Performance Characteristics of a Quantitative TaqMan Hepatitis CVirus RNA Analyte-Specific Reagent
James M. Barbeau,1 Jennifer Goforth,2 Angela M. Caliendo,1 and Frederick S. Nolte1*Department of Pathology and Laboratory Medicine, Emory University School of Medicine,1 and Emory
Medical Laboratories, Emory University Hospital,2 Atlanta, Georgia
Received 12 February 2004/Returned for modification 7 April 2004/Accepted 7 May 2004
We determined the dynamic range, reproducibility, accuracy, genotype bias, and sensitivity of the TaqManhepatitis C virus (HCV) analyte-specific reagent (ASR). Serum samples were processed using the MagNA PureLC instrument and run on the COBAS TaqMan 48 analyzer. The performance characteristics of the ASR werealso compared with those of the qualitative AMPLICOR and quantitative AMPLICOR MONITOR HCV tests.The ASR exhibited a >6-log10 linear dynamic range and excellent reproducibility, with a mean coefficient ofvariation of 14%. HCV RNA concentration measured with the ASR agreed within an average of 0.42 log10(2.6-fold) of the labeled concentration with members of a standard reference panel. HCV genotypes 1 to 4 wereamplified with similar efficiencies with the ASR. The ASR and AMPLICOR MONITOR viral load results weresignificantly correlated (r � 0.8898; P < 0.01), but the agreement was poor (mean difference, 0.45 � 0.35 log10)for 72 HCV RNA-positive clinical samples. However, 98.9% agreement between the ASR and qualitativeAMPLICOR test results was found with 60 positive and 29 negative samples. Limiting-dilution experimentsdemonstrated that the limits of detection for ASR and AMPLICOR tests were 84 and 26 IU/ml, respectively.The performance characteristics of the TaqMan HCV ASR are appropriate for all clinical applications of HCVRNA testing.
Both qualitative and quantitative hepatitis C virus (HCV)RNA tests are used in diagnosis and management of patientswith hepatitis C, because no single commercially available testcombines high analytical sensitivity with a broad dynamicrange. Qualitative nucleic acid amplification tests for detectionof HCV RNA in serum are used to confirm the diagnosis ofhepatitis C, distinguish active from resolved infection, assessvirological response to therapy, and screen blood donors (3, 18,23). Quantitative tests are used in evaluation of patients beingconsidered for therapy and to assess early response to therapy.A pretreatment viral load of less than 800,000 IU/ml is one ofseveral predictors of a sustained virological response (20, 24).Viral load testing has also been used in early assessment oftreatment response. Patients who fail to achieve at least a2-log10 decline in viral load after 12 weeks of treatment havelittle chance of a sustained response and can be spared the costand toxicity of a complete treatment course (9, 17). However,viral load does not predict the progression of hepatitis C and isnot associated with the severity of liver disease.
A variety of tests for detection and quantitation of HCVRNA based on different nucleic acid amplification technolo-gies are commercially available. The qualitative AMPLICORHCV and quantitative AMPLICOR HCV MONITOR version2.0 tests (Roche Diagnostics Corporation, Indianapolis, Ind.)are based on conventional reverse transcription-PCR in a het-erogeneous format (17). The VERSANT HCV RNA qualita-tive and VERSANT HCV RNA 3.0 quantitative assays (BayerHealthcare, Tarrytown, N.Y.) are based on transcription-me-
diated amplification and branched DNA signal amplification,respectively (10, 15).
These tests also differ in their lower limits of detection anddynamic ranges. The qualitative AMPLICOR and VERSANTtests have lower limits of detection of 50 and 5 IU/ml, respec-tively. Although the lower limits of detection for the quantita-tive AMPLICOR and VERSANT tests are both approximately600 IU/ml, the dynamic ranges differ by approximately 1 log10
and are 3.1 and 4.1 log10, respectively. Because of the differ-ences in sensitivity between the qualitative and quantitativeassays, many clinical laboratories use a quantitative test todetermine viral load and a sensitive qualitative test for diag-nosis and test-of-cure. A single test with sensitivity similar tothe qualitative tests that accurately quantitates high viral loadswould be beneficial for clinical laboratories.
