Fluorescent Hybridization Probes Research Articles

Association of Clinical Status of Follicular Lymphoma Patients after Autologous Stem Cell Transplant and Quantitative Assessment of Lymphoma in Blood and Bone Marrow as Measured by SYBR Green I Polymerase Chain Reaction

Molecular remission in the autograft and bone marrow after transplant are predictive of durable clinical remission in relapsed follicular lymphoma. Thus, a simple reliable method to quantify minimal residual disease (MRD) would improve prognostication in these patients. Fluorescent hybridization probes have been used in real-time quantitative polymerase chain reaction (RQ-PCR) to monitor MRD with a reproducible sensitivity of 0.01%; however, these techniques are expensive and require additional experiments to examine clonality. We describe a SYBR Green I detection method that is more universal, checks clonal identity, yields the same sensitivity for monitoring MRD, and is more economically attractive. Using this method to follow 14 follicular lymphoma patients treated with autologous stem cell transplantation, molecular markers were successfully defined for 12 patients. Median contamination of stem-cell grafts was 0.1% (range, 0 to 13%). Six patients with measurable graft contamination became PCR-negative in blood and bone marrow within 12 months after autologous stem cell transplantation. Three patients free of disease progression (median follow-up of 75 months) are in molecular remission. Increasing fractions of RQ-PCR-positive blood and bone marrow cells reliably predicted morphological and clinical relapse. In one case, both clinical relapse and spontaneous regression were reflected by changes in MRD levels. Thus, our RQ-PCR method reproducibly distinguishes different levels of MRD.

Multiplex Molecular Diagnosis of MEFV Mutations in Patients with Familial Mediterranean Fever by LightCycler Real-Time PCR

Familial Mediterranean fever (FMF) is an autoinflammatory disease inherited as an autosomal recessive condition and characterized by recurrent episodes of fever, abdominal pain, pleuritis, and arthritis (1)(2). The most dangerous manifestation is renal amyloidosis and end-stage renal failure. Many researchers have associated the M694V mutation (3)(4)(5) as well as the α allele of serum amyloid A protein I with significantly higher risk for amyloidosis (6). FMF is very prevalent in non-Ashkenazi Jews, Armenians, Arabs, and Turks, all of whom have ancestors of Mediterranean origin. The carrier frequency in these populations can reach up to 1:5, rendering it the most frequent autosomal recessive condition. Currently, it is considered frequent in other Mediterranean countries, including Cyprus (7)(8)(9)(10)(11). The gene associated with FMF, MEFV , which encodes pyrin/marenostrin, a protein of 781 amino acids (12)(13), belongs to the pyrin family of genes that play a role in autoinflammatory diseases and inflammatory pathways (14)(15). More than 40 mutations have been identified, but 4–6 mutations usually account for a high percentage of MEFV chromosomes in different ethnic populations, emphasizing the importance of molecular epidemiologic studies for identifying the various molecular defects in the population of interest. The detection of known mutations in MEFV can be accomplished by various methods, including direct DNA sequencing. These methods are time-consuming and prone to carryover PCR contamination because of the repetitive work in a diagnostic setting. Using fluorescence resonance energy transfer (FRET) on a LightCycler® instrument (Roche), we developed a rapid method that detects point mutations and small deletions by use of fluorescent hybridization probes. Because of the close proximity of several mutations on exon 10, we designed a single pair of fluorescent probes with multiplex melting profiles that permits …

Real time PCR assay with fluorescent hybridization probes for genotyping intronic polymorphism in presenilin-1 gene

Background Identification of all susceptibility loci for Alzheimer's disease has been a major goal in resolving the pathogenesis of this disease. Methods A PCR assay with fluorescently labeled oligonucleotide hybrinization probes with subsequent fluorescent probe melting point analysis was developed. Results Allelic discrimination of intronic polymorphism of presenilin-1 gene and the restriction fragment length polymorphism method yielded identical results, proving its usefulness for genotyping PS1 gene. Conclusions This method provides excellent robustness, speed, and accuracy, and is well suited for determination of the polymorphism in both small and large numbers of samples. This assay could help to overcome the controversy regarding the association between the PS1 s165932 intronic polymorphism and Alzheimer's disease.

