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

Abstract

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/μL 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.

LAMP-Based Point-of-Care Biosensors for Rapid Pathogen Detection

“…A clear band was visualized on the strip within 60 min for both species. Detection sensitivity reached 0.15 pg/µL for extracted genomic DNA [ 90 ].…”

“…( B ) A portable microfluidic platform integrated with a pregnancy test strip (PTSs) for POC diagnosis of COVID-19. Reproduced with permission from references [ 90 , 92 ]. Copyright 2021 American Chemical Society, 2022 MDPI.…”

Lateral flow assays for viruses diagnosis: Up-to-date technology and future prospects

“…Also, the specificity of the results is higher as many oligonucleotides are used in the reaction compared to RPA-LFA [ 151 ]. Another study using the LAMP-LFA technique for rapid malaria diagnosis reported a very low LOD value of 0.01 pg/mL, with assay time reduced to 42 minutes [ 152 ].…”

MiRNA: Biological Regulator in Host-Parasite Interaction during Malaria Infection

“…Diagnostic methods for malaria include microscopy, rapid diagnostic test (RDT), molecular diagnosis including polymerase chain reaction (PCR), quantitative PCR (qPCR) or reverse transcriptase PCR(RT-PCR), semi-nested PCR (n-PCR), multiplex PCR(m-PCR), indirect fluorescent antibody test and isothermal methods, viz, loop-mediated isothermal amplification (RPA) etc. [36]. While microscopy is the "Gold Standard" method for the detection of malaria in endemic regions, in the absence of well-trained technicians, it has low sensitivity and is time consuming compared to other methods.…”

“…Still, it is time-consuming and also requires fluorescence microscopy and trained technicians [2]. Other developed techniques for malaria diagnostic, including loop-mediated isothermal amplification(LAMP), RNA hybridization assays, DNA amplification method, laser desorption mass spectrometry(LDMS) and so forth, still have limitations to detect some parasite stages [36].…”