DETEKSI GEN ENTEROTOKSIN G dan I Staphylococcus aureus DARI SUSU NORMAL, MASTITIS SUBKLINIS DAN MASTITIS KLINIS DENGAN TEKNIK Multiplex Polymerase Chain Reaction

• Main Authors: , Lalita Prasiddhanti, , Dr. drh. Agnesia Endang Tri Hastuti Wahyuni, M.Si.
• Format: Theses and Dissertations NonPeerReviewed
• Published: [Yogyakarta] : Universitas Gadjah Mada 2014
• Subjects: ETD
• Online Access: https://repository.ugm.ac.id/130639/; http://etd.ugm.ac.id/index.php?mod=penelitian_detail&sub=PenelitianDetail&act=view&typ=html&buku_id=71065
• Institution: Universitas Gadjah Mada

Mastitis is an abnormality in mammary gland causing a great loss of milk production. One of the frequent bacteria that inflict this disease is Staphylococcus aureus, which can also produce toxin then lead to food toxicity and gastroenteritis. This toxin is one of important virulence factor in Staphylococcus aureus called Staphylococcal Enterotoxin (SE). In order to detect the presence of enterotoxin coding gene which can be in various type, the use of Multiplex Polymerase Chain Reaction which can detect more than one sequence target using two pair of primers or more in a reaction can detect the presence of enterotoxin coding gene faster than any other method. Various type of enterotoxin had been identified, SEA-A, and SEG-V, also SEG and SEI which had high morbidity rate in bovine mastitis case. Thirty two (32) isolates of Staphylococcus aureus from milk taken from dairy farm in Boyolali, Pacitan, and Ponorogo, were detected in presence of segandseigene. Reidentification of S. aureus by culturing the bacteria in Blood Agarose Plate (BAP), Gram stain, Catalase test, Coagulation Test, Mannitol Salt Agar (MSA), and VogesProuskauer (VP) Test. Molecular detection of 23S rRNA gene to identify S. aureus by Polymerase Chain Reaction (PCR) was done using specific primer 5�-ACGGAGTTACAAAGGACGAC-3� (forward) and 5�-AGCTCAGCCTTAACGAGTAC-3� (reverse) resulting in 1250 bp of amplification product. The primer for the detection of segand sei coding gene was designed for multiplex PCR method using MP Primer software. The primer for seg gene 5'-TGAATTATGTGAATGCTCAACCCG-3' (forward) and 5'-GCAGAACCATCAAACTCGTATAGC-3' (reverse) with amplification product of 541 bp.For the sei gene 5'-ACCTACCTATTGCAAATCAACTCG-3' (forward) and 5'-CCATATTCTTTGCCTTTACCAGTGT-3' (reverse) resulting in 373 bp of amplification product. The result showed thirty two (32) isolates were Staphylococcus aureus in 23s rRNA gene and from the sequence result. The results in the detection of seg and sei coding gene showed 28 (87.5%) positive sample in Boyolali, Pacitan, and Ponorogo. From 87.5% positive sample, 9.37% were seg positive, 34.37% were sei positive, and 43.75% were seg and sei positive. According to the result sequence, the seg gene had 99% similarity with S.aureus Accession number AY920262.1, and the sei gene had 99% similarity with S.aureus Accession number AY961382.1. Subclinical mastitis milk containing 3.7% seg gene, 37.08% sei, 44.44% se (g, i) and 14.8% negative, clinical mastitis milk containing 100% sei, and normal milk containing 50% seg and 50% se(g, i).