Comparison of the Isolation Methods of Viral Nucleic Acids

Year 2018, Volume: 14 Issue: 3, 351 - 355, 30.09.2018

Nagehan Şakrucu Uğur Sıdal , Tamer Şanlıdağ

https://doi.org/10.18466/cbayarfbe.350005

Abstract

In vitro amplification of the nucleic acids (DNA
or RNA) is used in the detection of microbial agents and thus in the diagnosis
of infectious diseases, as well as in the diagnoses of oncological and genetic
disorders and forensic medicine. The aim of the present study was to compare
the isolation methods of the nucleic acids of hepatitis B and C viruses,
causative agents of the two significant infections worldwide. Conventional
isolation methods were compared with the commercial kits that have been used
commonly in recent years, in terms of reliability, cost-effectiveness,
contamination risk and duration of the testing time. Five standards for the
isolation of the viral nucleic acids of both HBV DNA (Fluorion HBV QNP 2.0) and
HCV RNA (Fluorion HCV QNP 2.1) were used. The isolations of the viral nucleic
acids of HBV and HCV were done with the conventional methods, phenol-chloroform
and guanidine thiocyanate, and the commercial kits Roboscreen and NucleoSpin. The
resultant viral nucleic acid load was determined with a spectrophotometer (WPA
UV 1101, Biotech Photometer), and their amplification was conducted with
Real-Time PCR. The results of the assessments revealed that the highest nucleic
acid concentration were obtained with the conventional methods, while they
exhibited significant drawbacks such as long duration of the testing time,
difficulty in application, and higher contamination risk.

Keywords

HBV, HCV, PCR, spectrophotometer, amplification.

References

• 1. Saveci E. Prevalence Hepatitis B seroprevalence. Ministry of Health Taksim Training and Research Hospital Gynecology and Obstetrics Clinic. Family Medicine Specialization Thesis, 2006, İstanbul.
• 2. Hochberger, S., Althof, D., Gallegos de Schrott, R., Nachbaur, N., Röck, H., Leying, H. Fully automated quantitation of Hepatitis B virus (HBV) DNA in human plasma by the COBAS® AmpliPrep/COBAS® TaqMan® System. Journal of Clinical Virology, 2006, 35: 373-380.
• 3. Şanlıdağ, T., Akçalı, S., Özbakkaloğlu, B.Serum hepatitis B DNA: stability in relation to multiple freeze-thaw procedures.Journal of Virological Methods, 2005, 123: 49-52.
• 4. Kidd-Ljunggren, K., Holmberg, A., Blackberg, J., Lindqvist, B.High levels of hepatitis B virüs DNA in body fluids from chronic carriers. Journal of Hospital Infection, 2006, 64: 352-357.
• 5. Beguiristain N, Robertson DH, Gómez J. RNase III cleavage demonstrates a long range RNA: RNA duplex element flanking the hepatitis C virus internal ribosome entry site. Nucleic Acids Research, 2005, 33(16): 5250-5261.
• 6. Berger A, Preiser W, Doerr WH. The role of viral load determination for the management of human immunodeficiency virüs, hepatitis B virus and hepatitis C virus infection. Journal of Clinical Virology, 2001, 20:23-30.
• 7. Chien HJ, Lee SD, Cheng TY, Yeh HS, Chou PW, Chen HP. A novel fluorescence quantification method for polymerase chain reaction system. Optics Communications, 2006, 266: 744-750.
• 8. Leb V, Stöcher Valentine-Thon E, Hölzl G, Kessler H, Stekel H, Berg J. Fully Automated, Internally Controlled Quantification of Hepatitis B Virus DNA by Real-Time PCR by Use of the MagNA Pure LC and LightCycler Instruments. Journal of Clinical Microbiology, 2003, 42(2): 585-590.
• 9. Simpson RP, Yu HX, Redza MZ, Anson GJ, Chan HS, Lin Y. Quantification of hepatitis B virus DNA using competitive PCR and a scintillation proximity assay. Journal of Virological Methods, 1997, 69: 197-208.
• 10. Sun R, Schilling W, Jayakar H et al. Simultaneous extraction of hepatitis C virus (HCV), hepatitis B virus, and HIV-1 from plasma and detection of HCV RNA by a reverse transcriptase-polymerase chain reaction assay designed for screening pooled units of donated blood. Transfusion Complications, 1999, 39: 1111-1119.
• 11. Fawcett WT. Isolation of high-quality large plasmid DNA. Focus, 1999, 21(1).
• 12. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical Biochemistry, 1987, 162: 156-159.
• 13. Akin A, Wu CC, Lin LT. A comparison of two RNA isolation methods for double-stranded RNA of infectious bursal disease virus. Journal of Virological Methods, 1998, 74:179-184.
• 14. Kerur N, Jhala MK, Joshi CG. Genetic characterization of Indian pestedes petits ruminants virus (PPRV) by sequencing and phylogenetic analysis of fusion protein and nucleoprotein gene segments. Res Vet Sci, 2008, 85: 176-183,
• 15. Munir M, Zohari S, Saeed A, Khan QM, Abubakar M, LeBlanc N, Kanu S, Sankoh FA, Berg M, Barrie ML, Ståhl K. Genetic characterization of peste des petits ruminants virus, Sierra Leone. Emerg Infect Dis, 2012, 18:193-195
• 16. Luka PD, Erume J, Mwiine FN, Ayebazibwe C, Shamaki D. Molecular characterization and phylogenetic study of peste des petits ruminants viruses from North central states of Nigeria. BMC Vet Res, 2011, 7 :32
• 17. Kwiatek O, Ali YH, Saeed IK, Khalafalla AI, Mohamed OI, Obeida AA, Abdelrahman MB, Osman HM, Taha KM, Abbas Z, Harrak M, Lhor Y, Diallo A, Lancelot R, Albina E, Libeau G. Asian Lineage of Peste des Petits Ruminants Virus, Africa. Emerg Infect Dis, 2011, 17: 1223-1231
• 18. Munir M, Zohari S, Saeed A, Khan QM, Abubakar M, LeBlanc N, Berg M. Detection and phylogenetic analysis of peste des petits ruminants virus isolated from outbreaks in Punjab, Pakistan. Transbound Emerg Dis, 2012, 59: 85-93
• 19. De Nardi M, Lamin Saleh SM, Batten C, Oura C, Di Nardo A, Rossi D. First evidence of peste des petits ruminants (PPR) virus circulation in Algeria (Sahrawi Territories): Outbreak investigation and virüs lineage identification. Transbound Emerg Dis, 2012, 59: 214-222 20. 20. Sevik M. Molecular Detection of Peste des petits ruminants virüs from different organs/tissues of naturally infected animals. Kafkas Univ Vet Fak Derg, 2014, 20: 165-168