Probe Melting Research Articles

Use of lyophilized standards for the calibration of a newly developed real time PCR assay for human herpes type six (HHV6) variants A and B

A quantitative real time PCR assay utilizing an Eclipse minor groove binding hybridization probe was developed to detect and type human herpes 6. A 115 base pair product from the U67 gene was selected for amplification and the assay included a noncompetitive internal control. The probe's melting temperature from the amplified sequence differentiated between HHV6 variants (A and B). In this study, 120 samples (60 spiked and 60 negative) comprising CSF, plasma, and serum were tested at high and medium levels, and near the limit of quantitation. The use of stored standard curves for assay calibration was compared to curves run on each assay, and the stability of liquid frozen versus lyophilized frozen stocks for calibrators and controls was assessed. After 9 months of clinical testing, assay performance was examined to determine the percent positive rate and positive sample reproducibility, as well as to evaluate standard curve stability. We obtained 100% correlation to expected results for positive and negative samples. A stored curve proved easier, more cost effective, and more reliable than running a standard curve on each assay. The use of lyophilized standards contributes substantially to the maintenance of reproducible testing over an extended period of time.

Detection and typing of herpes simplex DNA in genital swabs by real-time polymerase chain reaction

The LightCycler polymerase chain reaction (PCR) is a sensitive assay for the detection of Herpes simplex virus (HSV) DNA in muco-cutaneous swabs. Software-based analysis of the probe melting temperature ( T m) can be used to discriminate between HSV types (HSV-1 and HSV-2). Among 76 HSV DNA positive genital swabs, atypical T ms were observed in 14 (18%). The 14 samples were all typed as HSV-2 by sequence alignment. In 4/14 samples, the atypical T m was associated with sequence variation at the probe-binding site. Among 10 samples with conserved sequences, T ms were influenced by the specimen preparation method prior to PCR. These findings indicate that multiple factors including, but not limited to sequence variation complicate melting curve analysis following real-time PCR. Alternative typing methods are recommended for specimens with atypical melting curves.

Detection of three common G6PD gene mutations in Chinese individuals by probe melting curves

Objective: In order to develop and validate an assay for rapid detection of three common G6PD gene mutations in Chinese individuals. Methods: In this report we design two sets of primers and fluorescently labeled hybridization probes recognizing adjacent sequences in the amplicon; after annealing, the fluorophores were in resonance energy transfer, providing real-time monitoring of the amplication process. At the completion of PCR, fluorescence was monitored as the temperature increased through the T m of the probe/product duplex, and a characteristic melting profile for each mutation was obtained. By using the fluorescence method and PCR/RE, a total of 57 samples obtained from two groups of G6PD-deficient individuals were studied. Result: A rapid method for detection of three common G6PD gene mutations in Chinese individuals by probe melting curves was developed. This method shows 100% accordance with the traditional method. Conclusion: This fluorescent melting curve analysis is a simple, rapid, and effective method for clinical diagnosis and screening of G6PD deficiency.

Diagnosis of genital herpes by real time PCR in routine clinical practice

Background: Virus isolation in cell culture is the recognised diagnostic gold standard for genital herpes. Although increasing evidence indicates that polymerase chain reaction (PCR) provides a more rapid and sensitive...

Rapid detection and simultaneous subtype differentiation of influenza A viruses by real time PCR

A real time RT-PCR, using the LightCycler, was developed and compared with rapid antigen enzyme immunoassay (AgEIA) and enhanced virus culture for rapid detection of influenza A viruses in stored and prospectively collected respiratory specimens. Specific hybridization probes were used for simultaneous detection and differentiation between H1N1 and H3N2 subtypes. The sensitivity of the RT-PCR for influenza A H1N1 was 120 copies and H3N2 350 copies of in vitro transcribed RNA. A specimen was considered positive for influenza A when it was culture positive or at least two methods yielded a positive test result. Using these criteria, with stored samples, the RT-PCR sensitivity, specificity, positive and negative predictive values were 82.9, 95.5, 98.9 and 52.5%, respectively. In specimens collected prospectively the RT-PCR sensitivity, specificity, positive and negative predictive values were 100, 87.9, 82.8 and 100%, respectively. There was complete concordance with subtype differentiation by hybridization probe melting temperature analysis and haemagglutination inhibition assay.

Real-time PCR.

