A New Rapid Method for the Authentication of Common Octopus (Octopus vulgaris) in Seafood Products Using Recombinase Polymerase Amplification (RPA) and Lateral Flow Assay (LFA)

The common octopus (Octopus vulgaris) is a highly valued cephalopod species which is marketed with different grades of processing, such as frozen, cooked or even canned, and is likely to be mislabeled. Some molecular methods have been developed for the authentication of these products, but they are either labor-intensive and/or require specialized equipment and personnel. This work describes a newly designed rapid, sensitive and easy-to-use method for the detection of Octopus vulgaris in food products, based on Recombinase Polymerase Amplification (RPA) and a detection using a Lateral Flow assay (LFA). After studying several gene markers, a system of primers and nfo-probe was designed in the COI (Cytochrome Oxidase I) region and was successfully tested in 32 reference samples (covering 14 species) and 32 commercial products, after optimization. The method was also validated in a ring trial with eight European laboratories and represents a useful tool for food authenticity control at all levels of the value chain.

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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

Parasitology ◽

10.1017/s0031182020002139 ◽

2020 ◽

pp. 1-8

Author(s):

Susan I. Jarvi ◽

Elizabeth S. Atkinson ◽

Lisa M. Kaluna ◽

Kirsten A. Snook ◽

Argon Steel

Keyword(s):

Lateral Flow ◽

Angiostrongylus Cantonensis ◽

Lateral Flow Assay ◽

Diagnostic Methods ◽

Qpcr Assay ◽

Recombinase Polymerase Amplification ◽

Intermediate Hosts ◽

Infection Level ◽

Specialized Equipment ◽

The Central Nervous System

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.

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Multiplex Assay of Viruses Integrating Recombinase Polymerase Amplification, Barcode—Anti-Barcode Pairs, Blocking Anti-Primers, and Lateral Flow Assay

Analytical Chemistry ◽

10.1021/acs.analchem.1c03030 ◽

2021 ◽

Author(s):

Alexandr V. Ivanov ◽

Irina V. Safenkova ◽

Anatoly V. Zherdev ◽

Boris B. Dzantiev

Keyword(s):

Multiplex Assay ◽

Lateral Flow ◽

Lateral Flow Assay ◽

Recombinase Polymerase Amplification

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Key significance of DNA-target size in lateral flow assay coupled with recombinase polymerase amplification

Analytica Chimica Acta ◽

10.1016/j.aca.2019.12.048 ◽

2020 ◽

Vol 1102 ◽

pp. 109-118 ◽

Cited By ~ 8

Author(s):

Irina V. Safenkova ◽

Alexandr V. Ivanov ◽

Elvira S. Slutskaya ◽

Alexey V. Samokhvalov ◽

Anatoly V. Zherdev ◽

...

Keyword(s):

Lateral Flow ◽

Lateral Flow Assay ◽

Recombinase Polymerase Amplification ◽

Target Size

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Recombinase Polymerase Amplification and Lateral Flow Assay for Ultrasensitive Detection of Low-Density Plasmodium falciparum Infection from Controlled Human Malaria Infection Studies and Naturally Acquired Infections

Journal of Clinical Microbiology ◽

10.1128/jcm.01879-19 ◽

2020 ◽

Vol 58 (5) ◽

Cited By ~ 3

Author(s):

Albert Lalremruata ◽

The Trong Nguyen ◽

Matthew B. B. McCall ◽

Ghyslain Mombo-Ngoma ◽

Selidji T. Agnandji ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Reverse Transcription ◽

Malaria Infection ◽

Lateral Flow ◽

Lateral Flow Assay ◽

Recombinase Polymerase Amplification ◽

Low Density ◽

Blood Samples ◽

Human Malaria ◽

Controlled Human Malaria Infection

ABSTRACT Microscopy and rapid diagnostic tests (RDTs) are the main diagnostic tools for malaria but fail to detect low-density parasitemias that are important for maintaining malaria transmission. To complement existing diagnostic methods, an isothermal reverse transcription-recombinase polymerase amplification and lateral flow assay (RT-RPA) was developed. We compared the performance with that of ultrasensitive reverse transcription-quantitative PCR (uRT-qPCR) using nucleic acid extracts from blood samples (n = 114) obtained after standardized controlled human malaria infection (CHMI) with Plasmodium falciparum sporozoites. As a preliminary investigation, we also sampled asymptomatic individuals (n = 28) in an area of malaria endemicity (Lambaréné, Gabon) to validate RT-RPA and assess its performance with unprocessed blood samples (dbRT-RPA). In 114 samples analyzed from CHMI trials, the positive percent agreement to uRT-qPCR was 90% (95% confidence interval [CI], 80 to 96). The negative percent agreement was 100% (95% CI, 92 to 100). The lower limit of detection was 64 parasites/ml. In Gabon, RT-RPA was 100% accurate with asymptomatic volunteers (n = 28), while simplified dbRT-RPA showed 89% accuracy. In a subgroup analysis, RT-RPA detected 9/10 RT-qPCR-positive samples, while loop-mediated isothermal amplification (LAMP) detected 2/10. RT-RPA is a reliable diagnostic test for asymptomatic low-density infections. It is particularly useful in settings where uRT-qPCR is difficult to implement.

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Development of a reverse-transcription recombinase polymerase amplification assay with a lateral flow assay for rapid detection of avian orthoavulavirus 1

Journal of Veterinary Diagnostic Investigation ◽

10.1177/1040638721990122 ◽

2021 ◽

Vol 33 (2) ◽

pp. 308-312

Author(s):

Jindai Fan ◽

Wenxian Chen ◽

Yuanyuan Zhang ◽

Zhixiang Liu ◽

Xiaoming Li ◽

...

Keyword(s):

Reverse Transcription ◽

Newcastle Disease ◽

Cross Reactivity ◽

Lateral Flow ◽

Lateral Flow Assay ◽

Recombinase Polymerase Amplification ◽

Control Measures ◽

Economic Losses ◽

Resource Limited ◽

Simple Detection

Newcastle disease is an avian infectious disease caused by avian orthoavulavirus 1, also known as Newcastle disease virus (NDV). This disease has caused significant economic losses to the poultry industry worldwide. The rapid and simple detection of NDV infection is crucial to inform the appropriate control measures. We developed a reverse-transcription recombinase polymerase amplification (RT-RPA) assay combined with a lateral flow assay (LFA) for NDV detection. The RPA assay can be completed at 37°C within 20 min, and the RPA result can be visualized by the LFA within 5 min. The NDV RT-RPA-LFA detected NDV specifically with no cross-reactivity with other pathogens. The detection limit of NDV cDNA with our RT-RPA-LFA was 3.34 × 10−3 ng/μL. Consequently, the RT-RPA-LFA showed good potential for the detection of NDV infection in the field, especially in resource-limited settings.

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