A number of homogeneous TaqMan reverse transcription-PCR assays for detection and quantitation of HCV RNA havebeen described (12, 13, 19, 21, 29). These tests are very sensi-tive, have broad dynamic ranges, and provide precise quanti-tation of viral load. These tests also generate results morerapidly than the earlier heterogeneous tests and are not proneto amplicon carry-over contamination, since the amplificationand detection steps are combined in a single closed tube.
Roche Diagnostics Corp. recently developed a TaqManHCV analyte-specific reagent (ASR). It was designed for thenewly released COBAS TaqMan analyzer, a real-time PCRinstrument developed for the clinical laboratory. An ASR maybe sold to clinical laboratories regulated under the ClinicalLaboratory Improvement Amendments (CLIA) of 1998 asqualified to do high-complexity testing. The laboratory is re-sponsible for verifying and validating the test, and the reportsshould be appended with a standard disclaimer stating that thetest was developed by and its performance characteristics de-
termined by the laboratory and that the test has not beencleared or approved by the U.S. Food and Drug Administra-tion (FDA).
This study is the first to report the dynamic range, sensitivity,reproducibility, accuracy, and genotype bias of the ASR. Theperformance characteristics of the ASR were also comparedwith those of the qualitative AMPLICOR and quantitativeAMPLICOR MONITOR HCV version 2.0 tests.
MATERIALS AND METHODS
Samples. Serum samples were selected from stored samples that had beensubmitted to Emory Medical Laboratories for HCV RNA testing. The sera wereremoved from clots within 4 h of collection and stored at �70°C until needed. Astandard reference panel calibrated against the World Health Organization 1stInternational Standard for HCV RNA (25) was purchased from AcroMetrix,Benicia, Calif. The panel members were assigned concentrations of 50, 500,5,000, 200,000, 500,000, and 2,000,000 IU/ml by the manufacturer as previouslydescribed (11). All dilutions of clinical samples were made in human plasmaobtained from a single outdated unit from our blood bank.
Sample preparation. The starting sample volume for all of the tests was 200 �l.All samples tested in the AMPLICOR MONITOR HCV test were diluted 1:10in normal human plasma prior to processing. Samples for the other tests were notdiluted. HCV RNA was extracted from samples for the AMPLICOR MONI-TOR HCV test using the manual method according to the manufacturer’s in-structions. HCV RNA was extracted from serum samples for the AMPLICORHCV test using the MagNA Pure LC instrument and the total nucleic acidreagent set (Roche Applied Science, Indianapolis, Ind.) as previously described(7).
The MagNA Pure LC instrument and total nucleic acid reagent set were alsoused to extract HCV RNA for the ASR. The instrument and reagents were usedas recommended by the manufacturer with the following exceptions. The ASRquantitation standard (QS) was added to the lysis buffer to achieve a finalconcentration of 5 copies/�l (0.125 ml of HCV QS to 10 ml of lysis buffer). Thepurified RNA was eluted in 65 �l of elution buffer. The nominal concentrationof the HCV QS in the eluant was 22 copies/�l, as recommended for the TaqManHCV ASR by the manufacturer.
HCV RNA tests. The microwell plate AMPLICOR and AMPLICOR MON-ITOR HCV version 2.0 tests were performed according to the manufacturer’sinstructions. The ASR consists of an HCV master mix, a QS, and a 50 mMsolution of manganese acetate. The master mix contains upstream and down-stream primers to the 5� untranslated region of the HCV genome, fluorescentlylabeled HCV- and QS-specific oligonucleotide probes, Z05 DNA polymerase,deoxynucleotide triphosphates (dATP, dCTP, dGTP, and dUTP), AmpEraseuracil-N-glycosylase, potassium acetate, dimethyl sulfoxide, glycerol, and sodiumazide in tricine buffer. The HCV QS contains a noninfectious, protein-encapsu-lated RNA with the HCV primer binding sequences and a unique probe bindingregion at a concentration of 400 copies/�l, poly(rA) RNA, EDTA, amaranth dye,and ProClin 300 in sodium phosphate buffer. Single lots of master mix and QSwere used throughout the study.
