TABLE 20.

Comparison of rapid-cycle real-time PCR assays for the detection of hepatitis virus types A, B, C, D, and E in clinical specimensa

Hepatitis virus typeReferenceSpecimenTest platform/probe chemistryTargetSpecimen volume inputRange of quantitative resultsComparison to other assaysComments
HAVCosta-Mattioli et al. 2002 (83)SerumABI Prism 7700/TaqMan probe87-bp fragment in 5′-noncoding region140 μl360 to 3.6 × 108 copies/ml100% sensitivity and specificity in 41 patient sera and 200 healthy blood donor sera tested by anti-HAV IgM.CV varied from 0.98% to 6.46%; HAV RNA remained detectable for >60 days after clinical diagnosis.
Rezende et al. 2003 (411)SerumLightCycler/TaqMan probe77-bp fragment in 5′ noncoding region140 μl100 to 108 copies/ml72% sensitivity in 50 patient sera tested by anti-HAV IgM.
Roche Applied Science (http://www.roche-applied-science.com/lightcycler-online/ )Plasma, serumLightCycler/FRET hybridization probes5′ noncoding region300 μl serum5,000 to 108 geq/mlCommercially available for research use only.
HBVAbe et al. 1999 (1)SerumABI Prism 7700/TaqMan probe174- or 241-bp fragment of S gene or 331-bp fragment of X gene100 μl200 to 2× 109 copies/mlCorrelated with Quantiplex HBV DNA 1.0 Assay (R2 ranged from 0.93 to 0.97 for the three targets) and quantified eight clinical sera more than Quantiplex among 46 patient sera; 100% specificity in 23 healthy donor sera.Interassay CV varied from 0.7% to 9.1%.
Aliyu et al. 2004 (5)SerumLightCycler/FRET hybridization probes259-bp fragment of S gene200 μl250 to 5 × 108 copies/mlDetected 26 positive sera more than conventional laboratory-developed PCR assay among 89 patient seraIntra- and interassay CV varied from 2.6% to 75.3% and from 9.2% to 70.2%, respectively.
Brechtbuehl et al. 2001 (44)Plasma, serumLightCycler/SYBR Green I100-bp fragment of S gene150 μl1.3 × 104 to 1.3 × 1010 copies/ml for single-round PCR; 400 to 1.3 × 108 copies/ml for nested PCRQuantification of HBV DNA levels in 21 (58%) of 36 HBsAg-positive blood donor sera were higher than those obtained by Amplicor HBV Monitor test, with mean titer difference of 0.64 log (range, 0.14 to 0.96 log) among samples of> 106 copies/ml.Interassay CV ranged from 1.2% to 2.9%.
Candotti et al. 2004 (59)PlasmaStratagene Mx4000/TaqMan probe81-bp fragment of S gene200μ l30 (95% detection rate) to 105 IU/mlCorrelated with single-virus real-time PCR assay (R2 = 0.94) among 24 samplesIntra- and interassay CV ranged from 0.8% to
with HBV DNA titers ranging from 15 to 105 IU/ml; 100% specificity among 266 healthy blood donor sera tested by HBsAg EIA.    1% and from 3.4 to 3.6%, respectively; equivalent detection of HBV genotypes A to F; a multiplex assay which also detects HCV RNA and HIV-1 RNA.
Cane et al. 1999 (60)SerumLightCycler/FRET hybridization probes104-bp fragment of polymerase gene10 μl105 copies/ml minimum100% agreement with direct DNA sequence analysis and/or cloning assay in 20 sera from 8 patients (HBV genotypes A to H).Detect polymerase gene mutations encoding resistance to lamivudine in HBV minor species comprising 20% of total population.
Hennig et al. 2002 (172)PlasmaABI Prism 7700/TaqMan probe132-bp fragment of C gene1 or 2 ml28 IU/ml for 1-ml sample; 14 IU/ml for 2-ml sampleDetected 14 of 16 (88% sensitivity) HBsAg-positive sera and 3 of 189 (98.4% specificity) HBsAg-negative sera among 205 anti-HBc-positive patientsDeveloped for screening of blood donors.
