Simple Bead Assay for Detection of Live Bacteria (Escherichia coli)

ARTICLE pubs.acs.org/ac

Simple Bead Assay for Detection of Live Bacteria (Escherichia coli) Philip Butterworth,† Henrique T. M. C. M. Baltar,† Martin Kratzmeier,‡ and Ewa M. Goldys*,† † ‡

Macquarie University, North Ryde 2109 NSW, Australia Liquid Phase Analysis Division, Agilent Technologies, Hewlett-Packard-Strasse 8, 76337 Waldbronn, Germany ABSTRACT: Bead assays are an important rapid microbial detection technology suitable for extremely low pathogen levels. We report a bead assay for rRNA extracted from Escherichia coli K12 that does not require amplification steps and has readout on an Agilent 2100 Bioanalyzer flow cytometry system. Our assay was able to detect 125 ng of RNA, which is 16 times less than reported earlier. The specificity was extremely high, with no binding to a negative control organism (Bacillus subtilis). We discuss challenges faced during optimization of the key assay components, such as varying amounts of RNA in the samples, number of beads, aggregation, and reproducibility.

B

ead assays are among one of the most promising microbial detection technologies. They offer rapid detection and data collection2-4 and can be used in multiplex analysis.2-7 Because of superior removal of background,8 bead assays are suitable for complex real samples, such as food and body fluids. Furthermore, they are suitable for low volume samples and trace amounts of analytes.4,6 Bead assays for detecting pathogens, such as E. coli,1,9 Salmonella enterica subsp. enterica,1,9 Yersinia enterocolitica,1 Bacillus cereus,1 Listeria monocytogenes,9 Campylobacter jejuni,9 Trichosporon spp.,10 human immunodeficiency virus (HIV),11 and hepatitis C virus (HCV),11 have been described in the literature. The general approach has been established reasonably well, including binding chemistries of microspheres to either antibodies or oligonucleotide probes and suitable fluorescent staining methods for the target analytes. Despite that, the development and optimization of the key assay steps remains challenging, especially in the case of RNA detection. RNA assays are important for several reasons. First, they have an advantage over antibody-based assays because the specificity of antibodies is generally restricted to species or subspecies.12 Moreover, RNA is a convenient molecule to detect in the case of microbial assays because it is abundant in cells.12 For example, the RNA content in one E. coli organism is about 58 fg, and our target,16S rRNA, constitutes approximately 27% of this amount.13 Furthermore, RNA is easily degraded,14 so it can be used to detect live pathogens in a nonquiescent state.15 Cells in quiescent state that have low physiological activity, may produce weak or even undetectable hybridization signals.15 The complete RNA assay procedure broadly consists of the following steps (Figure 1). First, RNA from microorganisms is extracted and fluorescently labeled. Then, nucleic acid probes are attached to the beads. Finally, these probe-bead complexes are hybridized with the target RNA (Figure 1a) and the complex analyzed by a biparametric flow cytometer (Figure 2b). For comparison, a similar hybridization with nontarget RNA from control organisms is analyzed as a control. The assessment of nonspecific binding is carried out as well. Each of these steps involves r 2011 American Chemical Society

Figure 1. Overview of assay procedures.

decisions that have a critical influence on the outcome of the assay. The most important decision is the selection of the beads, which requires attention to spectral properties, binding efficiency of probes to their surfaces, and nonspecific binding. The probes may be biotinylated for coupling with a streptavidin-coated bead surface4 or may be amino-modified for coupling with carboxyl groups.1,9,16 The performance of each type of bead needs to be Received: November 25, 2010 Accepted: January 6, 2011 Published: January 25, 2011 1443

dx.doi.org/10.1021/ac103109v | Anal. Chem. 2011, 83, 1443–1447

Analytical Chemistry

ARTICLE

Figure 2. (a) Schematics of the oligonucleotide probe-bead complex with attached RNA. (b) The principle of detection of beads in a 2100 Bioanalyzer: coregistration of a bead signal excited in blue and the assay signal excited in red. (c-e) Examples of results with Dragon Green beads. From left to right: (c) analysis of pure beads, (d) assay challenged with B. subtilis RNA, and (e) assay with E. coli RNA. A gate at 15 units of green fluorescence separates beads from debris and a gate at 1 unit of red fluorescence indicates the upper limit of the pure bead signal.

