Real-time Polymerase Chain Reaction Machine Research Articles

An Interdisciplinary Design Project for Undergraduate Engineering Training – Portable Real-Time Polymerase Chain Reaction System

Our world has been permanently changed by the pandemic outbreak of COVID-19 starts around the end of 2019. In the first few months of 2020, the whole world was in urgent need of an effective, easy, and quick method for the identification of the infection of the new virus. Polymerase Chain Reaction (PCR) machine, which can test DNA samples by rapidly making millions of copies of a specific DNA sample through the PCR process, including the COVID-19 virus, can perfectly fit this demand. In this study, a design project on PCR is introduced for undergraduate education in electrical and mechanical engineering. The objective of this project is to develop a low-cost, ease-of-use, wallet-size, portable real-time PCR (RT-PCR) machine for accurate testing of various bacteria or viruses. The key function of the PT-PCR system is to precisely control and maintain the temperature of the biosample solution within a range between 55°C and 95°C. This project provides students opportunities in studying and practicing a wide range of engineering technics and skills, including mechanical design, electronics design, microcomputer programming, data acquisition and processing, etc. Students can gain comprehensive understanding of the design of multiphysics system after they overcome various challenges emerging in the project. From the view of engineering education, the process of this project development has demonstrated the importance and benefits of adopting complex interdisciplinary engineering problems for student teams to solve, especially those involve contemporary issues.

COVID-19 Testing Pipeline: Lesson Learned

The transmission of severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) in Indonesia is seen to be uncontrollably increasing that urges the government to leverage the capacity for the disease detections. Real-time polymerase chain reaction (RT-PCR), rapid test and computed tomography (CT) scan are the most common methods to determine if one has been infected regardless of whether or not the common symptoms of such Corona Virus Disease 2019 (COVID-19) surface. Among these three, RT-PCR is considered the gold standard for qualitative and quantitative assessment of SARS CoV-2 detection. The present paper aims at elaborating the framework of Roche’s RT-PCR machine employed specifically for SARS CoV-2 detection performed by Genetics Indonesia which is deemed to be efficient and relatively quicker than other detection kits. RT-PCR machine detected SARS Cov-2 with RNA amplification curve equals to 10 copies RNA below the cut off value of Crossing point (Cp) positive control. Also elucidated in the paper is the implementations of EAV RNA and LightCycler® 96 RT-PCR System through which analysis time, amounts of individual required sample, as well as the reagents, can be accordingly reduced.

Multiplex Solid-Phase Real-Time Polymerase Chain Reaction without DNA Extraction: A Rapid Intraoperative Diagnosis Using Microvolumes

Current polymerase chain reaction (PCR) methods for the diagnosis of infections are time consuming and require large sample volume and skilled technicians. We developed a novel, easy-to-use, and rapid (processing time, 1 minute; total time, 33 minutes) multiplex real-time PCR test (Direct Strip PCR) that did not require DNA extraction to detect 9 pathogens that could cause uveitis in 20-μl samples. Multicenter prospective evaluation of a diagnostic PCR test. A total of 511 participants (patients with infectious uveitis and controls) were examined at 18 institutes worldwide. After validation, intraocular fluid samples were subjected to etiologic or exclusive diagnosis, including intraoperative rapid diagnosis. The concordance and correlations between Direct Strip PCR and quantitative PCR (qPCR) results. Direct Strip PCR exhibited rapid detection, good repeatability and specificity, long storage stability, and detection ability equal to that of qPCR. It also showed low interinstitutional variability compared with qPCR, even when PCR beginners used various real-time PCR machines. The Direct Strip PCR for 9 pathogens exhibited high concordance against the qPCR (positive concordance rate, 98.8%-100%; negative concordance rate, 99.8%-100%; κ coefficient, 0.969-1.000; P < 0.001-0.031). Additionally, results obtained using Direct Strip PCR and qPCR were highly correlated (ρ= 0.748; P < 0.001). This assay was used for rapid intraoperative diagnosis. The Direct Strip PCR test may improve the prognosis of various infectious diseases because it facilitates rapid etiologic evaluation at the first hospital visit and can be used for intraoperative diagnosis.

