scholarly journals Penentuan Patogenesitas Molekuler Virus Newcastle Disease yang Diisolasi dari Ayam Komersial Tahun 2013-2016

2018 ◽  
Vol 5 (2) ◽  
pp. 105-119
Author(s):  
Sarwo Edy Wibowo ◽  
Michael Haryadi Wibowo ◽  
Bambang Sutrisno
Keyword(s):  
Polymerase Chain Reaction ◽  
Avian Influenza ◽  
Newcastle Disease ◽  
Infectious Bursal Disease ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Infectious Bronchitis ◽  
Bursal Disease ◽  
Infectious Laryngotracheitis ◽  
Polymerase Chain

Newcastle Disease (ND) atau yang dikenal dengan “Tetelo” masih menjadi masalah di peternakan unggas komersil, meskipun telah dilakukan vaksinasi ND secara rutin, namun wabah ND masih tetap terjadi. Penelitian ini bertujuan untuk mengetahui patogenesitas molekuler dan genotipe virus ND berdasarkan analisis sekuen fragmen gen F, serta melihat hubungan kekerabatan antara isolat dalam penelitian dengan isolat ND di Indonesia sebelumnya serta strain vaksin pada peternakan ayam yang menerapkan vaksinasi ND secara berkala. Penelitian ini menggunakan primer yang didesain dengan konsensus fragmen gen F dari GenBank dan desain dengan aplikasi amplifX pada posisi 91-800 nt dengan panjang 710bp. Urutan basa pada primer kemudian di cek dengan BLAST primer dan di uji spesifisitas dengan beberapa virus penyakit unggas yaitu vaksin infectious bronchitis (IB) 120, virus vaksin infectious laryngotracheitis (ILT), virus vaksin avian influenza (AI), dan virus vaksin infectious bursal disease (IBD). Delapan sampel paru diperoleh dari delapan peternakan ayam komersial di Yogyakarta, Semarang, Jakarta, Magelang dan Muntilan. Tiga sampel diisolasi pada telur ayam berembrio dan diidentifikasi menggunakan uji HA dan HI. Lima sampel lain dilakukan ekstraksi secara langsung dari gerusan organ paru. Sampel di identifikasi dengan metode reverse transciptase-polymerase chain reaction (RT-PCR) untuk mendeteksi gen F menggunakan  primer forward 5’ TCT CTT GAT GGC AGG CCT CTT G ‘3 dan reverse 5’ CCG CTA CCG ATT AAT GAG CTG AGT’3 dengan panjang produk 710 bp. Hasil penelitian menunjukkan bahwa seluruh sampel positif virus ND. Analisis hasil sekuen dengan menggunakan perangkat lunak MEGA v.7 didapatkan susunan asam amino penyusun cleavage site 112RRQKR↓F117 dan 112RRRKR↓F117yang menunjukkan bahwa virus ND tersebut digolongkan strain velogenik. Berdasarkan analisis pohon filogenetik isolat ND-Layer/GK-SR1/2013, ND-Lay/Pullet-80/ 27/16 (N), ND-Bro/Lingga 2L/24/2 (N), dan ND-Lay/Smg-P/2015 merupakan genotip VIIi, sedangkan 3 isolat ND yaitu ND-Bro/Yog-P/2015, JKT/P1/2016 (Jakarta), dan JKT/P2/2016 (Jakarta) merupakan ND genotip VIIh. Jarak genetik isolat yang diteliti dengan virus ND Indonesia yang pernah dilaporkan sebelumnya pada fragmen gen F posisi 91-798 berkisar 0,4 – 9,6 % dengan tingkat homologi mencapai 90,4 – 99,5%.

scholarly journals Development of multiplex reverse transcription-polymerase chain reaction for differentiation of strains of infectious bursal disease virus and primary screening of the virus in Thailand

2021 ◽  
pp. 3105-3110
Author(s):  
Nataya Charoenvisal
Keyword(s):  
Polymerase Chain Reaction ◽  
Disease Virus ◽  
Reverse Transcription ◽  
Infectious Bursal Disease Virus ◽  
Bursa Of Fabricius ◽  
Infectious Bursal Disease ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Bursal Disease ◽  
Polymerase Chain

