| Yazarlar |
Ersin Karataylı
Türkiye |
Prof. Dr. Yasemin ÇELİK ALTUNOĞLU
Kastamonu Üniversitesi, Türkiye |
|
Senem Ceren Özen Karataylı
Ankara Üniversitesi, Türkiye |
|
S Gökçe K Alagöz
|
|
Kubilay Çınar
Ankara Üniversitesi, Türkiye |
|
Kendal Yalçın
Dicle Üniversitesi, Türkiye |
|
Ramazan İdilman
Ankara Üniversitesi, Türkiye |
|
Süleyman Cihan Yurtaydın
Ankara Üniversitesi, Türkiye |
|
Abdurrahman Mithat Bozdayı
Ankara Üniversitesi, Türkiye |
| Özet |
| Background: Hepatitis delta virus (HDV) RNA viral load measurement is critical in diagnosis and monitoring the response to antiviral treatment. Objectives: Our aim is to design a real time PCR method for accurate quantitation of HDV RNA in clinical specimens using an armored RNA as external standard, and an intrinsic internal control. Study design: A plasmid bearing delta antigen region of genotype I HDV genome was used to develop an armored RNA. Serial dilutions of the armored HDV RNA standard with 1012copy/mL were used as standards for quantitation. A primer-probe set derived from HDAg region was used in one step EZ RT PCR kit chemistry which uses rTth enzyme allowing reverse transcription and polymerization in the same tube. The kit also uses the advantage of uracil-N-glycosylase (UNG) enzyme treatment to prevent PCR contamination. Results: The established assay has a dynamic range of 102-1011copy/mL with a PCR efficiency of 96.9%. Detection limit was 858±32copy/mL with 95% confidence interval. Intra- and inter-assay variabilities were low for high, medium and low levels of viremia. Incorporation of freely circulating GAPDH in serum into the assay as an intrinsic internal control prevented false negative results and failures in PCR amplifications due to inhibitors, inefficient extraction procedures or enzymatic reactions. Conclusion: In conclusion, this study defines a novel assay for sensitive and reliable quantification of HDV RNA using an armored HDV RNA as a standard and GAPDH in plasma or serum as an intrinsic internal control in a single tube. © 2014 Elsevier B.V. |
| Anahtar Kelimeler |
| Armored RNA | HDV | Intrinsic control | Real time PCR |
| Makale Türü | Özgün Makale |
| Makale Alt Türü | SSCI, AHCI, SCI, SCI-Exp dergilerinde yayımlanan tam makale |
| Dergi Adı | Journal of Clinical Virology |
| Dergi ISSN | 1386-6532 |
| Dergi Tarandığı Indeksler | SCI-Expanded |
| Makale Dili | İngilizce |
| Basım Tarihi | 02-2014 |
| Cilt No | 60 |
| Sayfalar | 11 / 15 |
| Doi Numarası | 10.1016/j.jcv.2014.01.021 |
| Makale Linki | http://linkinghub.elsevier.com/retrieve/pii/S1386653214000419 |