The master mix was activated by the addition of 170 �l of 50 mM manganeseacetate, and the activated master mix was used within 60 min of preparation.Fifty microliters of processed sample or calibrator was added to 50 �l of acti-vated master mix. The amplification and detection reactions were started within30 min of the addition of the sample. Thermal cycle parameters were as follows:two precycles of 5 min at 50°C and 30 min at 59°C, two cycles of 15 s at 95°C and25 s at 58°C, and 60 cycles of 15 s at 91°C and 25 s at 58°C, followed by a postcyclehold at 40°C.
The ASR was run on the COBAS TaqMan 48 analyzer. It is a real-time PCRinstrument designed for the clinical laboratory that consists of two independentlyprogrammable, 24-sample thermal cyclers, a halogen light source, two 24-chan-nel fluorescence photometers with four different filter combinations, and Am-plilink software. It has a run size of 6 to 48 samples.
The ASR was calibrated by using serial 10-fold dilutions of a well-character-ized clinical specimen containing HCV genotype 1 that were tested at sevendifferent levels in quadruplicate in a single run. The starting concentration wasassigned using the AMPLICOR HCV MONITOR test. The threshold or elbowvalues (EVs) for both HCV and QS were stored and used by the instrumentsoftware to determine the lot-specific calibration coefficients that were used incalculation of the sample HCV RNA concentrations.
HCV genotyping. HCV genotypes were determined using a commerciallyavailable reverse hybridization, line probe assay (VERSANT HCV GenotypeAssay; Bayer Healthcare) according to the manufacturer’s instructions. Theamplicon from the 5� untranslated region used in the genotyping assay wasgenerated with the AMPLICOR HCV test.
Data analysis. Descriptive statistics, correlation coefficients, and regressionline equations were calculated with the data analysis tool pack of Microsoft Excel2000 (Microsoft Corp., Redmond, Wash.). Agreement between viral load valueswas assessed by the method of Bland and Altman (2). The limits of detectionwere determined using probit analysis (8).
RESULTS
The sample that was used to calibrate the ASR was assigneda starting concentration of 4 � 106 (6.58 log10) IU/ml with theAMPLICOR HCV MONITOR test. The undiluted sampleand six serial 10-fold dilutions were tested in 11 replicates inthree separate runs. The mean, standard deviation, and coef-ficient of variation (CV) of the EVs for each calibrator areshown in Table 1. The EVs were very reproducible over theentire concentration range, with CVs ranging from 0.48 to2.47%. The ASR detected 100% of the replicates containinggreater than or equal to 40 IU/ml and 73% of the replicatescontaining the lowest concentration tested, 4 IU/ml. The meanEVs and HCV RNA concentrations were highly correlated (r� 0.9961), and the assay showed a linear response over the6-log10 range of concentrations tested. The calibration coeffi-cients used to calculate the sample HCV RNA concentrationsin subsequent experiments were derived from these data.
A second set of six serial 10-fold dilutions was prepared fromanother clinical sample with a higher starting concentration of18,400,000 (7.27 log10) IU/ml as determined with the AMPLI-COR HCV MONITOR test. The undiluted sample and thedilutions were tested in quadruplicate in a single ASR run. Theplot of the measured values against the expected values isshown in Fig. 1. The measured and expected values were highlycorrelated (r � 0.9996) over the 6-log10 concentration range,and the equation for the linear regression line was y � 1.036x� 0.83. The slope of the line closely approximated the idealslope of 1. The measured values were consistently larger thanthe expected values, with an average bias of 0.24 log10 (range,0.1 to 0.37) or 1.7-fold. The average CV for the replicate viralload values in this dilution series was 13.9% (range, 6.6 to19.9).
