Real-Time Fluorescence Loop Mediated Isothermal Amplification for the Diagnosis of Malaria

被引：170

作者：

Lucchi, Naomi W. ^{[1
,2
]}

Demas, Allison ^{[1
]}

Narayanan, Jothikumar ^{[3
]}

Sumari, Deborah ^{[4
]}

Kabanywanyi, Abdunoor ^{[4
]}

Kachur, S. Patrick ^{[1
]}

Barnwell, John W. ^{[1
]}

Udhayakumar, Venkatachalam ^{[1
,2
]}

机构：

[1] Ctr Dis Control & Prevent, Malaria Branch, Div Parasit Dis & Malaria, Ctr Global Hlth, Atlanta, GA 30333 USA

[2] Atlanta Res & Educ Fdn, VA Med Ctr, Decatur, GA USA

[3] Ctr Dis Control & Prevent, Waterborne Dis Prevent Branch, Natl Ctr Emerging & Zoonot Infect Dis, Atlanta, GA USA

[4] Ifakara Hlth Inst, Dar Es Salaam, Tanzania

来源：

PLOS ONE | 2010年 / 5卷 / 10期

关键词：

FALCIPARUM-MALARIA; RAPID DETECTION; VIRUS; SYSTEM; ASSAY;

D O I：

10.1371/journal.pone.0013733

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

Background: Molecular diagnostic methods can complement existing tools to improve the diagnosis of malaria. However, they require good laboratory infrastructure thereby restricting their use to reference laboratories and research studies. Therefore, adopting molecular tools for routine use in malaria endemic countries will require simpler molecular platforms. The recently developed loop-mediated isothermal amplification (LAMP) method is relatively simple and can be improved for better use in endemic countries. In this study, we attempted to improve this method for malaria diagnosis by using a simple and portable device capable of performing both the amplification and detection (by fluorescence) of LAMP in one platform. We refer to this as the RealAmp method. Methodology and Significant Findings: Published genus-specific primers were used to test the utility of this method. DNA derived from different species of malaria parasites was used for the initial characterization. Clinical samples of P. falciparum were used to determine the sensitivity and specificity of this system compared to microscopy and a nested PCR method. Additionally, directly boiled parasite preparations were compared with a conventional DNA isolation method. The RealAmp method was found to be simple and allowed real-time detection of DNA amplification. The time to amplification varied but was generally less than 60 minutes. All human-infecting Plasmodium species were detected. The sensitivity and specificity of RealAmp in detecting P. falciparum was 96.7% and 91.7% respectively, compared to microscopy and 98.9% and 100% respectively, compared to a standard nested PCR method. In addition, this method consistently detected P. falciparum from directly boiled blood samples. Conclusion: This RealAmp method has great potential as a field usable molecular tool for diagnosis of malaria. This tool can provide an alternative to conventional PCR based diagnostic methods for field use in clinical and operational programs.

引用

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