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Two-Stage Detection of Plasmodium spp. by Combination of Recombinase Polymerase Amplification and Loop-Mediated Isothermal Amplification Assay

Meng Yee LaiDepartment of Parasitology, Faculty of Medicine, Universiti Malaya, Kuala Lumpur, Malaysia

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Yee Ling LauDepartment of Parasitology, Faculty of Medicine, Universiti Malaya, Kuala Lumpur, Malaysia

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ABSTRACT.

We developed a combination of recombinase polymerase and loop-mediated isothermal amplification methods (RAMP) for rapid screening of five human Plasmodium spp. simultaneously. RAMP is a two-stage isothermal amplification method, which consists of a first-stage recombinase polymerase amplification and a second-stage loop-mediated isothermal amplification. Under these two isothermal conditions, five Plasmodium spp. were amplified in less than 40 minutes. We demonstrated RAMP assay with 10-fold better limit of detection than a single (loop-mediated isothermal amplification) LAMP. As compared with microscopy, RAMP assay showed 100% sensitivity (95% CI: 95.65–100.00%) and 100% specificity (95% CI: 69.15–100.00%). The end products were inspected by the color changes of neutral red. Positive reactions were indicated by pink while the negative reactions remained yellow. The combination assay established in this study can be used as a routine diagnostic method for malaria.

Author Notes

Address correspondence to Yee Ling Lau, Department of Parasitology, Faculty of Medicine, Universiti Malaya, 50603 Kuala Lumpur, Malaysia. E-mail: lauyeeling@um.edu.my

Financial support: This study was supported by Long Term Research Grant Scheme (LRGS), LR002D-2018, LRGS/1/2018/UM/01/1/4 from the Ministry of Education, Malaysia.

Disclosure: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this manuscript.

Authors’ addresses: Meng Yee Lai and Yee Ling Lau, Department of Parasitology, Faculty of Medicine, Universiti Malaya, Kuala Lumpur, Malaysia, E-mails: mengylai11@um.edu.my and lauyeeling@um.edu.my.

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