We prepared serial dilutions of two other high-titer clinicalsamples containing 90,000,000 (7.95 log10) and 270,000,000(8.43 log10) IU/ml to test the upper limit of the dynamic rangeof the ASR. The initial concentrations were assigned with the
TABLE 1. Reproducibility of EVs for the TaqMan HCVASR calibratorsa
HCV RNA(IU/ml) No. detected/no. tested Mean EV SD % CV
ASR. The equations for the regression lines of the two dilutionseries were very similar, and the ASR demonstrated no devi-ation from the ideal response up to the highest concentrationtested (Fig. 2). The upper limit of the linear range was at least7.95 log10 IU/ml.
A standard reference panel was used to assess the accuracyof the ASR calibration. The panel consisted of seven memberswith concentrations ranging from 50 to 2,000,000 IU/ml. Thepanel was tested in quadruplicate in a single run. HCV RNAwas detected in all replicates at each concentration. The viralload values determined for all panel members containing �50IU/ml were very reproducible, with an average CV of 14%, andwere consistently greater than the labeled concentration by anaverage of 0.42 log10 (2.6-fold) (Table 2). Although HCV RNAwas detected in all the replicates of the 50-IU/ml panel mem-ber, the viral load value determined by the ASR, 6 IU/ml, wasalmost 1 log10 less than the labeled concentration and the assayat this concentration was poorly reproducible, with a CV of53%. The measured versus labeled concentrations for all of thepanel members except the 50-IU/ml member are plotted inFig. 3. The values were highly correlated (r � 0.9979), and theslope of the linear regression line was 1.002. The line shows aconsistent positive bias for the values determined with theASR.
We next examined the correlation and agreement of viralload values determined with the ASR and the AMPLICORMONITOR test for a total of 71 clinical samples: 61 withgenotype 1, 4 with genotype 2, 4 with genotype 3, and 1 with
genotype 4 HCV RNA. The population means (ranges) of theviral load values determined with the ASR and the MONITORtest were 6.5 (3.28) and 6.06 (2.43) log10, respectively. Thevalues were significantly correlated (r � 0.8898; P � 0.01), andthe linear regression analyses indicated that the slope andintercept were not significantly different from 1 and 0, respec-tively (Fig. 4). However, the agreement was poor, with a meandifference between values (ASR and MONITOR) of 0.45log10 0.35. The limits of agreement (mean difference 2standard deviations) were �0.25 and 1.15 log10.
A plot of the difference versus the average viral load valuesfor the 71 samples is shown in Fig. 5. We estimated the trueviral load value for each sample by averaging the results of thetwo tests, since the true values were not determined with anindependent reference test. The ASR values were consistentlygreater than the MONITOR values; however, the differencebetween the values measured by the two tests varied with theconcentration (r � 0.6156; P � 0.01). The differences betweenvalues were consistently less than the mean difference for sam-ples, with average viral load values of �6 log10 IU/ml, equallydistributed around the mean difference for samples with aver-age viral load values between 6 and 6.5 log10 IU/ml, and con-sistently greater than the mean difference for samples, withaverage viral load values of �6.5 log10 IU/ml.
Serial 10-fold dilutions of clinical samples containing HCVgenotypes 2, 3, and 4 were prepared and tested with the ASR.The EVs were plotted against the log10 of the HCV RNAconcentration, and data were analyzed by linear regression
FIG. 1. Measured versus expected concentrations of serial 10-fold dilutions of a sample with a starting concentration of 7.27 log10 IU/ml. Thestarting concentration was determined with the AMPLICOR HCV MONITOR test. Each point represents the mean of four replicates tested ina single run. The dashed line represents unity.
