A novel multiplex PCR assay for simultaneous detection of nine clinically significant bacterial pathogens associated with bovine mastitis.
For rapid and simultaneous detection of nine bovine mastitic pathogens, a sensitive and specific multiplex PCR assay was developed. The assay was standardized using reference strains and validated on mastitic milk cultures which were identified to species level based on 16S rRNA sequencing. Multiplex PCR assay also efficiently detected the target bacterial strains directly from milk. The detection limit of the assay was up to 50 pg for DNA isolated from pure cultures and 104CFU/ml for spiked milk samples. As estimated by latent class analysis, the assay was sensitive up to 88% and specific up to 98% for targeted mastitic pathogens, compared with the bacterial culture method and the 16S rRNA sequence analysis. This novel molecular assay could be useful for monitoring and maintaining the bovine udder health, ensuring the bacteriological safety of milk, and conducting epidemiological studies.
Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Shaykh Abdul Qadir Jilani Road, Lahore 54000, Pakistan.
Aqeela
A
Ashraf
Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Shaykh Abdul Qadir Jilani Road, Lahore 54000, Pakistan. Electronic address: muhammad.imran@uvas.edu.pk.
Muhammad
M
Imran
Department of Microbiology, University of Veterinary and Animal Sciences, Shaykh Abdul Qadir Jilani Road, Lahore 54000, Pakistan.
Tahir
T
Yaqub
Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Shaykh Abdul Qadir Jilani Road, Lahore 54000, Pakistan.
Muhammad
M
Tayyab
Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Shaykh Abdul Qadir Jilani Road, Lahore 54000, Pakistan.
Wasim
W
Shehzad
School of Life and Environmental Sciences, The University of Sydney, 425 Werombi Road, Camden 2570, NSW, Australia; ASLP Dairy Project, University of Veterinary and Animal Sciences, Shaykh Abdul Qadir Jilani Road, Lahore 54000, Pakistan.
Peter C
PC
Thomson
D000818:Animals; D001419:Bacteria / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification* / Q000472:pathogenicity; D002417:Cattle; D005260:Female; D008414:Mastitis, Bovine / Q000175:diagnosis* / Q000235:genetics / Q000382:microbiology; D008892:Milk / Q000382:microbiology; D060885:Multiplex Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012680:Sensitivity and Specificity
D012336:RNA, Ribosomal, 16S
Bovine mastitis; Latent class analysis; Multiplex PCR assay