Table 3.
The sensitivity evaluation of newly designed primers and probes using real-time qPCR and digital PCR.
| Leptospira species |
L. interrogans |
L. interrogans |
L. biflexa |
|||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| primer-probe sets |
lipl32_F&R_P |
fliG_F&R_P1b |
fliG_F&R_P2 |
|||||||||
| Assays | real-time qPCR | dPCR | real-time qPCR | dPCR | real-time qPCR | dPCR | ||||||
| Quantitya | Ct mean ± SDc | RSD (%)d | Total copies mean ± SDe | RSD (%)d | Ct mean ± SDc | RSD (%)d | Total copies mean ± SDe | RSD (%)d | Ct mean ± SDc | RSD (%)d | Total copies mean ± SDe | RSD (%)d |
| 20,000 | 29.53 ± 0.17 | 0.56 | 3,334.47 ± 110.85 | 3.32 | 28.99 ± 0.05 | 0.16 | 4,484.67 ± 163.12 | 3.64 | 31.45 ± 0.06 | 0.20 | 5,800 ± 448.54 | 7.73 |
| 2000 | 33.34 ± 0.08 | 0.24 | 372.50 ± 47.22 | 12.68 | 32.42 ± 0.15 | 0.46 | 464.47 ± 24.17 | 5.20 | 34.45 ± 0.78 | 2.27 | 602.20 ± 38.56 | 6.40 |
| 200 | 36.71 ± 0.45 | 1.21 | 35.92 ± 2.98 | 8.29 | 35.38 ± 0.23 | 0.66 | 55.07 ± 10.25 | 18.61 | 38.31 ± 0.30 | 0.79 | 57.88 ± 8.05 | 13. 90 |
| 20 | NA | NA | 3.93 ± 0.85 | 19.68 | 39.37 ± 0.10 | 0.24 | 8.25 ± 0.37 | 4.47 | NA | NA | 4.74 ± 0.95 | 20. 12 |
| 2 | NA | NA | NA | NA | NA | NA | NA | NA | NA | NA | NA | NA |
| 0 | NA | NA | NA | NA | NA | NA | NA | NA | NA | NA | NA | NA |
a
10-fold serial dilution (from 0.02 ng to 2 fg) of genomic DNA extracted from Leptospira species.
b
Developed in this study.
c
the cycle threshold (Ct) values (Ct < 40, positive; otherwise, negative); Mean ± SD, all the Ct values were the average of the three experiments ± standard deviation; NA: samples with no amplification detected.
d
RSD (%), relative standard deviation; used to analyze the precision of detection work, RSD (%) < 25 %, indicated this system has good repeatability.
e
Total number of copies detected by digital PCR (dPCR); Mean ± SD, all the average of the six experiments ± standard deviation; NA: samples with no amplification detected.