{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T14:04:40Z","timestamp":1788444280769,"version":"build-2803163510"},"reference-count":33,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2021,12,24]],"date-time":"2021-12-24T00:00:00Z","timestamp":1640304000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001411","name":"Indian Council of Medical Research","doi-asserted-by":"publisher","award":["3\/1\/3\/PDF (16)\/2017-HRD"],"award-info":[{"award-number":["3\/1\/3\/PDF (16)\/2017-HRD"]}],"id":[{"id":"10.13039\/501100001411","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Diagnostics"],"abstract":"<jats:p>Isothermal techniques with lateral flow detection have emerged as a point of care (POC) technique for malaria, a major parasitic disease in tropical countries such as India. Plasmodium falciparum and Plasmodium vivax are the two most prevalent malaria species found in the country. An advanced multiplex loop-mediated isothermal amplification (mLAMP) combined with a lateral flow dipstick (LFD) technique was developed for the swift and accurate detection of P. falciparum and P. vivax, overcoming the challenges of the existing RDTs (rapid diagnostic tests). A single set of LAMP primers with a biotinylated backward inner primer (BIP primer) was used for DNA amplification of both malaria species in a single tube. The amplified DNA was hybridized with fluorescein isothiocyanate (FITC) and digoxigenin-labelled DNA probes, having a complemented sequence for the P. falciparum and P. vivax genomes, respectively. A colour band appeared on two separate LFDs for P. falciparum and P. vivax upon running the hybridized solution over them. In total, 39 clinical samples were collected from ICMR-NIMR, New Delhi. Melting curve analysis, with cross primers for both species, was used to ascertain specificity, and the sensitivity was equated with a polymerase chain reaction (PCR). The results were visualized on the LFD for both species within 60 min. We found 100% sensitivity and specificity, when compared with a traditional PCR. Melting curve analysis of mLAMP revealed the lowest detection limit of 0.15 pg\/\u03bcL from sample genomic DNA. The mLAMP-LFD assays could be a potential point of care (POC) tool for early diagnosis in non-laboratory conditions, with the convenience of a reduced assay time and the simple interpretation of results.<\/jats:p>","DOI":"10.3390\/diagnostics12010032","type":"journal-article","created":{"date-parts":[[2021,12,24]],"date-time":"2021-12-24T08:38:46Z","timestamp":1640335126000},"page":"32","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":15,"title":["Advanced Multiplex Loop Mediated Isothermal Amplification (mLAMP) Combined with Lateral Flow Detection (LFD) for Rapid Detection of Two Prevalent Malaria Species in India and Melting Curve Analysis"],"prefix":"10.3390","volume":"12","author":[{"given":"Supriya","family":"Sharma","sequence":"first","affiliation":[{"name":"Parasite Host Biology, ICMR-National Institute of Malaria Research, New Delhi 110077, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sandeep","family":"Kumar","sequence":"additional","affiliation":[{"name":"Parasite Host Biology, ICMR-National Institute of Malaria Research, New Delhi 110077, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Md Zohaib","family":"Ahmed","sequence":"additional","affiliation":[{"name":"Parasite Host Biology, ICMR-National Institute of Malaria Research, New Delhi 110077, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nitin","family":"Bhardwaj","sequence":"additional","affiliation":[{"name":"Parasite Host Biology, ICMR-National Institute of Malaria Research, New Delhi 110077, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jaskirat","family":"Singh","sequence":"additional","affiliation":[{"name":"Parasite Host Biology, ICMR-National Institute of Malaria Research, New Delhi 110077, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sarita","family":"Kumari","sequence":"additional","affiliation":[{"name":"Parasite Host Biology, ICMR-National Institute of Malaria Research, New Delhi 110077, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Deepali","family":"Savargaonkar","sequence":"additional","affiliation":[{"name":"Parasite Host Biology, ICMR-National Institute of Malaria Research, New Delhi 110077, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anupkumar R.","family":"Anvikar","sequence":"additional","affiliation":[{"name":"Parasite Host Biology, ICMR-National Institute of Malaria Research, New Delhi 110077, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jyoti","family":"Das","sequence":"additional","affiliation":[{"name":"Parasite Host Biology, ICMR-National Institute of Malaria Research, New Delhi 110077, India"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,12,24]]},"reference":[{"key":"ref_1","unstructured":"World Health Organization (2019). World Malaria Report, World Health Organization."