Background and Aim: A new set of primers (400 base pairs partial of VP2) was designed and used for the infectious bursal disease virus (IBDV) screening test. Using this new primer set, the enzymes MboI and BstNI were unable to differentiate the field and vaccine strains. As a result, a new simple, cheap, and appropriate tool for strain differentiation is required. The objective of this study was to develop the appropriate restriction fragment length polymorphism (RFLP) and multiplex reverse transcription-polymerase chain reaction (RT-PCR) for the differentiation of classic IBDV (cIBDV) strains and very virulent IBDV (vvIBDV) strains in Thailand. Materials and Methods: Ninety seven bursa of Fabricius from 16 farms were collected from farms in the eastern and central regions of Thailand. RT-PCR screening showed that 82 samples were positive for IBDV and 15 samples were negative. Then, selected samples were sequenced from each farm with a positive test. Results: The sequencing results showed that samples from six of the farms were vvIBDV and samples from the other six farms were cIBDV. Although the whole genome sequencing was incomplete, both the sequencing results of segment A and segment B showed high similarity between cIBDV and vvIBDV. Restriction enzyme cutting site and primers for multiplex RT-PCR were hard to design. An RT-PCR-RFLP method was developed, but it failed to differentiate IBDV strains. However, the multiplex RT-PCR was able to differentiate cIBDV from vvIBDV. Four primers were used in the multiplex RT-PCR. Conclusion: These four primers were used together in one reaction at an annealing temperature of 45°C. Therefore, multiplex RT-PCR is a less complicated, cheaper, and less time-consuming method for the differentiation of cIBDV and vvIBDV strains.

scholarly journals COMPARISON OF AGAR GEL IMMUNODIFFUSION TEST, IMMUNOHISTOCHEMSITRY AND REVERSE TRANSCRIPTION - POLYMERASE CHAIN REACTION FOR DETECTION OF INFECTIOUS BURSAL DISEASE VIRUS

2013 ◽  
Vol 9 (2) ◽  
pp. 121-125 ◽  
Author(s):  
MT Islam ◽  
MN Islam ◽  
MZI Khan ◽  
MA Islam
Keyword(s):  
Polymerase Chain Reaction ◽  
Disease Virus ◽  
Sensitivity And Specificity ◽  
Infectious Bursal Disease Virus ◽  
Infectious Bursal Disease ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Agar Gel ◽  
Bursal Disease ◽  
Polymerase Chain

The objective of this study was to compare agar gel immunodiffusion test (AGID) and immunohistochemistry (IHC) with reverse transcriptase-polymerase chain reaction (RT-PCR) in terms of sensitivity and specificity for the detection of infectious bursal disease virus (IBDV). Thirty-five bursal samples collected from filed outbreak of IBD were evaluated by all 3 diagnostic tests. Sensitivity and specificity of both AGIDT and IHC with RT-PCR was 94.12% and 100%, respectively. Both AGIDT and IHC showed a 94.29% association with RT-PCR with a k value of 0.482, indicating a moderate degree of agreement. The Cochran’s Q value was 4.00, which is lower than the critical value, indicating that the methods did not differ significantly (p>0.05) from each other in detection of samples as positive or negative. However, RT-PCR had distinct edge over these two tests employed in the study.DOI: http://dx.doi.org/10.3329/bjvm.v9i2.13452

scholarly journals Detection of Infectious Bursal Disease Virus in Different Lymphoid Organs by Single-Step Reverse Transcription Polymerase Chain Reaction and Microplate Hybridization Assay

2002 ◽  
Vol 14 (3) ◽  
pp. 243-246 ◽  
Author(s):  
Darja Barlič-Maganja ◽  
Olga Zorman-Rojs ◽  
Jože Grom
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Infectious Bursal Disease Virus ◽  
Single Step ◽  
Lymphoid Organs ◽  
Infectious Bursal Disease ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Bursal Disease ◽  
Polymerase Chain

