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Isolation, detection and quantification of Hepatitis B Virus using magnetic nanobeads and chip electrophoresis
Author(s) -
Kukacka Jiri,
Huska Dalibor,
Pouch Milan,
Krizkova Sona,
Drbohlavova Jana,
Chomoucká Jana,
Adam Vojtech,
Hubálek Jaromír,
Klapková Eva,
Prusa Richard,
Kizek Rene
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.726.3
In this study, magnetic nanobeads coated with streptavidine were prepared to isolate ssDNA specific to Hepatitis B virus (HBV). DNA probes labeled by biotine molecules were designed to be complementary to specific HBV genes. Artificial synthetic oligonucleotides (25–35 nt), identical to HBV gene sequences, were used to mimic a real sample of human blood serum. The amount of nanoparticles utilized for isolation target molecules was within the range from 1 to 20 μL. It was found that already 1 μL of MNPs was sufficient for effective isolation of the molecule of the interest. Optimal hybridization temperature 15°C was determined and the hybridization process was monitored within the time range from 5 to 30 minutes. It was found that hybridization time did not influence significantly the isolation efficiency. The correlation coefficient of R 2 = 0.99 was obtained for all calibration curves determined for HBV oligonucleotides. Maximal efficiency up to 30 % was obtained under optimal conditions of DNA extraction. Detection limit of DNA was 0.1 ng/μL. The control of quality and quantity of extracted DNA was carried out with chip electrophoresis.