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"333","DOI":"10.1186\/s12936-018-2481-4","article-title":"Comparison of three diagnostic methods (microscopy, RDT, and PCR) for the detection of malaria parasites in representative samples from Equatorial Guinea","volume":"17","author":"Berzosa","year":"2018","journal-title":"Malar. J."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"104122","DOI":"10.1016\/j.meegid.2019.104122","article-title":"Novel molecular diagnostic technique for detecting the different species of Plasmodium","volume":"78","author":"Sharma","year":"2020","journal-title":"Infect. Genet. Evol."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Bharti, P.K., Chandel, H.S., Ahmad, A., Krishna, S., Udhayakumar, V., and Singh, N. (2016). Prevalence of pfhrp2 and\/or pfhrp3 Gene Deletion in Plasmodium falciparum Population in Eight Highly Endemic States in India. PLoS ONE, 11.","DOI":"10.1371\/journal.pone.0157949"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"277","DOI":"10.1016\/j.parint.2018.01.005","article-title":"Rapid and sensitive multiplex single-tube nested PCR for the identification of five human Plasmodium species","volume":"67","author":"Saito","year":"2018","journal-title":"Parasitol. Int."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"28","DOI":"10.1186\/s12936-015-0542-5","article-title":"Molecular detection of human Plasmodium species in Sabah using PlasmoNex\u2122 multiplex PCR and hydrolysis probes real-time PCR","volume":"14","author":"Lee","year":"2015","journal-title":"Malar. J."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"205","DOI":"10.1586\/erm.12.144","article-title":"Loop-mediated isothermal amplification test for the molecular diagnosis of malaria","volume":"13","author":"Han","year":"2013","journal-title":"Expert Rev. Mol. Diagn."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"3366","DOI":"10.1002\/mrm.28387","article-title":"Relaxometric studies of erythrocyte suspensions infected by Plasmodium falciparum: A tool for staging infection and testing anti-malarial drugs","volume":"84","author":"Ferrauto","year":"2020","journal-title":"Magn. Reson. Med."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1069","DOI":"10.1038\/nm.3622","article-title":"Micromagnetic resonance relaxometry for rapid label-free malaria diagnosis","volume":"20","author":"Peng","year":"2014","journal-title":"Nat. Med."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"034029","DOI":"10.1103\/PhysRevApplied.11.034029","article-title":"Diamond mag-netic microscopy of malarial hemozoin nanocrystals","volume":"11","author":"Fescenko","year":"2019","journal-title":"Phys. Rev. Appl."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Murzin, D., Mapps, D.J., Levada, K., Belyaev, V., Omelyanchik, A., Panina, L., and Rodionova, V. (2020). Ultrasensitive Magnetic Field Sensors for Biomedical Applications. Sensors, 20.","DOI":"10.3390\/s20061569"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"11425","DOI":"10.1038\/srep11425","article-title":"Enhancing malaria diagnosis through microfluidic cell enrichment and magnetic resonance relaxometry detection","volume":"5","author":"Kong","year":"2015","journal-title":"Sci. Rep."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"10098","DOI":"10.1002\/mds3.10098","article-title":"A sensitive on-chip probe\u2013based portable nuclear magnetic resonance for detecting low parasitaemia plasmodium falciparum in human blood","volume":"3","author":"Gupta","year":"2020","journal-title":"Med. Devices Sens."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Yamamoto, T., Hashimoto, M., Nagatomi, K., Nogami, T., Sofue, Y., Hayashi, T., Ido, Y., Yatsushiro, S., Abe, K., and Kajimoto, K. (2019). Field evaluation of a quantitative, and rapid malaria diagnostic system using a fluorescent Blue-ray optical device. bioRxiv, 721076.","DOI":"10.1101\/721076"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"969","DOI":"10.1038\/s41467-021-21110-w","article-title":"Magne-to-optical diagnosis of symptomatic malaria in Papua New Guinea","volume":"12","author":"Arndt","year":"2021","journal-title":"Nat. Commun."},{"key":"ref_16","first-page":"881","article-title":"A Photoacoustic-Surface-Acoustic-Wave Sensor for Ring-Stage Malaria Parasite Detection","volume":"67","author":"Wang","year":"2020","journal-title":"IEEE Trans. Circuits Syst. II Express Briefs"},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Hommel, M., and Kremsner, P.G. (2014). Malaria Diagnostic Platform, LAMP. Encyclopedia of Malaria, Springer.","DOI":"10.1007\/978-1-4614-8757-9"},{"key":"ref_18","unstructured":"Hommel, M., and Kremsner, P.G. (2016). Malaria Diagnostic Platform: Lateral Flow Assays. Encyclopedia of Malaria, Springer."