Search

Since the increment of the threat to public health caused by foodborne pathogens, researches have been widely studied on developing the miniaturized detection system for the on-site pathogen detection. In the study, we focused on the development of portable, robust, and disposable film-based polymerase chain reaction (PCR) chip containing a multiplex chamber for simultaneous gene amplification. In order to simply fabricate and operate a film-based PCR chip, different kinds of PCR chambers were designed and fabricated using polyethylene terephthalate (PET) and polyvinyl chloride (PVC) adhesive film, in comparison with commercial PCR, which employs a stereotyped system at a bench-top scale. No reagent leakage was confirmed during the PCR thermal cycling using the film PCR chip, which indicates that the film PCR chip is structurally stable for rapid heat cycling for DNA amplification. Owing to use of the thin film to fabricate the PCR chip, we are able to realize fast thermal transfer from the heat block that leads to short PCR amplification time. Moreover, using the film PCR chip, we could even amplify the target pathogen with 10 CFU mL -1 . The artificially infected milk with various concentration of Bacillus cereus was successfully amplified on a single film PCR chip. On the basis of the reliable results, the developed film PCR chip could be a useful tool as a POCT device to detect foodborne pathogens via genetic analysis.

A total integrated genetic analysis microsystem was developed, which consisted of solid phase extraction (SPE), polymerase chain reaction (PCR), and immunochromatographic strip (ICS) parts for multiplex colorimetric detection of pathogenic Staphylococcus aureus (S. aureus) and Escherichia coli O157:H7 (E. coli O157:H7) on a portable genetic analyzer. Utilizing a slidable chamber, which is a movable glass wafer, complex microvalves could be eliminated for fluidic control in the microchannel, which could simplify the chip design and chip operation. The integrated slidable microdevice was composed of 4 layers: a 4-point Pt/Ti resistance temperature detector (RTD) wafer, a micro-patterned channel wafer, a 2 μL volume slidable chamber, and an ICS. The entire process from the DNA extraction in the SPE chamber to the detection of the target gene expression by the ICS was serially performed by simply sliding the slidable chamber from one part to another functional part. The total process for multiplex pathogenic S. aureus and E. coli O157:H7 detection on the integrated slidable microdevice was accomplished within 55 min with a detection limit of 5 cells. Furthermore, spiked bacteria samples in milk were also successfully analysed on the portable genetic analysis microsystem with sample-in-answer-out capability. The proposed total integrated microsystem is adequate for point-of-care DNA testing in that no microvalves and complex tubing systems are required due to the use of the slidable chamber and the bulky and expensive fluorescence or electrochemical detectors are not necessary due to the ICS based colorimetric detection.

It has been very difficult to detect and quantify multiple somatic mutations simultaneously in single-tube qPCR. Here, a novel method for simultaneous detection of multiple mutations and a melting curve analysis was developed by using clamping PNA and detection PNA probes. Each PNA probe was designed to have a specific melting temperature by the introduction of γ-PNA monomer. This technique was successfully applied to the detection of six genotypes in two different mutations of K-RAS at the same time. Such simultaneous analysis of an amplified curve and a melting curve in qPCR can be widely used for the early diagnosis of cancer and determining the prognosis of drug treatments., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

An integrated allele-specific polymerase chain reaction (AS PCR) and microarray chip has been developed for multiplex single nucleotide polymorphism (SNP) typing on a portable genetic analyzer instrumentation. We applied the integrated PCR-microarray system for on-site Hanwoo (Korean indigenous beef cattle) identification. Eleven sets of primers were designed, among which ten sets of primers targeted ten SNP loci to discriminate Hanwoo from the imported beef cattle and one primer set was used as a positive PCR control. The AS PCR for multiplex SNP typing was conducted on a glass-based microchip consisting of four layers: a microchannel plate for microfluidic control, a Pt-electrode plate for a resistance temperature detector (RTD), a poly(dimethylsiloxane) (PDMS) membrane and a manifold glass for micropump and microvalve function. The resultant AS PCR products were mixed with a hybridization buffer in a micromixer channel through the micropumping operation, and then the microarray assay was performed in the downstream process. Eleven duplicate probes were spotted in a glass slide, which was connected at the end of the micromixer channel unit. When the mixed solution was injected into the disposable microarray chip, pneumatically actuated micropumping was executed to speed up the hybridization process by inducing the convective flow. The fluorescence signals on each spot were monitored by a miniaturized fluorescence scanner, and the Hanwoo was verified by detecting the number of fluorescent spots with three or fewer among eleven. An integrated portable PCR-microarray genetic analysis microsystem was first demonstrated for rapid, accurate, and on-site multiplex SNP typing to differentiate animal species.

