Abstract
Until recently, two clades of of avian influenza viruses (AIVs) designated as 2.3.2 and 2.2.3 havebeen circulating in Indonesia. Mutations of AIV genes have cretaed many more variants of the virus. It istherefore important to evaluate the appropriate methods used for the detection and diagnosis of AI virusin the field. Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) have been used as a standardmethod for detection of AIV in many laboratories in Indonesia. The success of RT-PCR for detection ofAIV virus is dependent on the nucleotide sequences of primer that match with the circulating of AIVs. Theaims of this study was to develop RT-PCR by designing primers for H5 subtype specific to the circulatingAIVs in the field. The primers were designed using Primer Design software, and optimization andvalidation of the primer were conducted using AIVs that have been characterized in the previous study.The primers were then used RT-PCR using AIV isolates from field samples and their sensitivity andspecificity were then determined. The results showed that the H5 primers designed in this study, H5-IDand H5-NLP, was able to detect the AIVs in field samples better than the H5-specific primers have beenused previously. In conclusion, H5 primers designed based on recent viruses in the field showed betterresults in the detection of AI virus as compared to the previous primers. As AIV-H5N1 subtype in the fieldwill continue to change and evolve, the use of primers designed in this study is recommended for diagnosisof H5 AIV.
Highlights
Virus influenza termasuk kelompok virus RNA berpolaritas negatif, famili Orthomyxoviridae yang diklasifikasikan menjadi tipe A, B, dan C berdasarkan pada mayoritas antigen protein internal yaitu nukleoprotein (NP) dan matriks (M1)
The results showed that the H5 primers designed in this study, H5-ID and H5-NLP, was able to detect the avian influenza viruses (AIVs) in field samples better than the H5-specific primers have been used previously
Isolasi dan Karakterisasi Virus Highly Pathogenic Avian Influenza subtipe H5 dari ayam asal Wabah di Indonesia
Summary
Virus influenza termasuk kelompok virus RNA berpolaritas negatif, famili Orthomyxoviridae yang diklasifikasikan menjadi tipe A, B, dan C berdasarkan pada mayoritas antigen protein internal yaitu nukleoprotein (NP) dan matriks (M1). Seiring dengan kemajuan teknologi molekuler, instrumen penting dalam mendeteksi AI berdasarkan asam nukleat virus dalam mendeteksi subtipe HA atau NA menggunakan metode Reverese Trancriptase-Polymerase Chain Reaction (RT-PCR) menjadi satu pilihan yang banyak digunakan untuk mendeteksi berbagai macam agen penyakit termasuk AI (Starick et al, 2000; Poddar, 2002). Ketidakberhasilan dalam mendeteksi subtipe virus AI dapat disebabkan oleh beberapa faktor, di antaranya adalah kemungkinan sampel tersebut adalah virus AI bukan subtipe H5 namun subtipe lainnya, atau primer yang digunakan dalam metode RT-PCR untuk mendeteksi virus AI sudah tidak cocok lagi. Hal tersebut tentunya akan menimbulkan banyak kegagalan dalam mendeteksi virus AI subtipe H5 sehingga diperlukan disain primer spesifik baru yang dapat mendukung meningkatnya keberhasilan dalam mendeteksi sirkulasi virus AI di lapang. Penelitian ini bertujuan untuk mengembangkan beberapa set primer spesifik subtipe H5 virus AI subtipe H5N1 asal Indonesia untuk meningkatkan keberhasilan deteksi virus AI di lapang
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