Small-Size Polymerase Chain Reaction Device with Improved Heat Transfer and Combined Feedforward/Feedback Control Strategy

We report improvements on the heat transfer and on the control strategy of a thermal cycler implemented on a novel device for performing a fast polymerase chain reaction (PCR). The reduction of the thermal mass of the sample holder and the direct contact with the sample allow for a significant reduction of the transition times, while the hybrid feedforward/feedback controller can rely on inexpensive components (actuators, sensors, processor) and still achieve minimal over/undershooting and good temperature stability. The design of the device has been improved by performing transient heat conduction analysis on the highly heat-conductive sample holder and on the solid metal body of the device which rapidly dissipates the excess heat produced during the thermal cycling. In the current setup, the sample holder hosts nine 1 μL samples covered with mineral oil, which can be simultaneously read in real time by the detection module designed in-house and installed on the device. An increase in speed of the PCR amplification was achieved with a reduction of the transition time of 63.8% when compared against a commercial real-time PCR machine. Our work shows that a complete, stand-alone, and ready-to-use quantitative PCR instrument can be fabricated from inexpensive and easily available components and it can achieve fast thermal cycling thanks to a hybrid control strategy.

A fully portable microchip real-time polymerase chain reaction for rapid detection of pathogen.

Point-of-care detection for pathogen is of critical need for wide epidemic warning and medical diagnosis. In this work, we have designed and developed a fully portable and integrated microchip based real-time polymerase chain reaction machine for rapid pathogen detection. The instrument consists of three functional components including heating, optical, and electrical modules, which are integrated into a portable compact box. The microchip is consumable material replaceable to meet various detection needs. Consequently, we demonstrated the outstanding performance of this portable machine for rapid detection of Salmonella and Escherichia coli O157:H7 with the advantage of time-saving (∼25min), less samples consumption, portability, and user-friendly operation.

Analysis and Normalization of Real-Time Polymerase Chain Reaction (PCR) Experimental Data.

In real-time polymerase chain reaction (PCR), also called quantitative real-time PCR [or simply quantitative PCR (qPCR)] or kinetic PCR, the amplification of DNA is monitored by the detection and quantitation of a fluorescent reporter signal, which increases in direct proportion to the amount of PCR product in the reaction. The fluorescent reporter is excited by light from the real-time PCR machine, a fluorescence-detecting thermocycler. By recording the amount of fluorescence emission at each cycle, the PCR can be monitored during the exponential phase when the first significant increase in the amount of PCR product correlates with the initial amount of target template. The ability to quantify the amplified DNA during the exponential phase of the PCR, when none of the components of the reaction is limiting, has resulted in dramatically improved precision in the quantitation of target sequences. In addition, because of the high sensitivity of fluorometric detection, real-time PCR is capable of measuring the initial concentration of target DNA over a vast dynamic range (up to eight or nine orders of magnitude) and with a high degree of sensitivity (as little as one copy of template DNA). Although it is a powerful technique, researchers often face challenges in reliability and reproducibility because of the lack of assay standardization. Therefore, it is critical to optimize the reagents and reaction conditions, include proper internal and external controls, and perform rigorous data analysis in order to generate accurate and reproducible results in real-time PCR experiments.

Quantitative Real-Time PCR Analysis of MicroRNAs and Their Precursors Regulated by TGF-β Signaling.

The signaling pathway of TGF-β and its family member BMP has been implicated in vascular development and maintenance of homeostasis by modulating expression of small noncoding microRNAs (miRNAs). MiRNAs repress target genes, which play a critical role in regulating vascular smooth muscle cell (VSMC) growth, phenotype, and function. To understand the mechanisms by which specific miRNAs control the TGF-β and BMP signaling pathway in VSMC, it is essential to quantitate levels of specific miRNAs and their precursors whose expression are controlled by TGF-β/BMP signaling. Here, we describe a real-time quantization method for accurate and sensitive detection of miRNAs and their precursors, such as primary transcripts of miRNAs (pri-miRNAs) and precursor miRNAs (pre-miRNAs). This method requires two steps; synthesis of single-stranded complementary DNAs (cDNAs) from total RNA samples and quantization of specific pri-, pre-, or mature miRNAs by quantitative polymerase chain reaction (PCR) using a real-time PCR machine.

