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Development of a recombinase polymerase amplification (RPA-EXO) and lateral flow assay (RPA-LFA) based on the ITS1 gene for the detection of Angiostrongylus cantonensis in gastropod intermediate hosts

Published online by Cambridge University Press:  04 November 2020

Susan I. Jarvi Open the ORCID record for Susan I. Jarvi [Opens in a new window] ,
Elizabeth S. Atkinson ,
Lisa M. Kaluna ,
Kirsten A. Snook  and
Argon Steel Open the ORCID record for Argon Steel [Opens in a new window]
Susan I. Jarvi*
Affiliation:
Department of Pharmaceutical Sciences, Daniel K. Inouye College of Pharmacy, University of Hawai‘i at Hilo, Hilo, Hawai‘i, USA
Elizabeth S. Atkinson
Affiliation:
Department of Pharmaceutical Sciences, Daniel K. Inouye College of Pharmacy, University of Hawai‘i at Hilo, Hilo, Hawai‘i, USA
Lisa M. Kaluna
Affiliation:
Department of Pharmaceutical Sciences, Daniel K. Inouye College of Pharmacy, University of Hawai‘i at Hilo, Hilo, Hawai‘i, USA
Kirsten A. Snook
Affiliation:
Department of Pharmaceutical Sciences, Daniel K. Inouye College of Pharmacy, University of Hawai‘i at Hilo, Hilo, Hawai‘i, USA
Argon Steel
Affiliation:
Department of Pharmaceutical Sciences, Daniel K. Inouye College of Pharmacy, University of Hawai‘i at Hilo, Hilo, Hawai‘i, USA
*
Author for correspondence: Susan I. Jarvi, E-mail: jarvi@hawaii.edu
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Abstract

Angiostrongylus cantonensis is a parasitic nematode known to infect humans through the ingestion of third stage larvae which can cause inflammation and damage to the central nervous system. Currently, polymerase chain reaction (PCR) is one of the most reliable diagnostic methods for detecting A. cantonensis in humans as well as in gastropod hosts, but requires expensive and specialized equipment. Here, we compare the sensitivity and accuracy of a recombinase polymerase amplification Exo (RPA-EXO) assay, and a recombinase polymerase amplification lateral flow assay (RPA-LFA) with a traditional quantitative PCR (qPCR) assay currently available. The three assays were used to test 35 slugs from Hawai‘i for the presence of A. cantonensis DNA. Consistent results among the three tests were shown in 23/35 samples (65.7%), while 7/35 (20%) were discordant in low infection level samples (<0.01 larvae per mg tissue), and 5/35 (14.3%) were equivocal. To evaluate sensitivity, a partial ITS1 gene was cloned, and serial plasmid dilutions were created ranging from 100 copies μL−1 to ~1 copy μL−1. All three assays consistently detected 50–100 copies μL−1 in triplicate and qPCR was able to detect ~13 copies μL−1 in triplicate. RPA-EXO was able to detect 25 copies μL−1 in triplicate and RPA-LFA was not able to amplify consistently below 50 copies μL−1. Thus, our RPA-EXO and RPA-LFA assays do not appear as sensitive as the current qPCR assay at low DNA concentrations; however, these tests have numerous advantages that may make them useful alternatives to qPCR.

Keywords

Angiostrongylus cantonensislateral flowLFAPCRrecombinase polymeraseRPARPA-EXORPA-LFA
Type
Research Article
Information
Parasitology , Volume 148 , Special Issue 2: 6th International Workshop on Angiostrongylus and Angiostrongyliasis, Hilo, Hawai'i, USA, January, 2020 , February 2021 , pp. 251 - 258
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Copyright
Copyright © The Author(s) 2020. Published by Cambridge University Press

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