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Detection of Newcastle Disease Virus of Poultry by Real Time Reverse Transcription-polymerase Chain Reaction

M M Rahman
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

L R Barman
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agrcultual University, Mymensingh-2202, Bangladesh

E H Chowdhury
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

M R Islam
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Abstract/ Executive Summary:

A real-time reverse transcription - polymerase chain reaction (rRT-PCR) was used for the detection of Newcastle disease virus (NDV) of poultry. A panel of seven known isolates of NDV in the form of allantoic fluid, obtained from a laboratory repository, was used for the development of the test. RNA was extracted from the allantoic fluid with a magnetic processor based automated RNA extraction system. The identity of the reference virus was first reconfirmed by a conventional RT-PCR specific for the Fusion (F) protein gene. Using these RNA, the rRT-PCR protocol was optimized with regard to the reaction mix and thermal profile using published primers and probes specific for M gene. The sensitivity of standardized rRT-PCR was compared to that of the conventional RT-PCR using serial 10-fold dilutions of the RNA of a selected sample. The thermal profile was modified from the published one; the annealing and extension steps were combined to a single step performed at 60ºC. The adopted rRT-PCR successfully amplified M gene from all the seven reference samples with a C_T value ranging from 15.28 to 32.68. The rRT-PCR for M gene was 100-fold more sensitive than the conventional RT-PCR for F gene. This is the first report of the use of rRT-PCR for the detection of NDV in Bangladesh. This test will be useful for virological surveillance, particularly for screening NDV in respiratory infections.

Keywords: Newcastle disease, Virus, rRT-PCR, Poultry, Sensitive

Location: Laboratory, Department of Pathology, Faculty of Veterinary Science, BAU

Start Date:

End Date:

Program Area: Animal Health and Management

Commodity: Poultry

Objectives:

The study aimed at the adoption of an rRT-PCR technique for the detection of Newcastle disease virus.

Methodology:

Virus isolates: A total of seven known isolates of NDV, previously detected by RT-PCR and propagated in embryonated chicken eggs, were obtained in allantoic fluid from a previous study. Primers and probes: The details of the primers and probes used in the conventional RT-PCR and rRT-PCR. The primers and probe were synthesized from a commercial source (Integreted DNA Technologies, Inc., USA). Extraction of RNA, RT-PCR and rRT-PCR protocols: Viral RNA was extracted from infected allantoic fluid with King Fisher ML automated RNA extraction machine using Ambion MagMAXTM-96 Viral RNA Isolation Kit (Life Technologies, USA) following manufacturer’s instructions. As the kit was designed for 96-well format, the volumes of reagents were adjusted for tube format of the RNA extraction machine.For amplification of the F gene fragment by conventional RT-PCR, SuperScript® III One-Step RT-PCR System with Platinum® Taq DNA polymerase (Life Technologies, USA) was used as recommended by the manufacturer. The thermal profile consisted of RT at 50ºC for 30 min followed by initial denaturation at 94ºC for 2 min, 40 cycles of denaturation at 94ºC for 30 sec, annealing at 55ºC for 30 sec, extension at 72ºC for 1 min, and final extension at 72ºC for 1 min. The amplified cDNA was analyzed by electrophoresis on 1.5% agarose gel. The rRT-PCR was performed in AB 7500 Fast real time PCR machine (Applied Biosystems, USA) using AgPath–IDTM One Step RT-PCR Kit (Life Technologies, USA) as recommended by the manufacturer. The reaction was performed in 25 µL volumes in 0.1 mL MicroAmp Fast PCR 8-tube strips; 5 µL RNA templates were added to 20 µL reaction mix. The thermal profile used by Wise et al. (2004) was modified considering the requirements of the present kit and the real-time PCR machine. Comparison of the sensitivity of real time and conventional RT-PCR: To compare the sensitivity of conventional and real-time RT-PCR, RNA extracted from a selected sample was serially diluted 10-fold from undiluted to 10-9. All the dilutions were tested in conventional and real time RT-PCR. The highest dilution at which the test gave positive result was considered as the detection limit

Source of Information: The Bangladesh Veterinarian (2016) 33(1): 16 – 22

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

Newcastle disease is endemic in Bangladesh. With the emergence of highly pathogenic and low pathogenic avian influenza, differential diagnosis between NDV and avian influenza is often required because of the clinical and pathological similarity. This is the first report of the application of rRT-PCR for the detection of NDV in Bangladesh. Availability of this modern, sensitive and quick diagnostic tool will be very useful in confirmation or ruling out NDV from suspected avian influenza outbreaks. This test will be useful for initial screening of samples in NDV surveillance.

Knowledge Management: Journal