Assessment of free fetal DNA concentration in maternal plasma during the first trimester of pregnancy: comparative study between EDTA and PPT tubes – pilot study

Objective: To compare ethylenediamine tetraacetic acid (EDTA) tubes and plasma preparation tubes (PPT) for evaluating maternal plasma during the first trimester of pregnancy.Methods: A cross-sectional study was conducted on 24 male fetuses in women between 6 and 14 weeks of pregnancy. Blood samples (10 mL) were collected and stored in EDTA and PPT tubes. Subsequently, the samples were centrifuged and sent for free fetal DNA extraction by means of the polymerase chain reaction (PCR) technique. The reactions were performed in a real time PCR machine for detecting the amplification products. The genome region chosen for performing the PCR reactions was a target specific for the Y chromosome, in which the DYS-14 marker was amplified only when the DNA was of male sex. The free fetal DNA concentration was given by the threshold cycle (TC). To compare the tubes, the paired Student t-test was used.Results: The mean gestational age was 11.08 ± 2.30 weeks (range: 6–14). The mean TC for PPT was 30.08 ± 1.05 (range: 27.08–32.61) and for EDTA, 30.23 ± 0.96 (range: 28.01–32.09), but without statistical significance (p = 0.357).Conclusion: We did not observe any statistically significant difference in free fetal DNA concentration between the EDTA and PPT tubes.

초고속 Real-time PCR을 이용한 Tomato yellow leaf curl virus의 신속진단

Tomato yellow leaf curl virus (TYLCV), transmitted exclusively by the whitefly (Bemisia tabaci) in a circulative manner is one of the most important virus in tomato. Since the first report of TYLCV incidence in Korea in 2008, the virus has rapidly spread nationwide. TYLCV currently causes serious economic losses in tomato production in Korea. Early detection of TYLCV is one of the most important methods to allow rouging of infected tomato plants to minimize the spread of TYLCV disease. We have developed an ultra-rapid and sensitive real-time polymerase chain reaction (PCR) using a new designed real-time PCR system, GenSpectorTM TMC-1000 that is a small and portable real-time PCR machine requiring only a reaction volume on microchips. The new system provides ultra-high speed reaction (30 cycles in less than 15 minutes) and melting curve analysis for amplified TYLCV products. These results suggest that the short reaction time and ultra sensitivity of the GenSpector-based real-time PCR technique is suitable for monitoring epidemics and pre-pandemic TYLCV disease. This is the first report for plant virus detection using an ultra-rapid real-time PCR system.

