Rapid detection of Salmonella enterica in primary production samples by eliminating DNA amplification inhibitors using an improved sample pre-treatment method.

Sensitive detection of pathogens in livestock farms is an integral part of the One Health Action Plan of the European Union (EU). Ensuring this requires on-site testing devices that are compatible with complex matrices such as primary production samples. Among all, faeces are considered the most challenging matrix type that makes it difficult to identify pathogens because of complexity in sample preparation for molecular testing. We have developed a loop-mediated isothermal amplification (LAMP) based veterinary point-of-care (POC) device (VETPOD) and adapted it to detect Salmonella enterica in primary production samples. Three different sampling methods (semi-wet chicken faeces, boot socks collection and dust samples from poultry shed) were iteratively tested to assess their nature of complexity and possibility for adapting them as suitable sampling methods for on-site testing. During the study, the sample preparation method that included a two-step centrifugation combined with washing of the enriched Salmonella cells was found crucial in eliminating amplification inhibitors originating from the faecal matrices. A total of 90 samples were tested that included 60 samples for sensitivity study and 30 samples for relative level of detection (RLOD, a level of detection in comparison to ISO 6579:1 reference method). Overall, the VETPOD had a sensitivity of 90%, 84.62% and 81.82% for boot sock, faecal and dust samples, respectively. The RLOD was 2.23 CFU/25 g which was found to be 1.33 times higher than the ISO 6579:1. Performing with an excellent agreement with ISO 6579:1, the VETPOD proved as a promising alternative to detect Salmonella spp. in primary production and animal husbandry samples.

Validation of Point-of-Care Device for Rapid Detection of Salmonella enterica in Meat Products.

Accurate and rapid detection of pathogens in foods of animal origin has been a critical part of the One Health Action Plan of the European Union (EU). Biosensors have the potential in bringing required technologies to accomplish this on the field, wherein loop-mediated isothermal amplification (LAMP) and lab-on-a-chip have proven to be ideal. We have developed a LAMP-based point-of-care (POC) device, the VETPOD, as a solution to the contemporary challenges in the rapid detection of Salmonella spp. The core technology in the VETPOD is a ready-to-use cartridge that included an injection-molded polymer chip with pyramid-shaped optical structures embedded within the chip. These pyramid-shaped optical structures direct the incident light, due to total internal reflection (TIR), through the reaction chambers to the phototransistor. The VETPOD was validated against the ISO 6579-1 reference method. A total of 310 samples were tested that included 180 Salmonella spiked samples in 6 different meat categories and 130 strains to determine the specificity. The overall results were satisfactory, wherein the VETPOD had an acceptable sensitivity (96.51%) compared to the reference (98.81%) and near perfect agreement with ISO 6579-1 with an overall Cohen's kappa of 0.94. The relative level of detection (RLOD) for the VETPOD was 1.38 CFU/25 g that was found to be 1.17 times higher than the reference. The VETPOD showed 98% precision for inclusivity and 100% precision for the exclusivity samples. The VETPOD proved as a useful alternative to detect Salmonella spp. that can be adaptable to a broader spectrum of pathogens in future.

PATHPOD - A loop-mediated isothermal amplification (LAMP)-based point-of-care system for rapid clinical detection of SARS-CoV-2 in hospitals in Denmark.

Sensitive and rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a vital goal in the ongoing COVID-19 pandemic. We present in this comprehensive work, for the first time, detailed fabrication and clinical validation of a point of care (PoC) device for rapid, onsite detection of SARS-CoV-2 using a real-time reverse-transcription loop-mediated isothermal amplification (RT-LAMP) reaction on a polymer cartridge. The PoC system, namely PATHPOD, consisting of a standalone device (weight less than 1.2 kg) and a cartridge, can perform the detection of 10 different samples and two controls in less than 50 min, which is much more rapid than the golden standard real-time reverse-transcription Polymerase Chain Reaction (RT-PCR), typically taking 16-48 h. The novel total internal reflection (TIR) scheme and the reactions inside the cartridge in the PoC device allow monitoring of the diagnostic results in real-time and onsite. The analytical sensitivity and specificity of the PoC test are comparable with the current RT-PCR, with a limit of detection (LOD) down to 30-50 viral genome copies. The robustness of the PATHPOD PoC system has been confirmed by analyzing 398 clinical samples initially examined in two hospitals in Denmark. The clinical sensitivity and specificity of these tests are discussed.