(results not shown). The slope of the regression line is a mea-sure of reaction efficiency for real-time PCR. The slopes forgenotypes 2, 3, and 4 were �2.95, �2.65, and �3.13, respec-tively. The regression line slope for the genotype 1 calibratorwas �2.73 (Fig. 1). The slopes of the regression lines for thedifferent genotypes were not significantly different. The ASRamplified all of the different genotypes with similar efficiencies.
We tested the 89 clinical samples in parallel with the qual-itative AMPLICOR HCV test and the ASR. The results of thetwo tests agreed for 98.9% of the samples (Table 3). HCVRNA was detected by both tests in 59 samples and not detectedby both tests in 29 samples. HCV RNA was detected only withthe AMPLICOR test in one sample. The sensitivity and spec-
ificity of the ASR with respect to the AMPLICOR test were98.3 and 100%, respectively.
The limits of detection of the qualitative AMPLICOR HCVtest and the ASR were determined and compared using serialdilutions of a reference standard with a labeled concentrationof 5,000 IU/ml (Table 4). HCV RNA was detected in 100% ofthe replicates at a concentration of 500 IU/ml with the ASR. Atconcentrations of �500 IU/ml, the detection failure rate withthe ASR increased as the concentration of HCV RNA de-creased. The AMPLICOR test detected HCV RNA in 100%of replicates at concentrations of �50 IU/ml by the AMPLI-COR test, and proportionally fewer replicates were detected atlower concentrations. Probit analysis indicated that the con-centration at which 95% of the replicates should be positive(limit of detection) was 84 IU/ml for the ASR and 26 IU/ml forthe AMPLICOR test.
DISCUSSION
The TaqMan HCV ASR is an example of a reagent that canbe marketed to clinical laboratories under the ASR rule (5).Simply stated, an ASR is the active ingredient of an in-labo-ratory-developed test. The ASR rule was intended to facilitatethe transfer of new technology to clinical laboratories, partic-ularly to those high-complexity laboratories capable of usingthese reagents in the development of new diagnostic tests.
Under the ASR rule manufacturers are not required to seek
FIG. 2. Measured versus expected concentrations of serial dilutions of two high-titer samples. �, sample 1; ■, sample 2. The startingconcentrations were determined with the TaqMan HCV ASR (sample 1, 8.43 log10 IU/ml; sample 2, 7.95 log10 IU/ml). Each point represents theaverage of duplicates tested in a single run. The dashed line represents unity.
TABLE 2. Reproducibility and accuracy of the TaqMan HCV ASRdetermined with a standard reference panel of HCV RNA
FDA premarket approval for low-risk ASRs, which include allbut blood banking tests, and those used for the diagnosis ofpotentially deadly infectious diseases (e.g., tuberculosis andAIDS) and genetic disorders. To qualify for the regulatoryexemptions, the manufacturer cannot make analytical or clin-ical performance claims. It also cannot provide clinical labo-ratories with instructions on how to use the ASR or withappropriate calibrators and controls. In addition, the manufac-turer is required to register and list the ASR with the FDA, tomeet good manufacturing process standards, to report adverseevents, and to restrict the distribution of the ASR to CLIAhigh-complexity laboratories. The clinical laboratories are re-quired to develop and maintain the analytical performancecharacteristics of the test in which the ASR is used and toreport the test results with a standard disclaimer.
Roche also markets a research-use-only (RUO) version ofthe TaqMan HCV RNA test. The reagents in an RUO test arecalibrated by the manufacturer and come with additional con-trols and instructions for use. However, clinical laboratoriesthat use the RUO test are still required to determine its localperformance characteristics under CLIA 1998. Clinical labo-ratories may use either the ASR or the RUO kit for all theclinical applications of HCV RNA testing except testing ofblood donors.
