Table 3.
Standard curve, amplification efficiency, coefficient of correlation, LOD, repeatability, and reproductibility of the 13 SYBR® Green qPCR assays for detection and discrimination of pathogenic E. coli
| E. coli markers | EIEC and Shigella markers | EPEC and EHEC markers | STEC and EAggSTEC markers | EAEC and EAggSTEC markers | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Accepted range | uidA-3 | uidA-7 | ipaH-569 | ipaH-3 | eae-185 | eae-EBP-1 | stx1and2–4 | stx1–185 | stx2–81 | aggR-185 | aggR-2 | aaiC-EBP1 | aaiC-EBP2 | |||
| Standard curve equation | − 3.1 < slope < − 3.6 | − 3.1(logX) + 34.7 | − 3.2(logX) + 35.1 | − 3.3 (logX) + 32.1 | − 3.3 (logX) + 32.5 | − 3.1(logX) + 34.7 | − 3.2(logX) + 35.4 | − 3.2(logX) + 36.3 | − 3.3(logX) + 35.7 | − 3.3 (logX) + 35.3 | − 3.3 (logX) + 34.6 | − 3.4 (logX) + 35.0 | − 3.4(logX) + 35.7 | − 3.5 (logX) + 36.2 | ||
| Amplification efficiency (E) | 89.6–110.2% | 108.1% | 105.3% | 100.1% | 99.3% | 108.5% | 104.0% | 106.4% | 100.4% | 103.0% | 100.0% | 97.4% | 95.3% | 93.3% | ||
| Coefficient of correlation (R2) | > 0.98 | 0.99 | 0.99 | 1.00 | 1.00 | 1.00 | 0.99 | 0.97 (stx1)/0.98 (stx2) | 1.00 | 0.99 | 1.00 | 1.00 | 0.98 | 1.00 | ||
| LOD (n = 36) in copies | ≤ 10 | 2–5 | 5–10 | nt | nt | 5–10 | 2–5 | nt | nt | nt | nt | nt | 5–10 | 1–2 | ||
| Check LOD (n = 12) in copies | ≤ 10 | nt | nt | <1 | <1 | nt | nt | 5–2 | 10–5 | 5–2 | 5–2 | 5–2 | nt | nt | ||
| Repeatability | Isolate 1 | LOD Cq RSDr (%) | ≤ 25% | 2.4 | 2.5 | na | na | 2.1 | 3.5 | na | na | na | na | na | 1.9 | 3.1 |
| LOD Cq r | na | 2.2 | 2.2 | na | na | 1.9 | 3.2 | na | na | na | na | na | 1.7 | 2.9 | ||
| Tm value ± SD (°C) | na | 78.2 ± 0.26 | 76.75 ± 0.26 | na | na | 75.9 ± 0.21 | 74.04 ± 0.32 | na | na | na | na | na | 73.9 ± 0.18 | 74.7 ± 0.23 | ||
| Tm RSDr (%) | ≤25% | 0.3 | 0.3 | na | na | 0.3 | 0.4 | na | na | na | na | na | 0.2 | 0.3 | ||
| Tm r | na | 0.7 | 0.7 | na | na | 0.6 | 0.9 | na | na | na | na | na | 0.5 | 0.7 | ||
| Isolate 2 | LOD Cq RSDr (%) | ≤25% | 2.3 | 2.4 | na | na | 2.6 | 2.6 | na | na | na | na | na | 2.5 | 3.8 | |
| LOD Cq r | na | 2 | 2.2 | na | na | 2.3 | 2.4 | na | na | na | na | na | 2.2 | 3.7 | ||
| Tm value ± SD (°C) | na | 78.2 ± 0.23 | 77.04 ± 0.16 | na | na | 75.9 ± 0.23 | 74.41 ± 0.19 | na | na | na | na | na | 73.8 ± 0.23 | 74.7 ± 0.26 | ||
| Tm RSDr (%) | ≤25% | 0.3 | 0.2 | na | na | 0.3 | 0.3 | na | na | na | na | na | 0.3 | 0.4 | ||
| Tm r | na | 0.6 | 0.4 | na | na | 0.6 | 0.5 | na | na | na | na | na | 0.7 | 0.7 | ||
| Reproductibility | Maximum RSDR (%) | Cq | ≤ 35% | 6.23 | 6.89 | 5.24 | 5.95 | 2.54 | 4.36 | 1.75 | 3.16 | 2.72 | 3.19 | 3.64 | 2.58 | 3.91 |
| T m | ≤35% | 0.72 | 0.87 | 0.54 | 0.27 | 0.7 | 0.77 | 0.59 | 0.46 | 0.94 | 0.24 | 0.15 | 0.34 | 0.62 | ||
| Uncertainty at 99% | Cq (# positive sample/12 samples) | na | 2.96 (12) | 3.97 (12) | 1.20 (1) | 1.37 (1) | 1.01 (5) | 1.51 (5) | 0.92 (6) | 1.24 (5) | 1.38 (7) | 0.96 (1) | 1.14 (1) | 0.83 (1) | 1.23 (1) | |
| Tm (# positive sample/12 samples) | na | 1.09 (12) | 1.15 (12) | 0.4 (1) | 0.2 (1) | 0.7 (5) | 0.84 (5) | 0.59 (6) | 0.45 (5) | 1.09 (7) | 0.17 (1) | 0.10 (1) | 0.23 (1) | 0.44 (1) | ||
Standard curves, E, and R2 were obtained from two replicates for each concentration (from 10,000 copies to 1 copy) from two different strains. LOD of chromosomic targets is based on 18 repetitions of each dilution (10-5-2-1-0.5-0.2-0.1 genomic copies per well) with the two bacterial strains as DNA template (36 repeats in total). Check of LOD is performed for non-chromosomic targets, based on six repeats of dilution points (10-5-2-1 genomic copies per well) with two bacterial strains as DNA template. Repeatability is calculated on the LOD determination test
X copy number, LOD limit of detection, na not applicable, # number, nt not tested