STUDY ON DETECTION OF VIBRIO PARAHAEMOLYTICUS IN SHELLFISH BY USE OF LOOP-MEDIATED ISOTHERMAL AMPLIFICATION METHOD

ABSTRACT We developed a loop-mediated isothermal amplification (LAMP) technology for rapidly detecting Vibrio parahaemolyticus in shellfish. In this study, the gyrB gene of V. parahaemolyticus was chosen to design six primers, the target DNA was dectected with white precipitate; specificity of the primers were identified with 14 V. parahaemolyticus strain, 15 non-parahaemolyticus Vibrio and 22 non-Vibrio strains; 84 shellfish sampled at random from market were tested by using this method and the results were evaluated with the Food and Drug Administration Bacteriological Analytical Manual most probable number method. The results indicated that this method is easy for operation, with a high level of specificity and the sensitivity of 19 cfu/mL for bacterial liquid and 87 cfu/g for shellfish samples. Detection of an initial inoculum of 1 cfu V. parahaemolyticus per gram of shellfish tissue homogenate was possible after enrichment of 2 h. These results indicate that the LAMP can provide a reliable method for the detection of V. parahaemolyticus in shellfish. PRACTICAL APPLICATIONS The detection of Vibrio parahaemolyticus involve traditional culture-based methods; such methods are labor-intensive and time-consuming. Molecular-based methods such as DNA probe hybridization and polymerase chain reaction (PCR) assays yielded rapid and specific results. However, the probe hybridization procedure and the gel electrophoresis technique are tedious and time-consuming; several real-time PCR assays require a dedicated real-time PCR machine, which is rather expensive and not yet widely available. Because of carrying out under isothermal conditions, the amplification efficiency of the loop-mediated isothermal amplification (LAMP) method is extremely high; LAMP is highly specific for the target sequence because of its recognition of the target sequence by six primers. Furthermore, because the LAMP assay synthesizes a large amount of DNA, the products can be detected by simple turbidity; thus, expensive equipment is unnecessary for providing a high level of precision. These features allow for a simple, rapid and cost-effective detection of V. parahaemolyticus.

Disruption of a Ciliary B9 Protein Complex Causes Meckel Syndrome

Nearly every ciliated organism possesses three B9 domain-containing proteins: MKS1, B9D1, and B9D2. Mutations in human MKS1 cause Meckel syndrome (MKS), a severe ciliopathy characterized by occipital encephalocele, liver ductal plate malformations, polydactyly, and kidney cysts. Mouse mutations in either Mks1 or B9d2 compromise ciliogenesis and result in phenotypes similar to those of MKS. Given the importance of these two B9 proteins to ciliogenesis, we examined the role of the third B9 protein, B9d1. Mice lacking B9d1 displayed polydactyly, kidney cysts, ductal plate malformations, and abnormal patterning of the neural tube, concomitant with compromised ciliogenesis, ciliary protein localization, and Hedgehog (Hh) signal transduction. These data prompted us to screen MKS patients for mutations in B9D1 and B9D2. We identified a homozygous c.301A>C (p.Ser101Arg) B9D2 mutation that segregates with MKS, affects an evolutionarily conserved residue, and is absent from controls. Unlike wild-type B9D2 mRNA, the p.Ser101Arg mutation failed to rescue zebrafish phenotypes induced by the suppression of b9d2. With coimmunoprecipitation and mass spectrometric analyses, we found that Mks1, B9d1, and B9d2 interact physically, but that the p.Ser101Arg mutation abrogates the ability of B9d2 to interact with Mks1, further suggesting that the mutation compromises B9d2 function. Our data indicate that B9d1 is required for normal Hh signaling, ciliogenesis, and ciliary protein localization and that B9d1 and B9d2 are essential components of a B9 protein complex, disruption of which causes MKS.

Comparative detection of rabies RNA by NASBA, real-time PCR and conventional PCR

Five methods for the RNA detection of rabies virus were directly compared in this study. These included conventional nucleic acid sequence-based amplification with electrochemiluminescence (NASBA-ECL) assay, reverse transcription (RT)-heminested (hn) polymerase chain reaction (PCR) and TaqMan real-time RT-PCR using protocols as described previously. The first two methods have been routinely utilised for ante-mortem diagnosis of human rabies in Thailand and other rabies-endemic Asian and African countries. In addition, two real-time NASBA assays based on the use of a NucliSens EasyQ analyser (NASBA-Beacon-EQ) and LightCycler real-time PCR machine (NASBA-Beacon-LC) were studied in parallel. All methods target the N gene, whereas the L gene is used for RT-hnPCR. Using serial dilutions of purified RNA from rabies-infected dog brain tissue to assess sensitivity, all five methods had comparable degrees of sensitivities of detection. However, both real-time NASBA assays had slightly lower sensitivities by 10-fold than the other three assays. This finding was also true (except for TaqMan real-time RT-PCR due to a mismatch between the target and probe sequences) when laboratory-adapted (challenge virus standard-11) virus was used in the assays. Testing on previously NASBA-ECL positive clinical samples from 10 rabies patients (saliva [6] and brain [4]) and 10 rabies-infected dog brain tissues, similar results were obtained among the five methods; real-time NASBA assays yielded false-negative results on 2 saliva samples. None of the assays showed positive results on cerebrospinal fluid specimens of 10 patients without rabies encephalitis. Due to the unavailability of the NASBA-ECL assay, the results show that TaqMan real-time RT-PCR and RT-hnPCR can be useful for ante- and post-mortem diagnosis of rabies.

