Diagnosis of Brugian Filariasis by Loop-Mediated Isothermal Amplification

Abstract

In this study we developed and evaluated a Brugia Hha I repeat loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Brugia genomic DNA. Amplification was detected using turbidity or fluorescence as readouts. Reactions generated a turbidity threshold value or a clear visual positive within 30 minutes using purified genomic DNA equivalent to one microfilaria. Similar results were obtained using DNA isolated from blood samples containing B. malayi microfilariae. Amplification was specific to B. malayi and B. timori, as no turbidity was observed using DNA from the related filarial parasites Wuchereria bancrofti, Onchocerca volvulus or Dirofilaria immitis, or from human or mosquito. Furthermore, the assay was most robust using a new strand-displacing DNA polymerase termed Bst 2.0 compared to wild-type Bst DNA polymerase, large fragment. The results indicate that the Brugia Hha I repeat LAMP assay is rapid, sensitive and Brugia-specific with the potential to be developed further as a field tool for diagnosis and mapping of brugian filariasis.

Detection of Onchocerca volvulus in Skin Snips by Microscopy and Real-Time Polymerase Chain Reaction: Implications for Monitoring and Evaluation Activities

“…methods include the O-150 PCR-ELISA used here, qPCR-MCA of the O-150 repeat, TaqMan-based qPCR, loopmediated isothermal amplification, specific oligonucleotide capture with magnetic beads, nested PCR combining general and species-specific primers, and PCR with general filarial primers followed by restriction fragment length polymorphism analysis to determine taxonomic identity. 13,[15][16][17][18][19][20][21][22][23] However, with few exceptions, these procedures require at least one post-amplification step for final determination, are specific to only one genus, or require multiple specific reaction components. 16,17 The procedure used here allows for singletube assessment of both presence and identity in a single 90-minute reaction.…”

Loop-Mediated Isothermal Amplification for Rapid and Semiquantitative Detection of Loa loa Infection

“…Although LAMP assays (like qPCR) can be quantitated in real time using a sophisticated turbidimeter (38), having the ability to perform the assays at the point of care or with minimal instrumentation led us to monitor our assays using a colorimetric readout through the use of HNB dye, previously shown to be useful in endpoint LAMP assays (32). Thus, the HNB-based LAMP assay provides a potential point-of-care method of rapid amplification and easy detection of L. loa DNA that, when standardized, can accurately distinguish the levels of mf that are correlated with increased risk for SAEs (Ͼ30,000 mf/ml; LAMP assay positive at 15 min) from the levels for individuals who might not be at risk (Ͻ5,000 mf/ml; LAMP assay positive at 20 min).…”

“…In particular, adding HNB dye to the reaction tube before amplification has improved the capacity of the LAMP method to be used as a point-of-care diagnostic test, since it has made the assay easier to use in resource-limited settings (32,33). Recently, LAMP technology has been applied with high accuracy to detect infection with a wide array of pathogens, including bacteria (34,35), viruses (36), and parasites (28,37,38).…”

Advances in the Diagnosis of Human Strongyloidiasis

“…It has been used in the diagnosis of several parasitic infections, including malaria, leishmaniasis, and cysticercosis [68][69][70]. It has several advantages: it uses stable reagents, is simple to use, and does not require a complex infrastructure.…”