Thermal factors influencing detection of Vibrio vulnificus using real-time PCR

Five thermal factors, including initial denaturation temperature, cycling denaturation temperature, annealing temperature, extension temperature and the temperature at which the intensity of the fluorescent signal is read, were evaluated for their effects on the detection of Vibrio vulnificus via real-time PCR. Fluorescent signal detection after extension was set between the Tm value of the primer-dimers (79 degrees C) and that of the PCR target amplicons (84 degrees C). This effectively eliminated the overestimation of the yield of PCR amplicons due to the presence of primer-dimers which otherwise led to erroneously lower Ct values (1.91+/-0.22 cycles lower). The annealing and extension steps were combined to convert a three-step PCR to a two-step PCR. This consisted of initial denaturation at 95 degrees C for 3 min, cycling denaturation at 94 degrees C for 15 s and a combined annealing and extension step at 60 degrees C for 5 s in each PCR cycle. One genomic target per real-time PCR reaction was detected with the simplified two-step PCR.     

Rapid nested-PCR for tyrosinase gene detection on chip

The availability of non-invasive, fast and sensitive technologies for detection of circulating cancer cells is still a critical need of clinical oncology, particularly for diagnosis of aggressive and highly metastatic tumors, like malignant melanoma. Here we present the first nested polymerase chain reaction process carried out by a microfabricated, hybrid plastic-glass microfluidic chip on the tyrosinase gene, a predictive marker for melanoma diagnosis. The device is a hybrid system consisting of a glass microchannel embedded in an elastomeric matrix, and operating in flow-oscillating modality on a droplet of biological sample. The convection heat transfer and the temperature distribution inside the carrier fluid in the device are investigated. The oil responds to temperature changes with a characteristic time around 53 s, and exhibits three different thermal gradients along the capillary, with temperature variations below 4°C in correspondence of heater electrodes. The sample heating/cooling rates in the chip are as high as 16°C/s, allowing rapid processes. The nested polymerase chain reaction process is performed in less than 50 min, namely more than four times faster than in a standard thermocycler. The rapidity of the analysis method, combined with the simple and low-cost fabrication, reduced sample evaporation, and flexibility of the overall microfluidic platform, make it promising for the detection of events of tumor spreading.     

硅-玻璃聚合酶链式反应微芯片对β-葡糖苷酸酶基因的扩增

聚合酶链式反应（PCR）微芯片是基于微机电系统（MEMS）制作,在微芯片上进行PCR反应,实现生物样品扩增的一项新技术.介绍了硅-玻璃PCR微芯片的设计和制作、微反应腔的清洗和表面处理、借助外置温度控制系统进行PCR扩增反应以及扩增产物在琼脂糖凝胶电泳下的检测分析,实现了对β-葡糖苷酸酶（GUS）基因的有效扩增,扩增时间由原来的90 min缩短到现在的37 min.     

Use of multiplex polymerase chain reactions to indicate the accuracy of the annealing temperature of thermal cycling

A condition for multiplex polymerase chain reactions (PCRs) of which outcomes sensitively indicate the actual annealing temperature of thermal cycling is reported. The multiplex reaction was designed to produce four different amplicons of 200, 300, 400, and 480 bp. However, the degree of amplification of each amplicon sensitively responds to a small change in the annealing temperature, by which one can predict the actual annealing temperature of thermal cycling. Deviations between the actual and the designated annealing temperatures as small as 0.5 degrees C were manifested by the banding patterns of the multiplex PCRs in simple agarose gel electrophoresis. For prediction of temperatures in a more objective manner, capillary electrophoresis was also applied to obtain numerical expressions of the relative intensities of the amplicons. By optimizing the multiplex PCR conditions, where concentrations of buffer, dNTPs, and primer pairs were major factors, satisfactory sensitivity and reproducibility of the band patterning were achieved. Blind tests demonstrated the accuracy of the prediction of actual annealing temperatures within +/-0.5 degrees C. The multiplex PCR approach will be further refined and tested for realization of an easily accessible alternative to a physical temperature measurement device in testing the performance of thermal cyclers for PCR.     