Ho et al. 2003 (180)SerumLightCycler/FRET hybridization probes130-bp fragment of C gene200μ l250 to 2.5 × 1010 copies/mlDetected 114 of 120 (95% sensitivity) HBsAg-positive sera and none of 45 (100% specificity) HBsAg-negative sera; correlated with Digene Hybrid Capture II HBV DNA test (R2 = 0.90) on 67 patient sera.Intra- and interassay CV, were 6% and 16%, respectively.
Ide et al. 2003 (192)SerumABI Prism 7700/SYBR Green I52-bp fragment of C gene150μ l50 to 316 copies/mlUtilizes amplified products from the Amplicor HBV Monitor test as templates; intra-assay CV was 1.4% at 316 copies/ml.
Jardi et al. 2001 (199)SerumLightCycler/FRET hybridization probes144-bp fragment of C gene200μ l103 to 108 copies/mlDetected 128 of 193 (66%) HBsAg-positive sera vs. 84 detected by Quantiplex HBV DNA 1.0 assay; correlated with Quantiplex assay (R2 = 0.94) in 30 patient sera.Intra- and interassay CV, varied from 2% to 4% and from 4% to 6%, respectively.
Leb et al. 2004 (253)PlasmaLightCycler/FRET hybridization probes139-bp fragment of C gene200μ l200 to 8 × 108 copies/mlQuantified 107 vs. 100 samples by COBAS Amplicor HBV Monitor test among 123 clinical plasma simples, with good correlation (R2 = 0.95) between the two assays in 97 samples.Intra- and interassay CV, were 0.7% and 1.5%, respectively.
Loeb et al. 2000 (283)SerumABI Prism 7700/TaqMan probe105-bp fragment of overlapping region between X and polymerase genes200μ l10 to 109 copies/mlQuantified 119 vs. 55 sera by Quantiplex HBV 1.0 assay among 157 patient sera; 100% specificity in 119 sera negative for all HBV serologic markers.Detected HBV DNA in 10 (9%) of 109 patients who were positive only for anti-HBc total antibodies in 84 (78%) of 108 HBsAg-positive/HBeAg-negative patients.
Mercier et al. 1999 (314)Plasma, serumABI Prism 7700/TaqMan probe113-bp fragment of C gene200 ul50 copies/ml (analytical sensitivity)100% sensitivity and specificity in 50 patients and 50 blood donors tested by Quantiplex HBV 1.0 (bDNA) assay; 1.5% false-positive rate in screening 274 healthy blood donors.A multiplex assay which also detects HCV RNA
Mukaide et al. 2003 324)SerumABI Prism 7700/TaqMan probe120-bp fragment of overlapping region between core and polymerase genes300 ul3 to 108 IU/mlCorrelated with Amplicor HBV Monitor test at R2 = 0.94 with a commercially available transcription-mediated amplification-hybridization protection assay (Chugai Diagnostics Science, Tokyo, Japan) at R2= 0.94 in 156 and 100 patient sera, respectively.Intra- and interassay CVs varied from 1.1% to 7.8% and from 1.6% to 4.8%, respectively.
Pang et al. 2004 (364)SerumABI Prism 7700/3′ minor groove binder-conjugated TaqMan probe108-bp fragment of core promoter gene, or 107-bp fragment of precore gene?200 to 109 copies/mlCorrelated with COBAS Amplicor HBV Monitor Test in 110 sera containing wild-type/core promotor mutant HBV (R2 = 0.86) and in 71 sera containing wild-type/precore mutant HBV (R2 = 0.85).Capable of quantifying HBV DNA in samples containing from 10% to 90% core promotor mutants or samples with 20% to 90% precore mutants
Paraskevis et al. 2002 (367)SerumLightCycler/FRET hybridization probes156-bp seqment of overlapping region of polymerase and S genes200 μl250 to 1011 copies/mlQuantified 292 vs. 282 sera by Amplicor HBV Monitor test among 302 HbsAg-positive patients (R2 = 0.86); quantified 63 vs. 36 sera by Quantiplex HBV DNA 1.0 assay among 66 HbsAg-positive patients (R2 = 0.88).Intra- and interassay CVs varied from 4.6% to 30.1% and from 5.1% to 53%, respectively.