Multiplexed Detection of Pathogens Using Solid-Phase Loop-Mediated Isothermal Amplification on a Supercritical Angle Fluorescence Array for Point-of-Care Applications.

Adaptations of new generation molecular techniques for multiplexed detection of pathogens are gaining interest in the field of point-of-care (POC) industry and onsite testing. Loop-mediated isothermal amplification (LAMP), an advanced molecular amplification technique, has proven promising due to its unique features that suits ideal for POC applications. However, application of LAMP for multiplexed detection of pathogens remains challenging because of the difficulty in the identification of specific LAMP amplicons that does not have a well-definite molecular size. In this study, we developed a solid-phase loop-mediated isothermal amplification (SP-LAMP) technique to address the challenge. Integration of LAMP with the supercritical angle fluorescence (SAF) micro-optic structures as a solid support (SS) in an array format enabled spatial separation of LAMP amplicons in a multiplexed configuration. Important parameters such as length of the SS primers, length of the primer-binding region, the effect of surface density of immobilized SS primers, and cross-reactivity among the primers of different targets were iteratively tested and optimized. With the combination of SP-LAMP and SAF techniques, it was possible to detect multiple pathogens that include Salmonella spp, Campylobater spp., Campylobacter coli, Campylobacter jejuni, avian influenza virus (AIV), and pan avian internal control (IC) under singleplex conditions. The multiplexing capacity of the SP-LAMP was demonstrated using AIV and IC with promising results. The success of SP-LAMP has opened a promising direction toward the development of a multiplex POC system for rapid detection of multiple pathogens.

Point-of-care system for rapid real-time detection of SARS-CoV-2 virus based on commercially available Arduino platforms.

The COVID-19 pandemic emphasized the importance of rapid, portable, and on-site testing technologies necessary for resource-limited settings for effective testing and screening to reduce spreading of the infection. Realizing this, we developed a fluorescence-based point-of-care (fPOC) detection system with real-time reverse transcriptase loop-mediated isothermal amplification for rapid and quantitative detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. The system is built based on the Arduino platform compatible with commercially available open-source hardware-software and off-the-shelf electronic components. The fPOC system comprises of three main components: 1) an instrument with integrated heaters, 2) optical detection components, and 3) an injection-molded polymeric cartridge. The system was tested and experimentally proved to be able to use for fast detection of the SARS-CoV-2 virus in real-time in less than 30 min. Preliminary results of testing the performance of the fPOC revealed that the fPOC could detect the SARS-CoV-2 virus at a limit of detection (LOD50%) at two to three copies/microliter (15.36 copies/reaction), which was comparable to reactions run on a standard commercial thermocycler. The performance of the fPOC was evaluated with 12 SARS-CoV-2 clinical throat swab samples that included seven positive and five negative samples, as confirmed by reverse transcription-polymerase chain reaction. The fPOC showed 100% agreement with the commercial thermocycler. This simple design of the fPOC system demonstrates the potential to greatly enhance the practical applicability to develop a totally integrated point-of-care system for rapid on-site screening of the SARS-CoV-2 virus in the management of the pandemic.

Elimination of Carryover Contamination in Real-Time Reverse Transcriptase Loop-Mediated Isothermal Amplification for Rapid Detection of the SARS-CoV-2 Virus in Point-of-Care Testing.

