Improved RNA-based metagenomic sequencing for rapid pathogen detection in pets

In veterinary medicine, particularly in pet clinics, the accurate and rapid detection of pathogens is crucial for effective disease diagnosis and treatment. Traditional diagnostic methods are often time-consuming and fall short in identifying a broad spectrum of pathogens. The newly developed metagenomic transcriptomics next-generation sequencing (mtNGS) technology is a promising tool for the rapid detecting RNA- and DNA-based pathogens. However, its application in pet clinics has been limited due to high costs, complex operational procedures, and the absence of unified protocols. Here we established a standardized mtNGS workflow for pathogen detection tailored to various clinical sample types from pets. This workflow involves the extraction of total RNA without rRNA depletion and sequencing using Illumina platforms. It also incorporates Bowtie2 to eliminate host genome sequences and MetaPhlAn3 to identify microbial compositions. Our mtNGS technology was evaluated in 16 diverse clinical cases involving body fluids, fecal samples, nasopharyngeal swabs, and tissue samples from dogs, cats, and parrots. Notably, it detected pathogens in all cases, including an identification of Mycobacterium intracellulare in a cat, highlighting its utility in diagnosing zoonotic diseases. These results, corroborated by traditional techniques, demonstrate that the mtNGS-based diagnostic approach is particularly advantageous in cases where conventional diagnostics are insufficient or when multiple co-infections are suspected. This method exhibits potential in diagnosing complex clinical diseases that are challenging to identify using traditional techniques, thus representing a promising tool and can be widely applied in veterinary diagnostics.