Discovering Rare Variants by Use of Melting Temperature Shifts Seen in Melting Curve Analysis

Two reports in this issue of Clinical Chemistry describe novel mutations in thrombophilia genes (1)(2). The authors of the reports describe variants detected by the identification of a melting temperature ( T m) different from the expected T m of the target mutation and from the T m of the wild-type sequence. The variants reported in this issue are A20218G in the prothrombin (factor II) gene and C1690T in the factor V gene. These cases add to the reported variants identified by fluorescent hybridization probes and melting analysis on the LightCycler. In addition to factor II and V variants, variants have been discovered similarly in genes coding for MTHFR and HFE (3)(4). Melting analyses, as performed on the LightCycler or similar instruments, can distinguish variants that lie within the region of the target probe. Differences in base composition, nucleotide position, and nearest-neighbor environments all affect T m. These differences are detected by monitoring fluorescence during an increase in temperature. Melting is visualized by a loss of fluorescence as the probes dissociate from the template. Differences in T m from the expected T m for wild-type and target mutations are indicative of a nontarget (a variant other than the mutation the assay was designed to detect) mutation or variant. In contrast, assays that use restriction enzymes, allele-specific amplification, or hybridization probes at a single detection temperature may not detect a variant or may show an indeterminate genotype (3)(4). With these 3 types of assays, when one allele carries the variant and the opposite allele is a wild type, the variant allele may be undetected, and the genotype would be reported as wild type. If the variant is in conjunction with a target mutation on the opposite allele, the genotype could be reported as a …

Refining chromosomal region critical for Down syndrome‐related heart defects with a case of cryptic 21q22.2 duplication

We report here a patient with features of Down syndrome and tetralogy of Fallot who had a 21q22 duplication. The extent of the duplication was defined using fluorescent hybridization probes that map to the critical region on chromosome 21. Included within the interval was the cell adhesion molecule DSCAM but not the collagen COL6A1. The present case provides further support to the concept that there exists Down syndrome-associated congenital heart disease gene(s) on chromosome 21q22 and that over-expression of DSCAM may contribute to the cardiac defects of Down syndrome.

Rapid presumptive identification of Burkholderia pseudomallei with real-time PCR assays using fluorescent hybridization probes

Burkholderia pseudomallei (the etiologic agent of melioidosis) can cause pyogenic or granulomatous lesions in almost any organ. Septicemia has a case fatality rate of >40%. Early diagnosis and appropriate antibiotic therapy are crucial for survival, but cultivation, biochemical identification, and conventional PCR of B. pseudomallei are time consuming. We established real-time PCR assays using fluorescent hybridization probes targeting the 16S rDNA, the flagellin C (fliC) and the ribosomal protein subunit S21 (rpsU) genes. The test sensitivity and specificity were assessed with a representative panel of 39 B. pseudomallei, 9 B. mallei, 126 other Burkholderia strains of 29 species, and 45 clinically relevant non-Burkholderia organisms. The detection limit for the 16S rDNA, fliC, and rpsU assay was 40, 40, and 400 genome equivalents per reaction, however, in spiked blood samples it was 300, 300, and 3000, respectively. Specificity, positive and negative predictive value of the assays was 100%. In conclusion, we recommend the use of the 16S rDNA and/or fliC real-time PCR assays for the rapid identification of B. mallei and B. pseudomallei in positive blood cultures or from suspicious bacterial colonies.