An integrated allele-specific (AS) polymerase chain reaction (PCR) and capillary electrophoresis (CE) microdevice has been developed for multiplex single nucleotide polymorphism (SNP) genotyping on a portable instrumentation, which was applied for on-site identification of HANWOO (Korean indigenous beef cattle). Twelve sets of primers were designed for targeting beef cattle's eleven SNP loci for HANWOO verification and one primer set for a positive PCR control, and the success rate for identification of HANWOO was demonstrated statistically. The AS PCR and CE separation for multiplex SNP typing was carried out on a glass-based microchip consisting of four layers: a microchannel plate for microfluidic control, a Pt-electrode plate for a resistance temperature detector (RTD), a poly(dimethylsiloxane) (PDMS) membrane and a manifold glass for microvalve function. The operation of the sample loading, AS PCR, microvalve, and CE on a chip was automated with a portable genetic analyzer, and the laser-induced fluorescence detection was performed on a miniaturized fluorescence detector. The blind samples were correctly identified as a HANWOO by showing one or two amplicon peaks in the electropherogram, while the imported beef cattle revealed more than five peaks. Our genetic analysis platform provides rapid, accurate, and on-site multiplex SNP typing., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Objectives: Percutaneous fine-needle aspiration cytology or biopsy has been used for pathological confirmation in pancreatic cancer. Sometimes, it is difficult to approach the mass because of surrounding major vessels, and there is a risk of seeding. Although endoscopic retrograde cholangiopancreatography (ERCP)-guided pancreatic duct brush cytology is less invasive, its reliability is very low. Recently, aberrantly methylated genes were reported in pancreatic cancer tissue. This study was to develop a novel molecular diagnostic approach based on epigenetic characteristics., Methods: We enrolled pathologically proven 33 pancreatic cancer patients and 22 benign pancreaticobiliary disease patients. The ERCP-guided pancreatic duct brush cytology samples were obtained. Genomic DNA was extracted, and NPTX2 CpG island hypermethylation was examined quantitatively by real-time polymerase chain reaction amplification after chemical modification., Results: Pancreatic cancer cytology samples had statistically significant higher levels of NPTX2 methylation compared with benign diseases, and the optimal cutoff value of NPTX2 methylation was 1.2%. The sensitivity was 87%, and specificity was 80%, whereas pathological examination by ERCP-guided pancreatic duct brush cytology had a sensitivity of 38%., Conclusions: The quantitative analysis of NPTX2 hypermethylation may play a role in making highly sensitive and less invasive diagnosis of pancreatic cancer. Therefore, NPTX2 hypermethylation could be a promising molecular diagnostic marker.

Real-time TRAP assay was developed to improve the sensitivity and quantitative detection of telomerase activity in the body fluids of cancer patients. The sensitivity and clinical significance of the real-time TRAP assay was investigated. Real-time PCR protocol was modified by using ACX primer and SYBR green mixture from the process of TS primer extension. Real-time TRAP showed high correlation (r2=0.843, p=0.001) and sensitivity (25 times higher) compared to conventional TRAP. Of 164 samples, there were 8 positives in cytology (4.9%), 7 (4.3%) using the conventional TRAP, and 41 (25%) using real-time TRAP. In cytology positive samples, real-time TRAP showed a higher positivity than conventional TRAP (75% vs 63%) suggesting a higher sensitivity in the body fluids. There was a tendency towards a longer progression-free duration in telomerase negative patients than in positive patients, as determined by conventional and real-time TRAP. The diagnostic interval between the first positivity documentation and clinical progression was short in the order of real-time TRAP, conventional TRAP and cytology. In conclusion, real-time TRAP assay can detect telomerase activity at an earlier phase of cancer progression and can replace conventional TRAP assay for detecting the telomerase activity in body fluids.

