Samples were homogenized prior to nucleic acid extraction. Total DNA was extracted from the collected tissues and blood samples using a DNeasy Blood and Tissue Kit, and total RNA was extracted using an RNeasy Mini Kit and a QIAamp RNA Blood Mini Kit (QIAGEN, Valencia, CA, USA). A QIAamp DNA Stool Mini Kit and a QIAamp Viral RNA Mini Kit (QIAGEN) were used to extract DNA and RNA, respectively, from rectal swabs.

The manufacturer’s recommended procedures were followed for nucleic acid extraction. Reverse transcription of total RNA to cDNA was performed using an iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) following the manufacturer’s instructions.

We selected consensus primer sets for each viral pathogen to avoid possible nucleotide mutations at the primer annealing sites [20]. The detection limit of RT-PCR was 10–100 gene copies/µL (Table (Table2).2). Samples that were PCR-positive for the virus were then subjected to sequencing for confirmation.

Sensitivity of specific PCR assays for detecting protoparvoviruses, FeLV, FIV, CoV, and CDV. The target genes were cloned into a plasmid vector, and the plasmid was diluted to 100 to 109 gene copies/µL for each detection assay

PCR amplicons were sequenced in an ABI377 sequencer using an ABI PRISM dye-terminator cycle sequencing ready reaction kit with AmpliTaq DNA polymerase (Perkin-Elmer, Applied Biosystems, MA, USA). To identify sequences similar to those of the amplicons, a BLAST search of the GenBank nt/nr database, available on the BLAST website (https://blast.ncbi.nlm.nih.gov/Blast.cgi), was performed.

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