A LightCycler real-time PCR hybridization probe assay for detecting food-borne thermophilic Campylobacter

In a previous study, we reported the performance of a PCR assay amplifying 287-bp of the 16S rRNA gene of thermo-tolerant Campylobacter ( C. jejuni, C. lari, C. coli) through an international ring-trial involving 12 participating laboratories. Based on the validated set of primers, a LightCycler real-time PCR assay (LC-PCR), which used fluorescent hybridization probes was developed. The test incorporated an internal amplification control co-amplified with the 16S rRNA gene of Campylobacter to monitor potential PCR inhibitors and ensure successful amplifications. The specificity study involving 39 Campylobacter and nine strains of other species indicated that the LC-PCR test was highly specific, giving cross-reactivity with only one strain of C. upsaliensis (CCUG19559). The sensitivity of the LC-PCR assay, evaluated in 32 spiked poultry-rinse or pork carcass-swab samples, was determined at 10 CFU/ml carcass-rinse. The prevalence of samples positive for thermo-tolerant Campylobacter was 58.8% in 68 naturally contaminated poultry rinse samples tested by LC-PCR and the data were in good concordance with those of bacteriological method. The Ct values of the three replicates obtained for each sample tested in three different runs demonstrate that the LC-PCR was highly reproducible and afford a powerful tool for rapid detection of the thermo-tolerant Campylobacter strains.

Quantitative real-time PCR with automated sample preparation for diagnosis and monitoring of cytomegalovirus infection in bone marrow transplant patients.

In bone marrow and stem cell transplant patients, the widespread use of preemptive cytomegalovirus (CMV) antiviral therapy necessitates faster, more precise, and more sensitive quantitative laboratory methods for serial viral load monitoring. We developed a novel CMV viral load assay using real-time PCR of plasma DNA prepared by an automated robotic workstation. Fluorescent hybridization probes directed at the glycoprotein B (gB) gene (or EcoRI D region) of CMV were used to detect and quantify PCR products. The beta-globin gene was amplified in parallel to control for the efficiency of the extraction and PCR steps. The assay was linear (R = 0.999) from a lower detection limit of 125 copies/mL to 5 x 10(9) copies/mL with a PCR efficiency of 1.975 (gB) or 2.02 (EcoRI D). The viral loads determined by PCRs directed at these two different viral targets were no different (n = 53; R = 0.928). The interassay CV was 3.5%, and the intraassay CV was 1-4%. Compared with a commercially available quantitative competitive PCR assay (Roche MONITOR; R = 0.59), the mean CMV viral load by real-time PCR was 3.1 times higher (mean ratio; P = 0.002). The diagnostic sensitivity and specificity of the real-time assay were 96% and 100%, respectively (n = 147), compared with 74% and 98% for a qualitative PCR assay (Roche AMPLICOR). On a subset of samples, the diagnostic sensitivity of viral culture was no greater than 50% (n = 44). Of 1115 clinical referral samples from 252 patients, 10% of the samples and 18% of the patients had low-level CMV viremia (median, 500 copies/mL). In this predominantly (85%) bone marrow transplant testing cohort, serial CMV viral load results were the predominant clinical trigger for the initiation, monitoring, and cessation of preemptive antiviral therapy. The combination of automated DNA preparation and semiautomated real-time fluorescent PCR detection allows for a sensitive, precise, and accurate high-throughput assay of CMV viral load that can be used as the laboratory trigger for preemptive antiviral therapy.

Retention of the Arginine Allele in Codon 72 of the p53 Gene Correlates with Poor Apoptosis in Head and Neck Cancer