Loop-mediated isothermal amplification (LAMP) is being used as a robust rapid diagnostic tool to prevent the transmission of infectious diseases. However, carryover contamination of LAMP-amplified products originating from previous tests has been a problem in LAMP-based bio-analytical assays. In this study, we developed a Cod-uracil-DNA-glycosylase real-time reverse transcriptase LAMP assay (Cod-UNG-rRT-LAMP) for the elimination of carryover contamination and the rapid detection of SARS-CoV-2 in point-of-care (POC) testing. Using the Cod-UNG-rRT-LAMP assay, the SARS-CoV-2 virus could be detected as low as 2 copies/µl (8 copies/reaction) within 45 min of amplification and 2.63 ± 0.17 pg (equivalent to 2.296 × 109 copies) of contaminants per reaction could be eliminated. Analysis of clinical SARS-CoV-2 samples using the Cod-UNG-rRT-LAMP assay showed an excellent agreement with a relative accuracy of 98.2%, sensitivity of 97.1%, and specificity of 95.2% in comparison to rRT-PCR. The results obtained in this study clearly demonstrate the feasibility of the use of the Cod-UNG-rRT-LAMP assay for applications toward the POC diagnosis of SARS-CoV-2 and on-site testing of other pathogens.

Development of Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Rapid and On-Site Detection of Avian Influenza Virus.

Avian influenza virus (AIV) outbreaks occur frequently worldwide, causing a potential public health risk and great economic losses to poultry industries. Considering the high mutation rate and frequent genetic reassortment between segments in the genome of AIVs, emerging new strains are a real threat that may infect and spread through the human population, causing a pandemic. Therefore, rapid AIV diagnostic tests are essential tools for surveillance and assessing virus spreading. Real-time reverse transcription PCR (rRT-PCR), targeting the matrix gene, is the main official standard test for AIV detection, but the method requires well-equipped laboratories. Reverse transcription Loop-Mediated Isothermal Amplification (RT-LAMP) has been reported as a rapid method and an alternative to PCR in pathogen detection. The high mutation rate in the AIV genome increases the risk of false negative in nucleic acid amplification methods for detection, such as PCR and LAMP, due to possible mismatched priming. In this study, we analyzed 800 matrix gene sequences of newly isolated AIV in the EU and designed a highly efficient LAMP primer set that covers all AIV subtypes. The designed LAMP primer set was optimized in real-time RT-LAMP (rRT-LAMP) assay. The rRT-LAMP assay detected AIV samples belonging to nine various subtypes with the specificity and sensitivity comparable to the official standard rRT-PCR assay. Further, a two-color visual detection RT-LAMP assay protocol was adapted with the aim to develop on-site diagnostic tests. The on-site testing successfully detected spiked AIV in birds oropharyngeal and cloacal swabs samples at a concentration as low as 100.8 EID50 per reaction within 30 minutes including sample preparation. The results revealed a potential of this newly developed rRT-LAMP assay to detect AIV in complex samples using a simple heat treatment step without the need for RNA extraction.

A Sensitive, Specific and Simple Loop Mediated Isothermal Amplification Method for Rapid Detection of Campylobacter spp. in Broiler Production.

Campylobacteriosis is one of the most common foodborne diseases worldwide. Two Campylobacter species - C. jejuni and C. coli in poultry and poultry products are considered to be the main source of human campylobacteriosis. Therefore, studying Campylobacter status in poultry flocks is needed to prevent transmission of disease and reduce human risk, health cost, and economic losses. In this study, we adapted and used a Loop-Mediated Isothermal Amplification (LAMP) assay for specific, sensitive, simple and cost-effective rapid detection of C. jejuni and C. coli in the poultry production chain. Amplified LAMP products were detected using a small, low-cost portable commercial blue LED transilluminator and a direct visual detection strategy was demonstrated. By using optimized conditions for amplification a limit of detection (LOD) of 50 CFU/ml was achieved for testing of C. jejuni and C. coli in spiked chicken feces without enrichment. The method took 60-70 min from receiving the samples to the final results (including 30 min for amplification). The optimized LAMP showed a relative accuracy of 98.4%, a specificity of 97.9%, and a sensitivity of 100% in comparison to real-time PCR method. Cohen's kappa index also showed an excellent agreement (0.94) between the two methods. The results showed that the method is specific, sensitive and is suitable to develop for rapid detection of Campylobacter spp. at poultry production.

