Validation of a Real-Time RT-PCR Method to Quantify Newcastle Disease Virus (NDV) Titer and Comparison with Other Quantifiable Methods

2011 ◽  
Vol 21 (1) ◽  
pp. 100-108 ◽  
Author(s):  
Juno Jang
Keyword(s):  
Newcastle Disease Virus ◽  
Real Time ◽  
Disease Virus ◽  
Newcastle Disease ◽  
Rt Pcr ◽  
Pcr Method

scholarly journals Real-Time Reverse Transcription-Polymerase Chain Reaction Detection of Newcastle Disease Virus Using Light upon Extension Fluorogenic Primers

2007 ◽  
Vol 19 (4) ◽  
pp. 400-404 ◽  
Author(s):  
Márta Antal ◽  
Tibor Farkas ◽  
Péter Germán ◽  
Sándor Belák ◽  
István Kiss
Keyword(s):  
Newcastle Disease Virus ◽  
Real Time ◽  
Disease Virus ◽  
Newcastle Disease ◽  
Plasmid Copy Number ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Plasmid Copy ◽  
Infectious Dose ◽  
Efficient Detection

A real-time reverse transcriptase (RT)-PCR assay, applying light upon extension (LUX) fluorogenic primers, was developed for rapid and efficient detection of Newcastle disease virus (NDV). The method, which targets the fusion (F) protein gene of the viral genome, gave positive signal with all NDV isolates tested (32/32), while negative results were obtained with heterologous pathogens (35/35), including 13 avian influenza virus isolates. The detection limit of the assay was approximately 10+1.2 egg infectious dose (EID)50/0.2 ml and 10+2.2 EID50/0.2 ml for virus suspensions and spiked chicken fecal samples, respectively. As expressed in plasmid copy number, the procedure has a sensitivity of approximately 20 copies of the plasmid harboring the target gene. Due to its high specificity, sensitivity, and relative simplicity, the LUX RT-PCR assay provides a novel, rapid, and practical tool for the detection of NDV.

scholarly journals Development of Strand-Specific Real-Time RT-PCR to Distinguish Viral RNAs during Newcastle Disease Virus Infection

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Xusheng Qiu ◽  
Yang Yu ◽  
Shengqing Yu ◽  
Yuan Zhan ◽  
Nana Wei ◽  
...  
Keyword(s):  
Newcastle Disease Virus ◽  
Real Time ◽  
Disease Virus ◽  
Newcastle Disease ◽  
Copy Number ◽  
Viral Rna ◽  
Quantitative Detection ◽  
Rt Pcr ◽  
Detection Range ◽  
Infected Cells

Newcastle disease virus (NDV) causes large losses in the global fowl industry. To better understand NDV replication and transcription cycle, quantitative detection methods for distinguishing NDV genomic RNA (gRNA), antigenomic RNA (cRNA), and messenger RNA (mRNA) in NDV-infected cells are indispensible. Three reverse transcription primers were designed to specifically target the nucleoprotein (NP) region of gRNA, cRNA, and NP mRNA, and a corresponding real-time RT-PCR assay was developed to simultaneously quantify the three types of RNAs in NDV-infected cells. This method showed very good specificity, sensitivity, and reproducibility. The detection range of the assay was between5.5×102and1.1×109copies/μL of the target gene. These methods were applied to investigate the dynamics of the gRNA, cRNA, and mRNA synthesis in NDV La Sota infected DF-1 cells. The results showed that the copy numbers of viral gRNA, cRNA, and NP mRNA all exponentially increased in the beginning. The viral RNA copy number then plateaued at 10’h postinfection and gradually decreased from 16 h postinfection. No synthesis priority was observed between replication (gRNA and cRNA amounts) and transcription (mRNA amounts) during NDV infection. However, the cRNA accumulated more rapidly than gRNA, as the cRNA copy number was three- to tenfold higher than gRNA starting from 2 h postinfection.Conclusion. A real-time RT-PCR for absolute quantitation of specific viral RNA fragments in NDV-infected cells was developed for the first time. The development of this assay will be helpful for further studies on the pathogenesis and control strategies of NDV.

