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 EDTA-blood 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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Preview
Unable to display preview. Download preview PDF.
References
Mullis KB, Faloona FA (1987) Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol 155:50–335
Espy MJ et al (2006) Real-time PCR in clinical microbiology: applications for routine laboratory testing. Clin Microbiol Rev 19(1):165–256
Lo YM et al (1999) Quantitative abnormalities of fetal DNA in maternal serum in preeclampsia. Clin Chem 45(2):184–188
Bussani C et al (2007) Prenatal diagnosis of common aneuploidies in transcervical samples using quantitative fluorescent-PCR analysis. Mol Diagn Ther 11(2):117–121
Aboud M, Oh HH, McCord B (2013) Rapid direct PCR for forensic genotyping in under 25 min. Electrophoresis 34(11):1539–1547
Park SJ et al (2008) Direct STR amplification from whole blood and blood- or saliva-spotted FTA without DNA purification. J Forensic Sci 53(2):335–341
Hansen TV et al (2007) Collection of blood, saliva, and buccal cell samples in a pilot study on the Danish nurse cohort: comparison of the response rate and quality of genomic DNA. Cancer Epidemiol Biomarkers Prev 16(10):2072–2076
Al-Soud WA, Joensson LJ, Radstroem P (2000) Identification and Characterization of immunoglobulin G in blood as a major inhibitor of diagnostic PCR. J Clin Microbiol 38(1):345–350
Al-Soud AW, Radstrom P (2000) Effects of amplification facilitators on diagnostic PCR in the presence of blood, feces, and meat. J Clin Microbiol 38(12):4463–4470
Al-Soud WA, Radstrom P (2001) Purification and characterization of PCR-inhibitory components in blood cells. J Clin Microbiol 39(2):485–493
Morata P, Queipo-Ortuno MI, de Dios J (1998) Colmenero, Strategy for optimizing DNA amplification in a peripheral blood PCR assay used for diagnosis of human brucellosis. J Clin Microbiol 36(9):2443–2446
Satsangi J et al (1998) Effect of heparin on polymerase chain reaction. The Lancet 343:1509–1510
Schrader C et al (2012) PCR inhibitors–occurrence, properties and removal. J Appl Microbiol 113(5):1014–1026
Opel KL, Chung D, McCord BR (2010) A study of PCR inhibition mechanisms using real time PCR. J Forensic Sci 55(1):25–33
Technologies A (2015) SureDirect Blood PCR Kit Protocol. 2015: Agilent Technologies, p 31
Nederhand RJ et al (2003) Logistics and quality control for DNA sampling in large multicenter studies. J Thromb Haemost 1(5):91–987
Mercier B et al (1990) Direct PCR from whole blood, without DNA extraction. Nucleic Acids Res 18(19):5908
Zhang Z, Kermekchiev MB, Barnes WM (2010) Direct DNA amplification from crude clinical samples using a PCR enhancer cocktail and novel mutants of Taq. J Mol Diagn 12(2):152–161
Technologies A (2015) Agilent SureDirect Blood PCR Protocol
Hue NT et al (2012) Extraction of human genomic DNA from dried blood spots. International Journal of Bioscience, Biochemistry and Bioinformatics 2(1):21–26
Acknowledgements
We would like to thank SucSong Poly-Clinic and all the generous volunteers for supporting sample to this study.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Nature Singapore Pte Ltd.
About this paper
Cite this paper
Truc-Chi, L.H., Thanh, N.D.G., Hue, N.T. (2018). Evaluate the Efficiency of AGILENT SureDirect Blood PCR Kit. In: Vo Van, T., Nguyen Le, T., Nguyen Duc, T. (eds) 6th International Conference on the Development of Biomedical Engineering in Vietnam (BME6) . BME 2017. IFMBE Proceedings, vol 63. Springer, Singapore. https://doi.org/10.1007/978-981-10-4361-1_39
Download citation
DOI: https://doi.org/10.1007/978-981-10-4361-1_39
Published:
Publisher Name: Springer, Singapore
Print ISBN: 978-981-10-4360-4
Online ISBN: 978-981-10-4361-1
eBook Packages: EngineeringEngineering (R0)