Neuro-genetic optimization of temperature control for a continuous flow polymerase chain reaction microdevice

In this study, a hybridized neuro-genetic optimization methodology realized by embedding finite element analysis (FEA) trained artificial neural networks (ANN) into genetic algorithms (GA), is used to optimize temperature control in a ceramic based continuous flow polymerase chain reaction (CPCR) device. The CPCR device requires three thermally isolated reaction zones of 94 degrees C, 65 degrees C, and 72 degrees C for the denaturing, annealing, and extension processes, respectively, to complete a cycle of polymerase chain reaction. The most important aspect of temperature control in the CPCR is to maintain temperature distribution at each reaction zone with a precision of +/-1 degree C or better, irrespective of changing ambient conditions. Results obtained from the FEA simulation shows good comparison with published experimental work for the temperature control in each reaction zone of the microfluidic channels. The simulation data are then used to train the ANN to predict the temperature distribution of the microfluidic channel for various heater input power and fluid flow rate. Once trained, the ANN analysis is able to predict the temperature distribution in the microchannel in less than 20 min, whereas the FEA simulation takes approximately 7 h to do so. The final optimization of temperature control in the CPCR device is achieved by embedding the trained ANN results as a fitness function into GA. Finally, the GA optimized results are used to build a new FEA model for numerical simulation analysis. The simulation results for the neuro-genetic optimized CPCR model and the initial CPCR model are then compared. The neuro-genetic optimized model shows a significant improvement from the initial model, establishing the optimization method's superiority.     

Microfluidic bead-based enzymatic primer extension for single-nucleotide discrimination using quantum dots as labels

This study reports the development of an on-chip enzyme-mediated primer extension process based on a microfluidic device with microbeads array for single-nucleotide discrimination using quantum dots as labels. The functionalized microbeads were independently introduced into the arrayed chambers using the loading chip slab. A single channel was used to generate weir structures to confine the microbeads and make the beads array accessible by microfluidics. The applied allele-specific primer extension method employed a nucleotide-degrading enzyme (apyrase) to achieve specific single-nucleotide detection. Based on the apyrase-mediated allele-specific primer extension with quantum dots as labels, on-chip single-nucleotide discrimination was demonstrated with high discrimination specificity and sensitivity (0.5 pM, signal/noise > 3) using synthesized target DNA. The chip-based signal enhancement for single-nucleotide discrimination resulted in 200 times higher sensitivity than that of an off-chip test. This microfluidic device successfully achieved simultaneous detection of two disease-associated single-nucleotide polymorphism sites using polymerase chain reaction products as target. This apyrase-mediated microfluidic primer extension approach combines the rapid binding kinetics of homogeneous assays of suspended microbeads array, the liquid handling capability of microfluidics, and the fluorescence detection sensitivity of quantum dots to provide a platform for single-base analysis with small reagent consumption, short assay time, and parallel detection.     

Rapid detection of genetically modified organisms on a continuous-flow polymerase chain reaction microfluidics

The ability to perform DNA amplification on a microfluidic device is very appealing. In this study, a compact continuous-flow polymerase chain reaction (PCR) microfluidics was developed for rapid analysis of genetically modified organisms (GMOs) in genetically modified soybeans. The device consists of three pieces of copper and a transparent polytetrafluoroethylene capillary tube embedded in the spiral channel fabricated on the copper. On this device, the P35S and Tnos sequences were successfully amplified within 9 min, and the limit of detection of the DNA sample was estimated to be 0.005 ng μl⁻¹. Furthermore, a duplex continuous-flow PCR was also reported for the detection of the P35S and Tnos sequences in GMOs simultaneously. This method was coupled with the intercalating dye SYBR Green I and the melting curve analysis of the amplified products. Using this method, temperature differences were identified by the specific melting temperature values of two sequences, and the limit of detection of the DNA sample was assessed to be 0.01 ng μl⁻¹. Therefore, our results demonstrated that the continuous-flow PCR assay could discriminate the GMOs in a cost-saving and less time-consuming way.     

Cell-free protein synthesis in microfluidic array devices

We report the development of a microfluidic array device for continuous-exchange, cell-free protein synthesis. The advantages of protein expression in the microfluidic array include (1) the potential to achieve high-throughput protein expression, matching the throughput of gene discovery; (2) more than 2 orders of magnitude reduction in reagent consumption, decreasing the cost of protein synthesis; and (3) the possibility to integrate with detection for rapid protein analysis, eliminating the need to harvest proteins. The device consists of an array of units, and each unit can be used for production of an individual protein. The unit comprises a tray chamber for in vitro protein expression and a well chamber as a nutrient reservoir. The tray is nested in the well, and they are separated by a dialysis membrane and connected through a microfluidic connection that provides a means to supply nutrients and remove the reaction byproducts. The device is demonstrated by synthesis of green fluorescent protein, chloramphenicol acetyl-transferase, and luciferase. Protein expression in the device lasts 5-10 times longer and the production yield is 13-22 times higher than in a microcentrifuge tube. In addition, we studied the effects of the operation temperature and hydrostatic flow on the protein production yield.     