scholarly journals Identification of isolated viral strains of atypical avian influenza using molecular methods of virological diagnostics

2008 ◽  
Vol 62 (3-4) ◽  
pp. 167-177
Author(s):  
Dejan Vidanovic ◽  
Milanko Sekler ◽  
Nikola Vaskovic ◽  
Aleksandar Zarkovic ◽  
Kazimir Matovic ◽  
...  
Keyword(s):  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Newcastle Disease ◽  
Virus Genome ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Rt Pcr ◽  
Rna Sequences ◽  
Sequencing Method ◽  
Pcr Method

In addition to the implementation of standard methods of virological diagnostics used for the isolation of the Newcastle disease virus from suspect material, as well as for its identification, nowadays there is increasing use of molecular diagnostic methods, primarily reverse transcription polymerase chain reaction (RT-PCR) and the sequencing method. The objective of this work was to examine possibilities for the implementation of the above methods in the diagnosis of poultry infection caused by the Newcastle disease virus. The presence of hem agglutination antigens for the Newcastle disease virus was established in samples of allantoises liquid from 62 poultry embargoed eggs 72 h after inoculation, whose titers ranged from 1:16 to 1:2048, while the hem agglutination inhibition test (HI test) with the implementation of a referent immuno serum against the given cause provided the identification of isolated viruses in serum dilutions of 1:128 to 1:1024. The RT-PCR method and the PCR established that in eight examined samples one fragment each of viral RNA is formed in agars gel of a size of 254bp, which is characteristic for the Newcastle disease virus genome according to its nucleotide sequence. On the grounds of a comparative analysis of RNA sequences obtained from eight isolated NDV strains and the genome sequences of referent atypical poultry influenza viral strains using Mega 40 and BLAST programmers, it was established that the isolated strains of the Newcastle disease virus were highly virulent.

Evaluation of RT-PCR for Rapid Detection of Sudanese Isolates and Vaccine Strains of Newcastle Disease Virus

2005 ◽  
Vol 8 (3) ◽  
pp. 418-420
Author(s):  
Mohamed A.M. Yousof . ◽  
I.E. Aradaib . ◽  
K.M.S. Khairalla . ◽  
M.A. Abdalla . ◽  
A.R.E. Karrar . ◽  
...  
Keyword(s):  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Newcastle Disease ◽  
Rapid Detection ◽  
Rt Pcr

scholarly journals First Genome Sequence of Newcastle Disease Virus of Genotype VIIi from Jordan

2018 ◽  
Vol 7 (23) ◽  
Author(s):  
Mustafa Ababneh ◽  
Helena L. Ferreira ◽  
Mohammad Khalifeh ◽  
David L. Suarez ◽  
Claudio L. Afonso
Keyword(s):  
Reverse Transcriptase ◽  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Genome Sequence ◽  
Illumina Sequencing ◽  
Newcastle Disease ◽  
Rt Pcr ◽  
Total Rna ◽  
Reverse Transcriptase Pcr ◽  
Chicken Spleen

Newcastle disease virus (NDV) was detected by reverse transcriptase PCR (RT-PCR) from total RNA isolated from a chicken spleen of a backyard flock in Jordan. The complete coding genome sequence of NDV/chicken/Jordan/J11-spleen/2018 was obtained with MiSeq (Illumina) sequencing.

