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Abstract - Development of simplex-PCR assays for accurate identification of nine Staphylococcal species at genus and species levels
Bibek Ranjan Shome, Krithiga Natesan, Susweta Das Mitra, Nimita Venugopal, Bhuvana Mani, Feroze Ganaie, Rajeswari Shome, Habibur Rehman

Development of Simplex-PCR Assays for Accurate Identification of Nine Staphylococcal Species at Genus and Species Levels

Bibek Ranjan Shome1, Krithiga Natesan1, Susweta Das Mitra1,2, Nimita Venugopal1, Bhuvana Mani1, Feroze Ganaie1, Rajeswari Shome1, Habibur Rahman3

1ICAR-National Institute of Veterinary Epidemiology and Disease Informatics, Bengaluru, India

2Department of Biotechnology, Dayanand Sagar University, Bengaluru, India

3International Livestock Research Institute, New Delhi, India



Objective: Species identification of Staphylococcus is the prerequisite for precise assessment of microbial dynamics including their transmission and pathogenic significance in the dairy herd environment. The present study aimed to develop simplex PCR assays for rapid and specific identification of nine different Staphylococcus species.

Methods: Specific primers targeting sodA gene for S. aureus, S. chromogenes, S. hominis, S. haemolyticus, S. hyicus; gap gene for S. sciuri, S. auricularis, S. simulans; and rdr gene for S. epidermidis were designed. The PCR assays were evaluated against 28 ATCC reference strains and 209 composite milk samples. Partial 16S rRNA sequencing was performed to reconfirm the results.

Results: The PCR assays allowed species level identification of 348 staphylococcal field isolates recovered from 209 milk samples. The identification pattern was S. aureus (n=101), S. chromogenes (n=89), S. epidermidis (n=57), S. sciuri (n=43), S. haemolyticus (n=34), S. hyicus (n=13), S. hominis (n=5), S. auricularis (n=3) and S. simulans (n=3). The PCR based species identification was in 100% concordance with the partial 16S rRNA gene sequencing approach.

Conclusion: The simplex PCR assays can be used as a precise tool for routine identification of Staphylococcus species from bovine milk as Staphylococcus species including coagulase-negative staphylococci is recognized a major cause of bovine mastitis in different parts of the world including India. J Microbiol Infect Dis 2018; 8(3):120-127

Keywords: 16S rRNA gene sequencing, Staphylococcus, Species-specific PCR

Volume 08, Number 03 (2018)