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Digital LAMP on a Commercial Membrane
摘要： In this work, we report digital loop-mediated isothermal amplification (LAMP) or reverse-transcription LAMP (RT-LAMP) on a commercial membrane, without the need for complex chip fabrication or use of specialized equipment. Due to the pore size distribution, the theoretical error for digital LAMP on these membranes was analyzed, using a combination of Random Distribution Model and Multi-volume Theory. A facile peel-off process was developed for effective droplets formation on the commercial track-etched polycarbonate (PCTE) membrane. Each pore functions as an individual nanoreactor for single DNA amplification. Absolute quantification of bacteria genomic DNA was realized with a dynamic range from 11 to 1.1 × 10^5 copies/μL. One-step digital RT-LAMP was also successfully performed on the membrane for the quantification of MS2 virus in wastewater. With the introduction of new probes, the positive pores can be easily distinguished from negative ones with 100 times difference in fluorescence intensities. Finally, the cost of a disposable membrane is less than $0.1/piece, which, to the best of our knowledge, is the most inexpensive way to perform digital LAMP. The membrane system offers opportunities for point-of-care users or common laboratories to perform digital quantification, single cell analysis, or other bioassays in an inexpensive, flexible and simplified way.
关键词： Digital LAMP,RT-LAMP,PCR,Droplets,Virus,Microfluidic,Membrane,Nucleic acid
更新于2025-09-23 15:23:52
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Assessing UV Inactivation of Adenovirus 41 Using Integrated Cell Culture Real-Time qPCR/RT-qPCR
摘要： Enteric adenoviruses are among most UV-resistant viruses in water. Cytopathic effects (CPE)-based cell culture TCID50 assay as a conventional virus assessment approach has major drawbacks for enteric adenovirus since it is selective on cell lines and takes longer time to show CPE. Integrated cell culture real-time quantitative PCR (ICC-qPCR) and reverse transcriptase (RT)-qPCR were applied in this study, in comparison with TCID50, to assess UV inactivation of adenovirus type 41 (Ad41) in water. Adenovirus type 41 was exposed to UV doses of 40, 80, 160, and 320 mJ/cm2 using a collimated beam apparatus. There was no significant difference of inactivation at conducted UV doses between measurements using TCID50 assay and ICC-RT-qPCR. Both assays fitted the Chick–Watson model at 95% confidence level. The inactivation measured by ICC-qPCR did not fit the Chick–Watson model. In summary, ICC-RT-qPCR is the most appropriate alternate to CPE-based assay for assessing UV inactivation of enteric adenoviruses. Water Environ. Res., 89, 323 (2017).
关键词： cytopathic effects,cell culture,real-time quantitative PCR/RT-PCR,adenovirus type 41,water,UV inactivation
更新于2025-09-23 15:22:29
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Fluorescence detection test by black printed circuit board based microfluidic channel for polymerase chain reaction
摘要： This paper proposes the optimal structure of a PCB-based micro PCR chip constructed on a PCB substrate using commercial adhesive tapes and plastic covers. The solder mask of the PCB substrate was coated black, and the area where the reaction chamber is attached was legend printed with white silk to minimize the noise during fluorescence detection. The performance of the PCR and fluorescence detection was compared using 6 types of reaction chambers, each made with different double-sided tapes. Three of the chambers were unsuccessful in completing the PCR. The performance of the other three chambers that successfully amplified DNA was compared using Taqman probe for Chlamydia Trachomatis DNA. The amplified product was illuminated diagonally with a blue LED to excite the product just before imaging, and the LED was turned off when the image was captured to prevent quenching of the probe. The images were taken 10 seconds prior to the last extension step for each cycle using a DSLR camera. The experiments were run as a quartet for each three chambers made with different double-sided tape. The results showed that there were significant difference between the three tapes.
关键词： microfluidic channel,polymerase chain reaction,fluorescence detection test,acrylic adhesive,micro-PCR chip,black PCB,double-sided tape
更新于2025-09-23 15:22:29
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Determination of Effective Dose of Ultraviolet Irradiation to Influent Water for Prevention of <i>Kudoa yasunagai</i> Infection
摘要： We determined the minimum ultraviolet (UV) irradiation dose to influent water to prevent Kudoa yasunagai infection, using microscopy and qPCR. The infection prevalence in Seriola lalandi reared in untreated water reached 45% while that in fish reared in UV-treated water at 5 mJ/cm2 remained below 10%. Additionally, 5 mJ/cm2 UV irradiation significantly reduced spore formation in the brain. No infection was detected when the water was treated with UV at the doses 15 and 30 mJ/cm2. These results indicate that K. yasunagai actinospores are relatively vulnerable to UV irradiation and the minimum effective dose lies between 5 and 15 mJ/cm2.
