(314f) Integrated Microfluidics for Serotype Identification of Foot and Mouth Disease Virus

This work reports an integrated microfluidics-based biosensor for Foot-and-mouth-disease virus (FMDV) detection. FMDV is highly contagious and prevalent in the most economically important animals worldwide [1]. FMDV exists in seven different types of serotypes and early identification of which is important in understanding the disease and its potential spread in the population.

Our laboratory is developing a field-based analysis tool for rapid FMDV serotype identification from clinical samples that range from vesicular fluid, feet or nose epithelium to whole blood. Specifically, this work shows the integration of an automated microfluidic extraction unit with a spinning disk platform for RT-PCR that allows us to analyze the different FMDV serotypes with high sensitivity in about an hour with current analysis protocol.

Figure 1 is a photograph of the microfluidic extraction system in its current format. The top portion consists of a PDMS microfluidic platform complete with on-chip valves (31 pneumatic valves), reservoir pumps (9 chambers that also work as pumps), and a disposable extraction filter. The system is controlled by a LabView program that operates a sequence of solenoid valves to run the RNA extraction protocol, which uses the same chemical sequence as the Qiagen RNeasy Mini spin kit. The initial experiments have been carried out with E. coli bacterial samples while the appropriate testing conditions for FMDV at our facilities are established. Figure 2 shows the comparison of extraction output with spin kit for stock E. coli RNA sample. The automatically extracted RNA elutant is compared to the output of the manual Qiagen RNA spin kit (RNEasy Kit) as the gold standard for extraction efficiency. RNA extraction results are determined using a Ribogreen RNA quantification kit obtained from Invitrogen. Fluorescent data is obtained using a 96-well plate reader. The latest version of our on-chip protocol has approached the efficiency of the commercial spin kit, and we anticipate further improvement and stabilization.

The disk for RT-PCR is based on the use of centrifugal force to move fluid along 7 short spiral channels with individual inlets located at the center to run separate chemistries for different viral serotypes. This device is fabricated from thin film plastics to create an inexpensive disposable, requiring only centrifugation for fluid control. The entire disk is 120 mm in diameter, the size of a CD, and is 375 µm thick (see Figure 3). The channel layer disks were manufactured using the process of xurography [2].

This platform has already been used to amplify and analyze a 172 bp E. Coli RNA product with encouraging results (using one-step Qiagen RT PCR kit). A rapid air thermocycler with proper cycle times and temperatures was used to perform RT-PCR in single run [3]. Work is underway to further optimize the PCR cycle times, extraction protocol and efficiency improvement (both extraction and PCR) for integrated analysis.

Figure Captions:

Figure 1 Picture of an integrated microfluidic extraction chip

Figure 2 Comparison plot showing ng RNA extracted with standard spin kit and microfluidic chip.

Figure 3 Schematic diagram of the RT-PCR disk showing individual serotype loading chambers and lanes.

References:

1. C. Carrillo, E. R. Tulman, G. Delhon, Z. Lu, A. Carreno, A. Vagnozzi, G. F. Kutish, and D. L. Rock, Comparative Genomics of Foot-and-Mouth Disease Virus. Journal of Virology, 79(10): 6487-6805, 2005.

2. Bartholomeusz DA, Boutte RW and Andrade JD. Xurography: rapid prototyping of microstructures using a cutting plotter. JMEMS, 14(6): 1364-1374, 2005.

3. Sundberg SO, Wittwer CT, Gao C and Gale BK. Spinning disk platform for microfluidic digital polymerase chain reaction. Anal Chem, published ASAP online, accepted December 31, 2009.