GENETIC DIVERSITY OF THE 16S rRNA GENE OF Leptospira spp. CLINICAL ISOLATES FROM JAKARTA AND DEMAK

NAFILA FAKHRURRIZA, Prof. dr. Tri Wibawa Sp. MK (K), Ph.D.; Prof. dr. Muhammad Hussein Gasem, Ph.D, Sp.PD, KPTI, FINASIM

2018 | Tesis | MAGISTER ILMU BIOMEDIK

Latar Belakang: Leptospirosis, adalah penyakit terabaikan yang disebabkan oleh Leptospira patogenik, dan dikenal sebagai zoonosis terluas di dunia, endemik di daerah tropis dan subtropis. Metode yang dapat digunakan untuk mendiagnosis Leptospirosis: Microscopic Agglutination Test (MAT), Indirect Hemagglutinin Assay (IHA), Enzyme-linked Immunosorbent Assay (ELISA), kultur, dan Polymerase Chain Reaction (PCR). Gen 16S rRNA adalah salah satu gen yang paling banyak dipelajari dan dikarakterisasi. Melalui gen 16S rRNA, pohon filogenetik dapat dikembangkan dengan baik dengan berbagai informasi taksonomi yang tersedia di berbagai database. Keragaman genetik dalam penelitian ini berdasarkan anaisis amplifikasi gen 16S rRNA, metode sekuensing dan filogenetik. Metode: Sampel yang digunakan dalam penelitian ini berasal dari Subdit Zoonosis Balai BBTKLPP Jakarta dan Rumah Sakit Sunan Kalijaga di Demak. Sebanyak 94 sampel darah diekstraksi menggunakan kit ekstraksi QIAGEN. Metode PCR menggunakan LipL32 akan dilakukan untuk mendeteksi Leptospira patogenik dari isolat DNA. Setelah itu, isolat DNA yang memiliki hasil positif Leptospira patogenik dilanjutkan identifikasi gen 16S rRNA menggunakan primer 16S rRNA. Identifikasi Leptospira dari variasi gen 16S rRNA dilakukan dengan mengisolasi DNA, amplifikasi gen target 16S rRNA, sekuensing dan analisis sekuensing. Hasil: Hasil analisis penjajaran sekuens basa nukleotida menunjukkan beberapa variasi genetik dari gen Leptospira patogenik 16S rRNA karena proses mutasi seperti substitusi, insersi dan delesi di beberapa basa nukleotida. Semua mutasi ini ditemukan dalam sampel darah dari wilayah Jakarta dan Demak. Hasil rekonstruksi phon filogenetik berdasarkan gen 16S rRNA menunjukkan bahwa 7 isolat (Jakarta dan Demak) memiliki hubungan kekerabatan dengan spesies Leptospira spp. yang bersifat patogenik Kesimpulan: Ada beberapa variasi genetik dari Leptospira patogenik berdasarkan gen 16S rRNA. Hasil rekonstruksi pohon filogenetik berdasarkan 16S rRNA menunjukkan adanya hubungan kekerabatan antara isolat yang berasal dari Jakarta dan Demak.

Background: Leptospirosis, a neglected disease caused by pathogenic Leptospira, is well known as the worlds most widespread zoonosis, which is endemic to tropical and subtropical regions. The available methods that can be used to diagnose Leptospirosis: Microscopic Agglutination Test (MAT), Indirect Hemagglutinin Assay (IHA), Enzyme-linked Immunosorbent Assay (ELISA), culture, and Polymerase Chain Reaction (PCR). 16S rRNA gene is one of the most-well studied and characterized gene, the phylogenetic trees are well developed and taxonomic information is readily available in a variety of databases. The genetic diversity in this study is generally based on PCR-ampliï-cation of 16S rRNA gene, sequencing and phylogenetic analysis. Methods: The samples are from BBTKLPP Jakarta and Sunan Kalijaga hospital in Demak. The 94 whole blood was extracted using QIAGEN extraction kit. The PCR method using LipL32 was performed to detect pathogenic Leptospira of DNA isolates. In addition, DNA isolates that have positive result of pathogenic Leptospira was followed by PCR technique using 16S rRNA primers. Leptospira identification of 16S rRNA gene variation was performed by PCR; DNA isolation, 16S rRNA amplification, sequencing and the analysis of sequencing. Results: The results of multiple alignment showed that there is a nitrogen base which undergoes substitution, insertion and deletion. All these mutations are found in whole blood samples from the Jakarta and Demak regions. The results of phylogenetic tree reconstruction based on the 16S rRNA gene showed that 7 isolates (Jakarta and Demak) had a very close relationship with the pathogenic species Leptospira spp. Conclusion: There are several genetic variations of the 16S rRNA pathogenic Leptospira gene. The results of phylogenetic tree reconstruction based on the 16S rRNA showed that there are relationship between isolates from Jakarta and Demak.

Kata Kunci : Leptospira spp., LipL32, gen 16S rRNA, Variasi Genetik., Leptospirosis, Leptospira spp., LipL32, 16S rRNA gene, Genetic Variation.