assessed, since surface coverage in different commercial bead products may vary. The optimization of the coupling reaction of the probes to the beads and the hybridization reaction are also important but not always possible, as the assay is intended to be used in real samples with an unknown amount of RNA. Moreover, the analysis of the complex by using flow cytometry requires appropriate gating that is difficult to predict without detailed studies. Thus, the aim of this work is to demonstrate a sensitive rRNA assay for E. coli as well as to present some of the key challenges in the development of such an assay. These include bead selection and minimization of nonspecific binding. We also discuss the range of bead concentrations required to provide an accurate assay readout, aggregation of beads, and signal variability.

’ MATERIALS AND METHODS 2100 Bioanalyzer. The 2100 Bioanalyzer (Agilent Technologies Inc.) is a microfluidic-based assay platform with a desktop main unit connected to a computer. It can perform electrophoresis of up to 12 samples in 40 min and flow cytometric assays of up to 6 samples in less than 30 min. It is equipped with two excitation devices, a blue LED with a peak at 470 nm and a red laser at 633 nm. It has two detection windows: green, in the range of 510-540 nm, and red, in the range of 674-696 nm. The green channel is used for the detection of beads and the red one for the fluorochromes (Figure 2b). Despite large spectral separation between the bead and fluorochrome emission bands, there is usually some level of bleedthrough of the bead signals into the red channel. Assay Procedure. In order to perform an assay, first, the RNA was extracted from cultured cells (UNSW Microbiology Culture Collection, Australia) by lysis with TRIzol. This was

followed by a purification step with a spin column based on the PureLink Total RNA Purification System (Invitrogen). The quality (RIN, RNA integrity number17) and quantity of the RNA was evaluated using the electrophoresis function of the 2100 Bioanalyzer. The result of this measurement is encapsulated in an RNA integrity number (RIN), which describes the degree of RNA degradation,17 from 1 for fully degraded RNA to 10 in the absence of degradation. The RIN of all the RNA extractions varied from 8.2 to 10, indicating near-intact RNA. Further, the RNA was labeled with a red fluorochrome (Alexa Fluor 647, Molecular Probes). The labeling involved ethanol precipitation, the labeling reaction as described in the fluorochrome kit (ULYSIS Nucleic Acid Labeling Kit, Molecular Probes) and another purification step with a spin column based on the RNeasy MinElute Cleanup Kit (Qiagen). Finally, the labeled RNA was hybridized for 2 h with an oligonucleotide probe-bead complex. At the conclusion of this step, the beads with bound RNA were resuspended in the cell buffer from the cell kit (Agilent Technologies) and assayed on the 2100 Bioanalyzer operating in the flow cytometry mode. The control samples were prepared by hybridizing the labeled RNA with pure, unmodified beads without oligonucleotides. In measurements with pure beads, they were washed with a solution of PBS and Tween 20 and then resuspended in the cell buffer. Oligonucleotide Probes and Beads. We used 50 amino oligonucleotide probes attached to carboxy-coated beads and 50 biotin-modified oligonucleotide probes attached to streptavidincoated beads (Figure 2a). The oligonucleotide probe, Eco16S07C (Sigma-Aldrich), is a single-stranded DNA specific for E. coli spp.18,19 and Shigella spp.19 with the 50 -30 sequence ACTTTACTCCCTTCCTC. The coupling of carboxylated beads and 1444

dx.doi.org/10.1021/ac103109v |Anal. Chem. 2011, 83, 1443–1447

Analytical Chemistry

ARTICLE

Table 1. Properties of the Beads bead

coating

diameter (μm)

