To develop the 2-D multiplex qPCR assay, specific primers and probes for nine HHVs, as well as one human housekeeping gene GAPDH were used. The probes for HSV-2, HHV-7 and GAPDH were labeled with Texas red, for EBV, HHV-6 (covering HHV-6A and -6B) and KSHV with CY5 and for HSV-1, VZV and HCMV with HEX (Supplementary Table S1). To distinguish the targets detected by probes with same fluorescence dye, the specific products were designed to have different Tm values with a Tm interval of at least 1 ℃. To assess whether the presence of herpesvirus variants occurring in the amplicon area of different herpesvirus strains alters the expected Tm, we downloaded all available genome sequences of nine herpesviruses from GenBank (Oct 26, 2020), and predicted the Tm values of all unique sequences in amplicon area. Nine herpesviruses appear to be very conserved with few variants at least in the region of amplicon (data not shown), and predicted Tm value has very little variability (Supplementary Table S1). To obtain the actual Tm values of each herpesvirus, we performed amplification and melting curve analyses using each herpesvirus plasmid with ten replicates. The actual Tm values were determined as 87.9 ℃, 79.7 ℃, 82.0 ℃ for HSV-2, HHV-7, GAPDH; and 82.8 ℃, 79.0 ℃, 84.8 ℃ for EBV, HHV-6 (covering HHV-6A and -6B), KSHV; 83.1 ℃, 86.3 ℃ and 84.0 ℃ for HSV-1, VZV and HCMV, respectively (Fig. 1 and Supplementary Table S2). The actual Tm values of each amplicon were very stable with a very small variation (Supplementary Table S1), and well matched the predicted Tm values for most herpesviruses except HSV-1 and HHV-7 that had a slight difference in Tm values between the predicted and actual values (Supplementary Tables S2). Importantly, the herpesviruses detected by the same fluorescent channel can be well distinguished from each other by their corresponding Tm values.
We assessed the performance of the 2-D multiplex qPCR assay using nine plasmids containing specific genomic segments of eight HHVs (HHV-6A and -6B sharing the same sequence) and a human gene GAPDH. The targets bound by the probes with same fluorescence dye (measured by the same fluorescence channel) were well distinguished by their corresponding Tm values when pooled templates were used (Fig. 2A). On the contrary, three amplicons with similar Tm values were easily distinguished by different fluorescent colors of probes (Fig. 2B). When combining the fluorescent color of probe and Tm value of amplicon, each gene was clearly distinguished in this multiplex qPCR assay (Fig. 2).
Figure 2. Proof-of-concept validation of the 2D-multiplex qPCR assay. A The co-existing targets detected by the same color probe are distinguished by different Tms. B Amplicons with similar Tms are distinguished by different color probes. The fluorescence channels used are shown in each plot. NTC non-template control.
To evaluate the capacity of the single-tube multiple qPCR assay in detection of co-existing targets, we tested all possible combinations (total: 36) of any two of the 9 plasmids. The co-existing targets sharing the same (fluorescent color) amplification curve but having two different Tm peaks, or having two amplification curves with different fluorescent colors but sharing similar Tm values, was very easily to be distinguished (Supplementary Fig. S1A and 1B). For the co-existing targets having two different amplification curves and two different Tm peaks (Supplementary Fig. S1C), they can be preferentially determined by their Tm values. To facilitate the judgment of Tm values of specific targets, a pooled template containing HSV-2, HHV-7 and GAPDH, which have Tms of 87.9 ℃, 79.7 ℃, and 82.0 ℃, respectively, was used as control for Tm references. By comparison with the reference Tm values, all co-existing targets having different color amplification curves and different Tm peaks were able to be correctly determined (Supplementary Fig. S1C).
To evaluate the sensitivity of the multiplex qPCR assay, tenfold serial dilutions of each plasmid from 3 × 107–3 × 101 copies/μL were tested (Supplementary Fig. S2). The results showed that the sensitivity was 300 copies per 25 μL reaction for HSV-1, HSV-2, VZV, EBV, HCMV, HHV-6A/B and GAPDH, 30 copies per 25 μL reaction for HHV-7 and KSHV. Specificity test showed that except GAGDH, there was no amplification for other fourteen common human viruses including seven DNA viruses by the multiplex qPCR assay, indicating that the new assay is specific to HHVs (Supplementary Fig. S3). We further tested whether human genomic DNA (gDNA) affects the specificity and amplification of the assay. The results showed that human gDNA does not inhibit the amplification of the multiplex qPCR assay (Supplementary Fig. S4).
