Article Figures & Data
Figures
- FIG. 1.
Measurement of viral amounts by real-time PCR with the MGB probe and PNAs. The table shows patterns of combination of the MGB probe and noncomplementary PNAs.
- FIG. 2.
(a) PCR with a complementary MGB probe. During the extension phase with primers, the DNA polymerase cleaves the reporter dye from the probe, and the dye emits its characteristic fluorescence. When a PNA added to the reaction mixture is noncomplementary to the target sequence, it does not affect amplification with a complementary MGB probe. NFQ, nonfluorescent quencher. (b) PCR with a complementary PNA. The mismatch of an MGB probe noncomplementary to the target sequence is blocked by the PNA, which is preferentially bound at a higher melting temperature than the MGB probe.
- FIG. 3.
Calculation of values from the results obtained by real-time PCR. The threshold was set in the linearly increasing region of accruals of fluorescence signals (ΔRn), and the threshold cycle (CT ) was measured. The value of the target strain was calculated from the CT number and a standard curve for each strain. The measurement was performed separately with each MGB probe and noncomplementary PNAs for all sets of target strains. Solid lines, 10-fold serially diluted standards; dotted line, a sample of the target strain.
- FIG. 4.
Measurement of a target strain in mixed strains. Samples of a 10-fold serial dilution of the YVDD strain were measured under the condition of coexistence of 6 log10 copies/ml of the YIDD2 strain. Line A, MGB for YVDD; line B, MGB for YVDD with PNAs for YMDD, YIDD1, and YIDD2. Linearity was maintained even at the lower concentration range.
- FIG. 5.
Clamping effects of PNA on PCR. Delays in the CT with different concentrations of complementary (solid lines) and noncomplementary (dashed lines) PNAs were measured with the MGB probe for the LLAQ strain (filled circles) and the LMAQ strain (open triangles). PNAs used were complementary (A) or noncomplementary (B) to the LLAQ strain and complementary (C) or noncomplementary (D) to the LMAQ strain. A delay in the CT was observed even with noncomplementary PNAs when the concentration was high. The differences in delays between PNAs complementary and noncomplementary to the LLAQ strain are similar to those between PNAs complementary and noncomplementary to the LMAQ strain.
- FIG. 6.
Real-time PCR with the MGB probe and PNAs. Samples of a 10-fold serial dilution of the YIDD1 strain were measured under the condition of coexistence of 9 log10 copies/ml of YMDD strain with an MGB probe complementary to YIDD1 and PNAs complementary to an YMDD motif other than YIDD1 (YMDD, YIDD2, and YVDD). ΔRn, accruals of a fluorescence signal.
- FIG. 7.
(a) Course of a clinical case. HBV DNA was measured by a transcription-mediated amplification assay (logarithmic genome equivalent [LGE] per milliliter). L, M, I, and V of rt180 or rt204 represent the amino acids leucine, methionine, isoleucine, and valine, respectively. (b) Changes in viral amounts of the LLAQ and YMDD motifs. ALT levels were stable while the LLAQ/YIDD strain was dominant. When the LMAQ/YVDD strain became dominant over preexisting strains, breakthrough hepatitis occurred.