excitation peak (nm)

emission peak (nm)

manufacturer

Dragon Green

carboxy

5.9

480

520

Bangs Laboratories

Yellow

carboxy

5.2

455

480

Spherotech

Yellow Green

carboxy

6.3

441

486

Polysciences

Yellow Green

streptavidin

6.2

441

486

Polysciences

Figure 3. (a-f) Assays with different amounts of pure Dragon Green beads: (b) scattergram of 818 detected events in 0.6 μL of bead solution; (a, c) histogram of the corresponding red and green signal; (e) scattergram of 3505 detected events with 5 μL of bead solution; (d, f) histograms of the corresponding red and green signal. (g) Results of assays with 0.6 μL of Dragon Green bead solution and a varying amount of RNA. (h) Results of control samples with and without incubation with Tween 20 (5 samples without Tween, 1 sample per each Tween concentration). The central line in the box plot represents the median. The horizontal edges limit the first and third quartiles, and the maximum whiskers length is 1.5 interquartile range (http:// en.wikipedia.org/wiki/Box_plot and http://www.mathworks.com/help/toolbox/stats/boxplot.html).

probes was carried out by using EDC chemistry based on the PolyLink Protein Coupling Kit for COOH Microspheres (Polysciences). The coupling of streptavidin-coated beads and probes was based on the Technical Data Sheet 616, Streptavidin and Biotin Conjugated Microspheres (Polysciences). We examined four different kinds of off-the-shelf polystyrene-based beads with nominal diameters of 6 μm and strong green fluorescence. Three types were carboxylated beads and one type was streptavidincoated. Their characteristics are described in Table 1.

’ RESULTS AND DISCUSSION Assay Performance with Dragon Green Beads. First, we discuss the overall assay results by using Dragon Green beads with carboxyl-modified surfaces and amino-modified probes. The assay successfully detected E. coli RNA in almost all of about 100 attempts. The assay signal produced by the target, E. coli

RNA, was higher than the signal of the control sample (without the probes) by a factor of up to 7.2. We also carried out assays with RNA extracted from the challenge microorganism B. subtilis. In these assays, we observed that the signal from binding to B. subtilis was even smaller than the signal from nonspecific binding of E. coli RNA. Some typical results are shown in Figure 2c-e. Bead Quantity. Earlier works1,20,21 in which the Bioanalyzer was used in a flow cytometry mode, reported results with up to 2 500 or 3 000 bead detection events per 4 min assay. However, the manufacturer recommends maintaining a number of events between 500 and 1 000 per 4 min assay and the corresponding concentration of 2106 particles/mL.22 This recommendation is because a high concentration of particles may lead to the presence of more than one particle at a time in the detection window. We evaluated the impact of such higher than recommended concentration of beads. Figure 3a-f presents examples of pure 1445

dx.doi.org/10.1021/ac103109v |Anal. Chem. 2011, 83, 1443–1447

Analytical Chemistry

Figure 4. (a) Scatter plot showing two populations in the gated green fluorescence signal. (b) Laser scanning microscopy images of selected bead singlets and aggregates. (c, d) Box plots of results with different commercial beads: assay with E. coli (EC) and its respective control (EC ctrl), assay with B. subtilis (BS) and its control (BS ctrl), probe-bead complex (probes) and pure beads (beads) (2 or 3 samples for each box plot). The central mark is the median. The edges of the box are the lower and upper quartiles, and the maximum whisker length is 1.5 interquartiles range.

