4. DISCUSSION
A multiplex real-time PCR assay can detect and differentiate two or more pathogens in a single assay, which provides the capacity to diagnose and rule out multiple diseases in a sample containing multiple pathogens. However, the challenge of simultaneous amplification of multiple targets (template) is that asymmetric amplifications can occur, resulting weaker target(s) (lower AE) amplified less efficiently than stronger targets (higher AE). Therefore, it is important to evaluate the performance of the multiplex assays against the corresponding singleplex assays to ensure that there is no loss of sensitivity for each testing target.
In this study, multiplex TaqMan® assays were developed and optimized for the detection and differentiation of CaPV, PaPV and FMDV. The original (singleplex) assays corresponding to each virus were optimized and validated on Cepheid SmartCycler. The above assays were further optimized on high throughput PCR platform ABI 7500 Fast using serial dilutions of viral DNA/RNA as template in this study; and the results were compared against parallel assays ran on Cepheid SmartCycler. The sensitivity of detection (LOD) of the template (viral DNA/RNA) remained unchanged between the two PCR platforms (not shown), indicating comparable performances.
To ensure diagnostic applicability on CS, the newly developed multiplex assays included the host β-actin gene ACTB as the IPC to detect PCR inhibition and monitor accuracy of nucleic acid extractions. The β-actin is ubiquitously expressed and detectable in most commonly used CS (skin, tissue, blood and swabs) of all mammalian species including cattle, sheep and goats (Das et al. 2017a & 2017b), which justifies its inclusion as the IPC. To determine the LOD, the viral DNA/RNA were serially diluted in nuclease-free water (singleplex) or the DNA purified from skin of non-infected healthy animal (multiplex) and used as template. This allowed parallel comparison of the performance of the assays between the singleplex and the multiplex formats. The results (LOD studies) showed no apparent change in the sensitivity of detection of the template (viral DNA/RNA) between the two formats of the assay (singleplex and multiplex), indicating no interference of either the host DNA or the amplification of ACTB . The choice of using the DNA purified from skin as a diluent was due to the fact that skin/scab and skin lesions are the most common specimens used for the detection of all three viruses (CaPV, PaPV and FMDV).
Three multiplex assays were developed and optimized: CaP rule-out assays for simultaneous detection and differentiation of CaPV and PaPV; FMD rule-out assays for simultaneous detection and differentiation of FMDV and PaPV; and CaP/FMD rule-out assays for simultaneous detection and differentiation of CaPV, PaPV and FMDV. At the onset of the development, the multiplex assays were subjected to the following optimization criteria: concentration of the primers and probe of each target, selection of the reporter and quencher dyes of the TaqMan® probes used for each target, and finally, assessment of the optimized (multiplex) assays against the corresponding singleplex assays. The concentration of the primers and probe of each target used in the multiplex assays remained the same as the corresponding singleplex assays except for FMDV where the primers and probe concentrations were doubled. The selection of the reporter dyes of the TaqManTM probes was thoroughly evaluated to rule-out any cross-talk (interference) between the dyes that might adversely affect the sensitivity of the assay. The final selection of the reporter dyes used in the CaP and FMD rule-out 3-plex assays were FAM (CaPV or FMDV), Cy5 (PaPV) and VIC (ACTB ) and those used in the CaP/FMD rule-out 4-plex assays were FAM (CaPV), Cy5 (PaPV), Texas Red (FMDV) and VIC (ACTB ). The use of Texas Red along with FAM and Cy5 as reporter dyes in multiplex TaqMan™ assays was also reported by others (Fratamico et al. 2011). Preferential use of FAM, Cy5 and VIC in 3-plex assays was also reported by others (Diallo et al. 2011; Del Amo 2013). These dyes (FAM, Cy5 and VIC) have wide differences in the excitation and emission wavelengths that reduce the risk of cross-talk or overlapping signals and allow accurate and reliable detection of the template (Cirino et al. 2007; Gunson et al. 2008). The CaP and FMD rule-out (3-plex) assays reported in this study also used FAM, Cy5 and VIC as reporter dyes and exhibited comparable performances (LOD) as the corresponding singleplex assays. The CaP/FMD rule-out (4-plex) assays also exhibited similar performances (LOD) as the corresponding singleplex and CaP and FMD rule out (3-plex) assays for all viruses with the exception of the FMDV serotypes A and O, which showed a 10-fold drop in the LOD. Reduced sensitivity (LOD) of multiplex assays is not unusual as previously reported (Das et al., 2019; Elnifro et al. 2000; Gunson et al. 2008). Reasons for this could be due to preferential amplification of one target (higher AE) over another (lower AE), competition for the same reagents in the mastermix (dNPTs, Mg+2 ions etc.) and non-specific interactions between multiple primers sets (Hindson et al. 2008).
The efficacy of the multiplex assays were evaluated against the corresponding singleplex assays using both PACs and the viral DNA/RNA as template. The CaP and FMD rule-out assays exhibited 100% specificity against all serotypes/isolates of the target viruses (CaPV, PaPV and FMDV) tested, and had no cross-reactivity against other viruses including CaPV differentials BHV and PPRV, as well as FMDV differentials BVDV, VSV and BTV. Assessment of DSe of the multiplex assays revealed 100% DSe against CaPV (35/35) and PaPV (36/36) by CaP rule-out assays; and 100% DSe against PaPV (36/36) and 95% DSe against FMDV (37/39) by FMD rule-out assays. The two FMDV specimens tested negative by FMD rule-out assay were also tested negative by singleplex FMDV RT-qPCR assay (not shown), indicating comparable DSe between the singleplex and multiplex assays.
Several PCR-based multiplex assays have been reported by others for differential diagnosis of mixed infections involving CaPV, PaPV and FMDV, including conventional PCR/RT-PCR assays (He at al. 2017, Venkatesan et al. 2014a) and multiplex RT-PCR microsphere array assays (Hindson et al. 2008). A duplex (2-plex) TaqMan™ assay (Venkatesan et al. 2014b) was reported for simultaneous detection and differentiation of SPV, GPV and ORFV but not LSDV or FMDV and the assay was evaluated without using any CS of cattle or IPC. A 4-plex TaqMan™ assay (Xu et al. 2019) was reported for simultaneous detection and differentiation of SPV, ORFV and FMDV but not GPV or LSDV, and this assay also evaluated without using any CS of cattle or IPC. The newly developed multiplex assays described in this study were evaluated against all three differential viruses and their serotypes (CaPV, PaPV and FMDV) some of which were not included in the above studies. Furthermore, all assays included an IPC (ACTB ) to detect PCR inhibition and monitor accuracy of nucleic acid extractions to rule out any false negative results.
The United States has been free from CaP and FMD which is partly due to the implementation of a highly effective FAD surveillance program. The newly developed CaP and FMD rule-out assays can strengthen this program by providing the tools to monitor any incursion of CaPV or FMDV. FADDL has been designated as the only lab in the US allowed to perform FAD diagnosis. Specimens from animals showing clinical signs resembling FADs are routinely sent to FADDL for testing, including those from suspected ruminants infected with PaPV showing clinical signs resembling CaP or FMD. Currently, target specific singleplex assays are carried out to rule-out CaP or FMD, which are time consuming and costly. These singleplex assays potentially can be replaced with the newly developed CaP and FMD rule-out assays. The new assays may be more useful for diagnostics in countries with endemic CaP, PaP and FMD, where mixed infections are prevalent. Overall, the newly developed multiplex assays have the potential for use in both routine laboratory diagnosis as well as disease surveillance investigations.