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.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/ETD-TAMU-2012-08-11714 |
| Date | 2012 August 1900 |
| Creators | Cifci, Osman |
| Contributors | Han, Arum |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | thesis, text |
| Format | application/pdf |
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