The development of instruments that allow real-time monitoring of fluorescence within PCR reaction vessels is a significant advance in clinical bacteriology. The technology is very flexible, and many alternative instruments and fluorescent probe systems are currently available. Real-time PCR assays can be completed very rapidly, because no manipulations are required after amplification. Identification of amplification products by probe detection in real time is highly accurate compared with size analysis on gels. Analysis of the progress of the reaction allows accurate quantification of the target sequence over a very wide dynamic range, provided suitable standards are available. Finally, probe melting analysis can detect sequence variants including single base mutations.

Rapid detection of the 46C --> T polymorphism in the factor XII gene, a novel genetic risk factor for thrombosis, by melting peak analysis using fluorescence hybridization probes.

Factor XII (FXII) level is an important intermediate phenotype associated with thrombotic disease. The 46C --> T transition in the exon 1 of the Factor XII (F12) gene is a significant, prevalent, and independent genetic risk factor for thrombotic disease. It is also associated with interindividual variation of plasma FXII zymogen levels. The aims of this study were to develop a rapid, reproducible, and easy method for 46C --> T genotyping and to compare its reliability with the classical endonuclease digestion methodology. DNA samples from 100 subjects were genotyped for the 46C --> T transition using the classical endonuclease digestion method with Sfna I. The genotypes of three of them (each with a different 46C R T genotype) were confirmed by direct sequencing analysis. We then set out to construct a LightCycler PCR protocol to detect the 46C --> T polymorphism. This protocol was designed to combine a rapid-cycle polymerase chain reaction (PCR) with an allele-specific fluorescent probe melting for mutation detection. In the three sequenced samples, as well as in the remaining 97, the LightCycler PCR procedure unambiguously resulted in the same genotype previously observed by sequencing and endonuclease digestion. Characteristic fluorescent curves were obtained for each genotype; the first derivative of these curves had a maximum at an apparent hybridization temperature (Tm) that was specific for each probe/allele duplex. The whole process took less than 40 min. Thus, if this method is used with a rapid DNA extraction, the genotypes would be obtained within 60 min after receiving a blood sample. In conclusion, the technique presented allows for easy, reliable, and rapid detection of this polymorphism, and is suitable for typing both small and large numbers of DNA samples.

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.

Detection and discrimination of B. pertussis and B. holmesii by real-time PCR targeting IS 481 using a beacon probe and probe-target melting analysis

A beacon probe was designed to detect one of the two documented single nucleotide changes in IS 481 target allele of Bordetella holmesii genome as compared to Bordetella pertussis. PCR amplified product targeting a region of IS 481 in presence of the probe was subjected to a post-PCR hybridization and melting cycle. Hybrid of the probe with B. pertussis specific target had a different thermal stability than that with allele having the single nucleotide change in B. holmesii. The melting of B. pertussis-probe hybrid occurred in a single phase; while that of B. holmesii-probe hybrid was biphasic-one for allele identical to that in B. pertussis and the other for that with a single nucleotide change in B. holmesii genome, with a difference in melting temperature ( T m) of 6.5 °C. The characteristic melting profile and T m analysis was the basis for discriminatory detection of B. pertussis from B. holmesii. The method was applied in a representative set of clinical isolates of B. pertussis and B. holmesii and the result was in agreement with conventional culture method.

Rapid simultaneous genotyping of the frequent butyrylcholinesterase variants Asp70Gly and Ala539Thr with fluorescent hybridization probes

Background : The clinically important variants of butyrylcholinesterase (BChE) are the A- (Asp70Gly) and K-variants (Ala539Thr), which are common among Caucasians. These variants are associated with abnormal drug metabolism during anaesthesia, which leads to a prolonged neuromuscular block following administration of the neuromuscular blocking agents, mivacurium and succinylcholine. In addition, the K-variant has been proposed to be associated with Alzheimer's disease together with apolipoprotein E k 4. To facilitate diagnostics, we set out to establish a rapid and simple method for simultaneous genotyping of the A- and K-variants. Methods : Using the LightCycler‘, a rapid-cycle duplex PCR is combined with generation of allele-specific fluorescent probe melting profiles. This allows simultaneous detection of both of the mutations in the BChE gene. The results were compared with direct sequencing and phenotyping results. Results : Samples from 80 subjects were genotyped. The genotypes determined using the LightCycler‘ were identical to those obtained by direct sequencing of conventional polymerase chain reaction products and was more accurate than phenotyping based on biochemical assays. Conclusions : A high-speed and easy to perform mutation detection assay has been established for the two most common mutations, Asp70Gly and Ala539Thr, in BChE, using the LightCycler technology and melting curves.

