Evaluate the efficiency of agilent suredirect blood PCR KIT

Abstract

In medical fields, the interest for direct amplification from blood has been increased rapidly, leading to the growing demand for direct DNA amplification from blood However, this direct assay may encounter major obstacle from potential inhibitors that present as blood components, or be introduced during preservation such as anticoagulants. Among commercial direct PCR kits, recently, the AGILENT SureDirect Blood PCR Kit had been developed and recommended as a convenient tool with powerful inhibitor tolerance for rapid PCR directly on blood samples. Accordingly, the aim of this study is to obtain optimal conditions and evaluate the efficiency of direct PCR by the SureDirect Kit in used for genetic diagnosis purpose. Target to be amplified was a highly conserved 121-bp sequence located on miRNA196a2. The archived optimal conditions for the SureDirect Kit, 5% (vol/vol) of whole blood sample and 10μL final volume, were validated successfully on 44 EDTAblood samples frozen for 1 year and 29 EDTA-blood samples in 2 conditions: fresh and 1-month freezing. None of validation reactions were failed, making the successful rate of the SureDirect Kit, when perform with the archived optimal conditions, approach to 1.0. Besides, it was suggested that storage duration seems to have subtle effect on lowering PCR product quality but no significant effect on the success of direct amplification. In conclusion, the AGILENT SureDirect Blood PCR Kit is suggested as an applicable and economical direct PCR tool for genetic diagnosis purposes. Keywords: AGILENT SureDirect Blood PCR Kit PCR Direct amplification Blood inhibitor Genetic diagnosis miRNA196a2