Pengembangan Diagnosis Molekuler Toksoplasmosis Berdasar Sekuen Deoxyribonucleic Acid (DNA) Spesifik Stadium Takizoit dan Bradizoit

APSARI, Ida Ayu Pasti, Wayan T. Artama

2011 | Disertasi |

Genom Toxoplasma gondii memiliki gena fungsional seperti sag-1 dan bag-1. Gena sag-1 merupakan gena spesifik untuk stadium takizoit, sedangkan bag-1 merupakan gena spesifik untuk stadium bradizoit. Ekplorasi kedua gena tersebut secara bersama dapat dikembangkan untuk diagnosis. Penelitian ini bertujuan untuk mendapat probe berdasar fragmen DNA spesifik stadium takizoit dan bradizoit yang dapat memenuhi syarat sebagai probe untuk mendeteksi toksoplasmosis. Deoxyribonucleic acid (DNA) takizoit diisolasi dari takizoit Toxoplasma gondii isolat lokal dengan metode alkali lisis. Fragmen spesifik DNA takizoit gena sag-1 diamplifikasi dengan primer spesifik gena sag-1 dan gena bag-1 diamplifikasi dengan primer bag-1. Produk PCR disekuensing menggunakan Automatic DNA sequencer Applied Biosystem 3130/3130x Genetic Analyser di Eijkman Institute, Jakarta. Spesifisitas sekuen stadium takizoit (sag-1) dan bradizoit (bag-1) dianalisis menggunakan program BLAST. Kajian sekuen sebagai probe molekuler untuk aplikasi dot blot, dilakukan pelabelan menggunakan Dig high prime DNA labeling and detection starter kit (Roche). Hasil penelitian dapat disimpulkan bahwa primer spesifik gena sag-1 dan bag-1 dapat mengamplifikasi fragmen gena sag-1 dan bag-1 Toxoplasma gondii. Sekuen DNA spesifik stadium takizoit dan bradizoit memenuhi persyaratan sebagai probe molekuler. Probe takizoit (sag-1) dan bradizoit (bag-1) dapat menentukan komplementernya yang ada pada DNA Toxoplasma gondii ayam buras menggunakan metode dot blot, dengan sensitivitas 0,45ng/μl dan 0,23 ng/μl. Kata kunci: Takizoit, Toxoplasma gondii, Probe sag-1 dan bag-1, hibridisasi dot blot

Toxoplasma gondii has tachyzoite and bradyzoite stage-specific genes. Gene sag-1 is specific gene for tachyzoite stage, whereas bag-1 is specific gene for bradyzoite stage. Exploration both genes together needs to be developed for the diagnosis. The aim of this research was to find out a probe based on the specific DNA fragment tachyzoite and bradyzoite stage that can be qualify as a probe for toxoplasmosis detection. Deoxyribonucleic acid of T. gondii tachyzoite from the local isolate was isolated using alkaly lysis method. Target sequence of specific fragments DNA was amplified by polymerase chain reaction (PCR) method using specific primers sag-1 gene, bag-1 gene and the PCR reaction mix (Invitrogen). PCR products were sequenced using Automatic DNA sequencer Applied Biosystem 3130/3130x Genetic Analyser at the Eijkman Institute, Jakarta. Spesivicity tachyzoite stage sequence (sag-1) and bradyzoite (bag-1) were analyzed using the BLAST programe. Study sequences as molecular probes for hybridization method with dot blot applications, first performed using Dig high prime DNA labeling and detection starter kit (Roche). The results of this research can be concluded that the specific primers sag-1 gene and bag-1 were can amplified fragment sag-1 and bag-1 genes which are a specific stage of Toxoplasma gondii. There are sequences specific DNA tachyzoite stage dan bradyzoite that meets the requirements as molecular probes. Tachyzoite probe (sag- 1) and bradyzoite probe (bag-1) to determine the existing complementary Toxoplasma gondii in free-range chicken genome using the dot blot method, with a sensitivity of 0.45 ng/μl and 0.23 ng/μl. Key words: Tachyzoite, Toxoplasma gondii, sag-1 and bag-1 probe, dot blot hybridization

Kata Kunci : Takizoit, Toxoplasma gondii, Probe sag-1 dan bag-1, hibridisasi dot blot