DIAGNOSIS TOKSOPLASMOSIS KONGENITAL BERDASARKAN GEN SAG-1 Toxoplama gondii ISOLAT LOKAL (IS-1) MENGGUNAKAN METODE LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (L-AMP)

DWI PRIYOWIDODO, Prof. Dr. drh. Sri Hartati, SU.

2015 | Disertasi | S3 SAINS VETERINER

Toksoplasmosis kongenital memiliki peranan penting dalam penularan toksoplasmosis pada hewan dan manusia, sehingga diperlukan metode diagnosis yang sederhana, cepat dan akurat. Penelitian ini bertujuan untuk menerapkan metode L-AMP untuk mendiagnosis toksoplasmosis kongenital pada mencit dengan target gen surface antigen-1 (SAG-1) isolat lokal (IS-1) dari sampel darah, cairan amnion, fetus, dan plasenta. Sebanyak 15 ekor mencit umur 10 minggu bunting galur Balb/C digunakan sebagai hewan percobaan. Mencit diinfeksi dengan 103 takizoit T. gondii strain RH setiap ekor secara intra peritoneal pada umur kebuntingan sembilan hari. Sampel cairan amnion, darah induk, fetus, dan plasenta dari masing-masing tiga ekor mencit diambil mulai hari ke- 1, 2, 3, 4, dan 5 sesudah infeksi untuk isolasi DNA dan crude DNA. Isolasi DNA dari semua sampel dikerjakan dengan PureLinkTM Genomic DNA Kit (Invitrogen, Life Tecnologies, U.S), isolasi crude DNA dari sampel cairan amnion dikerjakan dengan metode seperti yang dikerjakan oleh Grover et al., (1990) sedangkan sampel fetus dan plasenta dikerjakan dengan metode seperti yang dikerjakan oleh Savva et al., (1990). DNA hasil isolasi diamplifikasi dengan L-AMP dan PCR menggunakan primer L-AMP yang dirancang berdasarkan gen SAG-1 isolat lokal dengan program PrimerExplorer V4. Hasil L-AMP positif terlihat pada semua sampel dengan ektraksi DNA mulai 1-5 hari sesudah infeksi sedangkan pada sampel dengan crude DNA hanya ditemukan pada sampel cairan amnion 4-5 hari sesudah infeksi. Hasil PCR positif terlihat pada sampel cairan amnion dan plasenta mulai 2-5 hari sesudah infeksi dan pada sampel fetus mulai 3-5 hari sesudah infeksi; sedangkan pada semua sampel darah negatif. Hasil uji sensitivitas L-AMP dan PCR pada penelitian memperlihatkan bahwa L-AMP memiliki sensitivitas yang lebih baik jika dibandingkan dengan PCR dalam mengamplifikasi DNA dari sampel darah, cairan amnion, fetus dan plasenta. Berdasarkan hasil penelitian disimpulkan bahwa metode L-AMP dengan target gen SAG-1 isolat lokal mampu mendiagnosis toksoplasmosis kongenital pada mencit secara dini (1 hari sesudah infeksi) dari sampel darah induk, cairan amnion, fetus, dan plasenta. Kata kunci: toksoplasmosis kongenital, loop-mediated isothermal amplification (L-AMP), gen SAG-1 T. gondii isolat lokal (IS-1).

Congenital toxoplasmosis has an important role in the transmission of toxoplasmosis in animals and humans. Thus, a simple, rapid, and an accurate diagnostic method is needed. The aim of this study was to conduct the diagnosis technique of congenital toxoplasmosis in mice based on surface antigen-1 (SAG- 1) gene of local isolates (IS-1) T. gondii using loop-mediated isothermal amplification (L-AMP). A total of 15 pregnant mice Balb/C strain with the aged of ten weeks were used as experimental animal. Mice were intra peritoneally infected with 103 tachyzoites of T. gondii RH strain at day 9th of gestation. Amniotic fluids, maternal blood, fetus, and placenta then were collected at day 1, 2 , 3, 4 and 5 post infection. DNA was extracted from the above samples using PureLinkTM Genomic DNA Kit (Invitrogen, Life Technologies, US). Samples without DNA extraction of amniotic fluids was isolated using previously procedure described by Grover et al., (1990), whereas samples of fetus and placenta was isolated using previously procedure described by Savva et al., (1990), and then DNA was amplified by L-AMP and PCR using specific L-AMP primer were designed with software PrimerExplorer V4 program based on SAG-1 gene of the local isolate T. gondii. This study shows that L-AMP positive results were seen in all samples with DNA extraction at day 1 up to day 5 post infection, whereas in samples without DNA extraction was found only in samples of amniotic fluids at day 4 up to 5 day post infection, while positive PCR result were seen in all samples of amniotic fluids at day 2 up to day 5 post infection. Fetus and placenta samples also show positive PCR result at day 3 up to day 5 post infection. Negative PCR result shows in blood samples, however. The sensitivity test result shows that LAMP method has better sensitivity to DNA amplification from various biological samples (blood, amnionic fluids, fetus, and placenta) compared to PCR. To conclude, L-AMP technique using SAG-1 gene of local isolates T. gondii as a target gene, could be used to early diagnosis of congenital toxoplasmosis from infected mouse samples such as, maternal blood, amnionic fluids, fetus, and placenta. Keywords: congenital toxoplasmosis, loop-mediated isothermal amplification (L-AMP), SAG-1 gene of local isolate (IS-1) T. gondii.

Kata Kunci : toksoplasmosis kongenital, loop-mediated isothermal amplification (L-AMP), gen SAG-1 T. gondii isolat lokal (IS-1)