Real-Time Multiplex PCR Assays

The ability to multiplex PCR by probe color and melting temperature (Tm) greatly expands the power of real-time analysis. Simple hybridization probes with only a single fluorescent dye can be used for quantification and allele typing. Different probes are labeled with dyes that have unique emission spectra. Spectral data are collected with discrete optics or dispersed onto an array for detection. Spectral overlap between dyes is corrected by using pure dye spectra to deconvolute the experimental data by matrix algebra. Since fluorescence is temperature dependent and depends on the dye, spectral overlap and color compensation constants are also temperature dependent. Single-labeled probes are easier to synthesize and purify than more complex probes with two or more dyes. In addition, the fluorescence of single-labeled probes is reversible and depends only on hybridization of the probe to the target, allowing study of the melting characteristics of the probe. Although melting curves can be obtained during PCR, data are usually acquired at near-equilibrium rates of 0.05–0.2°C/s after PCR is complete. Using rapid-cycle PCR, amplification requires about 20 min followed by a 10-min melting curve, greatly reducing result turnaround time. In addition to dye color, melting temperature can be used for a second dimension of multiplexing. Multiplexing by color and Tm creates a “virtual” two-dimensional multiplexing array without the need for an immobilized matrix of probes. Instead of physical separation along the X and Y axes, amplification products are identified by different fluorescence spectra and melting characteristics.

Real-Time PCR Assay with Fluorescent Hybridization Probes for Rapid Interleukin-6 Promoter (−174G→C) Genotyping

Interleukin-6 (IL-6) is a central protein in the regulation of the inflammatory and immunologic response (1). A G→C polymorphism at position −174 has been associated with osteoporosis (2), juvenile rheumatoid arthritis (3), and atherosclerosis (4) with increased risks in the absence of the CC genotype. Restriction fragment length polymorphism analysis has been used for IL-6 genotyping, but it is time-consuming and requires multiple manual steps. To improve throughput, we developed a rapid-cycle PCR method on the LightCyclerTM instrument (Roche Diagnostics) with fluorescent probe melting analysis. This assay is completed in 60 min. DNA from 115 German Caucasians was extracted from whole blood according to standard procedures. …

Rapid genotyping of human platelet antigen 5 with fluorophore-labelled hybridization probes on the LightCycler.

Genotyping of human platelet antigens (HPAs) can be useful for the diagnosis and therapy of alloimmune thrombocytopenic syndromes such as neonatal alloimmune thrombocytopenic purpura, post-transfusion purpura and refractoriness to platelet transfusion therapy. We developed a single-tube method for HPA-5 genotyping on the LightCycler that combines rapid-cycle polymerase chain reaction with allele-specific fluorescent probe melting for mutation detection. Our method is fast, robust and suitable for routine HPA-5 typing. This work extends recent studies on HPA-1 typing using the LightCycler.

Real-time polymerase chain reaction with fluorescent hybridization probes for the detection of prevalent mutations causing common thrombophilic and iron overload phenotypes.

We evaluated more than 450 patients with thrombophilia or iron overload for the presence of a factor V Leiden (R506Q), prothrombin G20210A, or HFE C282Y mutation using a standard method (polymerase chain reaction [PCR]-restriction fragment length polymorphism) and a comparative real-time PCR fluorescent resonance energy transfer (FRET) hybridization probe melting curve method. There was 100% concordance between the genotypes ascertained by the 2 methods (at each loci). In addition, phenotypic biochemical laboratory parameters measured on a subset of referred patients correlated with their respective genotypes. In the iron overload cohort, HFE C282Y homozygotes (n = 74) had significantly higher (P < .0001) transferrin saturation levels (74% +/- 25%) than did nonhomozygotes (n = 340; 51.4% +/- 28%), suggesting a genotype-dependent increase in body iron loads. In the thrombophilic cohort, the degree of activated protein C resistance (APCR), measured by a clotting time-based test, was associated significantly with the presence of 0 (n = 255; APCR = 2.59 +/- 0.26), 1 (n = 84; APCR = 1.61 +/- 0.13), or 2 (n = 5; APCR = 1.16 +/- 0.04) copies of the mutant factor V Leiden allele. As the fluorescent genotyping method required no postamplification manipulation, genotypes could be determined more quickly and with minimized risk of handling errors or amplicon contamination. In addition to these practical advantages, the FRET method is diagnostically accurate and clinically predictive of phenotypic, disease-associated manifestations.