Micromachined polymerase chain reaction system for multiple DNA amplification of upper respiratory tract infectious diseases

This paper presents a micro polymerase chain reaction (PCR) chip for the DNA-based diagnosis of microorganism genes and the detection of their corresponding antibiotic-resistant genes. The micro PCR chip comprises cheap biocompatible soda-lime glass substrates with integrated thin-film platinum resistors as heating/sensing elements, and is fabricated using micro-electro-mechanical-system (MEMS) techniques in a reliable batch-fabrication process. The heating and temperature sensing elements are made of the same material and are located inside the reaction chamber in order to ensure a uniform temperature distribution. This study performs the detection of several genes associated with upper respiratory tract infection microorganisms, i.e. Streptococcus pneumoniae, Haemopilus influenze, Staphylococcu aureus, Streptococcus pyogenes, and Neisseria meningitides, together with their corresponding antibiotic-resistant genes. The lower thermal inertia of the proposed micro PCR chip relative to conventional bench-top PCR systems enables a more rapid detection operation with reduced sample and reagent consumption. The experimental data reveal that the high heating and cooling rates of the system (20 and 10 degrees C/s, respectively) permit successful DNA amplification within 15 min. The micro PCR chip is also capable of performing multiple DNA amplification, i.e. the simultaneous duplication of multiple genes under different conditions in separate reaction wells. Compared with the large-scale PCR system, it is greatly advantageous for fast diagnosis of multiple infectious diseases. Multiplex PCR amplification of two DNA segments in the same well is also feasible using the proposed micro device. The developed micro PCR chip provides a crucial tool for genetic analysis, molecular biology, infectious disease detection, and many other biomedical applications.     

Identification of the Hind III polymorphic site in the PAI-1 gene: analysis of the PAI-1 Hind III polymorphism by PCR

The identification of the Hind III polymorphic site in the 3' end of the plasminogen activator inhibitor 1 (PAI-1) gene and a simple method to identify the Hind III polymorphism rapidly in the PAI-1 gene using PCR is described. The Hind III restriction site was identified by restriction site mapping and sequence analysis from a cosmid DNA clone. Genomic DNA was isolated from individual human umbilical cords and a 754-bp fragment of the human PAI-1 gene was amplified by PCR. Aliquots of the PCR products were digested with Hind III and analyzed by agarose gel electrophoresis. The presence of two fragments, 754 and 567 bp, was identified, and they were designated as 1/1 (750-bp band), 1/2 (754- and 567-bp bands), and 2/2 (567-bp band). The PCR method is considerably less time consuming than the conventional DNA genotyping using Southern blot analysis. To ensure that this new method identified the same PAI-1 genotypes as previously identified by Hind III restriction fragment length polymorphism (RFLP), samples were simultaneously genotyped by PCR and Southern blot analysis. Both methods identified the same Hind III genotypes in all the samples, confirming the reliability of this new PCR method for the rapid identification of the Hind III polymorphism in the human PAI-1 gene.     

Thirty-cycle temperature optimization of a closed-cycle capillary PCR machine

The performance of a novel thermal cycler has been characterized in a 30-cycle PCR. The device consists of a microcapillary equipped with bidirectional pressure-driven flow and in situ optical position sensors. A 1-microL droplet of reaction mixture moves between three heat zones in a 1-mm i.d., oil-filled capillary using a multi-element scattered light detector and active feedback. The design permits time and number of cycles to be changed without hardware modification, unlike other flow-in-capillary PCR systems. Temperature optimization has been performed on the three PCR heat steps. The optimal denaturation temperature is 94 degrees C-96 degrees C, which is identical to commercial machines. The optimal extension temperature of 62 degrees C-66 degrees C is lower than reported for Taq DNA polymerase (70 degrees C-80 degrees C) because of the high enzyme concentration and/or the absence of detergent in the PCR mixture. The optimal annealing temperature seems to be the same as the optimal extension temperature. This is because extension occurs when the sample is inside of the annealing heat zone. Annealing takes place as the sample travels between heat zones. Device speed (23 minfor 30 cycles without time optimization) is competitive with other rapid PCR designs for efficiencies comparable to a commercial machine.     

