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Dini Wahyu Yudianingtyas
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Biochemistry, Genetics & Molecular Biology Veterinary

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Journal : Jurnal Veteriner

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Diagnosis Molekuler Virus Flu Burung-A Subtipe H5 Berdasarkan Amplifikasi Gen M dan H5 dengan Metode Onestep Simplex RT-PCR (MOLECULAR DIAGNOSIS OF AVIAN INFLUENZA VIRUS TYPE A AND SUBTYPE H5 BY AMPLIFICATION OF ITS M AND H5 GENES USING ONE STEP SIMPLEX R Aris Haryanto; Bibiana Krisanti; Sri Handayani Irianingsih; Dini Wahyu Yudianingtyas
Jurnal Veteriner Vol 13 No 2 (2012)
Publisher : Faculty of Veterinary Medicine, Udayana University and Published in collaboration with the Indonesia Veterinarian Association

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Abstract

Influenza A viruses which belong to the Family of Orthomyxoviridae are a group of viruses withsegmented ssRNA genome. The viruses can be subgroupped into many subtypes on the basis of theirsurface glycoproteins, hemagglutinin (HA) and neuraminidase (NA) proteins. Among the HA subtypes, H5and H7 have been found to be the most pathogenic. Conventional diagnosis of the viruses is usuallyperformed by isolation of the viruses in embryonated eggs, and hemagglutination (HA) and hemagglutinationinhibition test. Although those methods are sensitive and accurate, they are time consuming and requirelaboratory facilities with high biosafety level. Commercial methods such as emzyme-linked immonosorbentassay (ELISA) and immunoflurescense assay also provide a rapid result but less sensitive and specificthan conventional methods. Molecular diagnosis by amplification of M and H5 genes using one strepsimplex reverse transcriptase-polymerase chain eraction (RT-PCR) provices a rapid and accurate diagnosisfor the viruses. A study was therefore conducted to evaluate the accurate and rapidity of such the moleculartests for diagnosis of avian influenza A virus, subtype H5. As many as 10 sample of the virus isolateswhich were available at the Animal Desease investigation Center in Wates, Yogyakarta, were uses in thisstudy. The virus isolates were firstly propagated in specific antigen negative (SAN) chicken embryos andtested by HA/HI test. The viruses were then subjected for the RT-PCR test with varying annealingtemperatures of 500C and 520C. The result showed that all 10 isolates were type A influenza virus and 8out of 10 were influenza A subtype H5 influenza virus. RT-PCR used in this study appears to be moresensitive, rapid and accurate as compared to those by serological and isolation of the virus in embryonatedeggs.
Penerapan Metode Diagnosis Cepat Virus Avian Influenza H5N1 dengan Metode Single Step Multiplex RT-PCR Aris Haryanto; Ratna Ermawati; Medania Purwaningrum; Dini Wahyu Yudianingtyas; Michael Haryadi Wibowo; Charles Rangga Tabbu
Jurnal Veteriner Vol 11 No 4 (2010)
Publisher : Faculty of Veterinary Medicine, Udayana University and Published in collaboration with the Indonesia Veterinarian Association

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Abstract

Avian influenza (AI) virus is a segmented single stranded (ss) RNA virus with negative polarity andbelong to the Orthomyxoviridae family. Diagnose of AI virus can be performed using conventional methodsbut it has low sensitivity and specificity. The objective of the research was to apply rapid, precise, andaccurate diagnostic method for AI virus and also to determine its type and subtype based on the SingleStep Multiplex Reverse Transcriptase-Polymerase Chain Reaction targeting M, H5, and N1 genes. In thismethod M, H5 and NI genes were simultaneously amplified in one PCR tube. The steps of this researchconsist of collecting viral RNAs from 10 different AI samples originated from Maros Disease InvestigationCenter during 2007. DNA Amplification was conducted by Simplex RT-PCR using M primer set. Then, bysingle step multiplex RT-PCR were conducted simultaneously using M, H5 and N1 primers set. The RTPCRproducts were then separated on 1.5% agarose gel, stained by ethidum bromide and visualized underUV transilluminator. Results showed that 8 of 10 RNA virus samples could be amplified by Simplex RTPCRfor M gene which generating a DNA fragment of 276 bp. Amplification using multiplex RT-PCRmethod showed two of 10 samples were AI positive using multiplex RT-PCR, three DNA fragments weregenerated consisting of 276 bp for M gene, 189 bp for H5 gene, and 131 bp for N1. In this study, rapid andeffective diagnosis method for AI virus can be conducted by using simultaneous Single Step Multiplex RTPCR.By this technique type and subtype of AI virus, can also be determined, especially H5N1.
Amplifikasi Gen Penyandi Protein Fusion Virus Tetelo dari Spesimen Lapangan dengan Metode OneStep RT-PCR. (AMPIFICATION OF FUSION PROTEIN ENCODING GENE OF NEWCASTLE DISEASE VIRUS FROM FIELD SPECIMENS BY ONESTEP RT-PCR METHOD) Aris Haryanto; David Kristiawan; Sri Handayani Irianingsih; Dini Wahyu Yudianingtyas
Jurnal Veteriner Vol 14 No 3 (2013)
Publisher : Faculty of Veterinary Medicine, Udayana University and Published in collaboration with the Indonesia Veterinarian Association

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Abstract

Tetelo or Newcastle Disease (ND)  virus is belong to the family Paramyxoviridae, which has a singlestranded RNA (ss RNA) genome and it has viral envelope.  The viral envelope consists of two majorproteins, namely Haemagglutinin/Neuraminidase (H/N) and Fusion (F) protein. Molecular diagnosticmethods OneStep RT-PCR is a commonly diagnostic tool that used to diagnose of Tetelo in poultry. In thisstudy the diagnosis of Tetelo is accomplished by amplification of F protein encoding gene of Tetelo virusdirectly from field specimens without inoculation and propagation of Tetelo virus into embryonated chickeneggs. The objective of this study is to conduct rapid diagnosis of Tetelo virus directly from the field specimensbased on the amplification of the F gene by a OneStep RT-PCR method, so that the results of this study canbe used to assist in the identification of Tetelo virus directly from the field specimens. The results showedthat from the 15 samples of virus which isolated from tracheal swabs of clinical poultry showing symptomsof Tetelo virus infection, a total of 12 samples from 15 tested sampels (80%) are positive tested for Tetelovirus infection. They were indicated by the amplification product of DNA fragments in size of 362 bp.OneStep RT-PCR is a method for rapid and effective diagnosis which can be used  to diagnose of Tetelovirus directly from field specimens.
Co-Authors Aris Haryanto Aris Haryanto Bibiana Krisanti Charles Rangga Tabbu David Kristiawan Isnaniah Beganda Medania Purwaningrum Michael Haryadi Wibowo Ratna Ermawati Sri Handayani Irianingsih Wiwik Dariani