Kajian metoda diagnosis toksoplasmosis secara polymerase chain reaction (PCR) dan pemeriksaan histologis

PRIYOWIDODO, Dwi, drh. Sumartono, SU.,DEA

2003 | Tesis | S2 Sain Veteriner

Penelitian ini bertujuan untuk mengkaji metode diagnosis toksoplasmosis secara histologis dan PCR (Polymerase Chain Reaction). Sebanyak 25 ekor mencit digunakan dalam penelitian ini, setiap ekor mencit diinfeksi dengan 1x103 takizoit Toxoplasma gondii isolat lokal secara intraperitoneal. Sampel cairan asites , serum darah dan jaringan (hati, paru-paru, otak) diambil dari masing-masing 5 ekor mencit setiap pengambilan sampel mulai 1, 3, 5, 7 dan 9 hari sesudah infeksi. Sampel cairan asites dan serum darah diambil untuk isolasi DNA, sedangkan sampel jaringan (hati, paru dan otak) diambil untuk isolasi DNA dan pembuatan preparat histologi. Isolasi DNA parasit dari sampel cairan asites dan serum darah dikerjakan dengan metode seperti yang dikerjakan oleh Hohlfeld dkk (1994), sedangkan sampel jaringan (otak, paru, hati) dikerjakan dengan metode seperti yang dikerjakan oleh Owen dkk. (1998). DNA hasil isolasi dari sampel diamplifikasi menggunakan kit komersial Ready To Go TM PCR Beads dengan primer gen B1 Toxoplasma gondii. Hasil PCR dielektroforesis menggunakan agarose 1%, 0,5 x TBE (Tris borate buffer) dengan pewarnaan ethidium bromide. Data hasil PCR yang dideteksi dengan elektroforesis dari sampel cairan asites, serum darah dan jaringan (otak, paru, hati) serta hasil histopatologi jaringan dianalisis secara deskriptif. Dari hasil penelitian ini dapat ditarik kesimpulan bahwa metode Polymerase Chain Reaction (PCR) dapat digunakan untuk diagnosis toksoplasmosis secara dini (3 hari sesudah infeksi) dari sampel cairan asites dan serum darah, sedangkan metode histologis jaringan tidak spesifik untuk diagnosis toksoplasmosis.

The purpose of the research was to study of the toxoplasmosis diagnosis methods by histhology and Polymerase Chain Reaction (PCR). Twenty-five of mice were used in the research, each mice was infected intraperitoneally with 1x103 of Toxoplasma gondii tachyzoites of the local strain. Ascites fluid, blood serume and tissue samples (lever, lung, brain), were collected on the 1st, 3rd, 5th, 7th and 9th day after infection (each 5 mices per collection). The ascites fluid and blood serume samples were collected to DNA extraction, whereas tissue samples (lever, lung, brain), were collected to DNA extraction and histhological examination. The Toxoplasma gondii DNA was extracted of the ascites fluid and blood serume samples using previously procedure described by Hohlfeld et al. (1994), whereas the T. gondii of the tissue samples (lever, lung, brain) was extracted using procedure described by Owen et al, (1998). The DNA of the samples, then were amplified using Ready To GoTM PCR Beads with primer specific of the T. gondii B1 gene. The PCR product were analyzed by electrophoresis on a 1% agarose, 0,5 x TBE (Tris borate buffer) with ethidium bromide stained. The data of the PCR products and histhological examinations was analyzed descriptively. The research concluded that the Polymerase Chain Reaction (PCR) method could be used to toxoplasma diagnosis early (3rd day after infection) whereas histhology method no specific to toxoplasma diagnosis.

Kata Kunci : Penyakit Hewan,Toksoplasmosis,Diagnosa,PCR, Polymerase Chain Reaction, Tachyzoite, Toxoplasma gondii