Ultra fast miniaturized real-time PCR: 40 cycles in less than six minutes

We have designed, fabricated and tested a real-time PCR chip capable of conducting one thermal cycle in 8.5 s. This corresponds to 40 cycles of PCR in 5 min and 40 s. The PCR system was made of silicon micromachined into the shape of a cantilever terminated with a disc. The thin film heater and a temperature sensor were placed on the disc perimeter. Due to the system's thermal constant of 0.27 s, we have achieved a heating rate of 175°C s⁻¹ and a cooling rate of −125°C s⁻¹. A PCR sample encapsulated with mineral oil was dispensed onto a glass cover slip placed on the silicon disc. The PCR cycle time was then determined by heat transfer through the glass, which took only 0.5 s. A real-time PCR sample with a volume of 100 nl was tested using a FAM probe. As the single PCR device occupied an area of only a few square millimeters, devices could be combined into a parallel system to increase throughput.     

Plastic microfluidic chip for continuous-flow polymerase chain reaction: simulations and experiments

A continuous flow polymerase chain reaction (CF-PCR) device comprises a single fluidic channel that is heated differentially to create spatial temperature variations such that a sample flowing through it experiences the thermal cycling required to induce amplification. This type of device can provide an effective means to detect the presence of a small amount of nucleic acid in very small sample volumes. CF-PCR is attractive for global health applications due to its less stringent requirements for temperature control than for other designs. For mass production of inexpensive CF-PCR devices, fabrication via thermoplastic molding will likely be necessary. Here we study the optimization of a PCR assay in a polymeric CF-PCR device. Three channel designs, with varying residence time ratios for the three PCR steps (denaturation, annealing, and extension), were modeled, built, and tested. A standardized assay was run on the three different chips, and the PCR yields were compared. The temperature gradient profiles of the three designs and the residence times of simulated DNA molecules flowing through each temperature zone were predicted using computational methods. PCR performance predicted by simulation corresponded to experimental results. The effects of DNA template size and cycle time on PCR yield were also studied. The experiments and simulations presented here guided the CF-PCR chip design and provide a model for predicting the performance of new CF-PCR designs prior to actual chip manufacture, resulting in faster turn around time for new device and assay design. Taken together, this framework of combined simulation and experimental development has greatly reduced assay development time for CF-PCR in our lab.     

High-throughput PCR in silicon based microchamber array

Highly integrated hybridization assay and capillary electrophoresis have improved the throughput of DNA analysis. The shift to high throughput analysis requires a high speed DNA amplification system, and several rapid PCR systems have been developed. In these thermal cyclers, the temperature was controlled by effective methodology instead of a large heating/cooling block preventing rapid thermal cycling. In our research, high speed PCR was performed using a silicon-based microchamber array and three heat blocks. The highly integrated microchamber array was fabricated by semiconductor microfabrication techniques. The temperature of the PCR microchamber was controlled by alternating between three heat blocks of different temperature. In general, silicon has excellent thermal conductivity, and the heat capacity is small in the miniaturized sample volume. Hence, the heating/cooling rate was rapid, approximately 16 °C/s. The rapid PCR was therefore completed in 18 min for 40 cycles. The thermal cycle time was reduced to 1/10 of a commercial PCR instrument (Model 9600, PE Applied Biosystems-3 h).     