The TaqMan HCV ASR demonstrated a very broad dy-namic range of at least 6 log10 IU/ml. The dynamic range forthe ASR may actually be even broader, since with at least onesample we were able to document linearity of the assay up to270,000,000 IU/ml. Even with the more conservative estimate
of 6 log10, the ASR has a much broader dynamic range thaneither of the other commercially available quantitative HCVassays, the AMPLICOR HCV MONITOR version 2.0 (3.1log10) and the VERSANT HCV RNA 3.0 (4.1 log10). Thedynamic range is comparable to that described for in-labora-tory-developed, real-time PCR assays for HCV RNA. We wereunable to adequately characterize the performance of the ASRat the upper end of the dynamic range due to the scarcity ofclinical samples and reference material with viral loads of inexcess of 108 IU/ml. Based on our data, we estimate that theASR can precisely determine viral loads from 500 to200,000,000 IU/ml. The broad dynamic range of the ASRmakes it well suited for assessing viral loads throughout acourse of treatment.
The user defines the calibrators for the ASR. We chose tocalibrate it using a clinical sample that was assigned a viral loadvalue with the AMPLICOR HCV MONITOR test. A clinicalsample was used rather than standard reference material be-cause of the lack of available high-titer reference material. Weused the AMPLICOR HCV MONITOR test to assign theinitial concentration because it is commonly used in clinicallaboratories and it is calibrated against the World Health Or-ganization HCV international standard. We found that thestandard curve was stable when using the same lot of reagents,with an average CV in the EVs of only 1.1% over three runs.A single standard curve can be run once in quadruplicate togenerate lot-specific calibration coefficients.
The accuracy of the calibration was checked with a commer-cially available standard reference panel. The values deter-
FIG. 3. Measured versus labeled concentrations for a standard reference panel of HCV RNA tested with the TaqMan HCV ASR. Each pointrepresents the mean of four replicates tested in a single run. The dashed line represents unity.
mined with the ASR were consistently 2.6-fold greater than thelabeled concentrations from 100 to 2,000,000 IU/ml. Agree-ment within threefold is generally considered acceptable whencomparing different methods for HCV viral load measurement(28).
The within-run precision of the ASR viral load measure-ments was assessed with a standard reference panel and serialdilutions of a clinical sample. In each case, the average CVover the dynamic range was approximately 14%. Given thislevel of variation, a greater-than-twofold (0.3 log10) differencebetween samples will be statistically significant (P � 0.05) overmost of the dynamic range of the assay. At either end of thedynamic range, threefold (0.5 log10) differences will be statis-tically significant (P � 0.05). The precision of the ASR com-pares favorably with the precision reported for the AMPLI-COR HCV MONITOR version 2.0 test (16) and is similar tothat reported for the VERSANT HCV RNA 3.0 assay (1, 30).
We found significant correlation but poor agreement be-tween the results obtained with the ASR and the AMPLICORMONITOR test for the same clinical samples. The averagedifference between the results was 0.45 log10, and the differ-ence increased as the viral load increased. This is best ex-plained by differences in the true dynamic ranges of the twotests. Although the upper limit of the linear range claimed bythe manufacturer for the AMPLICOR MONITOR test is850,000 IU/ml, other studies indicate that the test plateaus atconcentrations above 500,000 IU/ml (14, 16, 22). Significantvariation between values from diluted and undiluted sampleswas observed in those studies with samples in the range of
500,000 to 850,000 IU/ml. We diluted all samples 1:10 prior totesting with the AMPLICOR MONITOR to permit betterquantitation of samples with high viral loads, and we used8,500,000 IU/ml as the upper limit of the linear range. Thesharp increase in the magnitude of the differences between testresults with samples with mean viral loads greater than3,200,000 (6.5 log10) observed here suggests that the dynamicrange of the AMPLICOR MONITOR test is more limitedthan previously reported.