Pas et al. 2002 (369)SerumABI Prism 7700/TaqMan probe90-bp fragment of pre-S gene200μ l46 to 1.25 × 109 IU/ml
Punia et al. 2004 (388)SerumLightCycler/SYBR-Green Irt204- and rt180-containing fragments of polymerase gene100 μl-Detected HBV rt204 and rt180 mutants in two more patients than conventional DNA sequencing assay among 10 patients treated with lamivudine.Detect and quantify HBV rt204 and rt180 mutant populations at 0.01% of 105 to 109 copies of wild-type HBV DNA using amplification-refractory mutation system PCR.
Schaefer et al. 2003 (434)SerumLightCycler/SYBR-Green I336-bp fragment of S gene200μ l20 to 2 × 109 IU/mlYielded quantification differences of <0.5 log IU/ml in 25 of 30 (83.3%) sera tested by a similar LightCycler assay amplifying a X gene target.Primers amplified S gene fragments of all well-characterized HBVs in humans, primates, squirrels, and woodchuck.
Stelzl et al. 2004 (461)SerumLightCycler/FRET hybridization probes120-bp fragment of HBV genome (? gene)200 μl2.5 × 102 to 109 IU/ml100% sensitivity and specificity compared to COBAS Amplicor HBV Monitor test among 117 clinical sera; results of 76% (37 of 49) of positive sera were within ±0.5 log IU/ml by both tests and 18%, respectively.Commercially as RealArt HBV LC PCR. Reagents available from Artus GmbH, Hamburg, Germany; intra- and interassay variation ranged from 9% to 40% and from 16% to 73%, respectively.
Sum et al. 2004 (469)SerumABI Prism 7000/molecular beacon probe112-bp fragment of S gene200 μl102 to 109 copies/mlDetected 128 (73%) vs. 119 (68%) positive sera by COBAS Amplicor HBV Monitor test among 175 HBsAg-positive patients (R2= 0.90).<7% intra-assay and <5% interassay variation in testing HBV ad and ay subtype DNA standards.
Takaguchi et al. 2002 (470)Serum, liver tissueABI Prism 7700/TaqMan probe272-bp fragment of X-pre-C gene region100 μl serum, 10 mg tissue200 copies/ml (analytical sensitivity for serum)Detected HBV DNA in 12 (33%) of 36 patients with HBsAg- and anti-HCV Ab-negative chronic liver disease; 5 of 6 serum HBV DNA-positive patients also had detectable HBV DNA in liver tissue.Unknown analytical sensitivity of assay for liver tissue.
Weinberger et al. 2000 (534)SerumABI Prism 7700/TaqMan probe81-bp fragment of S gene200μ l102 to 109 geq/mlGood correlation of results with laboratory-developed semiquantitative conventional PCR assay (R2 = 0.83).
Whalley et al. 2001 (539)SerumLightCycler/FRET hybridization probes130-bp fragment of polymerase gene200 μl400 copies/ml (analytical sensitivity)Good agreement with results of direct DNA sequence analysis.Utilize melting curve analysis to detect and differentiate rtM204 mutants at ≥25% level of minor population.
Wightman et al. 2004 (544)SerumABI Prism 7700/molecular beacon probertM204-containing fragment in C domain of polymerase gene200 μl102 copies/ml (analytical sensitivity)Good agreement with results of direct DNA sequence analysis.Able to detect 1% minor populations of rtM204V and rtM204I mutants.
Yeh et al. 2004 (553)SerumLightCycler/FRET hybridization probes340-, 368-, and 416-bp fragments of S gene200μ l102 to 1011 copies/mlGood correlation with quantitative results obtained by Amplicor HBV Monitor test (R2 = 0.99), NGI SuperQuant assay (R2 = 0.98), and Quantiplex HBV bDNA assay (R2 = 0.99); good agreement of genotype results with direct DNA sequence analysis.Intra- and interassay CVs were 8.9% and 14.3%, respectively, for quantification of HBV DNA; able to differentiate HBV genotypes A to G and detect mixed genotype infections at 10% level of minor population.