Development and validation of a SYBR Green I–based real‐time polymerase chain reaction method for detection of haptoglobin gene deletion in clinical materials

Anhaptoglobinemic patients run the risk of severe anaphylactic transfusion reaction because they produce serum haptoglobin (Hp) antibodies. Being homozygous for the Hp gene deletion (HP(del)) is the only known cause of congenital anhaptoglobinemia, and clinical diagnosis of HP(del) before transfusion is important to prevent anaphylactic shock. We recently developed a 5'-nuclease (TaqMan) real-time polymerase chain reaction (PCR) method. A SYBR Green I-based duplex real-time PCR assay using two forward primers and a common reverse primer followed by melting curve analysis was developed to determine HP(del) zygosity in a single tube. In addition, to obviate initial DNA extraction, we examined serially diluted blood samples as PCR templates. Allelic discrimination of HP(del) yielded optimal results at blood sample dilutions of 1:64 to 1:1024. The results from 2231 blood samples were fully concordant with those obtained by the TaqMan-based real-time PCR method. The detection rate of the HP(del) allele by the SYBR Green I-based method is comparable with that using the TaqMan-based method. This method is readily applicable due to its low initial cost and analyzability using economical real-time PCR machines and is suitable for high-throughput analysis as an alternative method for allelic discrimination of HP(del).

Real-time PCR Machine System Modeling and a Systematic Approach for the Robust Design of a Real-time PCR-on-a-Chip System

Chip-based DNA quantification systems are widespread, and used in many point-of-care applications. However, instruments for such applications may not be maintained or calibrated regularly. Since machine reliability is a key issue for normal operation, this study presents a system model of the real-time Polymerase Chain Reaction (PCR) machine to analyze the instrument design through numerical experiments. Based on model analysis, a systematic approach was developed to lower the variation of DNA quantification and achieve a robust design for a real-time PCR-on-a-chip system. Accelerated lift testing was adopted to evaluate the reliability of the chip prototype. According to the life test plan, this proposed real-time PCR-on-a-chip system was simulated to work continuously for over three years with similar reproducibility in DNA quantification. This not only shows the robustness of the lab-on-a-chip system, but also verifies the effectiveness of our systematic method for achieving a robust design.

Chip-Oriented Fluorimeter Design and Detection System Development for DNA Quantification in Nano-Liter Volumes

The chip-based polymerase chain reaction (PCR) system has been developed in recent years to achieve DNA quantification. Using a microstructure and miniature chip, the volume consumption for a PCR can be reduced to a nano-liter. With high speed cycling and a low reaction volume, the time consumption of one PCR cycle performed on a chip can be reduced. However, most of the presented prototypes employ commercial fluorimeters which are not optimized for fluorescence detection of such a small quantity sample. This limits the performance of DNA quantification, especially low experiment reproducibility. This study discusses the concept of a chip-oriented fluorimeter design. Using the analytical model, the current study analyzes the sensitivity and dynamic range of the fluorimeter to fit the requirements for detecting fluorescence in nano-liter volumes. Through the optimized processes, a real-time PCR on a chip system with only one nano-liter volume test sample is as sensitive as the commercial real-time PCR machine using the sample with twenty micro-liter volumes. The signal to noise (S/N) ratio of a chip system for DNA quantification with hepatitis B virus (HBV) plasmid samples is 3 dB higher. DNA quantification by the miniature chip shows higher reproducibility compared to the commercial machine with respect to samples of initial concentrations from 103 to 105 copies per reaction.