Differential detection of vibrios pathogenic to shrimp by multiplex PCR

The research was focused on the multiplex polymerase chain reaction (PCR) differential detection of shrimp pathogens Vibrio harveyi, Vibrio campbellii and isolates from a variant strain of Vibrio (referred to as Philippine Vibrio isolates in this study) exhibiting characteristics distinct from these two species. Sequence alignment of the hemolysin gene from type strains Vibrio harveyi (NBRC 15634) and Vibrio campbellii (NBRC 15631), as well as 10 variant Philippine Vibrio isolates, was performed in order to design a set of hemolysin-targeted primers for the specific detection of the Philippine Vibrio isolates. Primer PNhemo amplified a 320-bp hemolysin gene fragment of the Philippine Vibrio isolates in PCR using 65 degrees C annealing temperature, but did not amplify the target gene fragment in type strains V. harveyi and V. campbellii. Another new primer (VcatoxR) targeting the toxR gene was designed for the specific detection of type strain V. campbellii under stringent 65 degrees C annealing temperature. PCR using VcatoxR primer resulted in the specific amplification of a 245-bp V. campbellii toxR fragment. The simultaneous use of three primer sets in PCR, including PNhemo and VcatoxR (the two new primers designed in this study), and a primer VhtoxR (previously reported for the specific detection of V. harveyi), resulted in differential profiles with 390-bp, 245-bp, and 320-bp amplicons for V. harveyi, V. campbellii, and variant Philippine Vibrio isolates, respectively. Presence of all three types of Vibrio shrimp pathogens in the sample could be detected with a multiplex PCR profile containing all the expected size amplicons.     

Performance evaluation of thermal cyclers for PCR in a rapid cycling condition

The performance of thermal cyclers for polymerase chain reactions (PCR) is of great concern in terms of the reliability of PCR-based assays, particularly when rapid cycling conditions are applied to small volume reactions. In this work, the precision of the temperature controls during rapid thermal cycling was measured in 19 commercial thermal cyclers of 8 different models. The temperatures of test solutions in specific locations in each thermal block were simultaneously monitored at 1 s intervals during thermal cycling. A temperature-sensitive multiplex PCR was run in parallel to assess undesirable PCR results caused by poor temperature control. Under the given conditions (20 s of annealing time and 20 microL reaction volume), a majority of the tested instruments showed prominent curving, undershooting, and/or overshooting in their temperature profiles, which substantially influenced the results of the temperature-sensitive multiplex PCR. Variations between wells were also observed in most instruments. It is strongly hoped that these problems will be addressed by manufacturers and that they will make substantial improvements in the precision and efficiency of thermal cyclers. In the meantime, users of thermal cyclers might be able to avoid unexpected poor outcomes of sensitive PCR-based assays by designing their PCR protocols with these findings in mind.     

Fluorescence-based temperature control for polymerase chain reaction

The ability to accurately monitor solution temperature is important for the polymerase chain reaction (PCR). Robust amplification during PCR is contingent on the solution reaching denaturation and annealing temperatures. By correlating temperature to the fluorescence of a passive dye, noninvasive monitoring of solution temperatures is possible. The temperature sensitivity of 22 fluorescent dyes was assessed. Emission spectra were monitored and the change in fluorescence between 45 and 95 °C was quantified. Seven dyes decreased in intensity as the temperature increased, and 15 were variable depending on the excitation wavelength. Sulforhodamine B (monosodium salt) exhibited a fold change in fluorescence of 2.85. Faster PCR minimizes cycling times and improves turnaround time, throughput, and specificity. If temperature measurements are accurate, no holding period is required even at rapid speeds. A custom instrument using fluorescence-based temperature monitoring with dynamic feedback control for temperature cycling amplified a fragment surrounding rs917118 from genomic DNA in 3 min and 45 s using 35 cycles, allowing subsequent genotyping by high-resolution melting analysis. Gold-standard thermocouple readings and fluorescence-based temperature differences were 0.29 ± 0.17 and 0.96 ± 0.26 °C at annealing and denaturation, respectively. This new method for temperature cycling may allow faster speeds for PCR than currently considered possible.     

