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

Abstract

The recent spread of highly pathogenic H5N1 avian influenza (AI) has made it important to develop highly sensitive diagnostic systems for the rapid detection of AI genome and the differentiation of H5N1 variants in a high number of samples. In the present paper, we describe a high-throughput procedure that combines automated extraction, amplification, and detection of AI RNA, by an already described TaqMan real-time reverse transcription-polymerase chain reaction (RRT-PCR) assay targeted at the matrix (M) protein gene of AI virus (AIV). The method was tested in cloacal and tracheal swabs, the most common type of samples used in AI surveillance, as well as in tissue and fecal samples. A robotic system (QIAGEN Biosprint 96) extracted RNA and set up reactions for RRT-PCR in a 96-well format. The recovery of the extracted RNA was as efficient as that of a manual RNA extraction kit, and the sensitivity of the detection system was as high as with previously described nonautomated methods. A system with a basic configuration (one extraction robot plus two real-time 96-well thermocyclers) operated by two persons could account for about 360 samples in 5 hr. Further characterization of AI RNA-positive samples with a TaqMan RRT-PCR specific for H5 (also described here) and/or N1 was possible within 2 hr more. As this work shows, the system can analyze up to 1400 samples per working day by using two nucleic acid extraction robots and a 384-well-format thermocycler.

Selective Isolation of Avian Influenza Virus (AIV) from Cloacal Samples Containing AIV and Newcastle Disease Virus

“…?show "fnote_aff1"$^! "content-markup(./author-grp [1]/aff|./author-grp [1]/dept-list)> Highly pathogenic Avian influenza virus (HPAIV) and velogenic strains of Newcastle disease virus (NDV) are included on the reportable disease list of the World Organization for Animal Health (OIE). 21,22 Several studies have reported the isolation of AIV 2,5 and NDV 12,17 from waterfowl.…”

Molecular detection of Clostridium perfringens toxinotypes, Enteropathogenic Escherichia coli, rotavirus and coronavirus in diarrheic fecal samples of neonatal goat kids

“…among the several chemistries available for real-time PCr assays, SYBr Green based assays are the most widely used. Previously, real-time PCr based methods have been reported for detection of many pathogens of veterinary importance (aGUerO et al, 2007). SYBr green based real time PCr assay was used earlier to differentiate ePeC, Verotoxic E. coli and other enteroaggregative E. coli, and it was concluded in that study that it may be used as an effective diagnostic tool compared to conventional PCr, for detection of E. coli strains (BISChOFF et al, 2005;GUION et al, 2008) Rotavirus is responsible for causing economically significant maladies in neonates of many domestic animals (kaPIkIaN and ChaNOCk, 1996;eSteS and kaPIkIaN, 2007).…”

Failure of Transmission of Low-Pathogenic Avian Influenza Virus Between Mallards and Freshwater Snails: An Experimental Evaluation

“…Step RT-PCR master mix and run on an ABI 7900 Real Time PCR Thermocycler (Life Technologies Corp., Carlsbad, California, USA) with thermocycler conditions developed by Agü ero et al (2007). Calibrated controls with known viral titers (10 2 -10 5 EID 50 /mL, or 0.5, 5, 50, and 500 EID 50 per RT-PCR reaction) were included on each plate to construct four-point standard curves.…”