Colorimetric tests for diagnosis of filarial infection and vector surveillance using non-instrumented nucleic acid loop-mediated isothermal amplification (NINA-LAMP)

Abstract

Accurate detection of filarial parasites in humans is essential for the implementation and evaluation of mass drug administration programs to control onchocerciasis and lymphatic filariasis. Determining the infection levels in vector populations is also important for assessing transmission, deciding when drug treatments may be terminated and for monitoring recrudescence. Immunological methods to detect infection in humans are available, however, cross-reactivity issues have been reported. Nucleic acid-based molecular assays offer high levels of specificity and sensitivity, and can be used to detect infection in both humans and vectors. In this study we developed loop-mediated isothermal amplification (LAMP) tests to detect three different filarial DNAs in human and insect samples using pH sensitive dyes for enhanced visual detection of amplification. Furthermore, reactions were performed in a portable, non-instrumented nucleic acid amplification (NINA) device that provides a stable heat source for LAMP. The efficacy of several strand displacing DNA polymerases were evaluated in combination with neutral red or phenol red dyes. Colorimetric NINA-LAMP assays targeting Brugia Hha I repeat, Onchocerca volvulus GST1a and Wuchereria bancrofti LDR each exhibit species-specificity and are also highly sensitive, detecting DNA equivalent to 1/10-1/5000th of one microfilaria. Reaction times varied depending on whether a single copy gene (70 minutes, O. volvulus) or repetitive DNA (40 min, B. malayi and W. bancrofti) was employed as a biomarker. The NINA heater can be used to detect multiple infections simultaneously. The accuracy, simplicity and versatility of the technology suggests that colorimetric NINA-LAMP assays are ideally suited for monitoring the success of filariasis control programs.

SARS-CoV-2 Detection Using an Isothermal Amplification Reaction and a Rapid, Inexpensive Protocol for Sample Inactivation and Purification

“…Furthermore, although the stranddisplacing polymerase has reverse transcriptase activity, a reverse transcriptase can be included to improve sensitivity within the reaction when detecting an RNA target (RT-LAMP), such as the SARS-CoV-2 RNA. LAMP assays have a variety of readouts due to the large amount of DNA generated, including fluorescence using an intercalating DNA dye, turbidity, or by a drop in the pH if the reaction is minimally buffered 1,3,4 . This change in pH, sufficient to cause a pH indicator dye to visibly change color, is the most applicable method for a point-of-care LAMP-based diagnostic.…”

Validation of Loop Mediated Isothermal Amplification for the Detection of Loa loa Infection in Chrysops sp in Experimental and Natural Field Conditions

“…In PCR, thermal cycling is required to denature the template, anneal primers and extend the amplicon. LAMP employs Bst DNA polymerase, which provides both strand displacement and target ampli cation at a single temperature in a simple heat block or water bath at 60-65 °C [25] or other portable device with a stable heat source [57]. In addition, LAMP tests have been reported to be signi cantly cheaper than PCR [58].…”

Loop‐mediated isothermal amplification (LAMP) for the identification of invasive Aedes mosquito species

“…LAMP assays can also be run in multiplex format and this could be exploited for the identification of several mosquito species in one reaction or for the combination of mosquito identification and detection of pathogens carried by them. LAMP assays for the detection of pathogens in insect vectors have already been described (Aonuma et al, 2010;Poole et al, 2017). Analyses of such multiplex LAMP assays can easily be achieved by lateral flow dipstick tests (Yongkiettrakul et al, 2017).…”