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

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. Therefore we developed a one-step reverse transcription real time PCR (RRT-PCR) with a Minor Groove Binder (MGB) probe for the detection of different subtypes of AIVs. This technique also includes an IPC. METHODS: RRT-PCR was developed using an improved TaqMan technology with a MGB probe to detect AI from reference viruses. Primers and probe were designed based on the matrix gene sequences from most animal and human A influenza virus subtypes. The specificity of RRT-PCR was assessed by detecting influenza A virus isolates belonging to subtypes from H1-H13 isolated in avian, human, swine and equine hosts. The analytical sensitivity of the RRT-PCR assay was determined using serial dilutions of in vitro transcribed matrix gene RNA. The use of a rodent RNA as an IPC in order not to reduce the efficiency of the assay was adopted. RESULTS: The RRT-PCR assay is capable to detect all tested influenza A viruses. The detection limit of the assay was shown to be between 5 and 50 RNA copies per reaction and the standard curve demonstrated a linear range from 5 to 5 x 108 copies as well as excellent reproducibility. The analytical sensitivity of the assay is 10-100 times higher than conventional RT-PCR. CONCLUSION: The high sensitivity, rapidity, reproducibility and specificity of the AIV RRT-PCR with the use of IPC to monitor for false negative results can make this method suitable for diagnosis and for the evaluation of viral load in field specimens.

Effect of 3'UTR length on the translational regulation of 5'-terminal oligopyrimidine mRNAs.

In Vertebrates, all genes coding for ribosomal proteins, as well as those for other proteins implicated in the production and function of translation machinery, are regulated by mitogenic and nutritional stimuli, at the translational level. A cis-regulatory element necessary for this regulation is the typical 5'UTR, common to all ribosomal protein mRNAs, which always starts at the 5' end with several pyrimidines. Having noticed that the 3'UTR of all ribosomal protein mRNAs is much shorter than most cellular mRNAs, we have now studied the possible implication of this 3'UTR feature in the translational regulation. For this purpose, we constructed a number of chimeric genes whose transcribed mRNAs contain: (1) the 5'UTR of ribosomal protein S6 mRNA or, as a control, of beta-actin mRNA; (2) the EGFP reporter coding sequence from the starting AUG to the stop codon; (3) different 3'UTRs of various lengths. These constructs have been stably transfected in human HEK293 cells, and the translation regulation of the expressed chimeric mRNAs has been analyzed for translation efficiency, in growing and in serum starved cells, by the polysome association assay. The results obtained indicate that, while the typical growth-associated translational regulation is bestowed on an mRNA by the pyrimidine sequence containing 5'UTR, the stringency of regulation depends on the short size of the 3'UTR.