Multiplex polymerase chain reaction (mPCR) assay is a nucleic acid amplification
method that is considered reliable and practical means for several pathogen detections in a
single reaction, especially when multiple pathogens are suspected. In this study, a novel
mPCR assay was validated for the detection of four notifiable diseases in cattle, including
foot and mouth disease (FMD), Bovine viral diarrhea (BVD), Bluetongue (BT), and
Hemorrhagic Septicemia (HS). The assay was operated in a two-step procedure. The first
one was a reverse transcription of viral RNA, then mPCR of viral cDNA and bacterial DNA.
The optimized mPCR was applied on blood (26) and vesicular epithelium (10) samples
collected from 26 clinically infected animals from three governorates (Qalubia, Sharkia, and
Gharbia). mPCR detected at least 10 pg of microbial nucleic acid extracted from the local
isolates. The mPCR results showed that 22/26 (84.6%) of clinically infected animals were
positively infected by single or dual infection. Mixed infection of FMDV and Pasteurella
multocida was recorded in 11 animals (42.3%), while single FMDV infection was recorded
in 5 animals (19.2 %). Single BVDV infection was detected in 5 animals (19. 2 %) and dual
infection with FMDV in 1 animal (3.8%). Notably, BTV was not detected in any of the
clinical samples. The assessed mPCR was a rapid, accurate, and sensitive test for diagnosing
single and mixed infections in cattle and could be used to screen the notifiable diseases
affecting cattle.
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