Classification of Multiple DNA Dyes Based on Inhibition Effects on Real-Time Loop-Mediated Isothermal Amplification (LAMP): Prospect for Point of Care Setting.

LAMP has received great interest and is widely utilized in life sciences for nucleic acid analysis. To monitor a real-time LAMP assay, a fluorescence DNA dye is an indispensable component and therefore the selection of a suitable dye for real-time LAMP is a need. To aid this selection, we investigated the inhibition effects of twenty-three DNA dyes on real-time LAMP. Threshold time (Tt) values of each real-time LAMP were determined and used as an indicator of the inhibition effect. Based on the inhibition effects, the dyes were classified into four groups: (1) non-inhibition effect, (2) medium inhibition effect, (3) high inhibition effect, and (4) very high inhibition effect. The signal to noise ratio (SNR) and the limit of detection (LOD) of the dyes in groups 1, 2, and 3 were further investigated, and possible inhibition mechanisms of the DNA dyes on the real-time LAMP are suggested and discussed. Furthermore, a comparison of SYTO 9 in different LAMP reactions and different systems is presented. Of the 23 dyes tested, SYTO 9, SYTO 82, SYTO 16, SYTO 13, and Miami Yellow were the best dyes with no inhibitory effect, low LOD and high SNR in the real-time LAMP reactions. The present classification of the dyes will simplify the selection of fluorescence dye for real-time LAMP assays in point of care setting.

The Use of a DNA-Intercalating Dye for Quantitative Detection of Viable Arcobacter spp. Cells (v-qPCR) in Shellfish.

The genus Arcobacter (Vandamme et al., 1991), comprised of Campylobacter-related species, are considered zoonotic emergent pathogens. The presence of Arcobacter in food products like shellfish, has an elevated incidence worldwide. In this study, we developed a specific viable quantitative PCR (v-qPCR), using the dye propidium monoazide (PMA), for quantification of the viable Arcobacter spp. cells in raw oysters and mussels. The high selectivity of primers was demonstrated by using purified DNA from 38 different species, 20 of them from the genus Arcobacter. The optimization of PMA concentration showed that 20 µM was considered as an optimal concentration that inhibits the signal from dead cells at different concentrations (OD550 from 0.2 to 0.8) and at different ratios of live: dead cells (50:50 and 90:10). The v-qPCR results from shellfish samples were compared with those obtained in parallel using several culture isolation approaches (i.e., direct plating on marine and blood agar and by post-enrichment culturing in both media). The enrichment was performed in parallel in Arcobacter-CAT broth with and without adding NaCl. Additionally, the v-qPCR results were compared to those obtained with traditional quantitative (qPCR). The v-qPCR and the qPCR resulted in c.a. 94% of positive detection of Arcobacter vs. 41% obtained by culture approaches. When examining the reduction effect resulting from the use of v-qPCR, samples pre-enriched in Arcobacter-CAT broth supplemented with 2.5% NaCl showed a higher reduction (3.27 log copies) than that of samples obtained directly and those pre-enriched in Arcobacter-CAT broth isolation (1.05 and 1.04). When the v-qPCR was applied to detect arcobacter from real shellfish samples, 15/17 samples tested positive for viable Arcobacter with 3.41 to 8.70 log copies 1g-1. This study offers a new tool for Arcobacter surveillance in seafood.

MicroRNA amplification and detection technologies: opportunities and challenges for point of care diagnostics.

