Development of Loop-Mediated Isothermal Amplification–Based Lateral Flow Device Method for the Detection of Malaria

Abstract

The present study aims to develop a method for rapid diagnosis of malaria using loop-mediated isothermal amplification (LAMP) combined with a lateral flow device (LFD). By adding the biotin-labeled and fluorescein amidite-labeled loop primers to the LAMP reaction solution, the end product can be visualized on a LFD. The entire procedure takes approximately 42 minutes to complete, LAMP assay exhibited high sensitivity, as the detection limit was 0.01 pg/μL for all five species. It was demonstrated that all ( = 90) and ( = 56) were positively amplified by LAMP-LFD assay, whereas healthy donor samples ( = 8) were negative. However, not all mixed infections were positive, and other infected nonmalaria samples were negative. Loop-mediated isothermal amplification-LFD represents a robust approach with potential suitability for use in resource-constrained laboratories. We believe that LAMP-LFD has a potential to be developed as point-of-care diagnostic tool in future.

Development and evaluation of LAMP-coupled lateral flow device for the detection of MAP in livestock at point of care resource-limited areas

“…The unamplified LAMP sample does not react at the test position. This result is in accordance with other studies employed in LAMP lateral flow dipstick and in analysis of LAMP amplicons [28,[38][39][40]. Use of LAMP-coupled lateral flow device in the detection of LAMP amplicons is not only to eliminate the post amplification manipulations and use of ethidium bromide but also to strengthen the specificity of LAMP assay by hybridization of the specific probe to the LAMP amplicons.…”

“…Several molecular studies proposed IS900 as the most specific and highly sensitive target sequence for the detection of MAP [35]. The 5′ end of these loop primers LPF and LPB was tagged with biotin and fluorescein isothiocyanate (FITC) respectively [28]. The LAMP-coupled LFD takes an hour which is much shorter compared with other PCR assays.…”

“…Out of six primers, two inner primers (FIP, BIP), two outer primers (F3 and B3), and the remaining two primers were loop primers. The 5′ end of these loop primers LPF and LPB was tagged with biotin and fluorescein isothiocyanate (FITC) [28] respectively as shown in Table 1.…”

“…Sample pad is for loading the amplified sample and the conjugate pad holds the conjugate matrix, contains gold nanoparticles conjugated with streptavidin and mouse IgG antibody (Arista Biologicals Inc., USA). Nitrocellulose membrane was coated with captured components such as anti-FITC antibody at test line and anti-mouse IgG at control line that are specific for detecting components [28]. The complete setup of test strip was kept in a plastic device having well for sample addition and detector window labeled with control "C" and test "T" positions.…”

Loop-mediated Isothermal Amplification (LAMP) for Identification of Pythium insidiosum

“…Besides, it does not require sophisticated equipment (i.e., a thermocycler machine), making it an appealing method for many applications in resource-limited laboratories (Notomi et al, 2015;Wong et al, 2018). The LAMP technique has been employed for the detection of many pathogens, such as foodborne or meningitis-causing bacteria (Seki et al, 2018;Yuan et al, 2018;Zhang et al, 2018), contagious viruses (Boldbaatar et al, 2009;Tun et al, 2014;Lee et al, 2016;Bartolone et al, 2018), malarial parasites (Mallepaddi et al, 2018), medically-important fungi (Han et al, 2012;Al-Sheikh, 2015;Yamamoto et al, 2018), and plant-pathogenic oomycetes (Feng et al, 2018;Winkworth et al, 2020).…”