To evaluate the accuracy of the single-tube multiplex qPCR assay for the detection of nine HHVs, 170 clinical samples collected from First Affiliated Hospital of Kunming Medical University and Taizhou Fourth People's Hospital were tested. From 149 whole-blood samples, the multiplex qPCR assay detected 30 positive samples for only one of nine HHVs, including 19 EBV, 6 HCMV, 1 HHV-7, and 4 KSHV (Table 1). In addition, 14 samples were detected as coinfection by two HHVs, including 5 by HSV-2 and HCMV, 8 by EBV and HCMV, and 1 by HHV-1 and KSHV (Table 1). From 21 vesicular fluid samples, the multiplex qPCR assay detected 14 positive samples for only one of nine HHVs, including 4 HSV-1, 3 HSV-2 and 7 VZV (Table 2). In addition, 5 samples were detected as coinfection by two HHVs, including 1 by HSV-2 and EBV, 1 by HSV-2 and HCMV, 1 by HSV-2 and HHV-7, 1 by VZV and EBV, and 1 by VZV and KSHV (Table 2). In particular, we detected one sample to be infected by three HHVs (HSV-1, HSV-2 and HCMV) and one sample with four HHVs (HSV-2, VZV, HHV-7 and KSHV) (Table 2). To evaluate the performance of the single-tube multiplex qPCR assay, all these samples were also tested by the single qPCR assay. The results of the single-tube multiplex qPCR assay were completely consistent with those of the single qPCR assay, indicting a 100% consistence rate with the latter (Tables 1 and 2). Among these samples, 73 (42.9%) samples were positive for at least one of all nine HHVs, and the EBV was the most commonly detected HHVs (Table 3).
Herpesvirus 2-D Multiplex qPCR assay (%) Single qPCR assay (%) Concordance rate (%) Positive EBV 19/149 (12.8) 19/149 (12.8) 100 HCMV 6/149 (4.0) 6/149 (4.0) 100 HHV-6A/B 0/149(0) 0/149 (0) 100 HHV-7 1/149 (0.7) 1/149 (0.7) 100 KSHV 4/149 (2.7) 4/149 (2.7) 100 HSV-2 & CMV 5/149 (3.4) 5/149 (3.4) 100 HSV-2 & EBV 8/149 (5.4) 8/149 (5.4) 100 EBV & HCMV 8/149 (5.4) 8/149 (5.4) 100 HHV-7 & KSHV 1/149 (0.7) 1/149 (0.7) 100 Negative 97/149 (65.1) 97/149 (65.1) 100 Total 149 /149 (100) 149 /149 (100) 100
Table 1. Evaluation of the single-tube nonuple qPCR assay for nine human herpesviruses and the reference gene GAPDH using 149 whole-blood samples.
Herpes virus 2-D Multiplex qPCR assay (%) Single qPCR assay (%) Concordance rate (%) Positive HSV-1 4/21 (19.0) 4/21 (19.0) 100 HSV-2 3/21 (14.3) 3/21 (14.3) 100 VZV 7/21 (33.3) 7/21 (33.3) 100 HSV-2 & EBV 1/21 (4.8) 1/21 (4.8) 100 HSV-2 & HCMV 1/21 (4.8) 1/21 (4.8) 100 HSV-2 & HHV-7 1/21(4.8) 1/21(4.8) 100 VZV & EBV 1/21 (4.8) 1/21 (4.8) 100 VZV & KSHV 1/21 (4.8) 1/21 (4.8) 100 HSV-1 & HSV-2 & HCMV 1/21 (4.8) 1/21 (4.8) 100 HSV-2 & VZV & HHV-7 & KSHV 1/21 (4.8) 1/21 (4.8) 100 Negative 0/21(0) 0/21 (0) 100 Total 21/21 (100) 21/21 (100) 100
Table 2. Evaluation of the single-tube nonuple qPCR assay for nine human herpesviruses and the reference gene GAPDH using 21 vesicular fluid samples.
Proportion in all samples (%) Proportion of positive samples (%) HHV single infection rate 25.9 60.3 HHVs double infection rate 15.9 37 HHV multiple infections rate 1.2 2.7 Common HHV single infection rate EBV 11.2 26 VZV 4.1 9.6 CMV 3.5 8.2 KSHV 2.3 5.5 HSV-1 2.3 5.5 HSV-2 1.7 4.1 High proportion of HHV among multiple infections HSV-2 10.6 24.7 EBV 10.6 24.7 CMV 8.8 20.5 VZV 1.8 4.1 HHV-7 1.8 4.1 KSHV 1.8 4.1 HSV-1 0.6 1.4 A total of 170 samples were tested
Table 3. Detection rates of various HHVs.
On the other hand, in order to determine a suitable cutoff of the reference gene for the assay, we analyzed the Ct values of GAGDH in clinical samples. Because HSV-2 and HHV-7 share same fluorescent channel with GAGDH, 146 tested samples (including 97 negative samples but excluding 24 samples positive for HSV-2 and HHV-7) were used. The mean Ct value of GAGDH gene was 26.06 ± 3.66 with a scope of 19.06–35.13. For caution, a Ct value of 36 is recommended as the cutoff of GAGDH gene for the assay.
We further analyzed the Tm values of amplicons of clinical samples. For each herpesvirus, the Tm values of clinical samples had small variation, and were very similar to those of plasmid standard (Supplementary Table S2). These indicate that the multiplex qPCR assay has enough discrimination for the herpesviruses detected by the same fluorescent channel.