Polymerase Chain Reaction (PCR) detection paves the way to reliable and rapid diagnosis of diseases and has been used extensively since its introduction. Many miniaturized PCR systems were presented by microfluidics and lab-on-a-chip community. However, most of the developed systems did not employ real-time detection and thus required post-PCR processes to obtain results. Among the few real-time PCR systems, almost all of them aimed for medical applications and those for plant pathogen diagnosis systems are almost non-existent in the literature. In this work, we are presenting a portable system that employs microfluidics PCR system with integrated optical systems to accomplish real-time quantitative PCR for plant pathogen diagnosis. The system is comprised of a PCR chip that has a chamber for PCR sample with integrated metal heaters fabricated by standard microfabrication procedures, an optical system that includes lenses, filters, a dichroic mirror and a photomultiplier tube (PMT) to achieve sensitive fluorescence measurement capability and a computer control system for Proportional Integral Derivative (PID) control and data acquisition. The optical detection system employs portable components and has a size of 3.9 x 5.9 x 11.9 cm which makes it possible to be used in field settings. On the device side, two different designs are used. The first design includes a single chamber in a 25.4 x 25.4 mm device and the capacity of the chamber is 9 micro-liters which is sufficient to do gel electrophoresis verification. The second design has three 2.2 micro-liter chambers squeezed in the same size device while having smaller volume to increase high throughput of the system. The operation of the system was demonstrated using Fusarium oxysporum spf. lycopersici which is a fungal plant pathogen that affects crops in the USA. In the presence of the plant pathogen, noticeable increases in the photomultiplier tube output were observed which means successful amplifications and detections occurred. The results were confirmed using gel electrophoresis which is a conventional post-PCR process to determine the existence and length of the amplified DNA. Clear bands located in the expected position were observed following the gel electrophoresis. Overall, we have presented a portable PCR system that has the capability of detecting plant pathogens.

Yes / This paper outlined our method for developing polymer-linked contact lens type materials for rapid detection and differentiation of Gram-positive, Gram-negative bacteria and fungi in infected corneas. It can be applied to both model synthetic or ex-vivo corneal models and has been successfully trialed in an initial efficacy tested animal study. First a hydrogel substrate for the swab material is selected, we have demonstrated selective swabs using a glycerol monomethacrylate hydrogel. Alternatively any commercial material with carboxylic acid functional groups is suitable but risks nonspecific adhesion. This is then functionalised via use of N-hydroxysuccinimide reaction with amine groups on the specified highly branched polymer ligand (either individually gram negative, gram positive or fungal binding polymers or a combination of all three can be employed for desired sensing application). The hydrogel is then cut into swabs suitable for sampling, used, and then the presence of gram positive, game negative and fungi are disclosed by the sequential addition of dyes (fluorescent vancomycin, fluorescein isothiocyanate and calcofluor white). In summary this method presents: Method to produce glycerol monomethacrylate hydrogels to minimize nonspecific binding Methods of attaching pathogen binding highly branched polymers to produce selective hydrogel swabs Method for disclosing bound pathogens to this swab using sequential dye addition