Picard-Meyer E.,Who Collaborating Center For Res And Management In Zoonoses Control |
Peytavin De Garam C.,Who Collaborating Center For Res And Management In Zoonoses Control |
Schereffer J.L.,Who Collaborating Center For Res And Management In Zoonoses Control |
Marchal C.,Who Collaborating Center For Res And Management In Zoonoses Control |
And 2 more authors.
BioMed Research International | Year: 2015
This study evaluates the performance of five two-step SYBR Green RT-qPCR kits and five one-step SYBR Green qRT-PCR kits using real-time PCR assays. Two real-time thermocyclers showing different throughput capacities were used. The analysed performance evaluation criteria included the generation of standard curve, reaction efficiency, analytical sensitivity, intra- and interassay repeatability as well as the costs and the practicability of kits, and thermocycling times. We found that the optimised one-step PCR assays had a higher detection sensitivity than the optimised two-step assays regardless of the machine used, while no difference was detected in reaction efficiency, R2 values, and intra- and interreproducibility between the two methods. The limit of detection at the 95% confidence level varied between 15 to 981 copies/μL and 41 to 171 for one-step kits and two-step kits, respectively. Of the ten kits tested, the most efficient kit was the Quantitect SYBR Green qRT-PCR with a limit of detection at 95% of confidence of 20 and 22 copies/μL on the thermocyclers Rotor gene Q MDx and MX3005P, respectively. The study demonstrated the pivotal influence of the thermocycler on PCR performance for the detection of rabies RNA, as well as that of the master mixes. © 2015 Evelyne Picard-Meyer et al.