# Development and Evaluation of Quadruplex Droplet Digital PCR Method to Multiplex Detection of Different Respiratory Pathogens of Chickens

## TL;DR

This study developed a fast and accurate quadruplex ddPCR method to detect four respiratory pathogens in chickens, improving diagnosis for a disease causing significant economic losses in the poultry industry.

## Contribution

A novel quadruplex ddPCR method was developed for simultaneous detection of four chicken respiratory pathogens with high sensitivity and specificity.

## Key findings

- The quadruplex ddPCR method detected four pathogens with minimum concentrations as low as 3.02-3.39 copies/μL.
- The method showed no cross-reactivity with 10 other pathogens and had a coefficient of variation below 9%.
- Clinical testing showed the ddPCR method had slightly higher sensitivity than qPCR and high coincidence rates between the two methods.

## Abstract

A specific respiratory disease, termed chicken bronchial obstruction, caused by multiple respiratory pathogens, poses a continuous threat to the poultry industry. This study successfully established a highly specific and rapid quadruplex ddPCR method targeting the HA gene of H9 subtype AIV, the M gene of IBV, the Pal gene of P. aeruginosa, and the UidA gene of E. coli. The method achieved accurate detection of these pathogens or opportunistic pathogens through carefully designed primers and probes, showing no cross-reactivity with 10 other pathogens. Results demonstrated that the developed assay exhibited high sensitivity and specificity, with a strong positive correlation compared to quadruplex qPCR.

Chicken respiratory diseases represent multifactorial conditions resulting from viral, bacterial, mycoplasmal pathogens, and environmental factors, causing significant economic losses within the poultry industry. A specific respiratory disease characterized by breathing difficulties and bronchial occlusion due to caseous exudates is termed chicken bronchial obstruction. However, the absence of rapid, precise, and highly sensitive diagnostic methods for differentiation of primary respiratory disease pathogens or opportunistic pathogens, including avian influenza virus (AIV), infectious bronchitis virus (IBV), Pseudomonas aeruginosa (P. aeruginosa), and Escherichia coli (E. coli), constitutes a substantial challenge. This study developed a quadruplex droplet digital polymerase chain reaction (ddPCR) method that targeted the HA gene of H9 subtype AIV, the M gene of IBV, the Pal gene of P. aeruginosa, and the UidA gene of E. coli. Following the optimization of annealing temperature, sensitivity, and repeatability, the minimum detectable concentrations were determined as 3.02 copies/μL for the HA gene of H9 subtype AIV, 3.08 copies/μL for the M gene of IBV, 3.19 copies/μL for the Pal gene of P. aeruginosa, 3.39 copies/μL for the UidA gene of E. coli. No cross-reactivity was observed with Newcastle disease virus (NDV), H5 subtype AIV, H7 subtype AIV, fowl adenovirus serotype 4 (FAdV-4), infectious laryngotracheitis virus (ILTV), Avibacterium paragallinarum, Streptococcus, Salmonella, Pasteurella multocida, and Staphylococcus aureus. The method demonstrated excellent repeatability, with a coefficient of variation (CV) below 9%. The 185 clinical samples collected in Hebei Province China are tested by both quadruplex ddPCR and quadruplex qPCR method and the results compared. The sensitivity of the quadruplex ddPCR method (57.30%; 106/185) slightly exceeded that of the quadruplex qPCR method (49.73%; 92/185). Pathogens or opportunistic pathogens positive rates obtained via the quadruplex ddPCR were 40.00% for H9 subtype AIV, 33.51% for IBV, 24.32% for P. aeruginosa, and 27.57% for E. coli. In comparison, the positive rates of H9 subtypes AIV, IBV, P. aeruginosa, and E. coli from the quadruplex qPCR were 36.22%, 30.81%, 21.62%, and 24.32%, respectively. The coincidence rates between the two methods were 96.22% for H9 AIV, 97.30% for IBV, 97.30% for P. aeruginosa, and 96.76% for E. coli. These results demonstrated that the quadruplex ddPCR method represented a highly sensitive, specific, and rapid technique for identifying H9 subtype AIV, IBV, P. aeruginosa, and E. coli.