Development of loop-mediated isothermal amplification (LAMP) assay using SYBR safe and gold-nanoparticle probe for detection of Leishmania in HIV patients

Abstract

Asymptomatic leishmaniasis cases have continuously increased, especially among patients with HIV who are at risk to develop further symptoms of cutaneous and visceral leishmaniasis. Thus, early diagnosis using a simple, sensitive and reliable diagnostic assay is important because populations at risk mostly reside in rural communities where laboratory equipment is limited. In this study, the highly sensitive and selective determination of Leishmania infection in asymptomatic HIV patients was achieved using dual indicators (SYBR safe and gold-nanoparticle probe; AuNP-probe) in one-step LAMP method based on basic instruments. The assay can be simply evaluated under the naked eye due to clear interpretation of fluorescent emission of LAMP-SYBR safe dye-complex and colorimetric precipitate of specific AuNP-probes. The sensitivities and specificities of fluorescent SYBR safe dye and AuNP-probe indicators were equal, which were as high as 94.1 and 97.1%, respectively. Additionally, detection limits were 102 parasites/mL (0.0147 ng/µL), ten times more sensitivity than other related studies. To empower leishmaniasis surveillance, this inexpensive one-step SYBR safe and AuNP-LAMP assay is reliably fast and simple for field diagnostics to point-of-care settings, which can be set up in all levels of health care facilities including resource limited areas, especially in low to middle income countries.

Plasmonic LAMP: Improving the Detection Specificity and Sensitivity for SARS‐CoV‐2 by Plasmonic Sensing of Isothermally Amplified Nucleic Acids

“…Different from our method, previous efforts in implementing AuNPs‐based plasmonic sensing of LAMP products rely on indirect methods, where the ionic strength change in a salt solution or molecule‐labeled on primers essentially induce the aggregation of AuNPs‐based sensors. [ 30 , 31 , 32 , 33 , 34 , 51 , 52 , 53 ] Those methods cannot differentiate the template versus non‐template amplification and lead to false‐positive results.…”

Distance-based paper device using combined SYBR safe and gold nanoparticle probe LAMP assay to detect Leishmania among patients with HIV

“…This highly sensitive LAMP determination could be selectively performed using dual indicators, fluorescent SYBR safe and colorimetric precipitate gold-nanoparticle probe, in the one-step LAMP method. The assay could be simultaneously visualized in prereaction detection using fluorescent SYBR safe for rapid screening, and in postamplification detection using AuNP-LAMP probes for specific colorimetric precipitate induction 15 and the dPAD for semi-quantification of LAMP amplicons using DNA immobilized cellulose Whatman No. 1 12 .…”

“…Alternately, to avoid ambiguous reading and mutagenic effect, the LAMP products can be traced with functionalized gold nanoparticles (AuNPs) and analyzed using colorimetric detection 15 , 20 – 23 . Based on bio-nanoprobes, Thiol-linking of DNA and chemical functionalization of AuNPs for specific protein/antibody binding, simple and inexpensive methods were commonly applied to detect specific DNA sequences and are presently being expanded to diverse fields of pathogen diagnosis 24 – 28 .…”

“…After hybridization with complementary DNA targets, the red solution indicating positive results remains because the complementary targets and AuNP probes form the polymeric network of cross-linked AuNPs preventing them from aggregation by salt induction. On the other hand, non-crosslinking hybridization induces aggregation of the AuNP probes by increasing salt concentration and results in a color change from red to blue (negative result) 29 , 30 for which unambiguous interpretation of LAMP targets can be clearly observed by the naked eye 13 , 15 .…”

Evaluation of the real-time fluorescence loop-mediated isothermal amplification assay for the detection of Ureaplasma urealyticum

“…Due to the characteristics of primers, the concentration of LAMP is higher than that in PCR reaction. The temperature of real-time fluorescence LAMP reaction ranged between 60 and 65 ℃, and it merely takes Bst DNA polymerase 30–60 min to amplify target DNA sequence (Ruang-Areerate et al 2021 ). Compared with conventional PCR, real-time LAMP increases the reaction rate two to three times (Guo et al 2018 ).…”