The mRT-qPCR is a rapid and efficient method for the detection and differentiation of BRSV, BoHV-1 and BPI3 and is thus an invaluable tool in the aetiological resolution of BRD. Aside of multiple pathogen detection, the assay has several advantages over conventional methods including higher sensitivity and specificity, decreased cost, smaller sample size, rapidity of processing and the possibility of laboratory automation to suit high throughput veterinary diagnostic laboratories. The multiplex format showed complete concordance with the corresponding mono-specific RT-PCRs. The application of mRT-qPCR for the detection of multiple pathogens provides a major contribution to efficiency, logistics, and cost-effectiveness of molecular diagnostics [14
A multiplex real time RT-PCR has been reported previously for BVDV (5'UTR), BoHV-1 (glycoprotein C) and BPI3 (matrix) [18
]. While BVDV can be important in the development of BRD, BRSV is a primary pathogen and is thus included in the mRT-qPCR described herein. The use of shorter length MGB and LNA probes is advantageous, typically confering stability, target specificity, greater sensitivity and discrimination for the target gene [19
As co-infections are a regular feature of BRD in field outbreaks [1
], careful optimisation is required to ensure that the molecular diagnostic test employed will not detect one target virus preferentially. Primers and probes for multiplex assays should be assessed both in silico
and in vitro
for evidence of cross amplification, competition or inhibition. The mRT-qPCR assay described here can detect viral co-infections both in technical validation experiments and in clinical samples. No cross reactivity between primers and probes was observed, nor was a reduction in sensitivity detected.
The sensitivity of the multiplex and uniplex assay was evaluated by testing 10-fold serial dilutions of in vitro transcribed RNAs for BRSV, BoHV-1 and BPI3 i and ii. The sensitivity, efficiency and detection limits of the individual RT-qPCRs were not affected by multiplexing the reactions. The standard curves and reaction efficiencies were very similar for the mRT-qPCR and mono-specific reactions (Figure ; Table ). A perfect amplification reaction has an efficiency of 2, but in reality, reactions often have efficiencies of less than2; the acceptable range is considered to be between 1.7 and 2.2 [21
]. The efficiencies obtained for both assays in this study were within this range.
For BRSV and BPI3, all samples in which these viruses were detected by other methods were detected by the mRT-qPCR. Additionally, the mRT-qPCR identified BRSV and BPI3i in 20 and 14 additional samples respectively, suggesting a higher sensitivity. For BoHV-1, this was the case for virus isolation and for the majority of FAT positive samples. However, three samples were positive on BoHV-1 FAT but negative on both virus isolation and mRT-qPCR; this suggests a lack of specificity in the FAT; further, the mRT-PCR identified BoHV-1 in 25 additional samples when compared to FAT results, suggesting higher sensitivity. The mRT-qPCR also detected dual infections (BoHV-1 and BPI3 i) in three samples; in virus isolation of these samples, BoHV-1 overgrew BPI3, masking detection. These results demonstrate that the m RT-qPCR is more specific and sensitive in respiratory viral diagnosis when compared to conventional tests, as has been shown in both veterinary and human clinical pathology settings [16
False negative results can occur due to RT- PCR inhibition, which was controlled in this sample set by the use of an endogenous internal control (β-actin). While this assay was run separately, it would be possible to consider including this control in the multiplex reaction by, for example, labelling both subgenotypes of BPI3 with the same fluorophore. The selection of target sequences is also critical factor that can contribute to false negatives; it is possible (especially for RNA viruses, due to the higher error rate of RNA polymerases) that mutations in the primer and probe regions may occur which compromise molecular detection at the target site (25). While sequences for molecular assays are selected in silico to ensure the target region is highly conserved, unusual or unexpected results from clinical samples should always trigger further investigation by either conventional methods or use of molecular assays with different target sequences or degenerate primers.
A final advantage of the use of molecular based testing in a clinical setting is the ability to include other targets e.g. as adenovirus, BVDV and bacterial pathogens. The limitations of real time PCR based multiplex detection and differentiation rest with the number of reactions which can be optimised in a single tube and the number of fluorophores which can be simultaneously detected. While real time PCR platforms can generally detect no more than 5 fluorophores, labelling strategies can be used to increase the number of targets detected and the development of fluorophore labelled bead based detection systems (e.g. Luminex assays) may extend target detection. Currently, the aetiology of many BRD outbreaks is undiagnosed, in some part due to the range of respiratory pathogens which must be sought and the cost of multiple pathogen detection by mono-specific assays. Adding further pathogen targets to molecular assays should improve aetiologic identification in investigation of BRD.