Design of Multiplexed Detection Assays for Identification of Avian Influenza A Virus Subtypes Pathogenic to Humans by SmartCycler Real-Time Reverse Transcription-PCR

Wang, Wei; Ren, Pengpeng; Mardi, Sek; Hou, Lili; Tsai, Cheguo; Chan, Kwok Hung; Cheng, Peter K.C.; Sheng, Jun; Buchy, Philippe; Sun, Bing; Toyoda, Tetsuya; Lim, Wilina; Peiris, Malik; Zhou, Paul; Denis, Vincent

doi:10.1128/jcm.01090-08

Cited by 31 publications

(22 citation statements)

References 53 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The assays had similar rates of H5N1 detection in animal specimens and all four methods could detect 100 copies/reaction; NASBA was the most sensitive and detected 10 copies/reaction. Wang et al developed four PCR tests for the detection of m and ha genes of H5N1 using the SmartCycler instrument 44 . This four-reaction amplification approach was able to detect 10 RNA copies of the ha gene of H1, H2, H3, H5 and H7 and 100 copies of H9, and 6 different clades of H5N1 in Southeast Asia.…”

Section: Influenza Virusmentioning

confidence: 99%

Molecular diagnosis of respiratory virus infections

Mahony¹,

Petrich²,

Śmieja³

2011

Critical Reviews in Clinical Laboratory Sciences

192

181

View full text Add to dashboard Cite

The appearance of eight new respiratory viruses, including the SARS coronavirus in 2003 and swine-origin influenza A/H1N1 in 2009, in the human population in the past nine years has tested the ability of virology laboratories to develop diagnostic tests to identify these viruses. Nucleic acid based amplification tests (NATs) for respiratory viruses were first introduced two decades ago and today are utilized for the detection of both conventional and emerging viruses. These tests are more sensitive than other diagnostic approaches, including virus isolation in cell culture, shell vial culture (SVC), antigen detection by direct fluorescent antibody (DFA) staining, and rapid enzyme immunoassay (EIA), and now form the backbone of clinical virology laboratory testing around the world. NATs not only provide fast, accurate and sensitive detection of respiratory viruses in clinical specimens but also have increased our understanding of the epidemiology of both new emerging viruses such as the pandemic H1N1 influenza virus of 2009, and conventional viruses such as the common cold viruses, including rhinovirus and coronavirus. Multiplex polymerase chain reaction (PCR) assays introduced in the last five years detect up to 19 different viruses in a single test. Several multiplex PCR tests are now commercially available and tests are working their way into clinical laboratories. The final chapter in the evolution of respiratory virus diagnostics has been the addition of allelic discrimination and detection of single nucleotide polymorphisms associated with antiviral resistance. These assays are now being multiplexed with primary detection and subtyping assays, especially in the case of influenza virus. These resistance assays, together with viral load assays, will enable clinical laboratories to provide physicians with new and important information for optimal treatment of respiratory virus infections.

show abstract

Section: Influenza Virusmentioning

confidence: 99%

Molecular diagnosis of respiratory virus infections

Mahony¹,

Petrich²,

Śmieja³

2011

Critical Reviews in Clinical Laboratory Sciences

192

181

View full text Add to dashboard Cite

show abstract

“…The origin of the viruses is indicated in Table 1. The amplified products were cloned in pGEM-T easy Vector (Promega, Madison, WI, USA) and in vitro-transcribed with T7 polymerase, as described previously (Wang et al, 2009). The RNA transcripts were quantified in a UV spectrophotometer (Biorad, Shanghai, China).…”

Section: Rna Extraction and Preparation Of Rna Transcriptsmentioning

confidence: 99%

Simultaneous detection of respiratory viruses in children with acute respiratory infection using two different multiplex reverse transcription-PCR assays

