A one step real time PCR method for the quantification of hepatitis delta virus RNA using an external armored RNA standard and intrinsic internal control.

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 10(12)copy/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 10(2)-10(11)copy/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.

Potential proteomic biomarkers in assessing liver fibrosis using SELDI-TOF MS.

BACKGROUND/AIMS: The accurate assessment of the severity of liver fibrosis is of paramount importance in determining treatment strategies, response to treatment and prognosis in patients with chronic liver disease. The aim of this study was to investigate potential proteomic biomarkers for assessing stages of hepatic fibrosis. METHODS: Serum samples of 83 patients with chronic liver disease (using METAVIR index, 17 F0, 30 F1, 6 F2, 9 F3, and 21 F4 patients) and 29 healthy controls were analyzed using surface-enhanced laser desorption/ionization time-of- flight mass spectrometry on IMAC30 ProteinChip arrays. Discriminatory peaks between groups were identified using Mann-Whitney U non-parametric test. Comparison of mild (F0, F1) and severe fibrosis (F2, F3, F4) was performed using tree classification (cross-validation) with the Biomarker Patterns Software, version 5.0 (Ciphergen Biosystems, US). RESULTS: No statistically significant discriminatory peak was evident between F0, F1 and F2 fibrosis. More than 30 peaks were found to be discriminatory between patients with cirrhosis (F4) and all other stages of liver fibrosis, including F2 and F3. Six surface-enhanced laser desorption/ionization proteomic features were found to be discriminative for mild (F0, F1) vs. advanced (F2, F3, F4) fibrosis (AUROC ≥0.8, p<0.05, Mann-Whitney test). The decision tree (m/z 4280, 10453 and 6376) yielded a sensitivity of 83.3% (30/36), a specificity of 85.1% (40/47), a positive predictive value of 81.1%, and a negative predictive value of 86.9%, with an AUROC of 0.94. CONCLUSIONS: The results of this study revealed discriminatory peaks between the protein profiles of patients with cirrhosis and other stages of liver fibrosis. Potential proteomic biomarkers can be notably determined for discriminating mild and advanced fibrosis using surface-enhanced laser desorption/ionization time-of- flight mass spectrometry.