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Reference Manager
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Song J , Liu C , Mauk MG , Rankin SC , Lok JB , Greenberg RM , Bau HH , 2017. Two-stage isothermal enzymatic amplification for concurrent multiplex molecular detection. Clin Chem 63: 714–722.
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Two-Stage Detection of Plasmodium spp. by Combination of Recombinase Polymerase Amplification and Loop-Mediated Isothermal Amplification Assay
Meng Yee Lai
Meng Yee Lai
Department of Parasitology, Faculty of Medicine, Universiti Malaya, Kuala Lumpur, Malaysia
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Yee Ling Lau
Yee Ling Lau
Department 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
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.
ProCite
RefWorks
Reference Manager
BibTeX
Zotero
EndNote
-
1.
Notomi T , Okayama H , Masubuchi H , Yonekawa T , Watanabe K , Amino N , Hase T , 2000. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28: e63.
-
2.
Vincent M , Xu Y , Kong H , 2004. Helicase-dependent isothermal DNA amplification. EMBO Rep 5: 795–800.
-
3.
Gusev Y et al.2001. Rolling circle amplification. A new approach to increase sensitivity for immunohistochemistry and flow cytometry. Am J Pathol 159: 63–69.
-
4.
Piepenburg O , Williams CH , Stemple DL , Armes NA , 2006. DNA detection using recombination proteins. PLoS Biol 4: e204.
-
5.
Compton J , 1991. Nucleic acid sequence-based amplification. Nature 350: 91–92.
-
6.
Tanner NA , Zhang Y , Evans TC , 2012. Simultaneous multiple target detection in real-time loop-mediated isothermal amplification. BioTechniques 53: 81–89.
-
7.
Kasahara K , Ishikawa H , Sato S , Shimakawa Y , Watanabe K , 2015. Development of multiplex loop-mediated isothermal amplification assays to detect medically important yeasts in dairy products. FEMS Microbiol Lett 357: 208–216.
-
8.
Curtis KA , Morrison D , Rudolph DL , Shankar A , Bloomfield LS , Switzer WM , Owen SM , 2018. A multiplexed RT-LAMP assay for detection of group M HIV-1 in plasma or whole blood. J Virol Methods 255: 91–97.
-
9.
Crannell Z , Castellanos-Gonzalez A , Nair G , Mejia R , White AC , Richards-Kortum R , 2016. Multiplexed recombinase polymerase amplification assay to detect intestinal protozoa. Anal Chem 88: 1610–1616.
-
10.
Larrea-Sarmiento A , Stack JP , Alvarez AM , Arif M , 2021. Multiplex recombinase polymerase amplification assay developed using unique genomic regions for rapid on-site detection of genus Clavibacter and C. nebraskensis. Sci Rep 11: 12017.
-
11.
Snounou G , Viriyakosol S , Zhu XP , Jarra W , Pinheiro L , do Rosario VE , Thaithong S , Brown KN , 1993. High sensitivity of detection of human malaria parasites by the use of nested polymerase chain reaction. Mol Biochem Parasitol 61: 315–320.
-
12.
Lau YL , Lai MY , Fong MY , Jelip J , Mahmud R , 2016. Loop-mediated isothermal amplification assay for identification of five human Plasmodium species in Malaysia. Am J Trop Med Hyg 94: 336–339.
-
13.
Zhang Y , Ren G , Buss J , Barry AJ , Patton GC , Tanner NA , 2020. Enhancing colorimetric loop-mediated isothermal amplification speed and sensitivity with guanidine chloride. Biotechniques 69: 179–186.
-
14.
Song J , Liu C , Mauk MG , Rankin SC , Lok JB , Greenberg RM , Bau HH , 2017. Two-stage isothermal enzymatic amplification for concurrent multiplex molecular detection. Clin Chem 63: 714–722.
-
15.
Huang Q et al.2021. Microfluidic chip with two-stage isothermal amplification method for highly sensitive parallel detection of SARS-CoV-2 and measles virus. Micromachines (Basel) 12: 1582.
-
16.
Singh R , Singh DP , Savargaonkar D , Singh OP , Bhatt RM , Valecha N , 2017. Evaluation of SYBR green I based visual loop-mediated isothermal amplification (LAMP) assay for genus and species-specific diagnosis of malaria in P. vivax and P. falciparum endemic regions. J Vector Borne Dis 54: 54–60.
-
17.
Barazorda KA , Salas CJ , Bishop DK , Lucchi N , Valdivia HO , 2020. Comparison of real time and malachite-green based loop-mediated isothermal amplification assays for the detection of Plasmodium vivax and P. falciparum. PLoS One 15: e0234263.
-
18.
Kersting S , Rausch V , Bier FF , Nickisch-Rosenegk MV , 2014. Rapid detection of Plasmodium falciparum with isothermal recombinase polymerase amplification and lateral flow analysis. Malar J 13: 99.
-
19.
Viana GMR et al.2018. Field evaluation of a real time loop-mediated isothermal amplification assay (RealAmp) for malaria diagnosis in Cruzeiro do Sul, Acre, Brazil. PLoS One 13: e0200492.
| Past two years | Past Year | Past 30 Days | |
|---|---|---|---|
| Abstract Views | 2262 | 1167 | 306 |
| Full Text Views | 235 | 93 | 1 |
| PDF Downloads | 128 | 19 | 0 |