Genotype bias was a significant problem with the first ver-sion of the AMPLICOR HCV MONITOR test, due to sec-ondary structures that could form in the target cDNA at therelatively low annealing and extension temperatures used. Thiswas addressed by reformulation of the PCR mixture and mod-ification of the thermal cycle parameters in the version 2.0 test.The version 2.0 test amplifies all HCV genotypes with similarefficiency (6, 16). A prototype of the TaqMan HCV ASR wasalso shown to amplify RNA transcripts from the different HCVgenotypes with comparable efficiencies (13). We demonstratedthat the ASR amplification efficiencies were similar for clinicalsamples with HCV genotypes 1 to 4. Like the AMPLICORMONITOR version 2.0 and prototype TaqMan tests, the ASRwas free of significant genotype bias.
The clinical and analytical sensitivities of the ASR werecompared with those for the qualitative AMPLICOR HCVtest. We found that the ASR detected all but 1 of the 60positive clinical samples tested in parallel (sensitivity, 98.3%)and that the limits of detection were similar: 84 IU/ml for theASR and 26 IU/ml for the AMPLICOR test. Since the same
FIG. 4. Correlation and linear regression analysis of viral load values obtained with the TaqMan HCV ASR and the AMPLICOR HCVMONITOR test. HCV genotypes are indicated as follows: �, genotype 1; ■, genotype 2; Œ, genotype 3; �, genotype 4.
sample volume, nucleic acid extraction protocol, and reactioninput volume were used for both tests, the difference in thelimits of detection may have resulted from small differences inPCR efficiencies between the tests. The small difference inanalytical sensitivity is unlikely to have a major impact on theperformance of the ASR when it is used to diagnose activeinfections, since it is rare for pretreatment viral load values tobe less than 100,000 IU/ml.
The analytical sensitivity required to adequately assess end-of-treatment virological responses in HCV infection is not wellestablished. The VERSANT HCV RNA assay, with a 5-IU/mllimit of detection, was able to detect residual serum viral RNAin some patients who had no detectable viral RNA in theAMPLICOR HCV test at the end of treatment with interferonand subsequently experienced a virological relapse (4, 27).However, no difference between the tests was observed withend-of-treatment samples from patients treated with thenewer, faster-acting, polyethylene glycol-modified interferon
(26). It is unlikely that small differences in analytical sensitivitywill be important in the assessment of end-of-treatment viro-logical responses in patients treated with the most effectiveregimens.
The manual extraction protocol given as an example by themanufacturer in the ASR package insert uses a sample inputvolume of 500 �l. We used automated processing and a 200-�linput. The larger sample volume could improve the limit ofdetection by as much as 2.5-fold.
The COBAS TaqMan 48 analyzer is the first real-time PCRinstrument designed for the clinical laboratory. The instrumentwas simple to use and was reliable over the 6-month evaluationperiod. The software allowed storage of patient demographic
FIG. 5. Agreement of viral load values obtained with the TaqMan HCV ASR and the AMPLICOR HCV MONITOR test. Difference log10IU/ml � ASR value � MONITOR value. Average log10/IU/ml � (ASR value � MONITOR value)/2. HCV genotypes are indicated as follows:�, genotype 1; ■, genotype 2; Œ, genotype 3; �, genotype 4.
TABLE 3. Qualitative results obtained with the AMPLICOR HCVtest and the TaqMan HCV ASR for 89 clinical samples
AMPLICORresult
ASR resultTotal
Detected Not detected
Positive 59 1 60Negative 0 29 29Total 59 30
TABLE 4. Limits of detection for the AMPLICOR HCV test andthe TaqMan HCV ASR
and order data, which is helpful for clinical laboratories. How-ever, unlike research instruments, the software did not gener-ate amplification plots or multicomponent views, which can bevery helpful in troubleshooting real-time PCR.
In conclusion, the TaqMan HCV ASR and the COBASTaqMan 48 analyzer are among the first real-time PCR assaysand platforms designed specifically for the clinical laboratory.Together they represent a powerful new tool for quantitationof HCV RNA and can provide a single assay platform that hasthe required combination of analytical sensitivity, dynamicrange, and precision for all the current clinical applications ofHCV RNA testing.
ACKNOWLEDGMENT
This study was supported in part by a grant from Roche DiagnosticsCorp.
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