Real-time Polymerase Chain Reaction Instrument Research Articles

Monitoring temperature with fluorescence during real-time PCR and melting analysis

Accurate control of the sample temperature during thermal cycling is critical for successful polymerase chain reaction (PCR). Direct sensor contact with the reaction is problematic, forcing measurements external to the sample and compromising accuracy during rapid temperature transitions. The widespread use of fluorescence in real-time PCR and melting analysis suggests another measure of temperature, the intrinsic fluorescence of temperature-sensitive passive dyes. Calibration curves correlating sulforhodamine B fluorescence to temperature on nine real-time PCR instruments were obtained by heating at 0.018–0.1°C/s between 50 and 95°C, with a twofold change in fluorescence. After instrument stabilization for 20min, no dye photobleaching was observed and thermal degradation was 2.2%/h at 80°C. During cycling, solution temperatures derived from fluorescence were well matched to thermocouples placed within samples, but not to temperatures recorded by the instrument. Solution temperatures lagged instrument temperatures by up to 8°C during cycling, often requiring 5–10s at target temperatures for equilibration. Melting curves were displaced by 0.2–1.1°C. Temperature inaccuracies were dependent on the instrument, the ramp rate, and the sample volume. The fluorescence of passive dyes can be used to accurately assess solution temperatures during PCR and should be particularly useful at fast cycling speeds.

Thalidomide combined with interferon in relapsed or refractory mantle cell lymphoma: two cases report and literatures review

To investigate JAK2 exon 12 mutations in patients with Philadelphia (Ph) chromosome-negative myeloproliferative neoplasms (MPN) and the clinical characteristics of patients with JAK2 exon 12 mutants.Allele-specific PCR (AS-PCR) was applied to identify JAK2 V617F mutation. Genomic DNA corresponding to exon 12 of JAK2 gene and epigenetic regulator gene (TET2, ASXL1, EZH2) were amplified by polymerase chain reaction (PCR). Identification of mutants was by direct sequencing and classification of mutation types by sequencing followed by plasmid cloning. SNP genotyping of two 46/1 tag SNPs, rs12340895 and rs10974944, was analyzed using commercially available Taqman assays on the 7500HT real-time PCR instrument according to standard protocols.No JAK2 exon 12 mutation was detected in patients with ET, PMF or JAK2 V617F positive PV. Among 13 JAK2 V617F negative PV patients, JAK2 exon 12 mutation was detected as N542-E543del in 2(15.4%) patients who presented with a phenotype of predominant erythrocytosis and erythroid colonic grown from their bone marrow samples in the absence of exogenous EPO, reduced serum erythropoietin (EPO) level, and no mutations in TET2, ASXL1 or EZH2 genes. One of the affected patients was heterozygous for 46/1 but the second was negative for this haplotype.There was no need to detect JAK2 exon 12 mutation in ET, PMF or MPN-U patients without JAK2 V67F mutation. Ph negative MPN patients with JAK2 exon 12 mutations had somewhat unique clinical and laboratory features.

Genetic Susceptibility to Periapical Disease: Conditional Contribution of MMP2 and MMP3 Genes to the Development of Periapical Lesions and Healing Response

IntroductionIt has been proposed that individual genetic predisposition may contribute to a persistent apical periodontitis condition. Matrix metalloproteinases (MMPs) are associated with levels of inflammation and are involved in caries, pulpal, and periapical tissue destruction. MMPs also play a major role in bone resorption. In this study, we hypothesized that polymorphisms in MMP genes and their regulators may contribute to an individual’s increased susceptibility to apical tissue destruction in response to deep carious lesions. MethodsSixteen hundred radiographic records obtained through the University of Pittsburgh School of Dental Medicine Dental Registry and DNA Repository were screened for subjects with deep carious lesions in dentin with or without periapical lesions (≥3 mm). DNA samples of 268 patients were sorted into 2 groups: 158 cases with deep carious lesions but no periapical lesions (controls) and 110 cases with periapical lesions and deep carious lesions (cases). Sixteen SNP markers in MMP2, MMP3, MMP9, MMP13, MMP14, and TIMP2, were selected for genotyping. Genotypes were generated by endpoint analysis in a real-time polymerase chain reaction instrument. Analyses were performed comparing cases and controls. Allele and genotypic frequencies and haplotype analysis were calculated using the PLINK program. ResultsAn association was found for MMP3 rs639752 (P = .03) and rs679620 (P = .004) genotypes in individuals with periapical lesions. We also observed altered transmission of MMP2 marker haplotypes (P = .000004) in these individuals. ConclusionsVariations in MMP2 and MMP3 are associated with periapical lesion formation in individuals with untreated deep carious lesions. Future studies could help predict host susceptibility to developing periapical lesions.