The evolution of real-time PCR machines to real-time PCR chips

Development of Micro-Electro-Mechanical Systems (MEMS) technology has recently allowed the migration of real-time polymerase chain reaction (PCR) machines to lab-on-a-chip systems. The miniaturization of biological instruments has been studied extensively, with several prototypes constructed and tested. In this study, the lab-on-a-chip system is evaluated; its DNA quantification is estimated by theorems, and the specifications of proposed chip prototypes are compared with the original machines. The analysis results suggest five hypotheses. Using experiments and the data collected from published papers, these hypotheses were either verified or rejected, and the advantages and shortcomings of real-time PCR chips were identified. The proven advantages of the lab-on-a-chip system are its compact size, low sample volume to nano-liter, and short analysis time—less than 10s to complete one PCR cycle and 370s for completing the whole quantification process. However, the detection limits, quantification uncertainties, and melting analysis ability of the chip prototypes are at best comparable to, and perhaps worse than, those of commercial instruments. Real-time PCR chips are not perfectly accurate diagnostic tools for a laboratory but they have advantages over traditional techniques for point-of-care testing.

Helicobacter pylori Gastritis in Children Is Associated With a Regulatory T-Cell Response

Helicobacter pylori infection in children infrequently causes gastroduodenal mucosal ulceration. Because H pylori induces T-cell dependent gastric inflammation in adults and T regulatory (Treg) cells suppress T-cell-dependent pathology, we evaluated gastric histopathology and Treg cell responses in H pylori-infected children and adults. Gastric tissue from 36 children and 79 adults with abdominal symptoms in Santiago, Chile, was evaluated prospectively for H pylori bacteria and histopathology using the Sydney classification and Treg responses using immunoassay, immunohistochemistry, and real-time polymerase chain reaction. Eighteen (50%) of the children and 51 (65%) of the adults were infected with H pylori. Children and adults were colonized with similar levels of H pylori. However, the level of gastritis in the children was reduced substantially compared with that of the adults (P < .05). Coincident with reduced gastric inflammation, the number of Treg cells and levels of Treg cytokines (transforming growth factor [TGF]-beta1 and interleukin-10) were increased markedly in the gastric mucosa of H pylori-infected children compared with that of infected adults (P < .03 and < .05, respectively). Also, H pylori infection in the children was associated with markedly increased levels of gastric TGF-beta1 and interleukin-10 messenger RNA. Importantly, gastric TGF-beta1 in H pylori-infected children localized predominantly to mucosal CD25(+) and Foxp3(+) cells, indicating a Treg source for the TGF-beta1. Gastric pathology is reduced and local Treg cell responses are increased in H pylori-infected children compared with infected adults, suggesting that gastric Treg cell responses down-regulate the inflammation and ulceration induced by H pylori in children.

Development of a temperature sensor array chip and a chip-based real-time PCR machine for DNA amplification efficiency-based quantification

A temperature sensor array chip was developed to monitor the thermal cycling profiles of a polymerase chain reaction (PCR). DNA amplification efficiency of each cycle was estimated through temperature data to fit the stochastic model. A fluorescence detector system was constructed to detect the PCR amplifications of latter cycles, at which the fluorescence intensity passed the optical detection threshold. Through monitoring of both temperature and fluorescence, DNA amplification efficiency curve was completed for quantification. The Förster resonance energy transfer (FRET) was employed to detect the measurements of the PCR product amount at the reaction endpoint. The chip-based, real-time PCR machine was constructed to perform the amplification efficiency curve-based quantification method. This novel method achieved the absolute quantification of the Hepatitis B virus (HBV) DNA using a single sample without the construction of the standard curve. The coefficient of variation (CV) of the 15 replicates inter assay experiments was less than 5.87%. Compared with the CV values obtained from the commercial machine in the range of 4.33–14.56%, it is noted that CV values of the prototype with respect to the samples of different initial concentration ranging from 10 7 to 10 3 copies/ml are almost equable.

