Amp-PCR: Combining a Random Unbiased Phi29-Amplification with a Specific Real-Time PCR, Performed in One Tube to Increase PCR Sensitivity

In clinical situations where a diagnostic real-time PCR assay is not sensitive enough, leading to low or falsely negative results, or where detection earlier in a disease progression would benefit the patient, an unbiased pre-amplification prior to the real-time PCR could be beneficial. In Amp-PCR, an unbiased random Phi29 pre-amplification is combined with a specific real-time PCR reaction. The two reactions are separated physically by a wax-layer (AmpliWax®) and are run in sequel in the same sealed tube. Amp-PCR can increase the specific PCR signal at least 100×106-fold and make it possible to detect positive samples normally under the detection limit of the specific real-time PCR. The risk of contamination is eliminated and Amp-PCR could replace nested-PCR in situations where increased sensitivity is needed e.g. in routine PCR diagnostic analysis. We show Amp-PCR to work on clinical samples containing circular and linear viral dsDNA genomes, but can work well on DNA of any origin, both from non-cellular (virus) and cellular sources (bacteria, archae, eukaryotes)

Quantitative Assessment of the Sensitivity of Various Commercial Reverse Transcriptases Based on Armored HIV RNA

The in-vitro reverse transcription of RNA to its complementary DNA, catalyzed by the enzyme reverse transcriptase, is the most fundamental step in the quantitative RNA detection in genomic studies. As such, this step should be as analytically sensitive, efficient and reproducible as possible, especially when dealing with degraded or low copy RNA samples. While there are many reverse transcriptases in the market, all claiming to be highly sensitive, there is need for a systematic independent comparison of their applicability in quantification of rare RNA transcripts or low copy RNA, such as those obtained from archival tissues.We performed RT-qPCR to assess the sensitivity and reproducibility of 11 commercially available reverse transcriptases in cDNA synthesis from low copy number RNA levels. As target RNA, we used a serially known number of Armored HIV RNA molecules, and observed that 9 enzymes we tested were consistently sensitive to ∼1,000 copies, seven of which were sensitive to ∼100 copies, while only 5 were sensitive to ∼10 RNA template copies across all replicates tested. Despite their demonstrated sensitivity, these five best performing enzymes (Accuscript, HIV-RT, M-MLV, Superscript III and Thermoscript) showed considerable variation in their reproducibility as well as their overall amplification efficiency. Accuscript and Superscript III were the most sensitive and consistent within runs, with Accuscript and Superscript II ranking as the most reproducible enzymes between assays.We therefore recommend the use of Accuscript or Superscript III when dealing with low copy number RNA levels, and suggest purification of the RT reactions prior to downstream applications (eg qPCR) to augment detection. Although the results presented in this study were based on a viral RNA surrogate, and applied to nucleic acid lysates derived from archival formalin-fixed paraffin embedded tissue, their relative performance on RNA obtained from other tissue types may vary, and needs future evaluation