Development of a novel real-time PCR-based strategy for simple and rapid molecular pathotyping of Newcastle disease virus

2012 ◽  
Vol 157 (5) ◽  
pp. 833-844 ◽  
Author(s):  
Alia Yacoub ◽  
Mikael Leijon ◽  
Michael J. McMenamy ◽  
Karin Ullman ◽  
John McKillen ◽  
...  
Keyword(s):  
Newcastle Disease Virus ◽  
Real Time ◽  
Disease Virus ◽  
Newcastle Disease ◽  
Real Time Pcr

scholarly journals Detection of avian influenza virus and newcastle disease virus by duplex one step RT PCR

2013 ◽  
Vol 8 (6) ◽  
pp. 520-526 ◽  
Author(s):  
Dawid Nidzworski ◽  
Krzysztof Smietanka ◽  
Zenon Minta ◽  
Bogusław Szewczyk
Keyword(s):  
Influenza Virus ◽  
Avian Influenza ◽  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Newcastle Disease ◽  
Egg Production ◽  
Influenza Viruses ◽  
Detection Methods ◽  
Rt Pcr ◽  
One Step

AbstractNewcastle disease Virus (NDV), a member of the Paramyxoviridae family, and Influenza virus, from the Orthomyxoviridae family, are two main avian pathogens that cause serious economic problems in poultry farming. NDV strains are classified into three major pathotypes: velogenic, mesogenic, and lentogenic. Avian influenza viruses (AIV) are also divided into: low pathogenic (LPAI) and highly pathogenic (HPAI) strains. Both viruses are enveloped, single stranded, negative-sense RNA viruses which give similar symptoms ranging from sub-clinical infections to severe disease, including loss in egg production, acute respiratory syndrome, and high mortality, depending on their level of pathogenicity. This similarity hinders diagnosis when based solely on clinical and post mortem examination. Most of the currently available molecular detection methods are also pathogenspecific, so that more than one RT-PCR is then required to confirm or exclude the presence of both pathogens. To overcome this disadvantage, we have applied a One Step Duplex RT-PCR method to distinguish between those two pathogens. The main objective of the project was to develop a universal, fast, and inexpensive method which could be used in any veterinary laboratory.

scholarly journals Prevalence of Newcastle Disease Virus and Avian Influenza Virus (H7N3) in Poultry at Karachi

2020 ◽  
Vol 11 (1) ◽  
pp. 1-6
Author(s):  
Amjad Ali Channa ◽  
Nazeer Hussain Kalhoro ◽  
Zaheer Ahmed Nizamani ◽  
Ayaz Hussain Mangi ◽  
Jamila Soomro
Keyword(s):  
Influenza Virus ◽  
Avian Influenza ◽  
Newcastle Disease Virus ◽  
Avian Influenza Virus ◽  
Disease Virus ◽  
Newcastle Disease ◽  
Virus Isolation ◽  
Economic Losses ◽  
Rt Pcr ◽  
Agar Gel

Background: Poultry is largest and rapidly growing sector of livestock in Pakistan. It is mainly influenced by viral pathogens such as Newcastle Disease Virus (NDV) and Avian Influenza Virus (H7N3). These viruses cause severe disease in poultry and leads to heavy economic losses throughout the world. The outbreaks of these pathogens have been increased in last few decades. Therefore, the study about antigenic prevalence is needed to know about the emergence of these pathogenic viruses, and to get rid of severe ailments associated with reduced poultry production. Objectives: To determine the prevalence of Newcastle Disease Virus (NDV), Avian Influenza Virus (H7N3) and co-infections in poultry flocks at Karachi. Methodology: For detection of NDV and H7N3, a total of 200 tracheal swabs were collected and tested through virus isolation (V.I); the sample with positive virus isolation were tested through agar gel precipitation (AGP) and then the RNA was isolated through TRI Reagent, which was further tested through reverse transcription polymerase chain reaction (RT-PCR). Results: The virus isolation showed that 58% of samples were positive for various viruses. Agar gel precipitation (AGP) revealed that the occurrence of NDV, H7N3 and ND+H7 were 50%, 8% and 38%, respectively. RT-PCR for F and HA gene of NDV and H7N3 confirmed the presence of NDV and H7N3 in the poultry. Conclusion: It is concluded that NDV and H7N3 are circulating in the flocks causing co-infections, therefore it is important to know the field challenge of viruses and to prepare vaccine of circulating serotype of virus to mitigate the rate of infection.

Sign in / Sign up

Export Citation Format

Share Document