Rapid and easy prenatal diagnosis of sickle cell anemia using double‐dye LNA probe technology

Taking advantage of the high specificity of double-dye hydrolysis-locked nucleic acid (LNA) probes (Ugozzoli et al, 2004), we developed a qualitative three-colour real-time polymerase chain reaction (PCR) assay for the genotyping of sickle cell disease (SCD). The term SCD encompasses a group of disorders associated with mutations in the Haemoglobin Beta gene (HBB). The most common forms are defined by the presence, in codon 6, of an E6V mutation (GAG > GTG; Haemoglobin S; Hb S) on both alleles (sickle cell anaemia) and co-inheritance of Hb S and Hb C (E6K mutation; GAG > AAG; Hb SC disease). The highest prevalence of SCD is found in sub-Saharan Africa (sickle-cell gene carrier prevalence varying between 5% and 40% of the population), and most affected children die in the early years of life without being diagnosed or because of non-adapted treatments (Weatherall & Clegg, 2001). The methodological approach presented here combined the use of a primer pair designed to amplify the region of interest in the HBB gene and three double-dye probes containing LNA nucleotides. Each probe was labelled with a different fluorochrome to enable the different alleles to be discriminated (Fig 1). Primers were designed with the freeware meltcalc, ver. 2.0 (http://www.meltcalc.de). The Tm of each double-dye LNA probe was calculated by using the Exiqon Tm prediction tool (http://lna-tm.com). Specificity of the probes used in this approach. 20 ng of genomic DNA from three patients homozygous for the wild-type (WT/WT), S (S/S) and C (C/C) alleles respectively was tested in this experiment. The results given by the each probe are illustrated on different panels. The Cy5 channel was applied for the analysis of the WT probe, while FAM and Yakima Yellow were used for the S and C probes, respectively. The three probes showed absolute specificity because there was no signal detected when amplified genomic DNA did not contain the nucleotide targeted by the probe. The no template control did not give any signal for the three probes either. Samples from patients that were homozygous for each of the three aforementioned alleles were used to demonstrate the specificity of the probes (Fig 1). We used unique PCR conditions on the ABI 7500 Fast machine (Applied Biosystems, Foster City, CA, USA), which generated specific results for each probe in <40 min (see Table SI, Fig S1). A cohort of 37 genomic DNA representing the different possible genotypes was used to compare our new technique with either a PCR followed by restriction endonuclease cleavage to detect the A and S alleles (Saiki et al, 1988), or an allele-specific PCR designed to detect A and C alleles (Fischel-Ghodsian et al, 1990). A 100% concordance was observed between our technology and the two published methods. Moreover, the deduced genotypes matched perfectly with the available phenotypic analysis (Gulbis et al, 2006). To investigate the sensitivity of this analysis, we tested different amounts of genomic DNA per PCR reaction. The results were perfectly interpretable from 100 to 1 ng of starting material (data not shown), and enabled the testing of prenatal diagnosis samples even when there was scarce fetal tissue (demonstrated on 18 gDNA obtained from fetal samples; data not shown). Finally, to demonstrate the robustness of our approach, we tested 10 representative samples on currently available instruments that are widely used for multicolour real-time PCR analysis. The results obtained on the LC480 (Roche, Basel, Switzerland), the iCycler (Bio-Rad, Hercules, CA, USA) and the M × 3000 p (Stratagene, La Jolla, CA, USA) were perfectly comparable from one machine to another (see Fig S1). Although a fast PCR protocol was not applicable on all these machines, a longer PCR protocol did not affect the results. In summary, we have developed a very robust and rapid method that takes advantage of the high specificity of double-dye LNA probes, which enabled the identification of the two main mutations in SCD in <40 min. The protocol developed in our laboratory is easily transferable to other real-time PCR machines. The high sensitivity of our approach represents a great advantage for the analysis of samples containing small amounts of DNA. Moreover, this single closed tube method offers another obvious advantage for prenatal diagnosis as it prevents risk of cross contamination. We gratefully acknowledge the skillful technical assistance of Sandrine Delbauve. Table SI. Oligonucleotides used for the three-colour real-time polymerase chain reaction (PCR) assay in this study. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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.