Wang

Ren

Sheng³

et al. 2009

Journal of Virological Methods

Self Cite

View full text Add to dashboard Cite

A 4-tube multiplex RT-PCR (mRT-PCR), which showed higher sensitivity over conventional methods, was previously developed for the diagnosis of 14 viral pathogens of the respiratory tract. Herein the mRT-PCR was compared to the commercial Luminex mPCR-microsphere flow cytometry assay (Resplex II) which allows the detection of 12 different viruses. Eleven different viruses were identified in 91 nasopharyngeal swabs of children with acute respiratory infection, influenza A (IAV) and B, respiratory syncytial virus (RSV), human rhinovirus (hRhV), human echovirus, parainfluenza viruses (PIV) 1, 2, 3 and 4, human metapneumovirus (hMPV), and human coronavirus NL63. The results of the two techniques showed 53 and 40 positive patients by the Resplex II assay and mRT-PCR, respectively, with a concordance in 35 positive and 33 negative patients (74.7%). Individual RT-PCR tests were performed to control viruses not simultaneously detected by the two multiplex assays. The major virus misdiagnosed by mRT-PCR was IAV whereas the major viruses misdiagnosed by Resplex II were PIV1, 3 and 4. The mRT-PCR remains a simple, rapid, and specific assay for the specific detection of respiratory viruses, and can be easily implemented with standards in clinical laboratories at a low cost.

show abstract

“…The method also could be implemented on portable PCR instruments that may become available in the future. Recently a detection assay for AIV subtypes known to infect humans, including H5 and H7, was designed for the portable SmartCycler real-time PCR instrument (Cepheid, CA) to enable diagnosis in remote hospitals and in the field (63). The method presented here can complement such sensitive type-specific detection assays with pathotyping, and thus provides complete NAIV diagnosis on a portable real-time PCR platform in the vicinity of outbreaks.…”

Section: Discussionmentioning

confidence: 99%

Rapid PCR-Based Molecular Pathotyping of H5 and H7 Avian Influenza Viruses

et al. 2011

View full text Add to dashboard Cite

While the majority of avian influenza virus (AIV) subtypes are classified as low-pathogenicity avian influenza viruses (LPAIV), the H5 and H7 subtypes have the ability to mutate to highly pathogenic avian influenza viruses (HPAIV) in poultry and therefore are the etiological agents of notifiable AIV (NAIV). It is of great importance to distinguish HPAIV from LPAIV variants during H5/H7 outbreaks and surveillance. To this end, a novel and fast strategy for the molecular pathotyping of H5/H7 AIVs is presented. The differentiation of the characteristic hemagglutinin (HA) protein cleavage sites (CSs) of HPAIVs and LPAIVs is achieved by a novel PCR method where the samples are interrogated for all existing CSs with a 484-plex primer mixture directly targeting the CS region. CSs characteristic for HP or LP H5/H7 viruses are distinguished in a seminested duplex real-time PCR format using plexor fluorogenic primers. Eighty-six laboratory isolates and 60 characterized NAIV-positive clinical specimens from poultry infected with H5/H7 both experimentally and in the field were successfully pathotyped in the validation. The method has the potential to substitute CS sequencing in the HA gene for the determination of the molecular pathotype, thereby providing a rapid means to acquire additional information concerning NAIV outbreaks, which may be critical to their management. The new assay may be extended to the LP/HP differentiation of previously unknown H5/H7 isolates. It may be considered for integration into surveillance and control programs in both domestic and wild bird populations.

show abstract

Design of Multiplexed Detection Assays for Identification of Avian Influenza A Virus Subtypes Pathogenic to Humans by SmartCycler Real-Time Reverse Transcription-PCR

Cited by 31 publications

References 53 publications

Molecular diagnosis of respiratory virus infections

Molecular diagnosis of respiratory virus infections

Simultaneous detection of respiratory viruses in children with acute respiratory infection using two different multiplex reverse transcription-PCR assays

Rapid PCR-Based Molecular Pathotyping of H5 and H7 Avian Influenza Viruses

Contact Info

Product

Resources

About