The volume of point of care (POC) testing continues to grow steadily due to the increased availability of easy-to-use devices, thus making it possible to deliver less costly care closer to the patient site in a shorter time relative to the central laboratory services. A novel class of molecules called microRNAs have recently gained attention in healthcare management for their potential as biomarkers for human diseases. The increasing interest of miRNAs in clinical practice has led to an unmet need for assays that can rapidly and accurately measure miRNAs at the POC. However, the most widely used methods for analyzing miRNAs, including Northern blot-based platforms, in situ hybridization, reverse transcription qPCR, microarray, and next-generation sequencing, are still far from being used as ideal POC diagnostic tools, due to considerable time, expertize required for sample preparation, and in terms of miniaturizations making them suitable platforms for centralized labs. In this review, we highlight various existing and upcoming technologies for miRNA amplification and detection with a particular emphasis on the POC testing industries. The review summarizes different miRNA targets and signals amplification-based assays, from conventional methods to alternative technologies, such as isothermal amplification, paper-based, oligonucleotide-templated reaction, nanobead-based, electrochemical signaling- based, and microfluidic chip-based strategies. Based on critical analysis of these technologies, the possibilities and feasibilities for further development of POC testing for miRNA diagnostics are addressed and discussed.

Microfluidic devices for sample preparation and rapid detection of foodborne pathogens.

Rapid detection of foodborne pathogens at an early stage is imperative for preventing the outbreak of foodborne diseases, known as serious threats to human health. Conventional bacterial culturing methods for foodborne pathogen detection are time consuming, laborious, and with poor pathogen diagnosis competences. This has prompted researchers to call the current status of detection approaches into question and leverage new technologies for superior pathogen sensing outcomes. Novel strategies mainly rely on incorporating all the steps from sample preparation to detection in miniaturized devices for online monitoring of pathogens with high accuracy and sensitivity in a time-saving and cost effective manner. Lab on chip is a blooming area in diagnosis, which exploits different mechanical and biological techniques to detect very low concentrations of pathogens in food samples. This is achieved through streamlining the sample handling and concentrating procedures, which will subsequently reduce human errors and enhance the accuracy of the sensing methods. Integration of sample preparation techniques into these devices can effectively minimize the impact of complex food matrix on pathogen diagnosis and improve the limit of detections. Integration of pathogen capturing bio-receptors on microfluidic devices is a crucial step, which can facilitate recognition abilities in harsh chemical and physical conditions, offering a great commercial benefit to the food-manufacturing sector. This article reviews recent advances in current state-of-the-art of sample preparation and concentration from food matrices with focus on bacterial capturing methods and sensing technologies, along with their advantages and limitations when integrated into microfluidic devices for online rapid detection of pathogens in foods and food production line.

Solid-phase PCR for rapid multiplex detection of Salmonella spp. at the subspecies level, with amplification efficiency comparable to conventional PCR.

Solid-phase PCR (SP-PCR) has attracted considerable interest in different research fields since it allows parallel DNA amplification on the surface of a solid substrate. However, the applications of SP-PCR have been hampered by the low efficiency of the solid-phase amplification. In order to increase the yield of the solid-phase amplification, we studied various parameters including the length, the density, as well as the annealing position of the solid support primer. A dramatic increase in the signal-to-noise (S/N) ratio was observed when increasing the length of solid support primers from 45 to 80 bp. The density of the primer on the surface was found to be important for the S/N ratio of the SP-PCR, and the optimal S/N was obtained with a density of 1.49 × 1011 molecules/mm2. In addition, the use of solid support primers with a short overhang at the 5' end would help improve the S/N ratio of the SP-PCR. With optimized conditions, SP-PCR can achieve amplification efficiency comparable to conventional PCR, with a limit of detection of 1.5 copies/µl (37.5 copies/reaction). These improvements will pave the way for wider applications of SP-PCR in various fields such as clinical diagnosis, high-throughput DNA sequencing, and single-nucleotide polymorphism analysis. Graphical abstract Schematic representation of solid-phase PCR.

Molecularly imprinted polymers for sample preparation and biosensing in food analysis: Progress and perspectives.

