LOW SPECIFICITY OF A NESTED REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION TO DETECT AVIAN INFLUENZA VIRUS NUCLEOPROTEIN GENE

2016 ◽  
Vol 43 (02) ◽  
pp. 75-79
Author(s):  
Lih-Chiann Wang ◽  
Wei-En Hsu ◽  
Wei Thong ◽  
Ting-Yen Chao ◽  
Ching-Ho Wang
Keyword(s):  
Polymerase Chain Reaction ◽  
Influenza Virus ◽  
Avian Influenza ◽  
Avian Influenza Virus ◽  
Reverse Transcription ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Nucleoprotein Gene ◽  
Polymerase Chain ◽  
Np Gene

Reverse transcription polymerase chain reaction (RT-PCR) was used routinely to detect the avian influenza virus (AIV) nucleoprotein (NP) gene. The purpose of the present study was to compare the correctness of a nested RT-PCR (nRT-PCR), one conventional RT-PCR with its outer primer (oRT-PCR) and the other conventional RT-PCR with its inner primer (iRT-PCR) to detect AIV NP gene. A total of 365 AI-free fecal swabs (73 pools), 7 tracheal swabs and anllantoic fluid from 25 chicken embryos were used to determine the analytic specificities of those tests. Compared with the iRT-PCR, the nRT-PCR was more sensitive for AIV detection. However, the specificities of nRT-PCR, oRT-PCR and iRT-PCR were 48.6% (35/72), 100% (67/67) and 91.3% (84/92), respectively. The amplifying band was sequenced and confirmed to be the AIV NP gene as the positive control. The specificity of this nRT-PCR is too low to be used for the AIV screening test.

A Fully Automated Procedure for the High-Throughput Detection of Avian Influenza Virus by Real-Time Reverse Transcription–Polymerase Chain Reaction

2007 ◽  
Vol 51 (s1) ◽  
pp. 235-241 ◽  
Author(s):  
Montserrat Agüero ◽  
Elena San Miguel ◽  
Azucena Sánchez ◽  
Concepción Gómez-Tejedor ◽  
Miguel Angel Jiménez-Clavero
Keyword(s):  
Polymerase Chain Reaction ◽  
Influenza Virus ◽  
Avian Influenza ◽  
Avian Influenza Virus ◽  
Real Time ◽  
High Throughput ◽  
Reverse Transcription ◽  
Chain Reaction ◽  
High Throughput Detection ◽  
Polymerase Chain

scholarly journals Pathological and Molecular Characterization of H5 Avian Influenza Virus in Poultry Flocks from Egypt over a Ten-Year Period (2009–2019)

Animals ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 1010
Author(s):  
Samah Mosad Mosad ◽  
Fatma A. El-Gohary ◽  
Hanaa Said Ali ◽  
Hanem El-Sharkawy ◽  
Ehab Kotb Elmahallawy
Keyword(s):  
Polymerase Chain Reaction ◽  
Influenza Virus ◽  
Avian Influenza ◽  
Avian Influenza Virus ◽  
Blood Vessels ◽  
Molecular Characterization ◽  
Histopathological Examination ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain

Avian influenza virus (AIV) remains one of the enzootic zoonotic diseases that challenges the poultry industry in Egypt. In the present study, a total of 500 tissue samples were collected from 100 chicken farms (broilers and layers) suspected to be infected with AIV through the period from 2009 to 2019 from Dakahlia governorate, Egypt. These samples were pooled in 100 working samples and screened for AIV then the positive samples were subjected to histopathological examination combined with real time-polymerase chain reaction (RRT-PCR). RRT-PCR positive samples were also subjected to conventional reverse transcriptase-polymerase chain reaction (RT-PCR) for detection of H5 AIV and some of these resulting positive samples were sequenced for detection of the molecular nature of the studied virus. Interestingly, the histopathological examination revealed necrotic liver with leukocytic infiltration with degenerative changes with necrotic pancreatitis, edema, and intense lymphoid depletion of splenic tissue and hyperplastic tracheal epithelium. Likewise, edema and congested sub mucosal blood vessels and intense bronchial necrosis with hyalinized wall vascular wall and heterophils infiltration were reported. Pneumonic areas with intense leukocytic aggregation mainly and vasculitis of the pulmonary blood vessels were also detected in lung. Collectively, these significant pathological changes in examined tissues cohered with AIV infection. Regarding the molecular characterization, 66 samples were positive for AIV by RRT-PCR and 52 of them were positive for H5 AIV by RT-PCR. The phylogenetic analysis revealed that the H5 viruses identified in this study were aligned with other Egyptian H5N1 AIVs in the Egyptian sub clade 2.2.1, while some of the identified strains were aligned with other Egyptian H5N8 strains in the new Egyptian sub clade 2.3.4.4. Taken together, our present findings emphasize the wide spread of AIV in Egypt and the importance of developing an efficient surveillance and periodical screening program for controlling such disease of public health concern.

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 Development of multiplex reverse transcription polymerase chain reaction (RT-PCR) for simultaneous detection of matrix, haemagglutinin and neuraminidase genes of H5N1 avian influenza virus

2012 ◽  
Vol 28 (2) ◽  
pp. 55-59 ◽  
Author(s):  
R. Mojumder ◽  
E. H. Chowdhury ◽  
R Parvin ◽  
J. A. Begum ◽  
M. Giasuddin ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Influenza Virus ◽  
Avian Influenza ◽  
Matrix Protein ◽  
Simultaneous Detection ◽  
High Fidelity ◽  
Rt Pcr ◽  
Chain Reaction ◽  
One Step ◽  
Polymerase Chain

Influenza A virus, subtype H5N1 causes a fatal disease in domestic poultry and could spread directly from poultry to humans. The aim of this study was to develop a multiplex reverse transcription polymerase chain reaction (mRT-PCR) for simultaneous detection of Type A influenza virus-specific matrix protein (M) gene as well as H5 and N1 genes of highly pathogenic avian influenza (HPAI) viruses. Finnzymes Phusion-Flash High- Fidelity PCR Master Mix (Finnzymes Oy, Finland) and Qiagen one-step RT-PCR enzyme mix (Qiagen, Germany) were used in a one-step RT-PCR. RNA was extracted from two known positive samples using Qiagen RNA extraction kit. RT-PCR was carried out with a mixture of primers specific for the Type A influenza virus matrix protein (M), and H5 and N1 genes of H5N1 HPAI viruses in a single reaction system. The mRT-PCR cDNA products were visualized by gel electrophoresis. The mRT-PCR yielded fragments of 245 bp for M, 545 bp for H5 and 343 bp for N1 genes of HPAI virus, which were clearly distinguishable. The mRT-PCR using the Finnzymes Phusion-Flash High-Fidelity PCR Master Mix (Finnzymes Oy, Finland) with Qiagen one-step RT-PCR Enzyme Mix (Qiagen, Germany) required only one hour and 20 minutes. (Bangl. vet. 2011. Vol. 28, No. 2, 55 – 59)DOI: http://dx.doi.org/10.3329/bvet.v28i2.10653

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