Validation of a polymerase chain reaction–oligochromatography test for detection of influenza A (H1N1) 2009 virus

The outbreak of pandemic influenza A (H1N1) 2009 virus caused the first influenza pandemic disease of the 21st century. In August 2010, the pandemic moved into the post-pandemic period. However, localized outbreaks of various magnitudes continued with a higher rate of disease severity. The aim of this study was to assess a new polymerase chain reaction (PCR)–oligochromatographic assay (Speed-Oligo) in the diagnosis of novel influenza A (H1N1) 2009. A total of 405 nasopharyngeal aspirate specimens from 400 pediatric and adults patients with suspected infection of pandemic influenza A (H1N1) 2009 were analyzed. The sensitivity and specificity values of the Speed-Oligo assay in comparison to reverse transcriptase–PCR assay developed by the Centers for Disease Control and Prevention were 86.5% and 92.2%, respectively. The new assay is simple, rapid, and provides a good sensitivity for detection of influenza A (H1N1) 2009. This assay might be a good alternative to real-time PCR assays for laboratories not equipped with real-time PCR instruments.

Wobble-enhanced ARMS Method for Detection of KRAS and BRAF Mutations

Monoclonal antibodies targeting the epidermal growth factor receptor have expanded the range of treatment options for patients with metastatic colorectal cancer. However, somatic mutations in the KRAS and BRAF genes have proven to be molecular predictors of resistance to treatment with anti-epidermal growth factor receptor therapy in these patients. Thus, we have developed a sensitive mutation assay, wobble-enhanced amplification refractory mutation system, for detecting the 8 most commonly reported mutations of clinical importance in the KRAS and BRAF genes; KRAS g.34G>C (p.G12R), g.34G>A (p.G12S), g.34G>T (p.G12C), g.35G>A (p.G12D), g.35G>C (p.G12A), g.35G>T (p.G12V), g.38G>A (p.G13D), and BRAF g.1799T>A (p.V600E). A total of 28 candidate setups were designed based on bioinformatics and primer/probe design. Eight candidate setups were thus selected using a synthetic oligonucleotide model. The setups were further validated through several experiments using formalin-fixed paraffin-embedded tissue and cell lines. The results confirm that the wobble-enhanced amplification refractory mutation system method is quick, cost effective, and sensitive. The method is optimized to handle a typical template input of 1 to 20 ng DNA per polymerase chain reaction and can be implemented in any laboratory with ease with a real-time polymerase chain reaction instrument capable of handling TaqMan techonology. The steps used to develop this method can be implemented to design assays for other mutations located in KRAS, BRAF, or other candidate genes.

A CCD-based fluorescence imaging system for real-time loop-mediated isothermal amplification-based rapid and sensitive detection of waterborne pathogens on microchips

Rapid, sensitive, and low-cost pathogen diagnostic systems are needed for early disease diagnosis and treatment, especially in resource-limited settings. This study reports a low-cost charge-coupled device (CCD)-based fluorescence imaging system for rapid detection of waterborne pathogens by isothermal gene amplification in disposable microchips. Fluorescence imaging capability of this monochromatic CCD camera is evaluated by optimizing the gain, offset, and exposure time. This imaging system is validated for 12 virulence genes of major waterborne pathogens on cyclic olefin polymer (COP) microchips, using SYTO-82 dye and real time fluorescence loop-mediated isothermal amplification referred here as microRT(f)-LAMP. Signal-to-noise ratio (SNR) and threshold time (Tt) of microRT(f)-LAMP assays are compared with those from a commercial real-time polymerase chain reaction (PCR) instrument. Applying a CCD exposure of 5 s to 10(5) starting DNA copies of microRT(f)-LAMP assays increases the SNR by 8-fold and reduces the Tt by 9.8 min in comparison to a commercial real-time PCR instrument. Additionally, single copy level sensitivity for Campylobacter jejuni 0414 gene is obtained for microRT(f)-LAMP with a Tt of 19 min, which is half the time of the commercial real-time PCR instrument. Due to the control over the exposure time and the wide field imaging capability of CCD, this low-cost fluorescence imaging system has the potential for rapid and parallel detection of pathogenic microorganisms in high throughput microfluidic chips.

