A mismatch-tolerant RT-quantitative PCR: application to broad-spectrum detection of respiratory syncytial virus

Quantitative PCR (qPCR) is widely used to detect viruses. However, mismatches occurring in the 3'-end of the primers reduce amplification efficiency of qPCR and limit its capacity in detection of highly variable viruses. Here, we reported a mismatch-tolerant RT-qPCR with a small amount of additional high-fidelity DNA polymerase for simultaneous detection of RSV-A and RSV-B. The novel assay had higher amplification efficiency for various variants forming mismatches with the primers than the conventional RT-qPCR, and showed good specificity and sensitivity. It demonstrateda good correlation coefficient with a commercial RSV detection kit and had relatively lower Ct values than the kit for 16 of 20 RSV-positive samples. The mismatch-tolerant qPCR technique is a promising approach for sensitive detection of highly variable viruses. METHOD SUMMARY We developed a mismatch-tolerant RT-quantitative PCR by simply adding a small amount of additional high-fidelity DNA polymerase to the standard reaction mixtures. The new method well tolerates mismatches between primers and templates and is especially suited to the detection of highly variable viruses.