关键词： quantitative PCR,ultraviolet irradiation,Seriola lalandi,prevention,Kudoa yasunagai,hatchery
更新于2025-09-23 15:21:21
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Biosensing Technologies for the Detection of Pathogens - A Prospective Way for Rapid Analysis || Development of HRPzyme-Integrated PCR Platform for Colorimetric Detection of Foodborne Pathogens
摘要： In recent years, foodborne illnesses have become the most significant public health issue in both developed and developing countries. The World Health Organization (WHO) reported that in 2010, around 1.8 million people died due to foodborne illness. Therefore, the development of a cost-effective, sensitive, and selective detection method for identifying and monitoring foodborne pathogens is necessary for improved public health. Here, we describe a simple and ultrasensitive colorimetric method for the detection of foodborne pathogens based on HRPzyme-integrated PCR using PC-based ImageJ software. We present insights into different aspects of this method such as the importance of 16S rRNA detection, the modification of traditional PCR primers with a unique functional sequence for generating a color signal, and the application of ImageJ in colorimetric image data acquisition. The performance of the proposed strategy in detecting various foodborne pathogens is comparable to that of the commercial UV-Vis spectrophotometer Tecan Infinite 200 Pro. This detection platform exhibits linearity over wide range, high sensitivity, and high selectivity. The diagnostic capability of this colorimetric system to detect foodborne pathogens was demonstrated with spiked fruit and vegetable samples. This low-cost and effective colorimetric method can be conveniently employed for the analysis of DNA sequences arising from pathogenic bacteria.
关键词： PCR,HRPzyme,primer,16S rRNA,colorimetric detection,foodborne pathogens
更新于2025-09-23 15:21:21
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Sensitive and Direct DNA Mutation Detection by Surface-enhanced Raman Spectroscopy using Rational Designed and Tunable Plasmonic Nanostructures
摘要： Efficient DNA mutation-detection methods are required for diagnosis, personalized therapy development, and prognosis assessment for diseases such as cancer. To address this issue, we proposed a straightforward approach by combining active plasmonic nanostructures, surface enhanced Raman spectroscopy (SERS), and polymerase chain reaction (PCR), with a statistical tool to identify and classify BRAF wild type (WT) and V600E mutant genes. The nanostructures provide enhanced sensitivity, while PCR offers the high specificity towards target DNA. A series of positively charged plasmonic nanostructures including gold/silver nanospheres, nanoshells, nanoflowers and nanostars, were synthesized with a one-pot strategy and characterized. By changing the shape of nanostructures, we are able to vary the surface plasmon resonance from 551 nm to 693 nm. The gold/silver nanostar showed the highest SERS activity, which was employed for DNA mutation detection. We reproducibly analyzed as few as 100 copies of target DNA sequences using gold/silver nanostars, thus demonstrating the high sensitivity of the direct SERS detection. By means of statistical analysis (principal component analysis-linear discriminant analysis, PCA-LDA), this method was successfully applied to differentiate the WT and V600E mutant both from whole genome DNA (gDNA) lysed from cell line and from cell-free DNA (cfDNA) collected from cell culture media. We further proved that this assay is capable of specifically amplifying and accurately classifying a real plasma sample. Thus, this direct SERS strategy combined with the active plasmonic nanostructures has the potential for wide applications as an alternative tool for sensitively monitoring and evaluating clinical important nucleotide biomarkers.
关键词： PCR,statistical analysis,plasmonic nanostructures,surface-enhanced Raman spectroscopy,DNA mutation detection
更新于2025-09-23 15:21:01
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[IEEE 2019 Compound Semiconductor Week (CSW) - Nara, Japan (2019.5.19-2019.5.23)] 2019 Compound Semiconductor Week (CSW) - Formation and Characterization of Si Quantum Dots with Ge Core for Electroluminescent Devices
摘要： We report on a distributed circuit model for multi-color light-actuated optoelectrowetting devices. The model takes into consideration the large variation of absorption coefficient (15×) of photoconductors in the visible spectrum and the nonuniform distribution of photogenerated carriers. With the help of this model, we designed opto-electrowetting devices with optimum thickness of photoconductors. This leads to significant improvement in performance compared with prior reports, including 200× lower optical power, 5× lower voltage, and 20× faster droplet moving speed. This enables the use of commercial projectors to create on-demand “virtual” electrodes for large-scale parallel manipulation of droplets. We have achieved simultaneous manipulation of 96-droplet array. Finally, we have demonstrated parallel on chip detection of Herpes Simplex Virus Type 1 within 45 min using a real-time isothermal polymerase chain reaction assay.
