A quantum dot-based double-antibody sandwich immunochromatographic strip for rapid detection of porcine deltacoronavirus targeting the nucleocapsid protein.

Abstract

Porcine deltacoronavirus (PDCoV) is an emerging enteropathogenic pathogen that causes severe diarrhea in suckling piglets and carries significant zoonotic spillover potential. Therefore, developing a rapid, sensitive, and specific diagnostic assay is crucial for effective PDCoV surveillance and control. Here, we developed a novel quantum dot-based immunochromatographic strip (QD-ICS) using a double-antibody sandwich format to detect the PDCoV nucleocapsid (N) protein. The strip employs QD-conjugated mouse anti-PDCoV-N monoclonal antibody (mAb) as the detection probe and rabbit anti-PDCoV-N polyclonal antibody as the immobilized capture antibody. Through systematic optimization, we determined the ideal parameters: 20 μL of QD solution activated with 300 μL 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, 60 μg mAb coupling at pH 7.0, and a conjugate spraying density of 5 μL/cm. The QD-ICS achieved optimal performance with 1.75 mg/mL polyclonal antibody on the test line and 1 mg/mL goat anti-mouse IgG on the control line. The strip exhibited high specificity without cross-reactivity with porcine epidemic diarrhea virus, transmissible gastroenteritis virus, porcine enteric alphacoronavirus, porcine rotavirus, porcine parvovirus, etc. It achieved a 95 % detection limit of 236.661 TCID/mL for PDCoV and maintained stable performance throughout six months of storage at 25 °C and 37 °C. Both intra- and inter-batch coefficients of variation for PDCoV detection were below 10 %, confirming good reproducibility. In clinical validation with 60 porcine rectal swabs, the QD-ICS showed 96.7 % concordance with reference real-time RT-PCR results. Offering comparable clinical performance, our QD-ICS detects faster than real-time RT-PCR and requires neither expensive equipment nor specialized training, making it an ideal field-deployable diagnostic tool.