Sensitivity of multiplex real-time PCR reactions, using the LightCycler and the ABI PRISM 7700 Sequence Detection System, is dependent on the concentration of the DNA polymerase

Detection of Biological Threat Agents by Real-Time PCR: Comparison of Assay Performance on the R.A.P.I.D., the LightCycler, and the Smart Cycler Platforms

“…Patients with Known MTHFR Genotypes, Anna Pastore, 1 Sandro De Angelis, 3 Stefania Casciani, 2 Rosalba Ruggia, 2 Gianna Di Giovamberardino, 1 Annalisa Noce, 3 Giorgio Splendiani, 3 Claudio Cortese, 2 Giorgio Federici, 2 12 . Samples were drawn before administration of either, after the first and second periods, and again 2 months after treatment.…”

“…In a study by Kaplan et al (9 ), vitamin B 12 supplementation alone, or in combination with folic acid, decreased tHcy concentrations, but full normalization was not achieved. Dierkes et al (10 ) reported that supplementation with vitamin B 12 decreases not only tHcy but also serum folate in patients with Clinical Chemistry 52, No. 1,2006 …”

Rapid Simultaneous Detection by Real-Time PCR of Cytomegalovirus UL97 Mutations in Codons 460 and 520 Conferring Ganciclovir Resistance

“…The reaction mixture utilized a master mix containing a LightCycler Fast Start hybridization probe kit (Roche Diagnostics, Mannheim, Germany) 1ϫ, 4.5 mM MgCl 2 , 0.6 M of each primer, 0.2 M of each hybridization probe, and an additional 2.5 U/reaction Fast Start Taq DNA polymerase (Roche Diagnostics, Mannheim, Germany) for increase of the sensitivity of the amplification reaction. An enhanced concentration of Fast Start Taq DNA polymerase (5 U/l) in the reaction mixture resulted in a higher sensitivity of our assay (8). The following program was used for cycling: initial denaturation, enzyme activation at 95°C for 10 min; denaturation step at 95°C for 15 s; annealing step at 50°C for 20 s, elongation step at 72°C for 20 s, repeat for 50 cycles; and melting curve analysis at 95°C for 0 s, 50°C for 15 s, and 85°C for 0 s, with a slope of 0.1 (°C/s).…”

Development of multiplex suspension microarrays for the detection of alphaviruses and flaviviruses of public health significance

“…Other diagnostic tests, such as TaqMan assays, demonstrate a loss of sensitivity when multiplexed, as decreased PCR efficiency can result due to the addition of multiple primers and probes in a tube, primer-dimer formation and potential competition from varying template concentrations (Exner & Lewinski, 2002). The few studies available at the time this project commenced suggested this problem was overcome with suspension microarray technology (Diaz & Fell, 2004, Diaz et al, 2006.…”

“…In general, it is recognised that multiplexed PCRs do show a loss of sensitivity (Exner & Lewinski, 2002). However, if time had permitted, we would have attempted additional work to overcome this problem.…”