Primer selection for the amplification of E. granulosus s.l. species sequences
To design PCR primers and TaqMan® PCR probes that allow diagnostic differentiation between the targeted species, mitochondrial genome sequences were bioinformatically analysed (Fig. 1). The mitochondrial genomes of species and intraspecific genotypes from the E. granulosus s. l. complex were screened for polymorphic regions that could be used as targets for specific PCR primer pairs and corresponding probes. Identified regions and verified primers and probes for all four species, E. granulosus s.s. (G1-G3), E. equinus (G4), E. ortleppi (G5), and E. canadensis (G6-10), are shown in Table 1 and Fig. 1.
We used reference samples of known tapeworm species and genotypes to test the newly identified primer pairs and probes. To ensure the identity of the reference material, tapeworm specific DNA was amplified by conventional PCR and the resulting amplicons were then analysed by Sanger sequencing. The conventional PCR and sequencing results confirmed the identity of the DNA reference samples in all cases (Fig. 2, sequencing results not shown).
The bioinformatically selected primer pairs were then examined by SYBR® green qPCR to test if they specifically amplified sequences from the genome of the targeted species. We found that the selected primer pairs for E. granulosus s.s. (G1-G3) and E. equinus (G4) specifically amplified their respective reference DNA and generated amplicons that were distinguishable by their melting peak temperatures from amplicons generated using samples with DNA from closely related Echinococcus spp. and Taenia spp. (Fig. 3A and B). Furthermore, with the E. granulosus s.s. (G1-G3) primers, the melting temperature of 81 °C was distinguishable from the melting temperatures observed in amplicons generated with the same primer pair in reference samples of E. equinus (G4), E. ortleppi (G5) and E. canadensis (G6-10) at 79–80 °C (Fig. 3A). The selected primer pair designed for E. equinus (G4) generated a specific PCR product and did not cross-react with samples of the other E. granulosus s.l. species or E. multilocularis (Fig. 3B, Table 3).
Table 3
Specificity test results of TaqMan® qPCR assays.
Reference DNA
|
qPCR
|
Species
|
Genotype
|
G1_3_qPCR
|
G4_qPCR
|
G5_10_qPCR
|
G5_10_qPCR
|
E. granulosus s.s.
|
G1
|
31.65
|
No Cq
|
No Cq
|
No Cq
|
E. granulosus s.s.
|
G3 (‚G2’)
|
28.53
|
No Cq
|
No Cq
|
No Cq
|
E. granulosus s.s.
|
G3
|
27.68
|
No Cq
|
No Cq
|
No Cq
|
E. granulosus s.s.
|
Gx
|
36.90
|
No Cq
|
No Cq
|
No Cq
|
E. granulosus s.s.
|
Gx
|
37.46
|
No Cq
|
No Cq
|
No Cq
|
E. equinus
|
G4
|
No Cq
|
39.06
|
No Cq
|
No Cq
|
E. equinus
|
G4
|
No Cq
|
24.02
|
No Cq
|
No Cq
|
E. ortleppi
|
G5
|
No Cq
|
No Cq
|
27.53
|
28.02
|
E. ortleppi
|
G5
|
No Cq
|
No Cq
|
35.96
|
35.54
|
E. ortleppi
|
G5
|
No Cq
|
No Cq
|
26.02
|
26.54
|
E. canadensis
|
G6
|
No Cq
|
No Cq
|
No Cq
|
37.56
|
E. canadensis
|
G6
|
No Cq
|
No Cq
|
No Cq
|
28.37
|
E. canadensis
|
G7
|
No Cq
|
No Cq
|
No Cq
|
25.57
|
E. canadensis
|
G8
|
No Cq
|
No Cq
|
No Cq
|
29.6
|
E. canadensis
|
G10
|
No Cq
|
No Cq
|
No Cq
|
27.11
|
E. canadensis
|
G10
|
No Cq
|
No Cq
|
No Cq
|
21.0
|
E. vogeli
|
|
No Cq
|
No Cq
|
No Cq
|
No Cq
|
E. felidis
|
|
No Cq
|
No Cq
|
No Cq
|
No Cq
|
E. cf. granulosus
|
G-Omo
|
No Cq
|
No Cq
|
No Cq
|
No Cq
|
T. saginata
|
|
No Cq
|
No Cq
|
No Cq
|
No Cq
|
T. saginata
|
|
No Cq
|
No Cq
|
No Cq
|
No Cq
|
T. hydatigena
|
|
No Cq
|
No Cq
|
No Cq
|
No Cq
|
E. multilocularis
|
|
No Cq
|
No Cq
|
No Cq
|
No Cq
|
T. hydatigena
|
|
No Cq
|
No Cq
|
No Cq
|
No Cq
|
s.s.: sensu stricto |
By contrast, primers designed to amplify sequences of E. ortleppi (G5), targeting a polymorphic sequence in Nad5, also yielded non-specific PCR products in the E. canadensis (G6-10) reference samples and vice versa, i.e. primers designed to generate E. canadensis (G6-10) amplicons from Cox3 amplified also E. ortleppi (G5) reference DNA. Whilst the selected primer pairs amplified PCR products in E. granulosus DNA samples, the resulting melting curves of all of the products were hardly distinguishable at the observed temperatures of 78 °C to 78.5 °C for E. canadensis (G6-10) and 79 °C for E. ortleppi (G5) (Fig. 3C and D). Nevertheless, the primers were selected for further testing in the TaqMan qPCR with their corresponding probes.