关键词： electrowetting,polymerase chain reaction (PCR),optoelectrowetting,light-actuated digital microfluidics,Droplet microfluidics
更新于2025-09-23 15:19:57
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A strategy for preparing non-fluorescent graphene oxide quantum dots as fluorescence quenchers in quantitative real-time PCR
摘要： In recent years, graphene oxide quantum dots (GOQDs) have emerged as novel nanomaterials for optical sensing, bioimaging, clinical testing, and environmental testing. However, GOQDs demonstrate unique photoluminescence properties, with GOQDs having quantum limitations and edge effects that often affect the accuracy of the test results in the sensory field. Herein, GOQDs with a large content of hydroxyl groups and low fluorescence intensity were first prepared via an improved Fenton reaction in this study, which introduces a large amount of epoxy groups to break the C–C bonds. The synthesized GOQDs show no significant variation in the fluorescence intensity upon ultraviolet and visible light excitations. We further utilized the GOQDs as fluorescence quenchers for different fluorescent dyes in real-time fluorescence quantitative polymerase chain reaction (qRT-PCR), and verified that the addition of GOQDs (5.3 mg ml?1) into a qRT-PCR system could reduce the background fluorescence intensity of the reaction by fluorescence resonance energy transfer (FRET) during its initial stage and its non-specific amplification, and improve its specificity. In addition, the qRT-PCR method could detect two different lengths of DNA sequences with a high specificity in the 104 to 1010 copies per ml range. It is of paramount importance to carry out further investigations to establish an efficient, sensitive, and specific RT-PCR method based on the use of GOQD nanomaterials as fluorescence quenchers.
关键词： Fenton reaction,fluorescence quenchers,DNA detection,quantitative real-time PCR,graphene oxide quantum dots
更新于2025-09-23 15:19:57
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Optimization of rDNA degradation in recombinant Hepatitis B vaccine production plant wastewater using visible light excited Ag-doped TiO2 nanophotocatalyst
摘要： As widespread distribution of recombinant DNA of genetically modified microorganisms is a threat to the environment, the aim of this research is to investigate the efficiency of photocatalytic degradation of recombinant DNA under visible light. Using response surface methodology, a comprehensive evaluation of Ag doped-TiO2 photocatalytic degradation of recombinant DNA in Hepatitis B surface antigen production plant wastewater was performed. Photocatalytic synthesis parameters including dopant content, calcination temperature, and heating rate were investigated to model and optimize the recombinant DNA degradation efficiency. The Ag doped-TiO2 nanoparticles synthesis validation was accomplished by XRD, UV-Vis diffuse reflectance spectra, FESEM and energy-dispersive X-ray spectroscopy. A quadratic polynomial equation, developed by response surface methodology, with the correlation coefficient (R2) of 0.969 ensured the good fitness of the predicted data with the experimental results. The sensitivity analysis of model indicates that the square of silver content and calcination temperature have the greatest effect on the response, while the heating rate is the least important parameter. Furthermore, the optimum conditions of Ag content of 2.1%, calcination temperature of 485 ?C, and heating rate of 8 ?C/min resulted in 80.7% rDNA degradation experimentally.
关键词： photocatalytic degradation,genetically modified microorganisms,Ag-doped TiO2,Pichia Pastoris,recombinant DNA,Real-time PCR
更新于2025-09-23 15:19:57
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The pulsed light inactivation of veterinary relevant microbial biofilms and the use of a RTPCR assay to detect parasite species within biofilm structures
摘要： The presence of pathogenic organisms namely parasite species and bacteria in biofilms in veterinary settings, is a public health concern in relation to human and animal exposure. Veterinary clinics represent a significant risk factor for the transfer of pathogens from housed animals to humans, especially in cases of wound infection and the shedding of faecal matter. This study aims to provide a means of detecting veterinary relevant parasite species in bacterial biofilms, and to provide a means of disinfecting these biofilms. A real time PCR assay was utilized to detect parasite DNA in Bacillus cereus biofilms on stainless steel and PVC surfaces. Results show that both Cryptosporidium and Giardia attach to biofilms in large numbers (100-1000 oo/cysts) in as little as 72 hours. Pulsed light successfully inactivated all test species (Listeria, Salmonella, Bacillus, Escherichia) in planktonic and biofilm form with an increase in inactivation for every increase in UV dose.
关键词： Giardia,Cryptosporidium,Biofilms,Veterinary,PCR
更新于2025-09-19 17:15:36