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.     

Localization of the expressed human p58 protein kinase chromosomal gene to chromosome 1p36 and a highly related sequence to chromosome 15.

The gene for the human p58 protein kinase, a cell division control-related gene, has been mapped by somatic cell hybrid analyses, in situ localization with the chromosomal gene, and nested polymerase chain reaction amplification of microdissected chromosomes. These studies indicate that the expressed p58 chromosomal gene maps to 1p36, while a highly related p58 sequence of unknown nature maps to chromosome 15. Assignment of a p34cdc2-related gene to 1p36 may have implications for numerous tumors that involve deletion of this region, including neuroblastoma, ductal carcinoma of the breast, malignant melanoma, Merkel cell carcinoma, and endocrine neoplasia.     

An integrated biosensor platform for extraction and detection of nucleic acids

The development of portable systems for analysis of nucleic acids (NAs) is crucial for the evolution of biosensing in the context of future healthcare technologies. The integration of NA extraction, purification, and detection modules, properly actuated by microfluidics technologies, is a key point for the development of portable diagnostic systems. In this paper, we describe an integrated biosensor platform based on a silicon–plastic hybrid lab-on-disk technology capable of managing NA extraction, purification, and detection processes in an integrated format. The sample preparation process is performed by solid-phase extraction technology using magnetic beads on a plastic disk, while detection is done through quantitative real-time polymerase chain reaction (qRT-PCR) on a miniaturized silicon device. The movement of sample and reagents is actuated by a centrifugal force induced by a disk actuator instrument. The assessment of the NA extraction and detection performance has been carried out by using hepatitis B virus (HBV) DNA genome as a biological target. The quantification of the qRT-PCR chip in the hybrid disk showed an improvement in sensitivity with respect to the qRT-PCR commercial platforms, which means an optimization of time and cost. Limit of detection and limit of quantification values of about 8 cps/reaction and 26 cps/reaction, respectively, were found by using analytical samples (synthetic clone), while the results with real samples (serum with spiked HBV genome) indicate that the system performs as well as the standard methods.     

A vertically stacked, polymer, microfluidic point mutation analyzer: rapid high accuracy detection of low-abundance K-ras mutations

Recognition of point mutations in the K-ras gene can be used for the clinical management of several types of cancers. Unfortunately, several assay and hardware concerns must be addressed to allow users not well trained in performing molecular analyses the opportunity to undertake these measurements. To provide for a larger user base for these types of molecular assays, a vertically stacked microfluidic analyzer with a modular architecture and process automation was developed. The analyzer employs a primary polymerase chain reaction (PCR) coupled to an allele-specific ligase detection reaction (LDR). Each functional device, including continuous flow thermal reactors for the PCR and LDR, passive micromixers, and ExoSAP-IT purification, was designed and tested. Individual devices were fabricated in polycarbonate using hot embossing and were assembled using adhesive bonding for system assembly. The system produced LDR products from a DNA sample in approximately 1 h, an 80% reduction in time compared with conventional benchtop instrumentation. Purifying the post-PCR products with the ExoSAP-IT enzyme led to optimized LDR performance, minimizing false-positive signals and producing reliable results. Mutant alleles in genomic DNA were quantified to the level of 0.25 ng of mutant DNA in 50 ng of wild-type DNA for a 25-μl sample, equivalent to DNA from 42 mutant cells.     

Evaluation of polymerase chain reaction for the detection of Paracoccidioides brasiliensis DNA on serum samples from patients with paracoccidioidomycosis

The aim of this study was to demonstrate the DNA of Paracoccidioides brasiliensis in human serum samples of patients with paracoccidioidomycosis using the polymerase chain reaction (PCR). The diagnosis of paracoccidioidomycosis (PCM) was defined by microscopic observation of the fungus on direct exam or histopathology, culture, and serological positivity. DNA from serum of 33 patients with PCM was extracted and submitted to nested-PCR using primers from the gp 43 gene. Only one sample was positive on nested-PCR. We conclude that the prevalence of fungemia in patients with different clinical forms of PCM is low, limiting the use of serum DNA detection as an alternative diagnostic tool.     