Additional primer and probe combinations that were initially selected in silico, but did not satisfactorily amplify sequences from the targeted species during SYBR® green, or in further TaqMan® qPCR experiments, are shown in the supplementary Table S1.
In summary, amplicon generating primer pairs were identified for all targeted species (Table 1). Conventional SYBR® green qPCR was sufficient to differentiate E. granulosus s. s. (G1-G3) and E. equinus (G4) using primers targeting polymorphic regions in Cox1 (Table 1) based on the generated amplicon alone. Using the same method, primers targeting regions in Nad5 in E. ortleppi (G5) and in Cox3 in E. canadensis (G6-8, G10) amplified successfully, but cross-reacted and required additional testing to separate the respective genotypes.
Detection of Echinococcus species by TaqMan® quantitative PCRs
To increase the specificity of Echinococcus spp. target DNA detection, the four selected primer pairs were combined with amplicon-specific TaqMan® DNA probes (Table 1), and used to analyse serially diluted DNA reference samples (Fig. 4). TaqMan® qPCR assays for E. granulosus s. s. (G1-G3) and E. equinus (G4) specifically identified target DNA in the corresponding reference samples. The selected probes for both subgroups did not bind DNA from the other E. granulosus s.l. species, from E. multilocularis spp. or from Taenia spp. (Table 3). Primers and probe for detecting E. granulosus s.s. (G1-G3) also amplified DNA from the Gx, strain sample, indicating that the assay is also applicable to other genotypes of E. granulosus s.s..
Addition of TaqMan® probes to qPCR reactions with selected G5_qPCR primer pairs for E. ortleppi (G5) also showed specific amplification of the DNA target region. No cross- reactions were recorded with DNA samples from the remaining Echinococcus spp. and Taenia spp. (Table 3). Thus, it appears that the addition of sequence-specific DNA probes ameliorated the cross-reactivity with the E. canadensis cluster observed in the qPCR system.
TaqMan® qPCR with the G6_G10_qPCR primer set targeting the E. canadensis (G6-8, G10) cluster resulted in amplification of a probe-binding PCR product (Fig. 4C and D). However, this assay also cross-reacted with the E. ortleppi (G5) DNA sample and thus did not allow differentiation of these two species (Table 3). To distinguish E canadensis (G6-8, G10) from G5 samples, we combined the G5_qPCR with the G6_G10_qPCR primer pairs and probes in a TaqMan® qPCR (G5_G10_qPCR) duplex format. With this format only the probe-binding product for E. canadensis (G6-8, G10) would be amplified and detected if E. canadensis was present in the diagnostic sample. Whereas, if E. ortleppi (G5) were present in the diagnostic sample, the probe-binding products for both E. ortleppi (G5) and E. canadensis (G6-8, G10) genotypes would be detected, as the primer and probe for E. canadensis (G6-8, G10) also amplified and detected E. ortleppi (G5). Therefore, the duplex TaqMan® qPCR format of G5_G10_qPCR primer and probes allowed diagnostic differentiation of E. ortleppi (G5) from the E. canadensis (G6-8, G10) cluster (Table 3).
To characterise the diagnostic TaqMan® qPCR assays further, the analytical efficiency and the LoD of the qPCR reactions for each subgroup were determined by testing DNA extracted from clinical samples as cloned PCR products (plasmid DNA) (Table 4, Fig. 4 and Fig. 5).
Table 4
Analytical sensitivity and efficiency of developed TaqMan® qPCRs.