The allele constitution at codon 72 of the p53 gene (CGC-arginine or CCC-proline) plays a major role in inducing apoptosis in p53 mutant cells. To verify this, we determined GC-status, p53-mutations, and p53-loss of heterozygosity (LOH) in a group of 54 squamous cell carcinomas of the head and neck (SCCHN). A novel approach, using a one-step real-time PCR analysis with fluorescent hybridization probes, was applied to detect the GC status in tumors and corresponding blood samples. p53 mutations in exons 4 to 8 were detected by PCR-SSCP-sequencing analysis. Apoptosis was determined immunohistochemically using antibodies against Fas, FasL, p53, Bcl2, and terminal deoxy-transferase-mediated dUTP nick end labeling (TUNEL) staining. The overall frequency of p53-LOH in SCCHN was 45.2%. In cases of LOH, there was a preferential loss of the proline allele, which was associated with an up-regulation of Bcl2 and lack of co-expression of Fas/FasL and, thus, impaired apoptosis (P < 0.001). Apoptosis was not observed in tumors carrying the arginine allele. p53 mutations were detected in 29.6% of SCCHN and preferentially occurred at the arginine allele (P = 0.01). p53 alterations were more frequently observed in tumors of the oral cavity, oropharynx and hypopharynx, whereas they were rare in larynx carcinomas (P = 0.07). The p53-LOH status was not found to be significantly correlated with sex, age, TNM-status, or tumor grading. We conclude that apoptosis is correlated with the allelic status of codon 72 in SCCHN. Homozygous proline 72 appears to be an important regulator of apoptosis via the Fas/FasL pathway in SCCHN.

Rapid detection and quantitation of hepatitis B virus DNA by real-time PCR using a new fluorescent (FRET) detection system.

The diagnosis of hepatitis B virus (HBV) has until recently been based on traditional serologic methods targeting viral antigens and antibodies to viral proteins. The development of molecular methods allowing for the quantitation of HBV DNA is proving clinically valuable for monitoring therapy and detecting early treatment failures. Here we report a new real-time (LightCycler) quantitative PCR for the detection of HBV DNA based on sequence specific hybridisation probes (designed in-house), targeting the HBV surface antigen. The assay was evaluated using a 10-fold dilution series of standard HBV DNA [Eurohep standard reference 1, genotype A, HBsAg subtype adw with a unitage of 10(6) WHO. i.u./ml] and 89 clinical serum samples. The performance was measured against a quantified standard HBV DNA working reagent (NIBSC code 98/780) and the sensitivity compared with our conventional thermal-block PCR. Real-time PCR detected HBV DNA in 45% (40/89) and thermal-block PCR in 16% (14/75) of clinical samples. Results for 26 samples were below the detection limit of the thermal-block PCR but could be quantified by real-time (LightCycler) PCR. The LightCycler assay was at least 5 logs more sensitive than thermal-block PCR and could detect HBV in a linear range between 5 and 10(7) i.u. per reaction. The broad generic nature of the PCR primers coupled with the enhanced sensitivity and specificity of the fluorescent hybridisation probes makes this assay potentially valuable for both routine diagnostic and epidemiological work.

Rapid identification of commonly encountered Candida species directly from blood culture bottles.

We report a rapid-cycle, real-time PCR method for identifying six Candida spp. directly from BACTEC blood culture bottles. Target sequences in the noncoding internal transcribed spacer regions of the rRNA operon were simultaneously amplified and interrogated with fluorescent probes to identify Candida albicans, C. glabrata, C. parapsilosis, C. tropicalis, C. krusei, and C. lusitaniae; these account for 88% of the yeast species isolated from positive blood cultures in our laboratory. Any of the first four species can be identified in a single reaction using two fluorescent hybridization probe sets. The antifungal-resistant species C. krusei and C. lusitaniae are detected in a second reaction, also with two probe sets. The assay was validated with DNA extracted from BACTEC blood culture bottles positive for yeasts (n = 62) and was 100% concordant with culture identification based on biochemical and morphological features of C. albicans (n = 22), C. parapsilosis (n = 10), C. tropicalis (n = 1) C. glabrata (n = 22), C. krusei (n = 2), and C. lusitaniae (n = 1). No cross-reactivity was observed in blood culture samples growing yeasts other than the above-mentioned species (n = 4), in those growing bacteria (n = 12), or in the absence of microbial growth. Our assay allows rapid (</=2 h) and specific detection of the most common Candida spp. directly from positive blood cultures and may facilitate delivery of optimal antifungal therapy.