Disposable real-time microPCR device: lab-on-a-chip at a low cost

We have designed, fabricated and tested a real-time micro polymerase chain reaction (microPCR) system. It consists of a microscope glass cover slip placed on top of a micromachined silicon chip integrated with a heater and a temperature sensor. A single microL of a sample containing DNA was placed on the glass and encapsulated with mineral oil to prevent the evaporation of water, thus forming a virtual reaction chamber (VRC). The PCR chip required half a second to heat up from 72 to 94 degrees C and two seconds to cool from 94 to 55 degrees C, corresponding to a cooling rate of -20 K s(-1). The real-time PCR yield was determined by a fluorescence method. The melting curve analysis method as well as capillary electrophoresis was performed to determine the purity of the PCR product. As the glass slip is disposable, cross-contamination from sample to sample is eliminated. The total cost of running the PCR is given by the value of the cover slip and its treatment.     

Isotachophoresis preconcentration integrated microfluidic chip for highly sensitive genotyping of the hepatitis B virus

The genotyping of hepatitis B virus (HBV) has become recently a valuable tool not only for epidemiological reasons but also for the clinical practice. Conventional methods for HBV genotyping typically include amplification of the target DNA sequences with a two-round nested PCR followed by separation of the amplified fragments by gel electrophoresis. A microfluidic chip that couples isotachophoresis (ITP) preconcentration and zone electrophoresis (ZE) separation may provide great advantages for sensitive, rapid and cost-effective clinical analysis. In this study, an HBV genotyping method with only one amplification round was developed by the application of the ITP-ZE chip. All the analysis steps of the ITP-ZE separation including sample injection, stacking and separation were performed continuously, controlled by sequential high-voltage switching. A 2.1cm sample plug was preconcentrated between discontinuous buffers in ITP process, followed by ZE separation. Sensitivity enhancement was obtained through the increase of sample loading volume. The average LOD value of the ITP-ZE microfluidic chip was determined to be 0.0021pg/muL. In a large-scale HBV genotyping test, single round PCR products were analyzed by ITP-ZE microfluidic chip, and the results were compared with that of the conventional method. Among the 200 cases studied, the classification rate obtained with microfluidic chip was 93%, which was 6% higher than that obtained with the conventional method. Method with ITP-ZE chip analysis provides HBV genotyping information in reduced PCR amplification time with higher detection rate when compared with conventional method. This method holds great potential for extrapolation to the abundance of similar molecular biology-based techniques in clinical diagnosis.     

A versatile oscillating-flow microfluidic PCR system utilizing a thermal gradient for nucleic acid analysis

We report the development of a versatile system based on the oscillating-flow methodology in a thermal gradient system for nucleic acid analysis. Analysis of DNA and RNA samples were performed in the device, without additional temperature control and complexity. The technique reported in this study eliminates the need for predetermined fluidic channels for thermocycles, and complexity involved with additional incubation steps required for RNA amplification. A microfluidic device was fabricated using rapid prototyping by simply sandwiching dual side adhesive Kapton tape and a polydimethylsiloxane spacer between glass microscope slides. Amplification of the 181-bp segment of a viral phage DNA (ΦX174) and B2M gene in human RNA samples was demonstrated using the system. The developed system enables simultaneous acquisition of amplification and melt curves, eliminating the need for postprocessing. A direct comparison between the oscillating-flow system and a commercial real-time polymerase chain reaction (PCR) instrument showed complete agreement in PCR data and improved sample-to-result time by eliminating an additional 30 min melt curve step required in commercial PCR systems.     

