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

PRASIDDHANTI, Lalita , Agnesia Endang Tri Hastuti Wahyuni

2014 | Tesis |

Mastitis merupakan kasus penyebab penurunan produksi susu dan S.aureus sebagai salah satu bakteri patogen utama, selain menimbulkan mastitis juga sebagai penyebab kasus keracunanmakanan. Salah satu faktor virulensi yang berperan adalah Staphylococcal enterotoxin yang berhubungan dengan kasus gastroenteritis. Jenis enterotoksin S.aureus yang tinggi tingkat kejadiannya pada kasus bovine mastitis yaitu SEG dan SEI. Deteksi keberadaan gen penyandi enterotoksin salah satunya menggunakan teknik multiplex PCR. Penelitian ini bertujuan untuk mendeteksi keberadaan gen penyandi enterotoksin G dan I pada susu menggunakan teknik multiplex PCR dengan primer yang didesain sendiri, mengetahui pola distribusi gen seg dan sei pada ketiga wilayah pada berbagai status susu. Sebanyak 32 isolat S. aureus asal susu sapi di Boyolali, Pacitan dan Ponorogo, dideteksi keberadaan gen seg dan sei. Identifikasi ulang dilakukan dengan mengkultur S.aureuspadaPAD, pengecatan Gram, uji katalase, koagulase, MSA dan uji VP. Deteksi molekuler Polymerase Chain Reaction (PCR) untuk mengidentifikasi S.aureus dengan deteksi gen 23S rRNA menggunakan primer spesifik 5’-ACGGAGTTACAAAGGACGAC-3’(forward) dan 5’- AGCTCAGCCTTAACGAGTAC-3’ (reverse) dengan produk amplifikasi sebesar 1250 bp. Deteksi gen penyandi seg dan sei dengan teknik multiplex PCR menggunakan primer yang didesain sendiri melalui software online MP Primer Output yang menghasilkan urutan primer seg 5'-TGAATTATGTGAATGCTCAACCCG-3'(forward) dan 5'-GCAGAACCATCAAACTCGTATAGC-3' (reverse)dengan produk sebesar 541 bp, dan sei 5'-ACCTACCTATTGCAAATCAACTCG-3' (forward) dan 5'-CCATATTCTTTGCCTTTACCAGTGT-3' (reverse) dengan produk sebesar 373 bp. Hasil penelitian sebanyak 32 isolat terdeteksi sebagai S.aureus dengan teknik PCR pada gen 23S rRNA. Tingkat kejadian ditemukannya gen seg dan sei pada ketiga wilayah yaitu Boyolali 25% mengandung gen seg, 8,33% gen sei, dan 50% kombinasi gen se(g,i); wilayah Pacitan 50% mengandung gen sei dan 40% terdapat kombinasi gen se(g,i); dan Ponorogo 50% isolat mengandung sei dan 40% kombinasi gen se(g,i).Susu mastitis subklinis mengandung 3,7% seg, 37,08% sei, 44,44% se (g,i) dan 14,8% negatif, susu mastitis klinis 100% mengandung sei, dan susu normal 50% mengandung seg dan 50% kombinasi se(g,i).

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).

Kata Kunci : Staphylococcus aureus, enterotoksin, seg, sei, multiplex PCR