DNA Base Bulge vs Unmatched End Formation in Probe-based Diagnostic Insertion/Deletion Genotyping: Genotyping the UGT1A1 (TA)n Polymorphism by Real-Time Fluorescence PCR

Gilbert syndrome is a clinically inconsequential entity of mild unconjugated hyperbilirubinemia caused by an A(TA)(n)TAA insertion polymorphism (UGT1A1*28) in the promoter region of the gene coding for the enzyme UDP-glucuronosyltransferase 1 (EC 2.4.1. 17; UGT1A1). Present methods for genotyping this polymorphism are laborious. Hybridization probes were designed complementary to the wild type (TA)(6) and to alleles with (TA)(7) and (TA)(8) repeats in the promoter region. Melting points were measured in samples representing all currently known alleles with (TA)(5) to (TA)(8) repeats. Probe melting points were predicted with a thermodynamic nearest-neighbor model for Watson-Crick paired probes. The dominant secondary structures resulting from probe hybridization were predicted by thermodynamic free energy calculations. Alternatively samples were genotyped based on amplicon size resolved by high-resolution polyacrylamide gel electrophoresis. Only short probes (22-24 bases) could be successfully used for genotyping this locus because of the very low stability of this TA repeat. Assays based on (TA)(7) or (TA)(8) genotype-compatible hybridization probes effectively discriminated five to eight TA repeats. The consecutive use of two different detection probes was necessary for better discrimination of some heterozygous genotypes. All results were in concordance with the alternative genotyping method. Of 100 investigated Caucasians (50 males, 50 females), 9 (9%) were homozygous for the (TA)(7) allele. The presented method for genotyping the (TA)(n) promoter polymorphism of the UGT1A1 gene with the LightCycler has the potential to genotype all currently known (TA)(n) repeats in a single assay and is sensitive toward possible new genotypes. Our findings also show that thermodynamic calculations are of practical value for the design of hybridization probe assays for the genotyping of insertion/deletion polymorphisms.

Rapid genotyping of varicella-zoster virus vaccine and wild-type strains with fluorophore-labeled hybridization probes.

We developed a single-tube rapid method for the detection and differentiation of varicella-zoster virus (VZV) vaccine and wild-type strains that combines rapid-cycle PCR with wild-type-specific fluorescent probe melting profiles for product genotyping. A region including the polymorphic site in VZV open reading frame (ORF) 62 was amplified in the presence of two fluorescence-labeled hybridization probes. During the annealing step of the thermal cycling, both probes bound to their complementary sequences in the amplicon, resulting in resonance energy transfer, thus providing real-time fluorescence monitoring of PCR. Continuous acquisition of fluorescence data during a melting curve analysis at the completion of PCR revealed that loss of fluorescence occurred in a strain-specific manner as the detection probe, which was fully complementary to the wild-type VZV ORF 62 region, melted off the template. Use of this method allowed genotyping of samples within minutes after the completion of PCR, eliminating the need for post-PCR sample manipulation. In addition to reducing the time required to produce a result, this method substantially reduces the risk of contamination of the final product as well as the risk of sample tracking errors. The genotypes of 79 VZV-positive samples determined by this fluorescent resonance energy transfer (FRET) method were identical to the genotypes obtained by conventional PCR and restriction fragment length polymorphism analysis. The genotyping of VZV strains by the FRET method is a rapid and reliable method that is suitable for typing and that is also practical for use for the processing of large numbers of specimens.

Simultaneous detection of C282Y and H63D hemochromatosis mutations by dual-color probes.

Hemochromatosis is a common genetic disease, affecting one in every 200 individuals in the United States. A PCR assay was designed using fluorescent melting curve analysis to simultaneously detect the G845-->A (C282Y) and C187-->G (H63D) mutations. The G845-->A and C187-->G loci are distinguished by color, and mutant alleles are distinguished from wild type by probe melting temperature (Tm). The probe sets used two fluorophore pairs, fluorescein with LCRed 640 for G845-->A and fluorescein with LCRed 705 for C187-->G. The probes, complementary to the mutant allele, dissociate from the product at specific Tms. Wild-type alleles form mismatches with the probes, reducing the Tms by 6 degrees C (G845-->A) and 10 degrees C (C187-->G). One of 133 samples had a Tm shift 4 degrees C less than the wild-type Tm for the G845-->A locus. Sequencing confirmed the sample to be homozygous for G845-->A and heterozygous for a C-->A substitution at position 842 (C842-->A), substituting lysine for threonine. Multiplexing by color and Tm allows for simultaneous genotyping of each mutation. A novel base-pair alteration was detected in cis with a G845-->A mutation.

