CICEET Progress Report for the period 9/01/08 Through 2/15/09

Project Title: High-throughput Quantitative Detection of Microbial Contaminants
Principal Investigator(s): Mara R. Diaz
Additional Investigator(s): Kelly Goodwin and Jack W. Fell
Project Start Date: March 2009

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Tasks to meet objectives
i) Optimization of assay conditions: Environmental samples were validated with current PCR assay conditions. A series of assay optimizations involved different, temperature conditions and/or PCR cycling conditions. The optimum conditions were determined by the median fluorescence intensity values.

ii) Sequence alignments of the ITS region used DNAstar Megalign and comprised closely and non-closely related dinoflagellate species. Multiple sequence alignments of divergent sequences were generated with CLUSTALW. Areas displaying sequence divergence among the species were selected and computer analyzed using the software program Oligo (Molecular Biology Insights, Inc).

iii) Data is currently being analyzed.

Progress on Tasks
Progress on tasks is described in section G (Preliminary data section).

Have the results/data gathered during this reporting period changed the project objectives when compared to your original proposal? Please explain.
The main objective of the project has remained the same.

Dissemination activities during this reporting period (please include the number of participants where applicable).
None but we expect to present our results in American Society for Limnology and Oceanography or Planet xMAP.

Difficulties
Not all the objectives established for this period were fulfilled due to other grant and administrative obligations of the PI.

Although, we were able to successfully test a wide range of FWRI field samples, they represented an archived collection (2002-2003). Thus, in order to have a better assessment of the technology, we intend to further validate the method with another "up to date" collection of field samples. These samples will be obtained as part of recently funded EPA grant that seeks to standardize and evaluate new Habs detection technologies.

Data Generated to date
i) Test Pk658lna, Pkmikilna2, Pkb2, Pkb2lna, Pkb658 and Pkb with environmental samples and optimize assay conditions
An archived collection of environmental samples was employed to test the performance of the probes. The samples, which were collected during bloom and non-bloomed events, were obtained from previous studies undertaken in Rookery Bay National Estuarine Research Reserve (NEER) in Naples, Fl, (collection sites: Caxambas Pass, Hendersen's Creek, Marco Pass). Sample collections were undertaken at various times per year (2002-2003) and were characterized and enumerated following the parameters established by the Fish and Wildlife Research Institute (FWRI). The FWRI classification system characterizes K. brevis pre-blooming and blooming conditions based on microscopy counts: not present, present (£1000 cells/L), very low a (>1,000 to <5,000 cells/L), very low b (5,000 to 10,000 cells/L), low a (>10,000 to <50,000 cells/L), low b (50,000 to <100,000 cells/L), medium (100,000 to <106 cells/L), and high (>106 cells/L).

Initial testing was undertaken with a set of environmental samples representing various FWRI levels. PCR reactions used 1 µl to 4 µl of environmental genomic DNA in a 50 µl reaction and employed the universal primers D2C and DIRF. Initial PCR cycling conditions consisted of an initial activation at 95°C for 15 min, followed by 40 cycles amplification: 30 sec of denaturating at 95°C, 30 sec annealing at 50°C and 60 sec extension at 69°C. A final elongation step was applied at 69°C for 25min. Using the above parameters, we were able to successfully amplify all the environmental samples. However, no amplification was documented when using more than 2µl of genomic DNA. This inhibition could be due to excess of DNA or the presence of inhibitory agents, which are known to occur in environmental material. Each run included two sets of negative controls: a blank (all chemical except PCR amplicon) and a PCR negative control (water and PCR reagents). Background signals were subtracted from actual MFI readings. No amplicons were produced from any of the negative control samples.

To compare the performance of LNA modified and unmodified probes, K. brevis probes were challenged with environmental samples classified as High and Medium (Figure 1). As seen in our previous studies, which were undertaken with amplicons obtained from culture cells, the signal performance of LNA probes consistently displayed higher fluorescent intensities than their non-LNA modified versions. For instance, at those FWRI levels, Pkb2lna and Pkb658lna showed fluorescence signal intensities that were ~ 27 to 44% higher than their un-modified versions, respectively. These results are consistent with previous studies that we undertook to elucidate the affinity of LNA probes with target DNA.

Figure 2 illustrates the performance of the probes with a set of environmental samples representing a wide spectrum of FWRI categories. Robust signal intensities were documented when LNA and non LNA probes were challenged with samples classified as high, medium, low B and low A. For instance, Pkb2lna displayed signal intensities ranging from 2065 (high) to 447 MFI (low a), whereas Pkb658lna showed fluorescence signal intensities ranging from 1598 (High) to 286 MFI (low a). In contrast, a significant reduction in signal intensity was documented when the probes were challenged with samples containing cell concentrations below 10,000 cell/L (very low b, very low b and present). At these levels, the documented fluorescence intensities were below 125 MFI. An exception was Pkb2lna, which displayed ~265 MFI with a sample classified as "very low a" (1000 to 5000 cells/L). On the other hand, samples containing less that 1000 cells/L (present) were barely detected under the present assay conditions. At these levels, the probe signal intensities ranged from 23-36 MFI, except for Pkb2lna, which displayed ~83 MFI. Pkmikilna2 and Pkmiki displayed dramatically lower signal intensities when challenged with field samples. However, when both K. mikimotoi probes were tested with their target DNA, higher signal intensities were observed (data not shown).

