Identification of enterovirus 71 hand, foot and mouth disease from other enteroviruses by microfluidic digital PCR

Fig. 1. Determination of Standard samples. (a) EV71 VP2 gene inserted into pET28a(+) vector to construct the recombinant plasmid pET28a(+)-EV71VP2. (b) Target gene of EV71 VP2 was 63bp in qPCR and cdPCR.

Fig. 2. Different concentrations of primers and the probe in quantitative PCR

Fig. 3. Evaluation of Quantitative PCR. (a) Standard regression equation of qPCR was y=−3.206x + 36.6 and R2 = 0.9944. (b) The CT value of EV71 is 13.785 while the CT values of other viruses were above 32. (c) and (d) The CV(%) values of qPCR were range from 0.32%−4.93%, all which were under 10.0%

Fig. 4. Standard curve of EV71 chip digital PCR. (a) to (e) The measured values of standard plasmid templates (105 copies/µL to 101 copies/µL) were 7.165copies/µL, 51.426copies/µL, 304.24copies/µL, 2511.3copies/µL, 25318.7copies/µL, respectively. The yellow dots was VIC and the blue dots was FAM. (f) The calibration curve equation of cdPCR was y = 0.8785x- 0.06496 and the kappa value was 0.9977 by Graphpad prims 7.0 analyzing. (Note: Blue dots represent FAM signals. Yellow dots denote no-amplications signals. Red dots represent VIC signals. And green dots represent FAM and VIC signals.)

Fig. 5. The specificity of EV71 chip digital PCR. The measured concentration values of CVA16 (a), CVA6 (b), CVA4 (c), CVA2 (d), CVA10 (e), CVB3 (f), H1N1 (g), H3N2 (h), herpangina virus (i) and SARS-COV-2 (j) were less than 0.123copies/µL. The EV71 positive plasmid was positive amplification (k). This results showed that EV71 cdPCR had excellent specificity. (Note: Blue dots represent FAM signals. Yellow dots denote no-amplications signals. Red dots represent VIC signals. And green dots represent FAM and VIC signals.)

Fig. 6. The repeatability of EV71 chip digital PCR. (a) The CV% of EV71 cdPCR. Using standard plasmids of different concentrations for cdPCR detection, the CV% values obtained were all less than 5%. (b) The comparison between the average CV% of EV71 cdPCR and the average CV% of EV71 qPCR. The average CV% of EV71 cdPCR was was 1.09%, which was lower than that of EV71 qPCR. It showed that EV71 cdPCR has good repeatability. (Note: The formula for calculating the coefficient of variation (CV) is: CV = Standard Deviation / Mean. Specifically, the approximate allowable range for the CV value of blood/biochemical quantitative items is 5% - 15%, while the approximate allowable range for semi-quantitative/qualitative/immunological/coagulation/serum items is 6% - 20%.)