High-speed genotyping of CYP1A2*1F mutation with fluorescent hybridization probes using the LightCycler.

The polymorphism CYP1A2*1F of the cytochrome 1A2 gene affects the inducibility of this cytochrome, which is involved in the metabolism of many commonly used drugs. We developed a rapid-cycle polymerase chain reaction (PCR) and fluorescent probe melting curve analysis on the LightCycler instrument for identification of this mutation. This new method was confirmed by genotyping 101 samples in parallel to restriction enzyme digestion. The 100% concordance of both methods showed the reliability of our high-speed genotyping assay, which is suitable for large epidemiological studies or routine clinical use.

Low copy number detection of herpes simplex virus type 1 mRNA and mouse Th1 type cytokine mRNAs by Light Cycler quantitative real-time PCR

The real-time principle of reverse transcriptase polymerase chain reaction (RT-PCR) provides a quantitative and reproducible method to detect low copy number transcripts. The quantitative detection of cytokines from tissue samples is complicated by the low expression rates and the short half-lives of the cytokine proteins. The methods have been insensitive and labor-intensive. The LightCycler technique provides a 30-min PCR system with continuous fluorescent detection, analysis of the melting points of the products and user-friendly software for the analysis of the unknown samples. External copy number standards enable the measurement of amounts of the desired targets. We demonstrate the dynamic range of the RT-PCR system from a 100 to 10 7 mRNA copies of the mouse Th1 cytokines interleukin- (IL-) 12p35, 12p40 and IL-23p19 as well as gamma interferon (IFN-γ) and the housekeeping gene β-actin, with the usage of fluorescent hybridization probes. The cytokine quantitation was exemplified in murine nervous system samples. A viral transcript, mRNA of alpha trans-inducing factor (αTIF), or VP16 gene, of herpes simplex virus (HSV) type 1 was used to quantitate the viral replication in infected cells and in murine nervous system samples. For this viral transcript the linear dynamic range spanned from ten copies to one million copies (highest tested). For all tested cytokine transcripts, the detection level with the dsDNA binding dye SYBR Green I was one log lower than with the hybridizing fluorescent probes. The viral transcript was detected even with the SYBR Green I system at the level of ten copies. The specificity of the PCR was reached with the use of TaqStart antibody, by careful design of primers and probes, by melting temperature analysis and comparison with the gel electrophoresis and Southern blot analysis.

Rapid detection of Yersinia pestis with multiplex real-time PCR assays using fluorescent hybridisation probes

The objective of the present study was to establish a system of real-time polymerase chain reactions (PCRs) for the specific detection of Yersinia pestis using the LightCycler™ (LC) instrument. Twenty-five strains of Y. pestis, 94 strains of other Yersinia species and 33 clinically relevant bacteria were investigated. Assays for the 16S rRNA gene target and the plasminogen activator gene (resides on the 9.5-kb plasmid) and for the Y. pestis murine toxin gene and the fraction 1 antigen gene (both on the 100-kb plasmid) were combined for the use in two multiplex assays including an internal amplification control detecting bacteriophage λ-DNA. Applying these multiplex assays, Y. pestis was selectively identified; other bacteria yielded no amplification products. The lower limit of detection was approximately 0.1 genome equivalent. Rat or flea DNA had no inhibitory effects on the detection of Y. pestis. The results obtained using the multiplex real-time assays showed 100% accuracy when compared with combinations of conventional PCR assays. We developed and evaluated a highly specific real-time PCR strategy for the detection of Y. pestis, obtaining results within 3 h including DNA preparation.

Combined factor V leiden (G1691A) and prothrombin (G20210A) genotyping by multiplex real-time polymerase chain reaction using fluorescent resonance energy transfer hybridization probes on the Rotor-Gene 2000.

