A sensitive one-step real-time PCR for detection of avian influenza viruses using a MGB probe and an internal positive control

Abstract

Background: Avian influenza viruses (AIVs) are endemic in wild birds and their introduction and conversion to highly pathogenic avian influenza virus in domestic poultry is a cause of serious economic losses as well as a risk for potential transmission to humans. The ability to rapidly recognise AIVs in biological specimens is critical for limiting further spread of the disease in poultry. The advent of molecular methods such as real time polymerase chain reaction has allowed improvement of detection methods currently used in laboratories, although not all of these methods include an Internal Positive Control (IPC) to monitor for false negative results.

Quantification of avian influenza virus in tissues of mute swans using TaqMan real time qRT-PCR

“…A comparison of the number of viral RNA copies detected by qRT-PCR with that of infectious virions in a VERO cell line, gave a ratio of one RNA copy to 0.00157 TCID 50 . Similar results have been reported by Di Trani et al (2006) who, using the qRT-PCR assay, could detect up to 0.001 TCID 50 of the reference virus -an equivalent to 0.08 EID 50 . The ratio calculated by us was used to estimate the amount of virus originally present in swan tissues and, to be able to compare the amount of virus in swan tissues with those in the organs of swans and other water and gallinaceous avian species reported in the literature, we converted the TCID 50 obtained to EID 50 values.…”

Development and Validation of a One-Step Real-Time PCR Assay for Simultaneous Detection of Subtype H5, H7, and H9 Avian Influenza Viruses

“…Recently, molecular diagnostic tests have proven themselves to be invaluable as a first step in the identification and control of disease outbreaks. Conventional RT-PCR and RRT-PCR have been applied successfully to the diagnosis of AI (7,8,12,14,15,19,21,23,27). In this study, we present data on the development and validation of a real-time hydrolysis probe-based RT-PCR assay for the simultaneous detection of AI viruses belonging to subtypes H5, H7, and H9.…”

“…Because of their rapidity and sensitivity, molecular tests, such as reverse transcription-PCR (RT-PCR) and real-time RT-PCR (RRT-PCR), are being used more and more by medical and veterinary diagnosticians for the diagnosis of AI (4,24). Recently, RRT-PCR assays have been developed for the detection of type A influenza viruses (7,23) and for the specific diagnosis of H5 and H7 viruses (8,12,14,15,19,21,23,27). To date, the only published RRT-PCR assay designed for subtype H7 was validated for viruses belonging to the American lineage, and no primer and probe sets are currently available for the identification of subtype H9 viruses.…”

Real-Time PCR-Based Pathotyping of Newcastle Disease Virus by Use of TaqMan Minor Groove Binder Probes

“…The assay utilizes a fluorescent 5Ј-nuclease PCR technology for this purpose, which has the potential to be used for quantitative measurements and eliminates postamplification procedures and thus reduces the risk of contamination and handling errors. The viability of the TaqMan platform has been proven by its numerous applications in PCR-based assays, e.g., to detect OIE-notifiable avian pathogens: avian influenza A/H5/H7 virus (2,20,33,41), infectious bronchitis virus (10), infectious laryngotracheitis virus (11), Mycoplasma gallisepticum (12), and infectious bursal disease virus (34). Applying MGB quenchers in the 3Ј ends of TaqMan probes has further advantages: (i) low background fluorescence due to the short distances between the reporters and quenchers increases the signal-to-noise ratio of the assay (31), and (ii) the small MGB molecules bind into the minor groove of double-stranded DNA, thus stabilizing the duplex, which results in higher melting temperatures and allows the application of probes that are shorter and more specific (13-to 18-mers) than standard probes (15-to 40-mers).…”