Xiaofeng Chen

Lactococcus garvieae is a major bacterial pathogen responsible for lactococcosis outbreaks in aquaculture, resulting in substantial economic losses worldwide. Accurate identification of L. garvieae remains challenging because of its genetic similarity to other Lactococcus species and the limited field applicability of many existing molecular diagnostic methods. Therefore, there is an urgent need for a rapid, highly specific, and field-deployable analytical method that enables accurate identification of L. garvieae outside conventional laboratory settings. In this study, a one-pot analytical strategy integrating enzymatic recombinase amplification (ERA) with CRISPR/Cas12a detection was developed, enabling fluorescence or lateral flow dipstick (LFD) readouts within a single closed reaction tube. The one-pot ERA-CRISPR/Cas12a assays achieved a detection limit of 10 copies/reaction. When combined with a rapid DNA release protocol, qualitative detection could be completed within 50 min without the need for sophisticated instrumentation. In parallel, a TaqMan quantitative PCR assay was established as an analytical benchmark, exhibiting a detection limit of 20 copies/reaction with high linearity and good reproducibility. Clinical evaluation using 136 diseased fish samples demonstrated full concordance between the one-pot ERA-CRISPR/Cas12a and qPCR assays, with both methods achieving a positive detection rate of 23.5% (32/136). In addition, the ERA-CRISPR/Cas12a platform was successfully validated under simulated field conditions using a portable reaction device. This study presents a rapid and field-deployable CRISPR-based platform for the early detection and epidemiological surveillance of lactococcosis.

Carpione rhabdovirus (CAPRV) is an emerging virus within the family Rhabdoviridae, posing potential threats to aquaculture species such as golden pompano (Trachinotus anak). However, since the 21st century, and for CAPRV strains isolated from marine fish, only a single CAPRV2023 sequence has previously been available in public databases, with no additional sequences reported. Because the virus undergoes genetic variation, relying on this single sequence likely introduced mismatches or off-target risks in earlier detection assay designs. Notably, the previously developed two-step N-targeting detection assay was designed based solely on that single CAPRV2023 sequence. Consequently, this study involved determining and analyzing the N gene sequences from CAPRV isolates gathered from 2023 to 2025, with the aim of pinpointing conserved regions for assay development, and sequence comparisons subsequently verified the existence of mismatches in the primer–probe binding sites of the previous assay. Since quantitative assays in aquatic virology often define copy numbers utilizing either plasmid DNA templates or RNA templates produced via in vitro transcription, which may lead to variations in amplification kinetics and sensitivity, this study compared both standards to ensure reliable quantification across different nucleic acid types. Based on these findings, a one-step TaqMan quantitative PCR (qPCR) assay was developed and validated using dual nucleic acid standards, namely plasmid DNA and in vitro–transcribed RNA. Compared with conventional two-step qPCR, the one-step format combines cDNA synthesis and subsequent DNA amplification in a single sealed tube, thereby effectively preventing cross-contamination, simplifying the workflow, and improving detection efficiency. The assay exhibited strong linearity (R² > 0.99) and consistent amplification efficiencies between 90% and 110%, demonstrating excellent quantitative performance. The detection limits were 2 copies per reaction for plasmid DNA and 20 copies for in vitro–transcribed RNA templates. No cross-reactivity was observed with other aquatic pathogens, and the assay showed strong repeatability and reproducibility (coefficients of variation below 2.0%), providing a sensitive and reliable tool for epidemiological surveillance and the analysis of CAPRV distribution in marine aquaculture systems of southern China.