Dual-color highly sensitive polyomavirus (BKV/JCV) detection

The AlloDx two-color highly sensitive polyomavirus detection project can obtain BKV and JCV virus results in one test, saving costs and time for patients.

1. Combined BKV and JCV specific sequence detection can simultaneously identify whether samples contain bothBKV and JCVtwo viruses

Positive samples of BKV, JCV, and SV40 with similar viral sequences were used as DNA templates to perform single-plex fluorescence quantitative PCR detection. In the BKV template, only the BK Taqman probe has an amplification curve (Figure 1A); in the JCV template, only the JC Taqman probe has an amplification curve (Figure 1B); in the SV40 template, there is no "S" type amplification curve (Figure 1C).

Figure 1

2. The minimum detection can reach 102 copies

BKV and JCV recombinant plasmids were1~5×107copies/µL as a template to perform single-plex fluorescence quantitative PCR to test the sensitivity of the system. From the amplification curve, it can be determined that 5 × 102copies/µL is the minimum detection volume. Figure 2A shows the BKV virus, and Figure 2B shows the JCV virus.

Figure 2

3. Multiple verifications to improve detection accuracy and reliability and prevent missed detections

Place 5×104、5×105、5×106 copies/µL Three different concentrations of BKV recombinant plasmid and JCV recombinant plasmid were used as templates to conduct three intra-batch and inter-batch parallel experiments to calculate the coefficient of variation (CV). The results are shown in Table 1. The coefficients of variation within and between batches were less than 5%.

4. Single reaction tube dual-color fluorescence PCR detection

In order to verify whether the double quantification results would affect the accuracy of quantification, different concentrations of BKV recombinant plasmids and JCV recombinant plasmids were mixed, and double fluorescence PCR and single-plex fluorescence PCR were performed respectively. The results are shown in Table 2. The Ct value of dual PCR is no different from that of single PCR (p>0.05). Dual fluorescence PCR can accurately quantify BKV and JCV in samples.