In order to enhance the detection levels, few modifications to Mirus Label IT step and hybridization reaction were explored. To this end, we titrated different amount of amplicon (2.5 to 8 µl) in the Mirus Label It reaction and study the effect of SAPE at various concentration (300-600ng). Figure 3 illustrates the effect of SAPE concentration on signal intensity. This experiment, which employed 5 µl of amplicon in the Mirus Label IT reaction and 300 to 600 ng of SAPE, clearly showed that probe signal intensity can be successfully enhanced by increasing the concentration of SAPE. However, the increase in signal intensity was only documented for samples that contained over 50,000 cells/L. These samples are depicted as "high, medium, low, and low b" (Figure 3 A, B, C, and D). At those FWRI levels, the documented increase in probe signal intensity was ~50.5%, 44%, 30.5% and 21.9%, respectively. In contrast, probes challenged with samples classified as: " low a, very low a, very low b and present" were barely affected by changes in SAPE concentration (Figure 3 A, B, C, and D) or by the addition of 8 µl of amplicon in the Label IT reaction (data not shown). An exception was Pkb2lna, which showed a ~50% increase in fluorescence intensity when challenged with a sample classified as "very low a" and 600 ng of SAPE (Fig 3D).

The assay was further evolved by testing the effect of different amount of biotin labeled amplicons in the bead suspension hybridization array. As expected, a proportional increase in signal intensity was observed as the amount of amplicon increased (data not shown). Data derived from this experiment, indicated that 15 µl of biotin labeled amplicon appeared to suffice for the detection of species in environmental samples that contained as low as ~ 1000 cells/L. These samples are depicted in the graph as samples #14, #15 and #17 (Figure 4). At these cell levels, the signal intensity values for Pkb658lna ranged from 132 to 279 MFI. Similar range values were also documented for Pkb2lna. With the exception of Pkb, which displayed signal intensity values of 130-230 MFI, lower signal intensities were documented for the non-modified probe versions, Pkb2 and Pkb658. On the other hand, samples classified as "not present" ie #22 and #19 did not yield any significant signal that would indicate the presence of K. mikimotoi or K. brevis.

In contrast to the robust signal intensities of K. brevis probes, Pkmikilna2 and Pkmiki displayed dramatically lower signal intensities. The documented low MFI values appeared to be related to a very low abundance of K. mikimotoi cells at the sites of field collection.

In order to make the assay more specific, we designed two forward primers (Kar183 and Kr130) that selectively amplify species within the Karenia cluster group. When used in combination with the universal primer, D2C, both set of primers produced amplicon sizes of ~490bp (Karl83) and ~540bp (Kar130). As opposed to universal primers, these primers were designed to enhance the detection of both species. The use of two universal primers can sometimes be a liability since they have the potential to amplify a wide range of organism. In this scenario, an excess of amplify non-target DNA can cause a "dilution factor" associated with target DNA. This unbalance ratio of nonspecific to specific target material not only can underscore the potential detection of under-represented species, but can add some noise to the reaction, making it more difficult to achieve sensitivity and specificity. Therefore to overcome this potential effect, which can be more noticeable in field samples containing less than 10,000 cell/L, we amplified the species using similar cycling conditions as described above. Figure 5A, depict the outcome of this analysis, which used the primer combination D2C and Kar130. Under the chosen conditions (40 cycles and 15 µl of amplicon/well) all the probes displayed significant higher MFI values than those previously documented with the universal primer set: D2C and DIRF. For instance, probe signal intensities as high as 4,000 MFI were documented when Pkb658 lna was challenged with samples containing below 1000cell/L (samples #15 and 17). In addition, signal intensities as high as 800 MFI were documented when Pkb658 lna was hybridized with sample #19, which according to FMRI is a "not present" sample. An interesting thing to note is that the MFI values of Pkmikilna2 were barely noticeable. Despite the enhanced level of K. brevis detection, the data clearly showed that under the chosen assay conditions the system reached a plateau and thus could not provide any insights on the semi-quantitative character of the assay. In view of this over-saturation, the hybridization reaction was carried out with lower amounts of amplicon. Reducing the amount of amplicon to 5 µl ameliorated to some extent the over-saturation effect and allowed the detection of K. brevis at normal background levels ("present"') (Figure 5B). At those levels, pkb658lna, Pkb2lna and Pkb2 detected the species with signal intensities ranging from ~1040 to 2426 MFI. In spite the excellent sensitivity levels, the data did not follow a quantitative trend.

