ZIKV sequences were downloaded from GenBank and aligned, and conserved sequences were identified. Primers were designed to amplify the NS5 gene while ensuring that the GC contents and Tm values were similar. The length of the target fragment was about 100 bp without hairpin structures to avoid the formation of stable dimers and mismatches at the 3' terminus. The designed primers are shown in Table 1.
Table 1. Primers and probes for ZIKV detection
Tenfold dilutions of ZIKV plasmid standards (100–108 copy/μL) were prepared. The relationship between the RTqPCR cycle threshold (Ct) value and copy number exhibited a good linear relationship as follows: Ct = - 3.524 log10 (copy)+38.04 (R2 = 0.999, amplification efficiency = 92.203%). Based on the standard curve, we found that the range of linearity for RT-qPCR was 101–108 copy/μL, and the lower detection limit for ZIKV was 101 copy/μL. However, at a concentration of 1 copy/μL, ZIKV could not be detected (Fig. 1A).
Figure 1. Sensitivity of RT-qPCR and ddPCR for detection of ZIKV. A Standard curve of the RT-qPCR method; B–E results of fivefold dilution series of ZIKV plasmid standards detected by ddPCR. B Scatter plot of the total number of events (droplets). Z1: plasmid standard, 2000 copy/lL; Z2–Z6: fivefold dilution series of Z1; N: negative control. The threshold is 1000. C Positive (blue) and total (green) number of events (droplets). D Genetic concentration of each diluted sample. E Comparison between ddPCR-detected concentration and theoretical concentration; #P>0.05 (not significant)
In comparison with RT-qPCR, the results from ddPCR showed that the method was not suitable for detecting samples containing more than 104 copy/μL due to poor repeatability (data not shown). Therefore, we prepared a fivefold dilution series of the standard of 104 copy/μL for ddPCR. The signal threshold of event numbers was set at 1000, with a value above 1000 being positive and a value below being negative (Fig. 1B), and the total numbers of droplets in the sample should ideally be above 10, 000. For our samples, the total event numbers were more than 10, 000 (except one sample is 9990) (Fig. 1C). High accuracy was obtained if the copy number of each diluted sample was in line with the theoretical value for the standard sample (Fig. 1D). Compared with RT-qPCR, ddPCR had a lower detection limit of 1 copy/μL (Fig. 1E).
To verify the repeatability of the two methods for ZIKV detection, we conducted each experiment three times and compared the results. The results of RT-qPCR showed that there was good repeatability in the dilution range of 101– 108 copy/μL but that the Ct value of 1 copy/μL was significantly different among experiments. This suggests that the RT-qPCR method is unstable for low-copy-number detection (100–101 copy/μL). However, ddPCR showed good repeatability for the detection of low copy numbers (100–101 copy/μL) (Fig. 2A, 2B).
Figure 2. Repeatability and specificity of RT-qPCR and ddPCR methods for ZIKV detection. A Construction of RT-qPCR standard curves using a tenfold dilution series of plasmid standard and repeated three times. Red circle: no Ct value; #P>0.05 (not significant), *P < 0.05 (significant difference). B Results from a fivefold dilution series of plasmid standard detected with ddPCR and repeated three times. #P>0.05 (not significant). C Scatter plot of the total number of events (droplets); the threshold for a positive result was 1000, and there were no positives. D Positive (blue) and total (green) number of events (droplets), with no positive events shown. DV1–4 represents four serotypes of dengue virus. N, negative control
Next, we evaluated the specificity of the assays using four dengue virus (DENV) serotypes. Each experiment was repeated three times, and the results were compared. All RT-qPCR results were negative. As can be seen from Fig. 2C and 2D, negative results were also obtained using ddPCR. The nucleic acid concentrations detected by the absolute quantification method were zero. Moreover, only negative micro-droplets were observed (Fig. 2C), and histogram analysis showed that the total number of droplets was more than 10, 000, among which none were positive (Fig. 2D).
Two serum samples from clinically positive patients (Sample A and Sample B) were tested using the two methods with three repeated wells each. According to RTqPCR analysis, the Ct value of sample A was 28.75, and the corresponding concentration of ZIKV was 432.5 copy/μL. There was no significant difference between this result and that obtained via ddPCR (Fig. 3A). For ddPCR, the three replicates of sample A exhibited good consistency (P>0.05) (Fig. 3B, 3C). By contrast, there was a significant difference between the results of the two detection methods for sample B (Fig. 3D). According to RT-qPCR, the Ct value of sample B was 38.868, which exceeded the detection range (range of Ct: 15–35). Comparison with the standard curve showed that no ZIKV was detected (Fig. 3D). However, ddPCR analysis showed that the concentration of sample B was 13.8–14.3 copy/μL (Fig. 3E), with more than 10, 000 total droplets, ensuring the accuracy of the results (Fig. 3F).
Figure 3. Results of ZIKV detection in positive clinical blood samples using RT-qPCR and ddPCR. A Comparison of Sample A concentration using the two methods; #P>0.05 (not significant); B, C results of ddPCR analysis of sample A in three repeated wells; D comparison of Sample B concentration using the two methods. Red circle: no virus detected; *P < 0.05 (significant difference); E, F results of ddPCR analysis of sample B in three repeated wells