Several methods have been developed to detect common single point mutations in the factor V and prothrobin genes that are risk factors for thrombophilia. Most are based on PCR followed by restriction enzyme digestion and electrophoresis (RFLP), but gel analysis has certain limitations, and alternative detection methods, including real-time PCR, have therefore been developed. In this study we developed and evaluated a combined factor V Leiden and prothrombin (G20210A) genotyping method based on multiplex real-time PCR with fluorescent resonance energy transfer (FRET) hybridization probes on the Rotor-Gene 2000. Two hundred subjects were screened for the two mutations. The FRET assay clearly discriminated among wild-type, homozygous and heterozygous status for the two mutations, and the results were in full agreement with those of the RFLP assay. This robust FRET probe-based assay also has a higher throughput capacity than conventional methods, handling up to 72 samples in 90 min.

Rapid quantification of hepatitis B virus DNA by real-time PCR using fluorescent hybridization probes.

A highly sensitive and rapid assay has been developed to quantify hepatitis B virus (HBV) DNA, based on the fluorescence resonance energy transfer principle and real-time PCR, using the LightCycler and a pair of specific fluorescent hybridization probes. This LightCycler real-time PCR assay (LC-PCR) detected HBV DNA in a linear range from 10(1) to 10(8) copies per reaction (250-2.5 x 10(9) copies ml(-1)), with a rapid PCR cycling time of 35 min. The assay was validated with two EUROHEP HBV DNA standards (ad and ay subtypes) and exhibited low intra-assay (< 6 %) and inter-assay (< 16 %) variation for both subtypes over the complete range of 7 orders of magnitude. The assay was evaluated clinically using serum samples from 120 HBsAg(+) individuals and 45 healthy controls who were negative for both HBsAg and anti-HBc. Levels of HBV DNA were measured in these samples using both the LC-PCR and Digene Hybrid Capture II HBV DNA (HCII) assays. The prevalence rates for HBV DNA in the HBsAg(+) serum samples were respectively 95 % (114/120) and 56 % (67/120) by LC-PCR and HCII (P < 0.01). All 67 HCII-positive samples tested positive with LC-PCR, while the 47 discordant samples showed low levels of HBV DNA (down to 265 copies ml(-1)), detectable only by the more sensitive LC-PCR assay. Levels of HBV DNA as measured by the two assays showed good correlation (r = 0.902; P < 0.001). The level of HBV DNA was significantly higher in HBeAg(+) than anti-HBe(+) samples (median 1.5 x 10(7) vs 4.6 x 10(4) copies ml(-1); P < 0.01). It is concluded that this LC-PCR assay is clinically useful for the rapid, sensitive and accurate measurement of HBV DNA.

Rapid genotyping of MBL2 gene mutations using real-time PCR with fluorescent hybridisation probes

In this study, we describe a real-time polymerase chain reaction (PCR) for genotyping all known polymorphisms of the human mannose-binding lectin 2 ( MBL2) gene. These comprised two variations in the 5′ regulatory region at positions −550 (H/L) and −221 (X/Y), one in the 5′ untranslated sequence at position +4 (P/Q) and three structural mutations within exon 1 at codons 52, 54, and 57, also known as the D, B and C variants, respectively. Three reactions with two different conditions were sufficient to genotype one individual unambiguously. The three mutations in exon 1 were detected in one capillary using a sensor probe covering the three mutations, whereas amplification of the variants located upstream of the coding sequence was performed in only two reactions. Single colour detection was used for detection of the (H/L) polymorphism and multiplexing by dual colour probes was used for simultaneous genotyping of (X/Y) and (P/Q). The reliability of the system was evaluated by comparison with a conventional PCR method with sequence-specific primers (PCR-SSP). For this study, 100 individuals of Danish and 30 of African descent were analysed, and the genotypes obtained were concordant in all cases. This new method is rapid and provides reliable results without ambiguities.

