Generation and detection of TaqMan probe hydrolysis in iiPCR.

<p>Target pWSSV1 and pIMNV plasmids (10³ copies) were subjected to WSSV (A) and IMNV (C) iiPCR, respectively, for 30 min in the presence of WSSVprobe, IMNVprobe, or no probe. Fluorescent signals (520 nm) of individual reactions were collected before and after iiPCR by the modified iiPCR and shown below the gel image. After the reactions were completed, amplicons were also analyzed on a 12% polyacrylamide gel in 1X TAE buffer. WSSV (B) and IMNV (D) real time PCR assays containing the same components as in iiPCR were carried out as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045278#s2" target="_blank">Materials and Methods</a>. The result shown here is a representative of at least three experiments with similar results. Arrows, iiPCR amplicons; M, DNA size markers; NTC, no template control (ddH₂O); “before”, fluorescent signals detected before reaction; “after”, fluorescent signals detected after reaction; S/N ratio, signal intensity_after/signal intensity_before for iiPCR; signal intensity_{cycle 40}/signal intensity_{cycle 1} for real time PCR.