Dried Blood Spot HIV-1 RNA Quantification Using Open Real-Time Systems in South Africa and Burkina Faso

There is an urgent need to assess the accuracy/feasibility of using dried blood spots (DBS) for monitoring of HIV-1 viral load in resource-limited settings. A total of 892 DBS from HIV-1-positive pregnant women and their neonates enrolled in the Kesho Bora prevention of mother-to-child transmission trial conducted in Durban (South Africa) and Bobo-Dioulasso (Burkina Faso) between May 2005 and July 2008 were tested for HIV-1 RNA. The combination Nuclisens extraction method (BioMérieux)/Generic HIV Viral Load assay (Biocentric) was performed using one DBS (in Durban) versus 2 DBS (in Bobo-Dioulasso) on 2 distinct open real-time polymerase chain reaction instruments. DBS HIV-1 RNA results were compared with plasma HIV-1 RNA and HIV serology results used as the gold standards. The limits of detection of assays on DBS were 3100 and 1550 copies per milliliter in Durban and Bobo-Dioulasso, respectively. DBS HIV-1 RNA values correlated significantly with plasma levels (n = 327; R = 0.7351) and were uniformly distributed according to duration of DBS storage at -20°C (median duration, 280 days). For early infant diagnosis, the sensitivity and specificity were 100% (95% confidence interval: 97.2 to 100.0 and 96.5 to 100.0, respectively). HIV-1 viral load kinetics in DNase-pretreated DBS were similar to those obtained in plasma specimens among 13 patients receiving antiretroviral treatment. HIV-1 RNA findings from serial infant DBS collected prospectively (n = 164) showed 100% concordance with HIV serology at 18 months of life. Our findings strongly advocate the implementation of DBS HIV-1 RNA testing in remote areas from low-income and middle-income countries.

A Real‐Time Multiplex SNP Melting Assay to Discriminate Individuals

A method that quickly and inexpensively differentiates crime scene samples from multiple donors would expedite casework analysis by allowing the selection of probative items requiring comprehensive testing. This new method need not be perfectly definitive nor give a complete 13 locus short tandem repeat (STR) profile; it simply must be able to differentiate between most victim and suspect samples. We describe the development of multiplex, single nucleotide polymorphism (SNP), fluorescence resonance energy transfer-based real-time polymerase chain reaction (PCR) assays to fulfill this need. Dual probes, one fluorescently labeled and the other labeled with a quencher, are monitored during a melt analysis to reveal an increase in fluorescence, which allows the assessment of the two SNP alleles. Two alternate 6-plex assays (with and without gender determination) have been developed for the six-color RG6000 real-time instrument (Corbett Robotics, Inc.) and one seven SNP plus gender assay (performed as two 4-plex assays, one with gender the other without) have been developed for use in four/five color real-time instruments. This technique can discriminate between 95% and 99% of samples from different individuals. This assay is fast (approximately 2 h), much less expensive than STR analysis, and uses a real-time PCR instrument which is found in most forensic and molecular biology labs.

Evaluation of 5′-MGB hybridization probes for detection of Bordetella pertussis and Bordetella parapertussis using different real-time PCR instruments

Rapid and accurate tests capable of distinguishing Bordetella pertussis from milder Bordetella parapertussis infection can aid in treating patients. We evaluated a novel real-time polymerase chain reaction (PCR) method that allows rapid and accurate diagnosis and distinction between B. pertussis and B. parapertussis infections. The method is based on a fluorescent 5′-minor groove-binding molecule hybridization probe that can clearly distinguish between B. pertussis and B. parapertussis by post-PCR melt curve analysis. The reagents worked equally well in several different real-time PCR instruments. The Bordetella detection reagents are combined with internal control components to account for PCR inhibition without compromising assay sensitivity.

