methods: Altogether, 102 Giemsa-stained slides were collected from different part of south-west of Iran between 2008 and 2011. All the Giemsa-stained slides were examined under light microscope. After DNA extraction, real-time PCR amplification and detection were conducted with fluorescent SYBR Green I. For identification, PCR products were analysed with melting curve analysis.

Results: One hundred and two archived slides from suspected lesion examined by microscopy and real-time PCR. The sensitivity of the real-time PCR on Giemsa-stained slid was 98% (96/102). The melting curve analysis (T(m)) were 88·3±0·2°C for L. tropica (MHOM/IR/02/Mash10), 86·5±0·2°C for L. major (MHOM/IR/75/ER) and 89·4±0·3°C for L. infantum (MCAN/IR/97/LON 49), respectively.

Conclusion: This study is first report in use of real-time PCR for diagnosis and identification of Leishmania spp. in Iran. Up to now, in Iran, the majority of identification of Leishmania species is restriction fragment length polymorphism (PCR-RFLP) of ITS1 and kinetoplast DNA. Our data showed that Giemsa-stained slides that were stored more than 3 years, can be use for Leishmania DNA extraction and amplification by real-time PCR. Compared to conventional PCR-based methods, the real-time PCR is extremely rapid with results and more samples can be processed at one time.

伊朗西南部吉姆萨染色玻片中利什曼原虫的实时PCR和熔融曲线分析诊断和鉴定。
背景:本研究的目的是描述一种实时PCR方法,用于诊断和直接鉴定伊朗西南部吉姆萨染色玻片上的利什曼原虫。材料和方法:2008年至2011年,在伊朗西南部不同地区收集了102份giemsa染色玻片。光镜下检查所有giemsa染色的载玻片。提取DNA后,用SYBR Green i荧光仪进行实时PCR扩增和检测,PCR产物用熔融曲线分析进行鉴定。结果:对102例疑似病变的存档切片进行了镜检和实时PCR检测。real-time PCR对giemsa染色滑动的灵敏度为98%(96/102)。热带l (MHOM/IR/02/Mash10)、主要l (MHOM/IR/75/ER)和infuml (MCAN/IR/97/LON 49)的熔解曲线(T(m))分别为88·3±0.2°C、86.5±0.2°C。结论:利用实时荧光定量PCR技术对伊朗利什曼原虫进行诊断鉴定尚属首次报道。到目前为止,在伊朗,利什曼原虫的鉴定主要是ITS1和着丝体DNA的限制性片段长度多态性(PCR-RFLP)。我们的数据显示,保存3年以上的giemsa染色载玻片可以用于利什曼原虫DNA的提取和实时PCR扩增。与传统的基于PCR的方法相比,实时PCR的结果非常快,并且一次可以处理更多的样品。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Annals of tropical medicine and parasitology
Annals of tropical medicine and parasitology 医学-公共卫生、环境卫生与职业卫生
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