Since the observed DNA over-saturation appeared to be associated to PCR amplification effects (ie. products reaching at saturation concentrations), a series of amplification reactions were carried out with lower number of cycles (ie. 30 and 33 cycles). Upon reducing the number of cycles to 33, the DNA over-saturation effect was significantly reduced, while the sensitivity of the assay was maintained (Fig 6A). For instance, excellent detection levels were documented for all K. brevis probes when tested with amplicons from 7 field samples classified as "present". The documented MFI values were as followed: Pkb658lna (1228 ­633 MFI); Pkb2lna (1395-739 MFI); Pkb2 (1143-442 MFI); Pkb (703-382 MFI); Pkb658 (489-205 MFI). In addition, all K. brevis lna probes confirmed the presence of K. brevis in three samples classified as "not present"(i.e. #30, #19g, #19c ) (Figure 7). Overall, a non-significant effect on the fluorescent signal intensity was documented when the number of cycles was reduced to 30 cycles. However, when Pkb658lna and Pkb2lna were challenged with samples containing less than 1,000 cells/L, the effect of reduction of signal became more significant. For instance, when Pkb2lna was challenged with sample # 17, a ~79% reduction in signal was documented (Figure 6B). This effect is not surprising since samples containing few target cells require higher number of cycles to produce a detectable yield. As previously observed, none of the K. mikimotoi probes confirmed a significant presence of the species in the group of analyzed samples.

Even though the data followed a semi-quantitative distribution, that appeared to correlate with cell densities, the data depicted several outlier samples that did not fully appear to correlate with both microscopic enumeration as those established by FMRI counts and Luminex data (Figure 6). Several factors could have contributed to this outcome: a) differential amplification efficiencies, b) differential DNA extraction efficiencies; c) inaccurate microscopic cell counts; d) presence of inhibiting substances. Although PCR based methods are known to suffer from differential amplification efficiencies, is interesting to note that fairly similar amplification patterns were obtained despite repeated amplifications and the use of various PCR cycles. Thus, the observed variability appeared to be more related to DNA extraction efficiencies that led to different DNA yields. One thing to note is that the collection of field samples used various extraction techniques i.e. BIO 101 and FastDNA SPIN Kit for soil (Qbiogene, Irvine, Ca), which could have exacerbated the observed variable pattern.

Herein, we developed a bead suspension assay that combined the specificity of LNA probes with a signal amplification system that uses a non-enzymatic direct chemical labeling system that allows the covalent attachment of multiple biotin molecules. Merging both technologies we were able to detect a wide range of concentration of K. brevis. The assay proved to be sensitive enough to detect K. brevis in field samples classified as "present" with very robust signal intensities. The method also led the detection of K. brevis in various samples that were below the detection limit by microscopy with MFI values of sufficient strength to allow differentiation of positive versus negative signal. This high throughput method, which represents the first molecular detection strategy to integrate both technologies, can be adapted for the detection of other Habs and is well suited for the monitoring red tide at pre-blooming and blooms conditions.

Project Objectives for Next Reporting Period

Objectives
A. Test and validate new developed ITS probes.

B. Manuscript writing.

Work Plan to Meet Objectives
A) The ITS probes will be validated with complementary target amplicons and genomic DNA. Optimization of assay conditions will follow standard protocol.

B) Data will be analyzed and organized for manuscript writing.

Expenditures
The money allocated for the expenditures was appropriate.

End User Advisor Feedback
End User Advisor: Dr. James Jacobson
Organization: LabNow, Inc.
Location: 2800 B Industrial Terrace, Austin, TX 78758
Phone number: 512-329-5525 x240
E-mail: jjacobson@labnow.com

At this stage, what are the potential applications for this research? Please discuss how you and others could potentially use the technology.
As discussed in previous progress reports, the applications of this research could have significance in a variety of areas including environmental monitoring. The ability to detect and identify organisms before obvious bloom condition emerge would, at the least, allow access restrictions to be enforced. Moreover, an understanding of what organisms are present and at what levels is critical to development of control and containment methods and approaches. The development of a self-contained, deployable point-of-need detection system for these organisms is an important contribution.

What are the key challenges to application of this technology? Please consider the technology itself as well as issues related to regulation, politics, socio-economic pressures, trends in the field etc.
Key challenges remain in the diverse biology of the organisms of interest. PCR-based assay development always appears straightforward when sequences are being aligned for probe and primer selection. In practice, however, one must contend with many other organismal and environmental components that can be inhibit or otherwise impact the outcomes. Development thus far is commendable and has provided a broad understanding of the many aspects to be considered when developing a new and novel assay system.

Has anything changed about this project's potential applicability since the last reporting period (not applicable to the first Progress Report)?
No changes.

Questions/comments/ suggestions for the researchers
Nice work. Your willingness to forge ahead and find solutions to the many challenges that have come with this project is a great example. I hope to see this work published soon.