Research
Adaptation of a visualized loop-mediated isothermal amplification technique for field detection of Plasmodium vivax infection
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* Corresponding authors: Jun Cao caojuncn@hotmail.com - Qi Gao gaoqi54@hotmail.com
1 Department of Parasitology, Medical College of Soochow University, Suzhou 215123, People's Republic of China
2 Jiangsu Institute of Parasitic Diseases, Key Laboratory on Technology for Parasitic Disease Prevention and Control, Ministry of Health, Meiyuan Yangxiang 117, Wuxi 214064, People's Republic of China
3 Department of Parasitology, Bengbu Medical College, 2600 Donghai Dadao Road, Bengbu 233030, People's Republic of China
4 Malaria Unit, Institute of Tropical Medicine, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan
5 Department of Parasitology, Kangwon National University College of Medicine, Chuncheon, Republic of Korea
6 Chenzhou Center for Disease Control and Prevention, Chenzhou, 423000, People's Republic of China
Parasites & Vectors 2011, 4:115 doi:10.1186/1756-3305-4-115
Published: 21 June 2011Abstract
Background
Loop-mediated isothermal amplification (LAMP) is a high performance method for detecting DNA and holds promise for use in the molecular detection of infectious pathogens, including Plasmodium spp. However, in most malaria-endemic areas, which are often resource-limited, current LAMP methods are not feasible for diagnosis due to difficulties in accurately interpreting results with problems of sensitive visualization of amplified products, and the risk of contamination resulting from the high quantity of amplified DNA produced. In this study, we establish a novel visualized LAMP method in a closed-tube system, and validate it for the diagnosis of malaria under simulated field conditions.
Methods
A visualized LAMP method was established by the addition of a microcrystalline wax-dye capsule containing the highly sensitive DNA fluorescence dye SYBR Green I to a normal LAMP reaction prior to the initiation of the reaction. A total of 89 blood samples were collected on filter paper and processed using a simple boiling method for DNA extraction, and then tested by the visualized LAMP method for Plasmodium vivax infection.
Results
The wax capsule remained intact during isothermal amplification, and released the DNA dye to the reaction mixture only when the temperature was raised to the melting point following amplification. Soon after cooling down, the solidified wax sealed the reaction mix at the bottom of the tube, thus minimizing the risk of aerosol contamination. Compared to microscopy, the sensitivity and specificity of LAMP were 98.3% (95% confidence interval (CI): 91.1-99.7%) and 100% (95% CI: 88.3-100%), and were in close agreement with a nested polymerase chain reaction method.
Conclusions
This novel, cheap and quick visualized LAMP method is feasible for malaria diagnosis in resource-limited field settings.