Rapid HLA-DR Fluorotyping Based on Melting Curve Analysis

Real-time polymerase chain reaction (PCR) has been used in the study of human leukocyte antigen (HLA) genotyping as a potential alternative for routinely used molecular methods such as PCR-sequence specific primers (PCR-SSP) and PCR-sequence specific oligonucleotide probes (PCR-SSO). Combined with fluorescent dye like SYBR GREEN I, it has more advantages such as low cost and consistent background. The aim of this study was to optimize the fluorescent dye-based method and introduce it into the fluorotyping for HLA-DR lotus. 24 pairs of allele-specific primers and 1 pair of internal control were optimized to discriminate HLA-DRB1, -DRB3/B4/B5 alleles. Additionally, conditions of real-time PCR amplifying and melting curve recording had been improved for convenient and clear readout. Forty-two clinical samples previously typed by conventional PCR-SSP or sequence based typing (SBT) were tested and all got identical results. With this technique, 15 DNA samples can be assayed in parallel within 2 hours on the Real-time PCR instrument. These data strongly suggest a rapid HLA-DR fluorotyping method based on melting curve analysis, which could be a more economic and automatic alternative for clinical HLA-DR typing.

DNA quantification using EvaGreen and a real-time PCR instrument

DNA quantiWcation is an important, frequently used technique, and inaccuracies can result in failures with ligation, restriction, polymerase chain reaction (PCR),1 ampliWed fragment length polymorphism (AFLP), Southern blotting, and other techniques. DNA is most commonly quantiWed using absorbance at 260 nm, but because of the existence of many impurities, this can be an imprecise measurement and DNA levels can be more than 10 times overestimated in some cases [1]. QuantiWcation by agarose gel electrophoresis with a known amount of standard DNA [1,2] can provide more accurate data, but the procedures are complicated, the data often still are not accurate enough, and the technique is impractical for routine or high-throughput DNA quantiWcation [3]. Fluorescence spectroscopy using various DNA intercalating dyes is the most widely accepted technique for accurate DNA quantiWcation [4]. However, if the analysis is to be carried out with a Xuorescence spectrophotometer, a relatively large assay volume (e.g., 2 ml) is required [5], and this is impractical for small DNA samples and expensive dyes. Fluorescence can also be measured with a smaller volume of DNA sample using other instruments such as Xuorescent microplate readers [6], microplate Xuorometers [7,8], and transilluminator–microplate–CCD camera systems [9], but the instruments might not be readily available in most molecular biology laboratories. Real-time PCR instruments are common in molecular biology laboratories and can be used to measure Xuorescence. DNA intercalated with a dye emits Xuorescence at 520 nm under excitation at 490 nm, and with real-time PCR instrumentation the assay is carried out in PCR tubes, equivalent to spectrophotometer cuvettes used with a Xuo-

Multiplexed Detection of Anthrax-Related Toxin Genes

Simultaneous analysis of three targets in three colors on any real-time polymerase chain reaction (PCR) instrument would increase the flexibility of real-time PCR. For the detection of Bacillus strains that can cause inhalation anthrax-related illness, this ability would be valuable because two plasmids confer virulence, and internal positive controls are needed to monitor the testing in cases lacking target-specific signals. Using a real-time PCR platform called MultiCode-RTx, multiple assays were developed that specifically monitor the presence of Bacillus anthracis-specific virulence plasmid-associated genes. In particular for use on LightCycler-1, two triplex RTx systems demonstrated high sensitivity with limits of detection nearing single-copy levels for both plasmids. Specificity was established using a combination of Ct values and correct amplicon melting temperatures. All reactions were further verified by detection of an internal positive control. For these two triplex RTx assays, the analytical detection limit was one to nine plasmid copy equivalents, 100% analytical specificity with a 95% confidence interval (CI) of 9%, and 100% analytical sensitivity with a CI of 2%. Although further testing using clinical or environmental samples will be required to assess diagnostic sensitivity and specificity, the RTx platform achieves similar results to those of probe-based real-time systems.

