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Adaptations of new generation molecular techniques for multiplexed detection of pathogens are gaining interest in the field of point-of-care (POC) industry and onsite testing. Loop-mediated isothermal amplification (LAMP), an advanced molecular amplification technique, has proven promising due to its unique features that suits ideal for POC applications. However, application of LAMP for multiplexed detection of pathogens remains challenging because of the difficulty in the identification of specific LAMP amplicons that does not have a well-definite molecular size. In this study, we developed a solid-phase loop-mediated isothermal amplification (SP-LAMP) technique to address the challenge. Integration of LAMP with the supercritical angle fluorescence (SAF) micro-optic structures as a solid support (SS) in an array format enabled spatial separation of LAMP amplicons in a multiplexed configuration. Important parameters such as length of the SS primers, length of the primer-binding region, the effect of surface density of immobilized SS primers, and cross-reactivity among the primers of different targets were iteratively tested and optimized. With the combination of SP-LAMP and SAF techniques, it was possible to detect multiple pathogens that include Salmonella spp, Campylobater spp., Campylobacter coli , Campylobacter jejuni , avian influenza virus (AIV), and pan avian internal control (IC) under singleplex conditions. The multiplexing capacity of the SP-LAMP was demonstrated using AIV and IC with promising results. The success of SP-LAMP has opened a promising direction toward the development of a multiplex POC system for rapid detection of multiple pathogens.

Avian influenza virus (AIV) outbreaks occur frequently worldwide, causing a potential public health risk and great economic losses to poultry industries. Considering the high mutation rate and frequent genetic reassortment between segments in the genome of AIVs, emerging new strains are a real threat that may infect and spread through the human population, causing a pandemic. Therefore, rapid AIV diagnostic tests are essential tools for surveillance and assessing virus spreading. Real-time reverse transcription PCR (rRT-PCR), targeting the matrix gene, is the main official standard test for AIV detection, but the method requires well-equipped laboratories. Reverse transcription Loop-Mediated Isothermal Amplification (RT-LAMP) has been reported as a rapid method and an alternative to PCR in pathogen detection. The high mutation rate in the AIV genome increases the risk of false negative in nucleic acid amplification methods for detection, such as PCR and LAMP, due to possible mismatched priming. In this study, we analyzed 800 matrix gene sequences of newly isolated AIV in the EU and designed a highly efficient LAMP primer set that covers all AIV subtypes. The designed LAMP primer set was optimized in real-time RT-LAMP (rRT-LAMP) assay. The rRT-LAMP assay detected AIV samples belonging to nine various subtypes with the specificity and sensitivity comparable to the official standard rRT-PCR assay. Further, a two-color visual detection RT-LAMP assay protocol was adapted with the aim to develop on-site diagnostic tests. The on-site testing successfully detected spiked AIV in birds oropharyngeal and cloacal swabs samples at a concentration as low as 10 0.8 EID 50 per reaction within 30 minutes including sample preparation. The results revealed a potential of this newly developed rRT-LAMP assay to detect AIV in complex samples using a simple heat treatment step without the need for RNA extraction., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Golabi, Flodrops, Grasland, Vinayaka, Quyen, Nguyen, Bang and Wolff.)

The endemic of Avian Influenza Virus (AIV) in Asia and epizootics in some European regions have caused serious economic losses. Multiplex reverse-transcriptase (RT) PCR has been developed to detect and subtype AIV. However, the number of targets that can be amplified in a single run is limited because of uncontrollable primer-primer interferences. In this paper, we describe a lab-on-a-chip device for fast AIV screening by integrating DNA microarray-based solid-phase PCR on a microfluidic chip. A simple UV cross-linking method was used to immobilize the DNA probes on unmodified glass surface, which makes it convenient to integrate microarray with microfluidics. This solid-phase RT-PCR method combined RT amplification of extracted RNA in the liquid phase and species-specific nested PCR on the solid phase. Using the developed approach, AIV viruses and their subtypes were unambiguously identified by the distinct patterns of amplification products. The whole process was reduced to less than 1 hour and the sample volume used in the microfluidic chip was at least 10 times less than in the literature. By spatially separating the primers, highly multiplexed amplification can be performed in solid-phase PCR. Moreover, multiplex PCR and sequence detection were done in one step, which greatly simplified the assay and reduced the processing time. Furthermore, by incorporating the microarray into a microchamber-based PCR chip, the sample and the reagent consumption were greatly reduced, and the problems of bubble formation and solution evaporation were effectively prevented. This microarray-based PCR microchip can be widely employed for virus detection and effective surveillance in wild avian and in poultry productions.

Endemic of avian influenza virus (AIV) in Asia and epizootics in some European regions have caused considerable public concern on a possible pandemic of AIV. A rapid method for virus detection and effective surveillance in wild avian, poultry production as well as in humans is required. In this article, a DNA microarray-based solid-phase polymerase chain reaction (PCR) approach has been developed for rapid detection of influenza virus type A and for simultaneous identification of pathogenic virus subtypes H5 and H7. This solid-phase RT-PCR method combined reverse-transcription amplification of RNA extract in the liquid phase with sequence-specific nested PCR on the solid phase. A simple ultraviolet cross-linking method was used to immobilize the DNA probes over an unmodified glass surface, which makes solid-phase PCR a convenient possibility for AIV screening. The testing of 33 avian fecal and tracheal swab specimens was completed in less than 2 h with 94% accuracy., (Copyright © 2011. Published by Elsevier Inc.)

Avian influenza virus (AIV) causes great economic losses for the poultry industry worldwide and threatens the human population with a pandemic. The conventional detection method for AIV involves sample preparation of viral RNA extraction and purification from raw sample such as bird droppings. In this study, magnetic beads were applied for immunoseparation and purification of AIV from spiked chicken fecal sample. The beads were conjugated with monoclonal antibodies against the AIV nucleoprotein, which is conserved in all the AIV. The bead-captured virus was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) without RNA extraction because of effective removal of RT-PCR inhibitors. The developed bead-based assay showed a similar detection limit comparable to the RNA extraction and the classic virus isolation method. Using ready-to-use antibody-conjugated bead, the method requires less than 5 h. Furthermore, the method has potential to integrate into a Lab-on-a-chip system for rapid detection and identification of AIV., (Crown Copyright © 2011. Published by Elsevier Inc. All rights reserved.)

In this paper, a coupling of fluorophore-DNA barcode and bead-based immunoassay for detecting avian influenza virus (AIV) with PCR-like sensitivity is reported. The assay is based on the use of sandwich immunoassay and fluorophore-tagged oligonucleotides as representative barcodes. The detection involves the sandwiching of the target AIV between magnetic immunoprobes and barcode-carrying immunoprobes. Because each barcode-carrying immunoprobe is functionalized with a multitude of fluorophore-DNA barcode strands, many DNA barcodes are released for each positive binding event resulting in amplification of the signal. Using an inactivated H16N3 AIV as a model, a linear response over five orders of magnitude was obtained, and the sensitivity of the detection was comparable to conventional RT-PCR. Moreover, the entire detection required less than 2 hr. The results indicate that the method has great potential as an alternative for surveillance of epidemic outbreaks caused by AIV, other viruses and microorganisms.