Molecularly imprinted polymers (MIPs) are biomimetics which can selectively bind to analytes of interest. One of the most interesting areas where MIPs have shown the biggest potential is food analysis. MIPs have found use as sorbents in sample preparation attributed to the high selectivity and high loading capacity. MIPs have been intensively employed in classical solid-phase extraction and solid-phase microextraction. More recently, MIPs have been combined with magnetic bead extraction, which greatly simplifies sample handling procedures. Studies have consistently shown that MIPs can effectively minimize complex food matrix effects, and improve recoveries and detection limits. In addition to sample preparation, MIPs have also been viewed as promising alternatives to bio-receptors due to the inherent molecular recognition abilities and the high stability in harsh chemical and physical conditions. MIPs have been utilized as receptors in biosensing platforms such as electrochemical, optical and mass biosensors to detect various analytes in food. In this review, we will discuss the current state-of-the-art of MIP synthesis and applications in the context of food analysis. We will highlight the imprinting methods which are applicable for imprinting food templates, summarize the recent progress in using MIPs for preparing and analysing food samples, and discuss the current limitations in the commercialisation of MIPs technology. Finally, future perspectives will be given.

A novel lab-on-chip platform with integrated solid phase PCR and Supercritical Angle Fluorescence (SAF) microlens array for highly sensitive and multiplexed pathogen detection.

Solid-phase PCR (SP-PCR) has become increasingly popular for molecular diagnosis and there have been a few attempts to incorporate SP-PCR into lab-on-a-chip (LOC) devices. However, their applicability for on-line diagnosis is hindered by the lack of sensitive and portable on-chip optical detection technology. In this paper, we addressed this challenge by combining the SP-PCR with super critical angle fluorescence (SAF) microlens array embedded in a microchip. We fabricated miniaturized SAF microlens array as part of a microfluidic chamber in thermoplastic material and performed multiplexed SP-PCR directly on top of the SAF microlens array. Attribute to the high fluorescence collection efficiency of the SAF microlens array, the SP-PCR assay on the LOC platform demonstrated a high sensitivity of 1.6 copies/µL, comparable to off-chip detection using conventional laser scanner. The combination of SP-PCR and SAF microlens array allows for on-chip highly sensitive and multiplexed pathogen detection with low-cost and compact optical components. The LOC platform would be widely used as a high-throughput biosensor to analyze food, clinical and environmental samples.

Direct PCR - A rapid method for multiplexed detection of different serotypes of Salmonella in enriched pork meat samples.

Salmonellosis, an infectious disease caused by Salmonella spp., is one of the most common foodborne diseases. Isolation and identification of Salmonella by conventional bacterial culture method is time consuming. In response to the demand for rapid on line or at site detection of pathogens, in this study, we developed a multiplex Direct PCR method for rapid detection of different Salmonella serotypes directly from pork meat samples without any DNA purification steps. An inhibitor-resistant Phusion Pfu DNA polymerase was used to overcome PCR inhibition. Four pairs of primers including a pair of newly designed primers targeting Salmonella spp. at subtype level were incorporated in the multiplex Direct PCR. To maximize the efficiency of the Direct PCR, the ratio between sample and dilution buffer was optimized. The sensitivity and specificity of the multiplex Direct PCR were tested using naturally contaminated pork meat samples for detecting and subtyping of Salmonella spp. Conventional bacterial culture methods were used as reference to evaluate the performance of the multiplex Direct PCR. Relative accuracy, sensitivity and specificity of 98.8%; 97.6% and 100%, respectively, were achieved by the method. Application of the multiplex Direct PCR to detect Salmonella in pork meat at slaughter reduces the time of detection from 5 to 6 days by conventional bacterial culture and serotyping methods to 14 h (including 12 h enrichment time). Furthermore, the method poses a possibility of miniaturization and integration into a point-of-need Lab-on-a-chip system for rapid online pathogen detection.

Miniaturization of a micro-optics array for highly sensitive and parallel detection on an injection moulded lab-on-a-chip.

