Rapid Nucleic Acid Extraction for Aquatic Animal DNA Virus Determination Using Chelex 100 Resin via Conventional PCR and Digital Droplet PCR Detection

SIMPLE SUMMARY: Convenient, fast, and high-quality nucleic acid extraction methods are urgently needed in molecular diagnostic testing for viral pathogens in aquaculture. We developed a viral DNA extraction method from diseased tissues and cells using the Chelex 100 resin solution workflow. The only extraction reagents required are the Chelex 100 resin and phosphate-buffered saline. The whole extraction process only takes about 15 min from the tissue homogenate to obtain the DNA. The concentration of extracted DNA is at least 100 ng/µL. This methodology has clear benefits in terms of cost and time saving compared to the commercial kit extraction for aquatic animal DNA virus determination by PCR in the laboratory. In addition, the simplified method using Chelex 100 resin with a pH value of 10–11 presented excellent results in PCR application and could be a standard for the DNA extraction for DNA virus testing in the future. ABSTRACT: Molecular diagnostic testing for viral pathogens is crucial in aquaculture. The efficient and convenient preparation of pathogenic microbial nucleic acids is the basis of molecular diagnosis. Here, we developed a simplified deoxyribonucleic acid (DNA) extraction method from aquatic animal DNA viruses using the Chelex 100 resin. The nucleic acid was extracted from infected tissues and cell culture for the detection of three common aquatic viral pathogens (CEV, CyHV-2, and GSIV). We compared the extraction effects of a current commercial kit extraction method and the Chelex 100 resin extraction method according to nucleic acid concentration, conventional polymerase chain reaction (PCR), and digital droplet PCR (ddPCR). The results indicated that both extraction procedures could obtain high-quality nucleotide samples. Extracting DNA using the Chelex 100 resin led to better detective efficiency for ddPCR molecular diagnostic testing. The whole process took less than 20 min, and only Chelex 100 resin solution was added to the tissues or cells without multiple tubes being transferred several times. The extracted DNA concentration and the detection sensitivity were high. These results indicated that the Chelex 100 resin solution has the advantages of speed, efficiency, and economy compared to the commercial kit. In addition, the higher pH value (10–11) of the Chelex 100 resin solution markedly improved the detection sensitivity compared to a lower pH value (9–10). In conclusion, the comparison of the Chelex 100 Resin and commercial viral DNA extraction kits revealed the good performance of the Chelex 100 resin solution at pH 10–11 in DNA extraction for PCR amplification from aquatic animal viral samples of tissues and cells in molecular diagnostic testing. It is both rapid and cost-effective.