PCR-amplification of GC-rich regions: 'slowdown PCR'

The polymerase chain reaction (PCR) technique has become an indispensable method in molecular research. However, PCR-amplification of GC-rich templates is often hampered by the formation of secondary structures like hairpins and higher melting temperatures. We present a novel method termed 'Slowdown PCR', which allows the successful PCR-amplification of extremely GC-rich (>83%) DNA targets. The protocol relies on the addition of 7-deaza-2'-deoxyguanosine, a dGTP analog to the PCR mixture and a novel standardized cycling protocol with varying temperatures. The latter consists of a generally lowered ramp rate of 2.5 degrees C s(-1) and a low cooling rate of 1.5 degrees C s(-1) for reaching an annealing temperature and is run for 48 cycles. We established this protocol as a versatile method not only for amplification of extremely GC-rich regions, but also for routine DNA diagnostics and pharmacogenetics for templates with different annealing temperatures. The protocol takes 5 h to complete.     

PCR amplification using electrolytic resistance for heating and temperature monitoring

An alternative method of rapid-cycle PCR for DNA amplification is demonstrated using electrolyte resistance for heating and temperature monitoring. The PCR amplification solution is electrically conductive and can be heated by passing an alternating current through the sample. The temperature of the solution is evaluated by monitoring its electrical resistance. Cooling is accomplished by forced air convection at ambient temperature. Heating and cooling rates of up to 20 degrees C/s were achieved. The 35 cycles of PCR were completed in less than 12 min with product yields equivalent to conventional temperature cycling. Electrolyte resistance provides a method for both direct heating and monitoring the temperature of PCR samples.     

Development characterization and use of a high-performance enzymatic time-temperature integrator for the control of sterilization process' impacts

A small sized single-component enzymatic time temperature integrator (TTI) was developed. It consisted of glass beads coated with Bacillus licheniformis alpha-amylase (BLA) and stabilizing additives in a dehydrated form. Post heating residual enzymatic activity was used as a response property of the TTI. Under isothermal conditions, different batches of the system were characterized by z(TTI)-values around 13.5 degrees C in the temperature range 100-130 degrees C as well as by their ability to provide a response within 5 min after thermal processing. When used under non-isothermal conditions in a model food (silicone spheres), the system allowed to measure process-values (zTTI)F(121.1 degrees C) up to 60 min with an average error of 10.9%. The capabilities of the system were validated in a real solid/liquid food matrix sterilized by retorting. The combination of F(TTI)-values with heat transfer simulations based on finite difference calculations allowed for the determination of process values, which evaluated actual process-values (10 degrees C)F(121.1 degrees C) up to 90 min with an average error of 11.4%. The good performances of the system as well as its easiness of preparation and use, make the latter a valuable biological device for thermal process assessment.     

Adaptor long-range PCR procedure for clone-specific characterization and chromosomal localization

An efficient adaptor long-range PCR (ALR-PCR) procedure was developed to detect genomic rearrangements in high-plasticity genomic regions between closely related strains of bacteria. The method was precisely optimized using a combination of high-speed experimental steps for the chromosomal localization and elucidation of deletions, inversions, duplications, or inserted sequences within a clone-specific flanking region. The advantages of this strategy are: (i) ready-to-use polymerase mixtures and Master mix (ready-to-use reaction mixtures with polymerase MasterAmp and buffer 2x Premix 4); (ii) a 5-min ligation procedure; (iii) rapid purification of DNA digests; (iv) optimized DNA template concentration protocol to avoid nonspecific amplification and high backgrounds; (v) long-range PCR protocol to obtain at least 9.6 kb single PCR products; (vi) two-step PCR cycling with the same annealing and extension temperature at 68 degrees C; (vii) simple design of the adaptors according to the preferred restriction endonuclease enzyme; and (viii) simple technology and equipment required. The application of this method for a tester-specific suppressive subtractive hybridization (SSH) clone of Brucella melitensis 16M revealed an 837-bp deletion and a 7255-bp DNA transfer from one chromosomal location to another for Brucella abortus 2308 used as a driver.