Simultaneous detection of multiple bacterial and viral aquatic pathogens using a fluorogenic loop‐mediated isothermal amplification‐based dual‐sample microfluidic chip

Abstract

Rapid and user-friendly diagnostic tests are necessary for early diagnosis and immediate detection of diseases, particularly for on-site screening of pathogenic microorganisms in aquaculture. In this study, we developed a dual-sample microfluidic chip integrated with a real-time fluorogenic loop-mediated isothermal amplification assay (dual-sample on-chip LAMP) to simultaneously detect 10 pathogenic microorganisms, that is Aeromonas hydrophila, Edwardsiella tarda, Vibrio harveyi, V. alginolyticus, V. anguillarum, V. parahaemolyticus, V. vulnificus, infectious hypodermal and haematopoietic necrosis virus, infectious spleen and kidney necrosis virus, and white spot syndrome virus. This on-chip LAMP provided a nearly automated protocol that can analyse two samples simultaneously, and the tests achieved limits of detection (LOD) ranging from 10 0 to 10-1 pg/μl for genomic DNA of tested bacteria and 10-4 to 10-5 pg/μl for recombinant plasmid DNA of tested viruses, with run times averaging less than 30 min. The coefficient of variation for the time-to-positive value was less than 10%, reflecting a robust reproducibility. The clinical sensitivity and specificity were 93.52% and 85.53%, respectively, compared to conventional microbiological or clinical methods. The on-chip LAMP assay provides an effective dual-sample and multiple pathogen analysis, and thus would be applicable to on-site detection and routine monitoring of multiple pathogens in aquaculture.

Rapid detection of multiple pathogens by the combined loop-mediated isothermal amplification technology and microfluidic chip technology

“…Microfluidics can be divided into multiple reaction units for the detection of multiple pathogens, which can ensure the independence of each reaction and the accuracy of the results (13)(14)(15)(16)(17)(18)(19). The combined LAMP and microfluidics technology may be able to leverage the advantages of both technologies.…”

Application of Lab-on-Chip for Detection of Microbial Nucleic Acid in Food and Environment

“…As mentioned above, the microfluidic chip integrated with real-time fluorescence loop-mediated isothermal amplification technology developed by Zhou et al (2021) has good sensitivity and specificity, but preprocessing of samples is required to enrich target DNA. The integrated chip designed by Park et al (2018a) for the detection of Staphylococcus aureus and E. coli in food integrates nucleic acid amplification and electrochemical detection, but also requires advance DNA extraction and purification.…”

“…Fluorescent probes are often used to detect nucleic acids in real-time PCR ( Ahn et al, 2018 ; Aufdembrink et al, 2020 ; Salman et al, 2020 ; Zhou et al, 2021 ). Some nucleic acids have a very weak internal fluorescence that is hardly detected.…”

“…However, the development of sample preparation technology is relatively slow, and most LOC equipment still needs sample pretreatment in advance, which is inconsistent with the concept of micro total analysis system and LOC. As mentioned above, the microfluidic chip integrated with real-time fluorescence loop-mediated isothermal amplification technology developed by Zhou et al (2021) has good sensitivity and specificity, but preprocessing of samples is required to enrich target DNA. The integrated chip designed by Park et al (2018a) for the detection of Staphylococcus aureus and E .…”

“…Zhou et al (2021) developed a microfluidic chip integrating real-time fluorescence and LAMP technology, which could simultaneously detect 10 pathogenic microorganisms. They used a universal genomic DNA extraction kit in advance to extract total DNA from the sample and then added it to the system for sample testing.…”