Detection of Cryptosporidium parvum oocysts using a microfluidic device equipped with the SUS micromesh and FITC-labeled antibody

Development of a microfluidic device equipped with micromesh for detection of Cryptosporidium parvum oocyst was reported. A micromesh consisting of 10 x 10 cavities was microfabricated on the stainless steel plate by laser ablation. Each cavity size, approximately 2.7 microm in diameter, was adopted to capture a single C. parvum oocyst. Under negative pressure operation, suspensions containing microbeads or C. parvum oocysts flowed into the microchannel. Due to strong non-specific adsorption of microbeads onto the PDMS microchannel surface during sample injection, the surface was treated with air plasma, followed by treatment with 1% sodium dodecyl sulfate (SDS) solution. This process reduced the non-specific adsorption of microbeads on the microchannel to 10% or less in comparison to a non-treated microchannel. This microfluidic device equipped with the SUS micromesh was further applied for the capture of C. parvum oocysts. Trapped C. parvum oocysts were visualized by staining with FITC-labeled anti-C. parvum oocyst antibody on a micromesh and counted under fluoroscopic observation. The result obtained by our method was consistent with that obtained by direct immunofluorescence assay coupled with immunomagnetic separation (DFA-IMS) method, indicating that the SUS micromesh is useful for counting of C. parvum oocysts. The newly designed microfluidic device exploits a geometry that allowed for the entrapment of oocysts on the micromesh while providing the rapid introduction of a series of reagents and washes through the microfluidic structure. Our data indicate that this microfluidic device is useful for high-throughput counting of C. parvum oocysts from tap water sample.     

Optimization of a microfluidic microarray device for the fast discrimination of fungal pathogenic DNA

A microfluidic microarray device, which has been developed for parallel DNA detection, is now further optimized for more rapid and sensitive DNA detection and for the single-base-pair discrimination of two fungal pathogenic PCR products. Two poly(dimethylsiloxane) (PDMS)-based microfluidic chips consist of radial and spiral microchannels in which flexible probe creation and convenient sample delivery have been achieved by centrifugal pumping. The microarray hybridizations occurred at the cross sections within the spiral channels intersecting the preprinted radial probe lines. The centrifugal pumping method showed advantages over the vacuum suction method in terms of parallel solution delivery and less signal variations between replicate samples. The effect of microchannel depth was studied, and hybridization time is predictable at a certain rotation speed. Cy5 dye labels were proved to show much higher hybridization efficiency as well as less photobleaching effect as compared with the fluorescein dye labels used in our previous work. With these optimized conditions, the method was applied to the detection of three fungal pathogenic polymerase chain reaction (PCR) products with a sample load of 0.2 ng (in 1 μl). Furthermore, the single-base-pair discrimination between the PCR products of two relevant Botrytis species (B. cinerea and B. squamosa) was achieved in a duration as short as 3 min.     

The Community Dynamics of Major Bioleaching Microorganisms During Chalcopyrite Leaching Under the Effect of Organics

To determine the effect of organics (yeast extract) on microbial community during chalcopyrite bioleaching at different temperature, real-time polymerase chain reaction (PCR) was employed to analyze community dynamics of major bacteria applied in bioleaching. The results showed that yeast extract exerted great impact on microbial community, and therefore influencing bioleaching rate. To be specific, yeast extract was adverse to this bioleaching process at 30°C due to decreased proportion of important chemolithotrophs such as Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans. However, yeast extract could promote bioleaching rate at 40°C on account of the increased number and enhanced work of Ferroplasma thermophilum, a kind of facultative bacteria. Similarly, bioleaching rate was enhanced under the effect of yeast extract at 50°C owing to the work of Acidianus brierleyi. At 60°C, bioleaching rate was close to 100% and temperature was the dominant factor determining bioleaching rate. Interestingly, the existence of yeast extract greatly enhanced the relative competitiveness of Ferroplasma thermophilum in this complex bioleaching microbial community.     

EWOD silicon biosensor for multiple nucleic acids analysis

In this paper, a miniaturized biosensor containing 96 silicon microchambers electroloaded with nano-volumes of liquid (EW-chip) is presented. The liquid electroloading is achieved by the appropriate modulation of interface properties. The surface chemistries have been studied to guarantee effective interface properties for both electrowetting on dielectric actuation and biocompatibility versus biochemical reactions. The silicon microchambers are 200 nl in volume and are connected to a specific system of electrodes able to deliver liquid sample on each well. The device also integrates temperature sensors and heaters to perform biochemical reactions. On that, the effectiveness of this device has been successfully proven towards the nucleic acids detection via real-time polymerase chain reaction amplification. Hepatitis B virus genome target has been used to assess the device performance. Results show very uniform amplification over the 96 microchambers without any cross-contamination process. These features make this system a very appealing potential solution for genetic point-of-care devices where a high level of parallelism of analysis is required.     