Sensitive and accurate capture, enrichment, and identification of drug-resistant bacteria on human skin are important for early-stage diagnosis and treatment of patients. Herein, we constructed a three-dimensional hierarchically structured polyaniline nanoweb (3D HPN) to capture, enrich, and detect drug-resistant bacteria on-site by rubbing infected skins. These unique hierarchical nanostructures enhance bacteria capture efficiency and help severely deform the surface of the bacteria entrapped on them. Therefore, 3D HPN significantly contributes to the effective and reliable recovery of drug-resistant bacteria from the infected skin and the prevention of potential secondary infection. The recovered bacteria were successfully identified by subsequent real-time polymerase chain reaction (PCR) analysis after the lysis process. The molecular analysis results based on a real-time PCR exhibit excellent sensitivity to detecting target bacteria of concentrations ranging from 102 to 107 CFU/mL without any fluorescent signal interruption. To confirm the field applicability of 3D HPN, it was tested with a drug-resistant model consisting of micropig skin similar to human skin and Klebsiella pneumoniae carbapenemase-producing carbapenem-resistant Enterobacteriaceae (KPC-CRE). The results show that the detection sensitivity of this assay is 102 CFU/mL. Therefore, 3D HPN can be extended to on-site pathogen detection systems, along with rapid molecular diagnostics through a simple method, to recover KPC-CRE from the skin. [ABSTRACT FROM AUTHOR]

A fully integrated slidable and valveless microsystem, which performs solid phase DNA extraction (SPE), micro-polymerase chain reaction (μPCR) and micro-capillary electrophoresis (μCE) coupled with a portable genetic analyser, has been developed for forensic genotyping. The use of a slidable chip, in which a 1 μL-volume of the PCR chamber was patterned at the center, does not necessitate any microvalves and tubing systems for fluidic control. The functional micro-units of SPE, μPCR, and μCE were fabricated on a single glass wafer by conventional photolithography, and the integrated microdevice consists of three layers: from top to bottom, a slidable chip, a channel wafer in which a SPE chamber, a mixing microchannel, and a CE microchannel were fabricated, and a Ti/Pt resistance temperature detector (RTD) wafer. The channel glass wafer and the RTD glass wafer were thermally bonded, and the slidable chip was placed on the designated functional unit. The entire process from the DNA extraction using whole human blood sample to identification of target Y chromosomal short tandem repeat (STR) loci was serially carried out with simply sliding the slidable chamber from one to another functional unit. Monoplex and multiplex detection of amelogenin and mini Y STR loci were successfully analysed on the integrated slidable SPE-μPCR-μCE microdevice by using 1 μL whole human blood within 60 min. The proposed advanced genetic analysis microsystem is capable of point-of-care DNA testing with sample-in-answer-out capability, more importantly, without use of complicated microvalves and microtubing systems for liquid transfer. [ABSTRACT FROM AUTHOR]

Objectives Implant osteotomy yields a substantial amount of bone in the form of bone chips entrapped within drill flutes, and can provide a promising cell source for tissue engineering. The aims of this study were to isolate human alveolar bone-derived stromal cells ( hABCs) obtained during implant osteotomy, and to evaluate osteogenic differentiation capacity of hABCs. Material and methods Bone chips were obtained by minimally irrigated implant drilling technique from 10 human donors. Isolated cells were studied with respect to their colony-forming efficiency, surface marker expression by immunofluorescence staining, fluorescence-activated cell sorting analysis and self-renewal potency. To verify the differentiation activity, in vitro osteogenic and adipogenic gene expressions were evaluated by reverse transcription-polymerase chain reaction, and in vitro formation of mineralized nodule and adipocytes was also evaluated. In vivo bone-forming activity was assessed by ectopic transplantation in immunocompromised mice ( n = 5). Results Human alveolar bone-derived stromal cells population with characteristics of mesenchymal stem cells was present in the isolated cells. Upon hABC transplantation, significant ectopic bone formation was induced with the characteristics of fully matured bone tissue. Conclusion The data support the feasibility of using hABCs as a source of stem cells for dentoalveolar bone tissue reconstruction. The cell source has an advantage that the hABCs can be easily acquired during implant surgery. [ABSTRACT FROM AUTHOR]