PCR name
|
Specificity
|
Efficiency with cloned PCR products
|
Efficiency clinical DNA
|
Detection limit (Copy number/µl at Cq) with cloned PCR products
|
Detection limit in Cq with clinical DNA
|
G1_3_qPCR (single-plex)
|
E. granulosus s.s. (G1, G3)
|
100.7% (R2 = 0.99, slope=-3.34)
|
96.3% (R2 = 0.99, slope=-3.41)
|
0.8/µl at Cq 36 (± 0.9)
|
38.24 (± 0.1)
|
G4_qPCR (single-plex)
|
E. equinus (G4)
|
106.7% (R2 = 0.99, slope=-3.32)
|
93.7% (R2 = 0.99, slope=-3.48)
|
0.6/µl at Cq 39 (± 0.6)
|
38 (± 1)
|
G5_G10_qPCR (duplex)
|
E. ortleppi (G5)
|
100.7% (R2 = 0.99, slope=-3.34)
|
97.3% (R2 = 0.99, slope=-3.39)
|
1.4/µl at Cq 36 (± 0.7)
|
37 (± 0.4)
|
G5_G10_qPCR (duplex)
|
E. canadensis (G6-8, G10)
|
101.6% (R2 = 0.99, slope=-3.28)
|
99.9% (R2 = 0.99, slope=-3.33)
|
1.4/µl at Cq 36 (± 0.8)
|
38.6 (± 0.17)
|
The analytical efficiency of TaqMan® qPCR assays varied between 94% and 107%. The LoD of the assays varied between 0.6 and 1.4 copies/µl (Table 4). The specificity in G1_3_qPCR, G4_qPCR and G5_qPCR was 100%. G6_10_qPCR cross-reacted with E. ortleppi (G5) samples (Table 3) and required a duplex assay design with G5_qPCR for diagnostic purposes.
In summary, sequence-specific DNA probe-based TaqMan® qPCR assays were established that identified four species within E. granulosus s.l. in reference samples. These samples were further differentiated from other Echinococcus and Taenia species. (Table 3).
Detection of E. granulosus s.l. species in faecal samples
The reference DNA samples used in this study for the development of qPCR assays that differentiate four Echinococcus species was derived from cyst wall material of metacestodes isolated from infected hosts. While cysts may serve as a sample matrix for the detection of E. granulosus s.l. species, it is diagnostically relevant that the assays also amplify target DNA extracted from other relevant matrices such as faecal matter. To test the performance of the TaqMan® qPCR assays when used with faecal samples, fox faeces were spiked with serially diluted reference DNA to simulate the testing of faeces from definitive hosts infected with members of E. granulosus s.l.. A known quantity of standardised, heterologous plasmid DNA with matching primers and probe was included as an internal control [IC] in the qPCR mixture [31] to control for PCR inhibition by faecal factors. Inclusion of the IC also allowed testing the assays in duplex (G1_3_qPCR or G4_qPCR in combination with IC-qPCR) and triplex (G5_G10_qPCR and IC-qPCR) TaqMan® qPCR formats (see Table 1). All four assays targeting E. granulosus s.s. (G1-G3), E. equinus (G4), E. ortleppi (G5) and E. canadensis (G6-8, G10) respectively, amplified the specific tapeworm DNA region from each analysed faecal sample type (Table 5). Combining E. ortleppi, E. canadensis as well as IC primers and respective probes in a triplex qPCR differentiated E. ortleppi (G5) and E. canadensis (G6-8, G10) DNA in faecal samples (Table 5). Probe recognition of IC DNA and quantification showed that no inhibition of the amplification process had occurred in these faecal samples and that the selected primer probe mixtures performed reliably with samples prepared in a matrix of faecal matter under duplex or triplex qPCR conditions. The Cq values generated from the re-extracted tapeworm DNA from faeces were much higher than those from the non-faecal control samples i.e. DNA extracted from hydatid cyst material. This suggests that the quantities of re-extracted tapeworm DNA from faeces were much lower than from non-faecal control samples, possibly due to loss of DNA during the faecal DNA extraction process (Table 5).
Table 5
Detection of E. granulosus s.l. species in faecal samples by duplex and triplex TaqMan® qPCRs
PCR Name
|
Specificity
|
1:100§
|
1:100*
|
1:1000*
|
1:10000*
|
1:100000*
|
G1_3_qPCR + IC_qPCR (duplex)
|
G1, G3
|
24.5
|
30.1 (± 0.6)
|
35.2 (± 1)
|
neg.
|
neg.
|
G4_qPCR + IC_qPCR (duplex)
|
G4
|
20.2
|
27.4 (± 0,5)
|
30.7 (± 1)
|
35.8 (± 0.06)
|
neg.
|
G5_10_qPCR + IC_qPCR (triplex)
|
G5
|
21,37
|
30.3 (± 0.17)
|
32.4 (± 0.22)
|
neg.
|
neg.
|
G5_10_qPCR + IC_qPCR (triplex)
|
G6-10
|
24.2
|
31.2 (± 0.02)
|
32.6 (± 0.22)
|
neg.
|
neg.
|
§Cq values from 1:100 pre-diluted DNA, extracted from cyst material |
*Cq-Values from re-extracted DNA prepared by logarithmic dilution of the corresponding DNA, used to spike faecal samples (± standard deviation) |
Taken together, we developed four sequence specific-DNA probe-based qPCR assays that allow differentiation of E. granulosus s.l. species detected in DNA samples derived from cyst material or spiked faecal matter, namely E. granulosus s.s. (G1-G3), E. equinus (G4), E. ortleppi (G5) and E. canadensis (G6-8, G10).