A rapid and sensitive method for the detection of infectious bursal disease virus (IBDV) RNA in different chicken lymphoid organs was developed. The method is based on a single-step reverse transcription polymerase chain reaction (RT-PCR) procedure and the enzyme-linked immunosorbent assay (ELISA) detection method of amplified products. Vaccinal IBDV strain and field isolates were used for the optimization of RT-PCR and for the determination of conditions for microplate hybridization and colorimetric detection of the amplicons. With this method, viral RNA could be detected in various stages of infection in samples of different lymphoid organs. Bursas and cecal tonsils were suitable organs for viral RNA detection at different times during IBDV infection. The RT-PCR/ELISA method can be applied for IBDV detection in routine diagnostic tests, which are not usually carried out because of the difficulties involved in isolating the virus.

scholarly journals Evaluation of a 384–Well Format for High-Throughput Real-Time Reverse Transcription Polymerase Chain Reaction for Avian Influenza Testing

2009 ◽  
Vol 21 (5) ◽  
pp. 679-683 ◽  
Author(s):  
Pamela J. Ferro ◽  
Jason Osterstock ◽  
Bo Norby ◽  
Geoffrey T. Fosgate ◽  
Blanca Lupiani
Keyword(s):  
Polymerase Chain Reaction ◽  
Avian Influenza ◽  
Real Time ◽  
High Throughput ◽  
Reverse Transcription ◽  
Virus Isolation ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Response Planning ◽  
Polymerase Chain

As concerns over the global spread of highly pathogenic avian influenza H5N1 have heightened, more countries are faced with increased surveillance efforts and incident response planning for handling a potential outbreak. The incorporation of molecular techniques in most diagnostic laboratories has enabled fast and efficient testing of many agents of concern, including avian influenza. However, the need for high-throughput testing remains. In this study, the use of a 384–well format for high-throughput real-time reverse transcription polymerase chain reaction (real-time RT-PCR) testing for avian influenza is described. The analytical sensitivity of a real-time RT-PCR assay for avian influenza virus matrix gene with the use of both 96– and 384–well assay formats and serial dilutions of transcribed control RNA were comparable, resulting in similar limits of detection. Of 28 hunter-collected cloacal swabs that were positive by virus isolation, 26 (92.9%) and 27 (96.4%) were positive in the 96– and 384–well assays, respectively; of the 340 hunter-collected swabs that were negative by virus isolation, 45 (13.2%) and 23 (6.8%) were positive in the 96– and 384–well assays, respectively. The data presented herein supports the utility of the 384–well format in the event of an avian influenza outbreak for high-throughput real-time RT-PCR testing.

scholarly journals DETEKSI VIRUS AVIAN INFLUENZA SUBTIPE H5N1 DI BEBERAPA PASAR UNGGAS HIDUP DALAM WILAYAH PROVINSI JAWA BARAT SEKITARNYA

2015 ◽  
Vol 9 (1) ◽  
Author(s):  
Atik Ratnawati ◽  
NLP Indi Dharmayanti
Keyword(s):  
Polymerase Chain Reaction ◽  
Avian Influenza ◽  
Reverse Transcriptase ◽  
Ribonucleic Acid ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain

Pada penelitian ini dilakukan identifikasi virus avian influenza (AI) subtipe H5N1 pada unggas dan lingkungan pasar untuk mengetahui peran pasar sebagai sumber penularan virus. Metode yang digunakan dalam penelitian ini adalah pengambilan sampel swab kloaka unggas dan lingkungan di beberapa pasar di wilayah Jawa Barat dan Tangerang. Sampel selanjutnya dilakukan isolasi ribonucleic acid (RNA) dan dilakukan reverse transcriptase polymerase chain reaction (RT-PCR) dengan menggunakan primer AI subtipe H5N1. Hasil penelitian menunjukkan bahwa virus AI/H5N1 terdeteksi pada unggas dan lingkungan pasar. Disimpulkan bahwa pasar dapat menjadi sumber penularan virus AI subtipe H5N1 terhadap unggas lainnya.

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