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.