A miniaturised array of supercritical angle fluorescence (SAF) micro-optics embedded in a microfluidic chamber was fabricated by injection moulding. The fabricated chip could enhance the fluorescence signal around 46 times compared to a conventional microscope. Collection of the fluorescence signal from the SAF array is almost independent of the numerical aperture, and the limit of detection was improved 36-fold using a simple and inexpensive optical detection system.

A lab-on-a-chip system with integrated sample preparation and loop-mediated isothermal amplification for rapid and quantitative detection of Salmonella spp. in food samples.

Foodborne disease is a major public health threat worldwide. Salmonellosis, an infectious disease caused by Salmonella spp., is one of the most common foodborne diseases. Isolation and identification of Salmonella by conventional bacterial culture or molecular-based methods are time consuming and usually take a few hours to days to complete. In response to the demand for rapid on line or on site detection of pathogens, in this study, we describe for the first time an eight-chamber lab-on-a-chip (LOC) system with integrated magnetic bead-based sample preparation and loop-mediated isothermal amplification (LAMP) for rapid and quantitative detection of Salmonella spp. in food samples. The whole diagnostic procedures including DNA isolation, isothermal amplification, and real-time detection were accomplished in a single chamber. Up to eight samples could be handled simultaneously and the system was capable to detect Salmonella at concentration of 50 cells per test within 40 min. The simple design, together with high level of integration, isothermal amplification, and quantitative analysis of multiple samples in short time, will greatly enhance the practical applicability of the LOC system for rapid on-site screening of Salmonella for applications in food safety control, environmental surveillance, and clinical diagnostics.

An oligonucleotide-tagged microarray for routine diagnostics of colon cancer by genotyping KRAS mutations.

Colorectal cancer (CRC) is one of the most prevalent types of cancer, causing significant morbidity and mortality worldwide. CRC is curable if diagnosed at an early stage. Mutations in the oncogene KRAS play a critical role in early development of CRC. Detection of activated KRAS is of diagnostic and therapeutic importance. In this study, KRAS gene fragments containing mutations in codon 12 were amplified by multiplex PCR using a 5'-Cy5-labeled reverse primer in combination with 3'-mutation-specific forward primers that were linked with four unique nucleotide-sequence tags at the 5'-end. The Cy5-labeled reverse primer was extended under PCR amplification to the 5'-end of the mutation-specific forward primers and thus included the complimentary sequence of the tag. PCR products were hybridized to tag-probes immobilized on various substrates and detected by a scanner. Our results indicate that all mutations at codon 12 of KRAS derived from cancer cells and clinical samples could be unambiguously detected. KRAS mutations were accurately detected when the mutant DNA was present only in 10% of the starting mixed materials including wild-type genomic DNA, which was isolated from either cancer cells or spiked fecal samples. The immobilized tag-probes were stable under multiple thermal cycling treatments, allowing re-use of the tag-microarray and further optimization to solid PCR. Our results demonstrated that a novel oligonucleotide-tagged microarray system has been developed which would be suitable to be used for detection of KRAS mutations and clinical diagnosis of CRC.

Pre-storage of gelified reagents in a lab-on-a-foil system for rapid nucleic acid analysis.

Reagent pre-storage in a microfluidic chip can enhance operator convenience, simplify the system design, reduce the cost of storage and shipment, and avoid the risk of cross-contamination. Although dry reagents have long been used in lateral flow immunoassays, they have rarely been used for nucleic acid-based point-of-care (POC) assays due to the lack of reliable techniques to dehydrate and store fragile molecules involved in the reaction. In this study, we describe a simple and efficient method for prolonged on-chip storage of PCR reagents. The method is based on gelification of all reagents required for PCR as a ready-to-use product. The approach was successfully implemented in a lab-on-a-foil system, and the gelification process was automated for mass production. Integration of reagents on-chip by gelification greatly facilitated the development of easy-to-use lab-on-a-chip (LOC) devices for fast and cost-effective POC analysis.