Rapid β-Globin Genotyping by Multiplexing Probe Melting Temperature and Color

Single-nucleotide polymorphisms have been identified by many different experimental approaches. The human β-globin gene has been genotyped by several methods, including PCR followed by restriction digestion (1)(2), denaturing gradient gel electrophoresis (3), allele-specific amplification during PCR, and the ligase chain reaction (4). These methods require several hours and sometimes days for diagnosis. Recently, rapid-cycle PCR has been combined with real-time fluorescence monitoring to detect mutations by fluorescent probe melting point analysis for homogeneous genotyping in 50 mutations, which produce various hemoglobinopathies (9). Hemoglobins S, C, and E are common and are routinely screened. Hemoglobin C (Hb C) results from a G-to-A mutation in the first nucleotide of codon 6, whereas hemoglobin S (Hb S) arises from an A-to-T mutation in the second nucleotide of this codon. Hemoglobin E (Hb E) results from a G-to-A mutation in the first nucleotide of codon 26. The close proximity of these three mutations allowed us to design a probe system that discriminated all genotypes using T m and two probe colors. The human β-globin gene sequence (GenBank accession no. U01317 …

Rapid, Homogeneous Genotyping of the 4G/5G Polymorphism in the Promoter Region of the PAI1 Gene by Fluorescence Resonance Energy Transfer and Probe Melting Curves

Many studies have convincingly shown that survivors of myocardial infarction have impaired fibrinolytic activity because of increased concentrations of plasma plasminogen activator inhibitor-1 (PAI-1). A single guanosine insertion/deletion polymorphism in the promoter region of the PAI1 gene, commonly called 4G/5G, has been shown to be associated with plasma PAI-1 activity. Our aim was to develop and validate a homogeneous assay for rapid genotyping of the 4G/5G polymorphism. In this report we present a single-tube method for genotyping of the 4G/5G polymorphism that combines both rapid-cycle PCR with real-time monitoring of the amplification process and generation of allele-specific fluorescent probe melting profiles on the LightCycler(TM). Two fluorescently labeled hybridization probes recognizing adjacent sequences in the amplicon were present in the reaction mixture. The shorter detection probe spanned the polymorphic site, perfectly matching the 5G allele. After annealing, the fluorophores were in resonance energy transfer, providing real-time monitoring of the amplification process. At the completion of the PCR, fluorescence was monitored as the temperature increased through the T(m) of the probe/product duplex, and a characteristic melting profile for each genotype was obtained. With this method, 32 samples were genotyped within 30 min without the need of any post-PCR sample manipulation. The genotypes of 100 DNA samples determined with the LightCycler were identical to those obtained with conventional PCR and restriction fragment length analysis. The genotyping of the 4G/5G polymorphism with the LightCycler is a rapid, reliable method that is suitable for typing both small and large numbers of samples.

Rapid genotyping of human platelet antigen 1 (HPA-1) with fluorophore-labelled hybridization probes on the LightCycler.

Genotyping of human platelet alloantigens (HPA) has become an important procedure in the diagnosis and prevention of disorders such as neonatal alloimmune thrombocytopenic purpura, post-transfusion purpura, and refractoriness to platelet transfusion therapy. We present a single-tube method for HPA-1 genotyping that combines rapid-cycle PCR with allele-specific fluorescent probe melting profiles for product genotyping. A fragment covering the polymorphic site is amplified in the presence of two fluorescently-labelled hybridization probes. During the annealing step of the thermal cycling, both probes bind to their complementary sequences in the amplicon resulting in resonance energy transfer, thus providing real-time fluorescence monitoring of PCR. Continuous aquisition of fluorescence data during a melting curve analysis at the completion of PCR revealed that loss of fluorescence occurred in an allele-specific manner as the detection probe, which was fully complementary to the HPA-1b allele, melted off the template. By determining the temperature at which maximum melting of the hybrids occurred, the two alleles were readily distinguishable. Using this method, genotyping of 32 samples was completed within 30 min without the need for any post-PCR sample manipulation, thereby eliminating the risks of end-product contamination and sample tracking errors. The genotypes determined with the LightCyclerTM were identical when compared with a conventional PCR and restriction fragment length polymorphism technique. The genotyping of HPA-1 on the LightCycler is a rapid and reliable method that is suitable for typing both small and large numbers of samples.