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

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.

Recombinase Polymerase Amplification for Rapid Detection of Zoonotic Pathogens

“…Like primers of traditional PCR, the GC content of the RPA primers should be between 30% and 70%, and long-chain guanines should be avoided at the 5' end, while guanine and cytosine nucleosides can be used at the 3' end to improve performance. The probe is not necessary in the common RPA assay, while is necessary when RPA is combined with various endpoint detection methods addressed below [16][17][18]. The procedure for primer and probe design is not standardized and no software is available, however, the selected primers and probes can be evaluated using the software for PCR primer design like Primer Premier 5.…”

Angiostrongylus cantonensis and neuroangiostrongyliasis (rat lungworm disease): 2020

“…The latest research on infection in non-human species is described (Feckova et al, 2021;Malik et al, 2021;Tsai and Chen, 2021;Wun et al, 2021a, b). Methods for preventing infection or reducing risks of human exposure (Howe et al, 2021;Steel et al, 2021), documentation of chronic sequelae (Meyer, 2021) and updates on research in disease diagnosis and treatment (Ansdell et al, 2021;Eamsobhana et al, 2021;Jacob et al, 2021;Jarvi et al, 2021) are provided. We hope these research papers will provide clinicians, researchers, educators and the general public with information that might help to reduce the risk of infection in humans and other animals, develop more efficient and targeted diagnostic methods, rationalise therapeutic approaches in acute and chronic cases, and direct future research on this fascinating but potentially destructive disease.…”

Rapid On-Site Detection of the Bursaphelenchus xylophilus Using Recombinase Polymerase Amplification Combined With Lateral Flow Dipstick That Eliminates Interference From Primer-Dependent Artifacts

“…In addition, RPA assays have been developed to detect some plant-parasitic and animal-parasitic nematodes, including B. xylophilus (Cha et al, 2019(Cha et al, , 2020, Meloidogyne enterolobii (Subbotin, 2019), Meloidogyne spp. (M. incognita, M. arenaria, M. javanica, and M. enterolobii) (Ju et al, 2019), Meloidogyne javanica (Chi et al, 2020), Meloidogyne hapla (Song et al, 2020;Subbotin and Burbridge, 2021), Angiostrongylus cantonensis (Jarvi et al, 2021), and Trichinella spp. (Li et al, 2019).…”