A Single-Tube Nucleic Acid Extraction, Amplification, and Detection Method Using Aluminum Oxide

A disposable 0.2-ml polymerase chain reaction (PCR) tube modified with an aluminum oxide membrane (AOM) has been developed for the extraction, amplification, and detection of nucleic acids. To assess the dynamic range of AOM tubes for real-time PCR, quantified herpes simplex virus (HSV) DNA was used to compare AOM tubes to standard PCR tubes. AOM PCR tubes used for amplification and detection of quantified HSV-1 displayed a crossing threshold (C(T)) shift 0.1 cycles greater than PCR tube controls. Experiments with HSV-1-positive cerebrospinal fluid (CSF) examined the extraction, amplification, and detection properties of the AOM tubes compared to the Qiagen DNA blood mini kit. The AOM extraction, amplification, and detection of HSV-1 in CSF displayed differences of less than one C(T) when compared to Qiagen-extracted samples. Experiments testing the AOM method using clinical CSF samples displayed 100% concordance with reported results. AOM tubes have no adverse effects on amplification or fluorescence acquisition by real-time PCR and can be effectively used for the extraction, amplification, and detection of HSV from CSF. The AOM single tube method is a fast, reliable, and reproducible technique for the extraction, amplification, and detection of HSV in CSF.

Use of duplex probes simulating TaqMan to detect hepatitis B virus

A novel method for duplex probes is designed to simulate the TaqMan probe during polymerase chain reaction (PCR). In this method, two partly complementary single-labelled oligonucleotide probes labelled with a fluorophore or a quencher, respectively, are used. At lower temperature the two probes can bind to each other and form a mismatched duplex, in which the fluorophore and quencher are in close proximity and the same energy transfer mechanism as in molecular beacons may occur between them; thus, a quenching efficiency better than conventional TaqMan probes is acquired. In the anneal-extend step of PCR, one single-labelled probe hybridises to the predetermined target and is cleaved by Taq DNA polymerase. Increased fluorescent signal can be observed at lower temperature. The fluorescent data analysis demonstrated that a significantly higher level of fluorescent signal and hence higher sensitivity of detection is obtainable using our duplex probes in place of conventional TaqMan probes. Combined with real-time PCR instruments, the assay can be used to quantify the input target molecules and the dynamic linear range is of at least six orders of magnitude.

Real-time PCR and its application for rapid plant disease diagnostics

Rapid-cycle real-time polymerase chain reaction (PCR) methods may revolutionize the manner in which plant pathogens are identified and diseases are diagnosed. As the genomics age progresses and more and more DNA sequence data become available, highly specific primers and fluorescent probe sequences can be designed to yield target amplicons to unique regions of a pathogen's genome. Portable real-time PCR instruments described here are now allowing for diagnostic assays to be run directly in the field or at remote locations other than the standard diagnostic laboratory. Rapid real-time PCR diagnosis can result in appropriate control measures and (or) eradication procedures more quickly and accurately than traditional methods of pathogen isolation. Disease losses are minimized and control costs reduced. Advantages and disadvantages of rapid real-time PCR for the detection of bacterial, fungal, and viral plant pathogens are described.

Blood group antigen profile predicted by molecular biology— use of real-time polymerase chain reaction to genotype important KEL, JK, RHD, and RHCE alleles

Abstract The most clinically important blood group systems in transfusion medicine, excluding the ABO system, are the RH, Kell, and Kidd systems. Alloantibodies to antigens of these systems may be produced following blood transfusion or during pregnancy and can result in serious hemolytic transfusion reactions and hemolytic disease of the newborn.We developed rapid and robust techniques for RHD, RHCE, KEL, and JK genotyping with the use of a real-time polymerase chain reaction instrument. Two fluorescence-based methods for the detection of amplification products were used: for KEL1/KEL2, JK1/JK2, and RHE/RHe (exon 5) we used the hybridization probes protocol; for RHC/RHc the analysis was done in sequences of exon 1 for RHC and exon 2 for RHc; and for RHD,analysis was done in sequences of intron 4, exon 7,and exon 4 pseudogene using the SYBR Green I protocol. The genotyping tests were validated with samples from 85 Caucasian Portuguese and 15 Black European blood donors. Complete phenotype-genotype correlations were obtained. The potential use of the presented methods can be predicted in clinical transfusion medicine, allowing appropriate monitoring, early intervention, and improved care. When blood group genotyping techniques are necessary, this methodology is highly competitive for a routine laboratory. Immunohematology 2002;18:59–64.