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Yongkiettrakul, S., Koli\u00e9, F.R., Kongkasuriyachai, D., Sattabongkot, J., Nguitragool, W., Nawattanapaibool, N., Suansomjit, C., Warit, S., Kangwanrangsan, N., and Buates, S. (2020). Validation of PfSNP-LAMP-Lateral Flow Dipstick for Detection of Single Nu-cleotide Polymorphism Associated with Pyrimethamine Resistance in Plasmodium falciparum. Diagnostics, 10.","DOI":"10.3390\/diagnostics10110948"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Morris, U., and Aydin-Schmidt, B. (2021). Performance and Application of Commercially Available Loop-Mediated Isothermal Amplifi-cation (LAMP) Kits in Malaria Endemic and Non-Endemic Settings. Diagnostics, 11.","DOI":"10.3390\/diagnostics11020336"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"26","DOI":"10.1186\/1475-2875-12-26","article-title":"A novel, single-amplification PCR targeting mitochondrial genome highly sensitive and specific in diagnosing malaria among returned travellers in Bergen, Norway","volume":"12","author":"Haanshuus","year":"2013","journal-title":"Malar. J."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Ahmed, M.Z., Bhardwaj, N., Sharma, S., Pande, V., and Anvikar, A.R. (2019). Transcriptional Modulation of the Host Immunity Mediated by Cytokines and Transcriptional Factors in Plasmodium falciparum-Infected Patients of North-East India. Biomolecules, 9.","DOI":"10.3390\/biom9100600"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1101","DOI":"10.1038\/nprot.2008.73","article-title":"Analyzing real-time PCR data by the comparative C T method","volume":"3","author":"Schmittgen","year":"2008","journal-title":"Nat. Protoc."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"151","DOI":"10.1016\/j.bios.2012.05.019","article-title":"Ambient temperature detection of PCR amplicons with a novel sequence-specific nucleic acid lateral flow biosensor","volume":"38","author":"Ang","year":"2012","journal-title":"Biosens. Bioelectron."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"2847548","DOI":"10.1155\/2017\/2847548","article-title":"Can mixed parasite infections thwart targeted malaria elimination program in India?","volume":"2017","author":"Singh","year":"2017","journal-title":"BioMed Res. Int."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"336","DOI":"10.4269\/ajtmh.15-0569","article-title":"Loop-Mediated Isothermal Amplification Assay for Identification of Five Human Plasmodium Species in Malaysia","volume":"94","author":"Lau","year":"2016","journal-title":"Am. J. Trop. Med. Hyg."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"20","DOI":"10.7883\/yoken.JJID.2009.20","article-title":"Evaluation of a new rapid molecular diagnostic system for Plasmodium falciparum combined with DNA filter paper, loop-mediated isothermal amplification, and melting curve analysis","volume":"62","author":"Yamamura","year":"2009","journal-title":"Jpn. J. Infect. Dis."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"2435","DOI":"10.1128\/JCM.43.5.2435-2440.2005","article-title":"Real-Time PCR for Detection and Identification of Plasmodium spp.","volume":"43","author":"Mangold","year":"2005","journal-title":"J. Clin. Microbiol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"50","DOI":"10.1099\/jmm.0.47216-0","article-title":"Development of a loop-mediated isothermal amplification method for diagnosing Pneumocystis pneumonia","volume":"57","author":"Uemura","year":"2008","journal-title":"J. Med Microbiol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"277","DOI":"10.1016\/j.mimet.2011.04.020","article-title":"Dry-reagent gold nanoparticle-based lateral flow biosensor for the simultaneous detection of Vibrio cholerae serogroups O1 and O139","volume":"86","author":"Yu","year":"2011","journal-title":"J. Microbiol. Methods"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"704","DOI":"10.4269\/ajtmh.18-0177","article-title":"Development of Loop-Mediated Isothermal Amplification\u2013Based Lateral Flow Device Method for the Detection of Malaria","volume":"99","author":"Mallepaddi","year":"2018","journal-title":"Am. J. Trop. Med. Hyg."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"777","DOI":"10.1016\/j.parint.2014.06.004","article-title":"Application of loop-mediated isothermal amplification assay combined with lateral flow dipstick for detection of Plasmodium falciparum and Plasmodium vivax","volume":"63","author":"Yongkiettrakul","year":"2014","journal-title":"Parasitol. Int."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"29","DOI":"10.1186\/s12936-016-1676-9","article-title":"Detection of Plasmodium knowlesi, Plasmodium falciparum and Plasmodium vivax using loop-mediated isothermal amplification (LAMP) in a co-endemic area in Malaysia","volume":"16","author":"Piera","year":"2017","journal-title":"Malar. J."}],"container-title":["Diagnostics"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2075-4418\/12\/1\/32\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T07:52:52Z","timestamp":1760169172000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2075-4418\/12\/1\/32"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,12,24]]},"references-count":33,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2022,1]]}},"alternative-id":["diagnostics12010032"],"URL":"https:\/\/doi.org\/10.3390\/diagnostics12010032","relation":{},"ISSN":["2075-4418"],"issn-type":[{"value":"2075-4418","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,12,24]]}}}