Zanella et al. 2002 (557)SerumGeneAmp 5700/TaqMan probe123-bp fragment of S gene200μ l102 to 1010 copies/mlDetected HBV DNA in 38 (35%) more samples than Digene Hybrid Capture HBV DNA test (R2 = 0.76)Intra- and interassay CVs ranged from 8.4% to 35.8% and from
among 108 HBsAg-positive sera; 100% specificity on sera from 20 healthy seronegative blood donors.6.2% to 53.1%, respectively.
Zanella et al. 2002 (558)Liver tissueGeneAmp 5700/TaqMan probe123-bp fragment of S gene200 mg tissue50 to 107 copies/mlNo comparison with other assays.Higher HBV DNA titers in liver tissues from HBsAg-positive than HBsAg-ngative patients (P< 0.01).
Zhang et al. 2002 (560)SerumLightCycler/FRET hybridization probes217-, 586-, and 650-bp fragments of basal core promoter, pre-C, and S genes, respectively200μ l100 copies/ml (analytical sensitivity)Good agreement with results of direct DNA sequence analysis.Utilize melting curve analysis to detect amd differentiate core/precore and HBsAg mutants at 5% level of minor population
HCVBullock et al. 2002 (53)SerumLightCycler/FRET hybridization probes324-bp fragment in 5′ noncoding region for initial PCR; 284-bp fragment for second-round PCR200μ l2 × 104 copies/ml minimumConcordance with line probe assay (INNO-LiPA) in 110 (99%) of 111 patient sera.HCV genotyping assay differentiating among genotypes 1, 1a, 1b, 2, 2a/c, 2b, 3a, and 4.
Candotti et al. 2004 (59)PlasmaStratagene Mx4000/TaqMan probe68-bp fragment in 5′ noncoding region200 μl167 (95% detection rate) to 105 IU/mlCorrelated with single virus real-time PCR assay (R2= 0.95) among 14 samples with HIV-1 RNA titers ranging from 300 to 105 IU/ml; 99% specificity among 266 healthy blood donor sera tested by anti-HCV EIA.Intra- and interassay CVs ranged from 0.8% to 2.4% and from 2.6 to 2.9%, respectively; equivalent detection of HCV genotypes 1 to 6; a multiplex assay which also detects HBV DNA and HIV-1 RNA.
Enomoto et al. 2001 (112)SerumABI Prism 7700/TaqMan probe161-bp fragment in 5′ noncoding region250μ l10 to 108 copies/mlCorrelated with Quantiplex HCV 2.0 (R2 = 0.84), Amplicor HCV Monitor v2.0 (R2 = 0.85), and HCV core
Ag EIA (R2 = 0.55) in 50 patient sera.
Kawai et al. 1999 (209)SerumABI Prism 7700/TaqMan probe199-bp fragment in 5′ noncoding region50μ l2 × 103 to 2 × 108 IU/mlCorrelated with Amplicor HCV Monitor v1.0 (R2 = 0.81) in 138 patient sera; 10- to 100-fold higher titers than those of Amplicor HCV Monitor.
Kleiber et al. 2000 (222)Plasma, serumABI Prism 7700/TaqMan probe250-bp fragment in 5′ noncoding region200 μl64 to 107 IU/ml100% specificity in 100 HCV-seronegative blood donor sera tested by Cobas Amplicor HCV test, v2.0.CVs varied from 21.6% to 30.4%; equivalent detection of HCV genotypes 1 to 5.
Komurian-Pradel et al. 2001 (231)SerumLightCycler/SYBR Green I220-bp fragment in 5′ noncoding region200 μl4 to 4.05 × 106 IU/mlCorrelated with Quantiplex HCV RNA 2.0 assay (R2 = 0.79) and quantified 2 more clinical serum specimens than Quantiplex among 33 patient sera.CVs varied from 0.7% to 3.7%.
