Tesis

Pengembangan Real-Time Reverse Transcription Polymerase Chain Reaction Berbasis Sybrgreen untuk Deteksi Virus Avian Influenza Subtipe H5N1 dan H9N2 = Development of Sybrgreen Real-Time Reverse Transcription Polymerase Chain Reaction test for detection of H5N1 and H9N2 subtype Avian Influenza virus.

Virus Influenza A (AIV) merupakan penyakit zoonosis yang mampu menginfeksi baik pada manusia dan hewan, dan berpotensi mengakibatkan epidemi periodik dan pandemi global. Dinamika virus AI di Indonesia menunjukkan adanya perubahan yang signifikan, mutasi virus, perubahan patogenitas, fenomena antigenic drift dan antigenic shift termasuk diantaranya escape mutant, reassortant sampai dengan introduksi jenis baru dari clade 2.3.2. Pada penelitian ini dilakukan studi terkait dengan pengembangan alat diagnostik berbasis real-time reverse transcription PCR (rRT-PCR) Sybrgreen. Tujuan dari penelitian ini adalah melakukan pengembangan desain primer secara in silico dan validasinya secara in vitro dengan uji rRT-PCR berbasis Sybrgreen untuk mendeteksi virus AI subtipe H5N1 dan H9N2. Penelitian ini berbasis penelitian in silico yang dikonfirmasi secara in vitro melalui eksperimen laboratorium. Hasil isolasi virus pada TAB umur 9-11 hari diekstraksi RNAnya menggunakan kit QIAmp RNA Mini kit. Dari hasil penelitian diperoleh bahwa uji rRT-PCR singlepleks dengan primer H5, N1, H9, dan N2 mampu mendeteksi keberadaan RNA virus AI subtipe H5N1 dan H9N2. Primer H5 dan N1 memiliki suhu annealing terbaik pada 55,7 oC, sedangkan primer H9 dan N2 memiliki suhu annealing trebaik pada 55 oC. Selain itu, keempat primer menunjukkan kondisi optimalnya pada konsentrasi 200 nM. Pada uji terhadap virus ND, dan IB serta uji silang antara virus H5N1 dan H9N2 ditemukan masing-masing primer hanya mampu mengamplifikasi virus target. Primer H5 dan N1 menunjukkan nilai LoD 10 1,416 EID50/0,1 mL, sedangkan primer H9 dan N2 menunjukkan nilai LoD 10 2.083 EID50/0,1 mL. Nilai ketertiruan dan keterulangan 100% dengan perbedaan minor yang mungkin disebabkan karena penggunaan mesin thermal cycler yang berbeda. Uji dupleks rRT-PCR H5N1 mampu mendeteksi dengan baik RNA virus AI subtipe H5N1, begitu juga dengan dupleks rRT-PCR H9N2 mampu mendeteksi RNA virus AI subtipe H9N2. Uji multipleks rRT-PCR H5N1-H9N2 (quadripleks) rRT-PCR mampu mendeteksi RNA virus AI subtipe H5N1 dan H9N2, namun masih membutuhkan optimasi lebih lanjut karena adanya amplifikasi produk non spesifik.
Kata kunci: subtyping; orthomyxoviridae; LPAI; HPAI; melt curve.


Influenza A virus (AIV) is a zoonotic disease capable of infecting both humans and animals and has the potential to cause periodic epidemics and global pandemics. The dynamics of the AI virus in Indonesia shows significant changes, virus mutations, changes in pathogenicity, antigenic drift and antigenic shift phenomena including escape mutants, reassortants and the introduction of new types from clade 2.3.2. In this research, a study was carried out related to the development of a Sybrgreen realtime reverse transcription PCR (rRT-PCR) based diagnostic tool. The aim of this research is to develop primer designs in silico and validate them in vitro with a Sybrgreen-based rRT-PCR test to detect H5N1 and H9N2 subtype AI viruses. This research is based on in silico research which was confirmed in vitro through experiments. The results of virus isolation in TAB aged 9-11 days were extracted for RNA using the QIAmp RNA Mini kit. From the research results, it was found that the singleplex rRT-PCR test with primers H5, N1, H9, and N2 were able to detect the presence of the H5N1 and H9N2 subtype AI RNA viruses. Primers H5 and N1 have the best annealing temperature at 55.7 oC, while primers H9 and N2 have the best annealing temperature at 55 oC. In addition, the four primers showed their optimal conditions at a concentration of 200 nM. In tests against ND and IB viruses as well as cross tests between H5N1 and H9N2 viruses, it was found that each primer was only able to amplify the target virus. Primers H5 and N1 showed a LoD of 10 1.416 EID50/0.1 mL, while primers H9 and N2 showed a LoD 10 2.083 EID50/0.1 mL. The replicability and repeatability values are 100% with minor differences which may be caused by using different thermal cycler machines. The H5N1 rRT-PCR duplex test was able to detect the H5N1 subtype AIV well, while the H9N2 rRT-PCR duplex was able to detect the H9N2 subtype AIV. The multiplex rRT-PCR H5N1-H9N2 (quadriplex) rRT-PCR test can detect the H5N1 and H9N2 subtype AI viruses, but still requires further optimization due to non-specific product amplification.
Keywords: subtype; orthomixoviridae; LPAI; HPAI; melting curve.