Background and Aims: Every country has standardized reprocessing guidelines for reducing the risk of microorganism transmission via reusable biopsy forceps. Sterilization is performed either by autoclaving or with the use of ethylene oxide (EO) gas. However, there are no clear standard global recommendations. The aim of this study was to determine whether EO gas or autoclaving is a safer and more effective method for the sterilization of reusable forceps. Methods: This was a prospective study conducted at multiple tertiary referral centers. Seventy reusable biopsy forceps that had been reused at least 20 times each were collected from six endoscopy centers. In all, 61 forceps from five centers were sterilized using EO gas, and the nine forceps from the remaining center were placed in an autoclave. We performed real-time polymerase chain reaction (RT-PCR) for Mycobacterium tuberculosis and hepatitis B virus and performed bacterial cultures on the reusable forceps, which were cut into 2- to 3-cm sections. The forceps were also scanned with an electron microscope (EM) to detect surface damage and contamination. Results: Escherichia coli bacteria were cultured from 2 of the 61 (3.3%) reusable biopsy forceps sterilized with EO gas. On EM scanning, abundant debris and tissue materials remained on the cup surfaces of the reused biopsy forceps and on their inner wires. No microorganisms were found on the autoclaved forceps. Conclusions: Sterilization with EO gas may be inadequate because the complicated structure of the forceps may interfere with sterilization. Therefore, for optimum safety, reusable biopsy forceps should be sterilized by autoclaving. [ABSTRACT FROM AUTHOR]

Correction for ‘An integrated slidable and valveless microdevice with solid phase extraction, polymerase chain reaction, and immunochromatographic strip parts for multiplex colorimetric pathogen detection’ by Yong Tae Kim et al., Lab Chip, 2015, 15, 4148–4155. [ABSTRACT FROM AUTHOR]

Accurate analysis of digital polymerase chain reaction (dPCR) images is a key to the successful miniaturization of a dPCR system, which would allow point-of-care testing, but has been hampered by several issues, mainly out-of-focus images, and variations in shape, size, and fluorescence intensities of the droplets. Herein, we performed semantic segmentation by using a deep learning model combined with the circle Hough transform (CHT). For the deep learning model, Attention DeepLabV3+ was modified and used to analyze positive and negative droplets in the low-quality dPCR images, while CHT helps to count the droplets. The modified Attention DeepLabV3+ was trained with augmented 4224 dPCR images, which were sufficient for deep learning model training. As a proof of concept, encapsulated genomic DNA (gDNA) of Escherichia coli O157:H7, a foodborne pathogen, was amplified, and the corresponding droplets were analyzed with the deep learning model to demonstrate its rapid, absolute quantification and accurate identification of the target gDNA. Our analysis method achieved similar accuracy to the commercial, bulky dPCR system, which is based on continuous-flow analysis methods. Furthermore, the deep learning-based approach exhibited a great sensitivity for detecting pathogenic bacteria from low-quality images with a low detection limit of 4.0 × 101 copies/μL. Compared with the commercially available instrument, the deep learning method demonstrated a similar molecular analysis performance. The presented results may lay the ground for efficient absolute quantification of target nucleic acids from the dPCR images as well as the establishment of a point-of-care analysis platform. • A deep learning-based accurate image analysis was proposed to apply in digital polymerase chain reactions. • The deep neural network-based semantic segmentation model was optimized to analyze unfocused images. • Infectious pathogenic bacteria were selected as a realistic model to demonstrate a accurate and absolute quantification. • The deep learning analysis shows high analysis accuracy similar to that of commercially available dPCR systems. [ABSTRACT FROM AUTHOR]