Martell et al. 1999 (304)SerumABI Prism 7700/TaqMan probe194-bp fragment in 5′ noncoding region140μ l330 to 107 copies/mlCorrelated with Quantiplex HCV RNA 2.0 assay (R2 of 0.71 to 0.88) and National Genetics Institute Superquant assay (R2 of 0.74 to 0.99) in 79 patient sera.CVs varied from 1% to 6.2%.
Mercier et al. 1999 (314)Plasma, serumABI Prism 7700/TaqMan probe168-bp fragment in 5′ noncoding region200 μl50 copies/ml (analytical sensivity)100% sensitivity and specificity in 50 patients and 50 blood donors tested by Quantiplex HBV 1.0 assay; 0.7% false-positive rate in screening 274 healthy blood donors.CVs varied from 8.7% to 74.7%; a multiplex assay which also detects HBV DNA.
Ratge et al. 2002 (398)SerumLightCycler/FRET hybridization probes235-bp fragment in 5′ noncoding region200 μl109 to 2,500 IU/ml100% agreement in results with a qualitative conventional assay for 156 patient sera.CVs varied from 11.5% to 12.9%; two rounds of rapid-cycle real-time PCR in this assay.
Schroter et al. 2001 (440)SerumLightCycler/SYBR Green I124-bp fragment in 5′ noncoding region200 μl900 to 7 × 106 copies/mlCorrelated with Amplicor HCV Monitor v1.0 and in-house conventional PCR assays in 81 patient sera and 30 healthy blood donor sera.
Schroter et al. 2002 (439)SerumLightCycler/FRET hybridization probes143-bp fragment in 5′ noncoding region200μ l103 IU/ml (analytical sensitivity)100% concordance with conventional HCV NS5B gene sequencing assay in 190 patient sera.HCV genotyping assay differentiating among genotypes 1 to 4 (not subtypes).
Takeuchi et al. 1999 (471)SerumABI Prism 7700/TaqMan probe161-bp vs. 256-bp fragment in 5′ noncoding region250 μl10 to 108 copies/ml256-bp target amplification correlated with Quantiplex HCV 1.0 assay (R2= 0.84) and Amplicor HCV Monitor v1.0 (R2 = 0.61) in 15 patient sera; 10- to 100-fold more sensitive than and Amplicor HCV Monitor v1.0, and 100% specificity in 50 patient sera.CVs varied from 0.4% to 4.7%; 161-bp target amplification showed higher sensitivity (e.g., higher RNA titers) than 256-bp target amplification.
White et al. 2002 (542)Serum, liver biopsy tissueLightCycler/SYBR Green I244-bp fragment in 5′ noncoding region200μ l serum; 0.8 to 8.1 mg liver tissue102 to 106 copies/ml serum or copies/ug of total RNA in tissueCorrelated with Amplicor HCV Monitor v2.0 (R2 = 0.86) in 16 patient sera.Assay conisted of an initial 15-cycle real-time PCR followed by 40-cycle real-time PCR.
Yang et al. 2002 (552)Plasma, serumABI Prism 7700/molecular beacon182-bp fragment in 5′ noncoding region100μ l104 (60% detection rate) to 109 copies/ml100% sensitivity and specificity in 17 patients and 14 healthy blood donors tested by anti-HCV EIA.Equivalent detection of HCV genotypes 1 to 6.
HDVYamashiro et al. 2004 (551)SerumLightCycler/SBYR Green I134-bp fragment; 98% homologous for genotypes I and IIa, 100% homologous for genotype Iib150 μl100 to 106 copies/mlGood correlation with HBV DNA levels in 48 patients.
HEVOrru et al. 2004 (357)FecesLightCycler/SYBR Green I77-bp fragment in ORF2 region300 μl of 10% stool suspension in PBS10 to 106 copies/PCRCorrelated with anti-HEV antibody by EIA and conventional real-time PCR in one clinical sample.
  • a HAV, HBV, HCV, HDV, and HEV, hepatis virus type A, B, C, D, and E, respectively; CV, coefficient of variation; geq, genome equivalent; EIA, enzyme immunoassay; Ab, antibody; PBS, phosphate-buffered saline.