Rapid Detection of Highly Pathogenic Avian Influenza H5N1 Virus by TaqMan Reverse Transcriptase–Polymerase Chain Reaction

Abstract

Highly pathogenic avian influenza (AI) H5N1 viruses have been spreading from Asia since late 2003. Early detection and classification are paramount for control of the disease because these viruses are lethal to birds and have caused fatalities in humans. Here, we described TaqMan reverse transcriptase-polymerase chain reaction assays for rapid detection of all AI viruses (influenza type A) and for identification of H5N1 of the Eurasian lineage. The assays were sensitive and quantitative over a 10(5)-10(6) linear range, detected all of the tested AI viruses, and enabled differentiation between H5 and H7 subtypes. These tests allow definitive confirmation of an AI virus as H5 within hours, which is crucial for rapid implementation of control measures in the event of an outbreak.

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

“…Recent techniques have developed a one-step TaqMan rRT-PCR specific for the detection of less than 1 PFU of H5 AIV from clades 0, 1, 2, and 3 (10, 27). The limit of detection in reaction 2 of 10 copies of RNA molecule and 0.01 50% lethal dose of virus was similar to limits obtained using one of the WHOrecommended protocols and to those previously published (13,22,55,56). Two H1 and 37 H3 virus subtypes positive for the M gene in rRT-PCR or RT-PCR (Table 6) were subtyped using reaction 3.…”

Molecular Analysis of H7 Avian Influenza Viruses from Australia and New Zealand: Genetic Diversity and Relationships from 1976 to 2007

“…The presence of avian influenza virus RNA in each of the samples was confirmed by a reverse transcription-PCR (RT-PCR) TaqMan assay targeted at the matrix (M) gene; this assay is capable of detecting all influenza A virus types (16).…”

Performance of six influenza rapid tests in detecting human influenza in clinical specimens

“…RNA extraction was performed using a MagnaPure extraction system (Roche) in accordance with manufacturer's instructions. Real-time Table 1 Comparison of the influenza A and B sensitivity, specificity, positive predictive value (PPV) and negative predictive values (NPV) for the six different rapid tests to RETCIF Rapid test (73) 127/128 (99) 36/37 (97) 127/140 (91) 3/10 (30) 167/167 (100) 3/3 (100) 167/174 (96) BD Directigen EZ Flu A + B 34/49 (69) 128/128 (100) 34/34 (100) 128/143 (90) 3/10 (30) 167/167 (100) 3/3 (100) 167/174 (96) Denka Seiken Quick Ex-Flu 35/49 (71) 128/128 (100) 35/35 (100) 128/142 (90) 3/10 (30) 167/167 (100) 3/3 (100) 167/174 (96) Fujirebio Espline Influenza A&B-N 33/49 (67) 128/128 (100) 33/33 (100) 128/144 (89) 3/10 (30) 167/167 (100) 3/3 (100) 167/174 (96) Rockeby Influenza A Antigen Test 5/49 (10) 128/128 (100 (67) 128/128 (100) 33/33 (100) 128/144 (89) 3/10 (30) 167/167 (100) 3/3 (100) 167/174 (96) RT-PCR detection was performed using Taqman One-Step RT-PCR Master Mix Reagents (Applied Biosystems, USA) and utilising separate influenza A primers and probe as described previously (Heine et al, 2007) and influenza B primers (Poddar, 2002) with SYBR green (Qiagen QuantiTect SYBR green RT-PCR kit) using an Applied Bio...…”