A miniPCR-Duplex Lateral Flow Dipstick Platform for Rapid and Visual Diagnosis of Lymphatic Filariae Infection

Abstract

Lymphatic filariasis (LF) is a neglected major tropical disease that is a leading cause of permanent and long-term disability worldwide. Significant progress made by the Global Programme to Eliminate Lymphatic Filariasis (GPELF) has led to a substantial decrease in the levels of infection. In this limitation, DNA detection of lymphatic filariae could be useful due to it capable of detecting low level of the parasites. In the present study, we developed a diagnostic assay that combines a miniPCR with a duplex lateral flow dipstick (DLFD). The PCR primers were designed based on the HhaI and SspI repetitive noncoding DNA sequences of Brugia malayi and Wuchereria bancrofti, respectively. The limits of detection and crossreactivity of the assay were evaluated. In addition, blood samples were provided by Thais living in a brugian filariasis endemic area. The miniPCR-DLFD assay exhibited a detection limit of 2 and 4 mf per milliliter (mL) of blood for B. malayi as well as W. bancrofti, respectively, and crossamplification was not observed with 11 other parasites. The result obtained from the present study was in accordance with the thick blood smear staining for the known cases. Thus, a miniPCR-DLFD is an alternative tool for the diagnosis of LF in point-of-collection settings with a modest cost (~USD 5) per sample.

Molecular and Antifilarial IgG4 Detection Using the miniPCR-Duplex Lateral Flow Dipstick and BmSxp-ELISA in Myanmar Immigrant Communities

“…The miniPCR-DLFD demonstrated a high sensitivity and specificity. The assay exhibited a detection limit of 2 and 4 mf per milliliter (mL) of blood for B. malayi as well as W. bancrofti, respectively, and no cross-amplification was observed with other parasites [23]. The duplex lateral flow dipstick (DLFD) format can detect specific reaction mix in as little as 15 min and use only 2 µL reaction mix, whereas agarose gel electrophoresis requires gel-analysis steps and imaging and use 10 µL sample.…”

“…4.3.2. Amplification of W. bancrofti DNA by Conventional PCR and by miniPCR DNA extracted from the 300 blood samples were amplified by conventional PCR and the miniPCR using previously published primers targeting SspI repetitive non-coding DNA sequences of W. bancrofti [23]. The Sequences were obtained from the National Center for Biotechnology Information database (www.ncbi.nlm.nih.gov accessed on 7 January 2022; accession number L20344.1; position [23].…”

“…Amplification of W. bancrofti DNA by Conventional PCR and by miniPCR DNA extracted from the 300 blood samples were amplified by conventional PCR and the miniPCR using previously published primers targeting SspI repetitive non-coding DNA sequences of W. bancrofti [23]. The Sequences were obtained from the National Center for Biotechnology Information database (www.ncbi.nlm.nih.gov accessed on 7 January 2022; accession number L20344.1; position [23]. The sequences of the forward and reverse primers recognize a 182-bp region of the SspI repetitive non-coding DNA sequence of W. bancrofti (forward 5 -CAAAGTAGCGTAAGGGAATTG and reverse 5 -CCCTCACTTACCATAAGACAAC).…”

“…The sequences of the forward and reverse primers recognize a 182-bp region of the SspI repetitive non-coding DNA sequence of W. bancrofti (forward 5 -CAAAGTAGCGTAAGGGAATTG and reverse 5 -CCCTCACTTACCATAAGACAAC). Conventional PCR and miniPCR were performed using the optimal PCR condition published recently [23]. Both conventional PCR and miniPCR assay used the same primer set except for miniPCR, the 5 ends of the forward primers of SspI were labeled with fluorescein isothiocyanate (FITC).…”

“…For both conventional PCR and miniPCR, the amplifications were performed in a 20 µL volume consisting of 10 µL of PCR Master Mix [23] (QuantaBio, Beverly, MA, USA), 0.2 µM of the forward and reverse primers, 7.2 µL of dH 2 O, and 2 µL DNA template. Nuclease-free water was used as a negative control.…”