Research Accomplishment Reports 2010

Rapid Assay, Probe Technologies, and Media for Monitoring Flora in Foodstuffs

C.L. Hicks
Department of Animal and Food Sciences

 

Non-Technical Summary

If rapid assays were developed that could be used in systems that contain some food solids, have high sensitivity, low preparation and assay time they would find immediate commercial application. A virtual nondestructive assay would have the greatest value in the marketplace. While there is zero tolerance for many pathogens in foods, usually enrichment methods are necessary to find these organisms using current assay procedures. Thus, if easy concentration methods were employed to collect bacteria from large samples, and an assay could measure 102 bacteria /ml or less and each assay costs less than $5.00 (including operator time), the rapid assay system would replace most other assays that require greater cost, greater preparation time, and higher skilled technicians.

One method being investigated is an Acoustic Emission (AE) assay, which appears to be specific for individual bacteria, including pathogens, that it can measure pathogens at a minimum concentration within a mixture of bacteria. This assay would have an initial up front cost for the equipment but the actual assay cost would be limited to operator time, a disposable sample vessel, and millipore filter, thus the actual cost may be far lower than most assays that are currently on the market. While this test would be considered a screening test, because it does not provide actual numbers of organisms present, it may be accurate enough to suggest contamination levels.

A second method being investigated is a rapid assay probe system that use bacteriophage peptide as a vector which is attached through a tether to a reporter molecule. While some commercial systems do use bacteriophage that have reporter molecules attached to them as rapid assay probes, this probe has the advantage of being much smaller, thus the number of probes that can saturate the host membrane goes up significantly, thus assay sensitivity increases with saturation. Also, once pathogen specific peptides from phage are characterized, highly specific peptides can be designed and synthesized to increase receptor binding and saturation. While this probe would be more expensive than AE assays, it may have some market application because of sensitivity. Again, this is a rapid screening technology and not a conformation technology.

Currently, companies pay identification laboratories from $19.00 (negative test) per assay for a standard plate assay that requires 7 days. ELISA tests cost about $25.00 per test and require 24 to 48 hours. PCR tests cost approximately $30.00 per test, take at least 12 hours, and are positive if dead cells are present. AE fingerprint assays would replace all PCR and ELISA tests (positive for non viable cells) currently being used by testing laboratories if assay times could be cut to 30 min because only viable cells numbers would be determined. Thus, several million assays/year would be performed for the food, regulatory, medical, and defense industries per year at a cost of greater than 120 million dollars per year (estimates derived from an average cost of $30/assay).

2010 Project Description

Lactococcus lactis ssp. lactis C2 bacteria in M17 medium, at 26̊C for 8 h were infected with phages sk1 or ml3, and monitored using contact piezoelectric sensors attached to the sides of the growth vessel. The two sensors (5 to 50 kHz range) had individual characteristic and internal amplification mechanisms that were calibrated and adjusted to minimize background noise. After the sensors had been calibrated, the M17 medium was inoculated with L. lactis ssp. lactis C2 culture (1 x 109 cfu/ml), stirred for 1 min, and allowed to grow for approx. 90 min prior to infection (stirred for 1 min) with phages sk1 or ml3. Infection time was set to correspond with the start of the log growth phase. Infection level was 105 pfu/ml for both phages sk1 and ml3.

Sound intensity from the growth chambers was measured in attojoules (aJ = 10-18 Joules) and plotted as the energy rate-per-detected acoustic wave. Acoustic peaks considered significant and beyond internal or external generated noise were those having greater than 3 times the sigma value of the general variation in acoustic intensity over the entire data set of each test. Energy rate data from control tests in which L. lactis ssp. lactis C2 was grown without phage sk1 or phage ml3 infections contained no acoustic peaks with intensities that exceeded the 3 sigma standard whereas phage sk1 or ml3 infected L. lactis ssp. lactis C2 culture contained multiple acoustic peaks with intensities that exceeded 3 sigma. The first peaks for phage sk1 appeared at 33.2 min whereas the first peak for phage ml3 appeared 40 min.

Thus, the acoustic data from phage sk1 or phage ml3 infected L. lactis ssp. lactis C2 were considered to be the result of phage infection. The timings of the acoustic peaks from phage sk1 were sufficiently different from phage ml3, that these two phage could probably be distinguished by acoustic emission monitoring during phage infection of the bacteria.

2010 Impact

Results should be important in the development of virtual assays.

2010 Publications

Wardani, A. K., C. L. Hicks, and J. M. Stencel. 2010. Acoustical emissions generated by bacteriophages sk1 and ml3 using Lactococcus lactis ssp. lactis C2 host. J. Dairy Sci. 93(Suppl. 1): 604 W64.