Rapid detection of Salmonella from poultry by real-time polymerase chain reaction with fluorescent hybridization probes.

Detection of Salmonella by bacteriologic methods is known to be time consuming. Therefore, we have developed a real-time probe-specific polymerase chain reaction (PCR) to rapidly detect Salmonella invA gene-based PCR products from chicken feces and carcasses by a fluorescence resonance energy transfer assay. The sensitivity and the specificity of this system were determined as 3 colony-forming units ml(-1) and 100%, respectively. Overnight tetrathionate broth enrichment cultures of chicken feces and carcass samples were used in template preparation for PCR. Also, a standard bacteriology was performed (National Poultry Improvement Plan-U.S. Department of Agriculture, Bacteriological Analytical Manual-Food and Drug Administration Center for Food Safety and Applied Nutrition) for confirmation. Seventy-two cloacal swab, 147 intestine, and 50 carcass (neck) samples were examined. Thirteen (8.8%) and 25 (17%) of the intestinal samples were found to harbor Salmonella by bacteriology and PCR, respectively. Forty-five of 50 (90%) carcass samples were Salmonella positive by both methods. Salmonella was not detected from cloacal swab samples. Results indicate that this assay has the potential for use in routine monitoring and detection of Salmonella in infected flocks and carcasses.

A one-step real-time PCR assay for rapid prenatal diagnosis of sickle cell disease and detection of maternal contamination.

Mutations at the codon 6 of the beta-globin gene (hemoglobin [Hb] S and HbC) can be routinely identified by various methods and prenatal diagnosis has been available to affected families for several years. However, the presence of maternal cells in fetal samples constitutes a serious potential source of prenatal misdiagnosis and most methods currently used to detect maternal contamination are based on the analysis of highly polymorphic loci. In addition, these methods are labor intensive and time consuming and risk carry-over contamination. We describe here a one-step method for mutation detection that uses fluorescent hybridization probes with melting curve analysis for both simultaneously prenatal diagnosis of sickle cell disease and potential maternal contamination. Retrospective and prospective prenatal diagnosis studies (conducted in 20 and 50 cases, respectively), using both the regular procedure and real-time PCR assay show perfect concordant results. We show in addition, that as little as 5% maternal contamination can be detected and that genotype determinations are unambiguous.

Rapid detection of hepatitis B virus mutations using real-time PCR and melting curve analysis

Current methods of detecting hepatitis B virus (HBV) mutations are time consuming, labor intensive, and not suitable for screening large numbers of samples. In the present study, we documented the advantages of a system that exploits differences in thermal stability between perfect match and mismatch hybrids, and thereby distinguishes between wild-type and mutants. Hybridization probes were designed complementary to specific wild-type HBV sequences in surface (S), precore, and basal core promoter (BCP) regions of the HBV genome (nt 587, 1896, and 1762/1764, respectively). Two probes were designed for each mutation: anchor probes were 3' labeled with fluorescein and sensor probes, 5' labeled with LC-Red 640, and 3' phosphorylated. Temperatures for each probe melted from amplification products were then determined in a melting program. Sera from 12 patients, each containing identified HBV mutants (6 S-escape, 1 precore, 1 BCP, and 4 mixed precore and BCP), and 5 control sera from patients with wild-type virus were analyzed. Genomic sequences of mutant and wild-type viruses were confirmed by direct sequencing. Real-time polymerase chain reaction (PCR) with fluorescent hybridization probes accurately identified each mutant and wild-type genome. Melting temperatures obtained from probe-product duplexes for the 3 mutants were distinguished from wild-type (>4.0°C, minimal) within 45 minutes. The sensitivity of the system was 100 copies/mL and as few as 5% of mutant among wild-type virus were detected. In conclusion, real-time PCR with fluorescent hybridization probes is a specific, sensitive, quantitative, and rapid means of detecting clinically relevant HBV mutants. (H EPATOLOGY 2002;36:723-728.)