Comparison of five PCR assays for detecting Chlamydophila pneumoniae DNA

We compared five different polymerase chain reaction (PCR) assays for the detection of Chlamydophila pneumoniae DNA using highly purified elementary bodies (EBs) and peripheral blood mononuclear cells (PBMCs) from healthy blood donors. The primers were as follows; two targeting the 16S rRNA gene, one targeting the ompA gene, one targeting the Pst-I gene, and one targeting the 53 kDa outer membrane protein gene. The 16S rRNA touchdown enzyme time release (TETR) PCR, the ompA nested PCR and the 53 kDa nested PCR were the most sensitive assays and could detect one or more EB per assay. These three PCRs also had the same reproducibility, but the minimal amount of C. pneumoniae that could be reproducibly detected (10 of 10 testing positive) was 20 EBs. In a sample of specimens from healthy blood donors, we found 5 of 77 (6.5%) PBMCs specimens to have C. pneumoniae DNA according to the nested ompA PCR. Specimens with the 16S rRNA TETR and 53 kDa nested assays were found to have C. pneumoniae DNA 7 of 77 (9.1%) and 18 of 77 (23.4%) specimens, respectively. The other two assays failed to detect even a single positive. However, the detection rate decreased with repeated testing of the same samples. Our newly designed 53 kDa nested PCR may be as useful as the other four recommended PCR assays and may be a more useful assay for the detection of C. pneumoniae DNA from PBMCs.     

Development of multisample detection system using a tag insertion primer and an electrochemical DNA chip

We have developed a novel multisample detection system by employing a technology combining a tag insertion primer and an electrochemical DNA chip. In the first application, Helicobacter species-infected mouse samples were detected. The primers that insert a different tag sequence in each sample were prepared, and loop-mediated isothermal amplification (LAMP) reaction was carried out. Then amplification products in which a part of the sequence was different in each sample could be obtained. The target sample in which these amplification products were mixed was injected into a cassette that included the DNA chip with immobilized probes. After the cassette was set in the DNA detection system, Genelyzer, the processes of hybridization, washing, and detection were performed by the system automatically. The positive and negative concordance rates of the existing nested polymerase chain reaction (PCR) method and this method were 100% (40/40 samples) and 97.3% (117/120 samples), respectively. This is a simple high-throughput method. Moreover, the cost per sample can be drastically lowered. Therefore, it is expected to contribute to the diagnosis of infectious agents in humans and animals.     

Detection of Epstein Barr virus in formalin-fixed paraffin tissues by fluorescent direct in situ PCR

Specific viral laboratory diagnosis of primary Epstein-Barr Virus (EBV) infection is usually based on antibody-detection assays. However, molecular detection is also considered the reference standard assay for diagnosis of central nervous system infections and of most cases of nasopharyngeal carcinoma (NPC). One-step or nested polymerase chain reaction (PCR) has rapidly replaced immunological assays based on virus-specific Ig antibodies for the laboratory diagnosis of Herpesvirus infections, even if serological methods are considered an additional tool for defining clinical diagnosis. In this article, we will present a rapid, sensitive and robust molecular tool for the viral detection of EBV (EBNA-1) within tissue specimens by making use of in situ PCR (IS-PCR).     

Nested double PCR法对献血者血浆中HCV-RNA的检出——兼评作为献血筛选试验的Anti-HCV抗体检查

本文报道了在位于5’端非编码区的引物的诱导下用Nested double PCR技术检测献血者血浆中HCV-RNA的方法。我们发现Nested double PCR敏感性及特异性均优于单次PCR。anti-HCV(C100-3)阳性血浆样品中只有35.2％(19/54)能同时被证实为PCR阳性。由于anti-HCV(C100-3)假阳性率太高,作为献血筛选试验检测方法不能令人满意,而本文报导的Nestel double PCR方法可以弥补anti-HCV试验的不足。     

A single-tube nested real-time polymerase chain reaction for sensitive contained detection of Cryptosporidium parvum

Aim:  A new real-time polymerase chain reaction (PCR) was developed for sensitive contained detection of Cryptosporidium parvum .
Methods and Results:  The method is a nested PCR targeting a specific region of rDNA of C. parvum , which takes place in one tube, using different annealing temperatures to control the first and the second rounds of PCR, with real-time fluorogenic probe-based detection of the second round of PCR. The DNA-based detection limit of the method was 2 fg, which corresponds to approx. one genome per reaction. The detection level determined using diluted samples of C. parvum oocysts was ten oocysts per millilitre.
Conclusions:  The method facilitates sensitive detection of C. parvum thanks to the nested format, while reducing the risk of laboratory contamination thanks to the single-tube, real-time fluorimetric format.
Significance and Impact of the Study:  The developed method may be useful for sensitive contained detection of C. parvum in environmental and food samples, after appropriate separation of oocysts.