Computational protocol: Analytical Performance of a Multiplex Real-Time PCR Assay Using TaqMan Probes for Quantification of Trypanosoma cruzi Satellite DNA in Blood Samples

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Protocol publication

[…] On the basis of a previously reported TaqMan procedure for detection of T. cruzi satellite DNA that showed high sensitivity and specificity in an international PCR study , we assayed the same T. cruzi primers and probe and designed a set of primers and probe for the IAC target (). The melting temperatures of IAC Fw and IAC Rv primers are similar to those of Cruzi 1 and Cruzi 2 primers (61.2°C, 60.9°C, 58.4°C and 59.5°C, respectively) using Oligo Calculator version 3.26 at http://www.basic.northwestern.edu/biotools/oligocalc.html.The qPCR reactions were carried out with 5 µL of re-suspended DNA, using FastStart Universal Probe Master Mix (Roche Diagnostics GmbHCorp, Mannheim, Germany) in a final volume of 20 µL. Optimal cycling conditions were a first step of 10 min. at 95°C followed by 40 cycles at 95°C for 15 sec. and 58°C for 1 min. The amplifications were carried out in a Rotor-Gene 6000 (Corbett, UK) or in an Applied Biosystems (ABI 7500, USA) device. Standard curves were constructed with 1/10 and 1/2 serial dilutions of total DNA obtained from a GEB sample spiked with 105 par. eq./mL of blood. TcI and TcVI based standard curves were used to quantify parasitic loads in G1 and in G2–G5 samples, respectively.In order to evaluate the influence of the concentrations of IAC template, primers and probe in the efficiency of T. cruzi DNA amplification in the multiplex format, DNA extracts from samples carrying 0.5 to 750 par. eq./mL as well as samples without T. cruzi were amplified by both simplex qPCR (only T. cruzi primers and probe) and multiplex qPCR formats.In order to assess the influence of T. cruzi load on the efficiency of IAC amplification in the multiplex format, T. cruzi DNA samples obtained to build the CL-Brener standard curve were amplified and the IAC was quantified. For this, a standard curve was built with DNA obtained from 300 µL of GEB spiked with 50 to 800 pg of linear IAC on duplicate as well as the PCR assay from each DNA lysate. [...] The LOQ was derived from a 20% threshold value for the coefficient of variation (CV) of measurements obtained in the precision experiments, following the recommendations of NCCLS document EP17-A . Assuming an exponential decrease in CV, a curve for the relationship between CV and log10 par. eq./10 mL was fitted using SigmaPlot version 10.0 for Windows (SPSS, Chicago, IL). […]

Pipeline specifications

Software tools Biotools, OligoCalc, SigmaPlot, SPSS
Applications Miscellaneous, Population genetic analysis, qPCR
Organisms Trypanosoma cruzi, Trypanosoma cruzi strain CL Brener, Homo sapiens
Diseases Infection, Chagas Disease
Chemicals Guanidine