The method developed in this study combines the multiplexing capability of PLP chemistry with microarray detection to identify samples positive for AIV with HA and NA subtyping simultaneously in approximately 4 h.
The assay presented contained the following reaction steps. During the ligation, the probes which were base paired to their appropriate target became circularized by DNA ligase and only these were amplified in the following RCA reaction. Since RCA is a linearly processing amplification, the probes were further amplified and fluorescently labeled in the following PCR. The PCR products were loaded onto a microarray to which oligonucleotides complementary to the tag sequences of the probes had been attached previously. Finally, the microarray slide was scanned to measure and quantify the intensity of the fluorescence of each spot.
This method is able to detect isolates positive for influenza virus and simultaneously subtype the HA and NA surface proteins. The PLP methodology was chosen because of its high multiplexing capacity, which constrains more conventional PCR methods (10
). PLPs also reduce the number of conserved regions needed on target nucleic acids because both probe arms bind to a single region (2
). Amplification is then based on the circularized PLP and is independent of the viral sequences. Furthermore, the DNA ligase only functions for precise matches that help avoid a false diagnosis (11
). However, the high variability of the influenza virus genome can produce false-negative results as a consequence of even a single nucleotide change at the site of the ligation. It may be difficult to find probes for all variations of the HA and NA genes, but the system incorporates redundant probes, targeting each gene in more than one position—this reduces the risk that mutations will result in assay failure during subtyping. The aim during the design of the probes was to cover all variants with the sequence information available, and this resulted in various numbers of probes for various subtypes. The main reason for that was the different genetic diversity of each subtype, which is strongly related to the number of accessible sequence data.
The use of orthogonal tag sequences amplified by common primers renders the assay flexible, as new probes can be added if new AIV variants emerge that are not covered by the current set of probes. This assay format also provides the option of broadening the assay's detection capability to include PLPs for differential diagnosis involving pathogens that produce similar clinical symptoms.
The sensitivity of the assay was evaluated with a dilution series of artificially synthesized target molecules. The detection limit was found to be 600 copies. To further investigate its sensitivity, the assay's performance was compared to that of a diagnostic PCR (17
) and classical virus isolation. The PLP assay gave reliable signals down to a 10−4
dilution, whereas the diagnostic PCR had a detection limit of 10−6
. The virus isolation gave barely readable signals after just one dilution step. This work, as another study (25
) has already suggested, showed that although the PLP assay cannot match the sensitivity of a real-time PCR, it still has reasonably high sensitivity. Furthermore, it offers advantages in multiplexing and target region selection that conventional PCR methods cannot, providing a powerful alternative for the determination of AIV subtypes.
For sample testing, mostly Scandinavian AIV strains were available in this study, but in the system design a worldwide collection of sequences was used, ensuring that the assay can be used on samples from any location. Allantois fluids containing cultivated viruses were used in this study, but five H5N1 clinical samples (V428 to V599) were also tried out with appropriate results. This study was focused on avian viruses, but during the design stage it was unavoidable to involve strains from other species as well. Preliminary test results suggest that the method, after further extension and evaluation, can be applicable for the detection of human and swine influenza viruses.
Although PLPs are long oligonucleotides (>100 bp), which cost more than regular primers, 1 nmol of probes allows almost a million reactions, which makes the individual assay cost very low. The other reagents involved in the reaction are standard molecular assay chemicals. Although the equipment used is currently uncommon in many diagnostic laboratories and the lack of automation of the assay is a certain limitation in this area, similar approaches are becoming widely accepted (5
The subtyping assay presented here takes 4 h to perform (excluding cDNA preparation), which is comparable to diagnostic PCRs. In summary, subtyping of AIV by PLPs can be accomplished in a comparably short and straightforward process that has the potential to be applied for the clinical diagnostic differentiation of AIV strains.