High-throughput real-time reverse transcription-PCR quantitation of hepatitis C virus RNA

Abstract

We describe a rapid and reproducible method for assessment of the hepatitis C virus (HCV) load in serum samples. The method combines Taqman technology (Roche) and the ABI Prism 7700 (Perkin Elmer) real-time sequence detection system. We have optimized a single-tube reverse transcription-PCR (RT-PCR) that contains a dual-labeled fluorogenic probe to quantify the 5' noncoding region (5' NCR) of HCV. The probe contains a fluorescent reporter at the 5' end and a fluorescent quencher at the 3' end. The use of such a probe combined with the 5'-3' nuclease activity of Taq polymerase allows direct quantitation of the PCR product by the detection of a fluorescent reporter released in the course of the exponential phase of the PCR. For accurate quantitation of the number of copies of HCV in samples containing unknown quantities, we have used serial dilutions of a synthetic 5' NCR RNA standard of HCV that was previously quantified with an isotopic tracer. The method has a 5-log dynamic range (10(3) to 10(7)). The coefficient of regression of the standard curve was, on average, 0.98. The intra-assay and the interassay coefficients of variation of the threshold cycle were 1% and 6.2%, respectively. Seventy-nine RNA samples from the sera of infected patients were quantified by this method. Comparison of the results with those obtained by other quantitation methods (the Quantiplex 2.0 branched-DNA assay and the Superquant assay from the National Genetics Institute) revealed a significant correlation with all of the results. The mean values were also statistically comparable. In conclusion, the high sensitivity, simplicity, and reproducibility of the real-time HCV RNA quantitation which allows the screening of large numbers of samples, combined with its wide dynamic range, make this method especially suitable for monitoring of the viral load during therapy and tailoring of treatment schedules.

FIG. 1

Typical amplification plot. The graph of the increment of fluorescence reporter signal (ΔRn) versus cycle number during PCR shows three stages: baseline, exponential phase, and plateau. The C_T value is calculated by determining the point at which the fluorescence exceeds an arbitrary threshold limit (usually 10 times the standard deviation of the baseline).

FIG. 2

Schematic representation of the transcription fragment from the 5′ NCR of HCV. Oligonucleotide primers C149 and C342 were used for amplification by RT-PCR. Primers C24 and C339 were hybridized to the RNA transcript to assess its integrity. FT275 is the dual-labeled probe. R, reporter; Q, quencher; nt, nucleotides.

FIG. 3

Standard curve of the input RNA concentration in serial dilutions of the first reference standard versus C_T. Each dot represents the result of triplicate PCR amplifications for each dilution. The stars represent the results of PCR amplification of samples containing unknown quantities of HCV RNA tested by this method. R was equal to 0.999, and the slope was −3.429.

FIG. 4

Assay of the quality of the RNA transcripts. Unlabeled RNA transcript was preheated at 90°C for 1 min, immediately mixed with oligonucleotides C24 (lane 2), C339 (lane 4), and both C24 and C339 (lane 6), and slowly cooled to room temperature. Reactions were conducted in reaction buffer (20 mM HEPES [pH 8], 50 mM KCl, 10 mM MgCl₂, 1 mM dithiothreitol) with 0.6 nM unlabeled transcript and 20 nM oligonucleotide. The reaction mixtures in lanes 3 and 5 were identical to those described above, but they lacked the RNA substrate. The arrow indicates a single band corresponding to the labeled RNA transcript (lane 1). Note that the bands in lanes 2, 4, and 6 are slightly higher than the band in lane 1, resulting from the increment in the size due to the hybridized oligonucleotides. Unincorporated oligonucleotides appear at the bottom of the gel in lanes 2 to 6.

FIG. 5

Spearman’s correlation plot between quantitation results determined by our RT-PCR protocol (ABI Prism 7700 assay) and both the bDNA assay (solid line and squares) and the NGI method (dashed line and dots) (P < 0.001). There is only no correlation between the ABI Prism 7700 assay and the bDNA assay and between the ABI Prism 7700 assay and the NGI method for two and three samples, respectively, all of which contained genotype 1 virus.

FIG. 6

Mean values of results (expressed as log₁₀ HCV RNA copies per milliliter of serum) determined by our RT-PCR protocol (ABI Prism 7700 assay), the bDNA assay and the NGI method are comparable. (A) Comparison of totals; differences in mean values are not significant (ns). The percentages of samples positive by each method are given in the box. (B) Comparison of mean values for groups of different genotypes; differences are not significant.