Dragon Green beads assayed at two different concentrations. Two groups of samples, one with 0.6 μL of beads solution in each sample (5 samples, 54 000 beads each) and another one, with 5 μL (6 samples, 450 000 beads each) were analyzed. The distributions in the scatter plots for these two groups are different. At a higher concentration, the beads are more spread in the green channel, suggesting that they are too dense to be counted individually. This observation is confirmed in the data: with 0.6 μL of beads solution producing a count of 818 beads, a count of around 6 800 is expected for 5 μL of beads solution. However, there were only 3 505 events, indicating that some beads were counted in groups and not as singles. We note that the amount of 0.6 μL of beads solution produces a concentration of 2.7  106 beads/mL, which is somewhat higher than the recommended value. Nevertheless, after the wash steps, the concentration of beads is reduced and this quantity is adequate to yield a number of events between 500 and 1 000. RNA Quantity. We took advantage of the gating feature in the 2100 Bioanalyzer that makes it possible to separate different populations in scatter plots and histograms. We noted that almost all the pure Dragon Green beads assayed had red fluorescence below 1 unit and green fluorescence above 15 units. Hence, gates were applied to separate the beads with the green signal over 15 units from the other particles detected with a lower green fluorescence. Additionally, a second gate at the red signal value of one unit was applied to separate pure beads with low red fluorescence from beads with attached fluorescent RNA. With

ARTICLE

such gating in place, we performed a sequence of assays with different amounts of RNA (Figure 3g), in which we could clearly distinguish the assay sample from the control sample down to 125 ng of RNA. This amount of RNA was used in subsequent assays. For comparison, the limit of detection in an RNA assay for E. coli reported in ref 1 was 16 times higher, which we tentatively attribute to their different type of commercial beads, with a likely different surface density of functional groups. Moreover they used a high concentration of beads, which distorts the signals. We also note ref 1 uses a different RNA probe. Reduction of Nonspecific Binding. Nonspecific binding is a crucial problem in immunoassays since it imposes a detection limit. It can depend on the specificity of the probe and on the adsorption of molecules to the surfaces.23 Oligonucleotide probes are highly specific to a determined sequence of RNA,18,19 but the adsorption of the molecules to the surfaces of Dragon Green beads was observed clearly through a red signal shift in the control samples. In order to address this problem, the control samples were incubated with a solution of PBS (10 mM phosphate buffer, 2.7 mM potassium chloride, 137 mM sodium chloride, pH 7.4 at 25 °C) with different amounts of Tween 20 for 1.5 h at room temperature. Tween has been used by other authors, since this nonionic detergent can block vacant sites on the surface free from the probes.23,24 After incubation, we carried out the assay procedures for control samples. These results as well as the results of samples without this incubation step are presented in Figure 3h. It is clear that the average red signal was lower with the incubation step, and a low concentration of Tween 20, as small as 0.1%, was sufficient for the blocking of nonspecific binding to take place. Accordingly, this procedure was included as part of the protocol to prepare beads for hybridization. Aggregation of Beads. In some assay samples with Dragon Green beads, we observed scatter plots with two closely related populations (see example in Figure 3a). Such double peaks indicate aggregation, which can severely distort the average signals observed. Laser scanning microscopy confirmed that bead aggregation was present in these different populations. A selection of such aggregated particles is presented in Figure 4b. Image analysis showed that, out of 158 observed particles, 66.5% were singlets, 31% were doublets, and 2.5% were triplets. The aggregation was not affected by a longer vortexing (3 min) or by bath sonicating the samples after each wash step in the hybridization procedure. However, such double peaks were not observed in the analysis of pure beads or probe-bead complexes. Comparison of Different Beads. The average red signals of assays performed with the four different types of beads listed in Table 1 are presented in box plots in Figure 4c,d. The comparison between Dragon Green and Yellow beads showed that the two beads produce signals of comparable value; however, the Dragon Green beads had a higher signal for the control samples with E. coli (EC ctrl). The carboxylated Yellow Green beads had a signal about 10 higher than the Dragon Green ones; nevertheless, its streptavidin-coated version did not show such a high specific signal (EC). These streptavidin-coated beads also showed a relatively high, nonspecific binding (EC ctrl, BS and BS ctrl) compared to its specific assay. The ratio of average specific signal to the average control signal was 24.4 for the Yellow beads and 19.6 for the carboxylated Yellow Green beads. These values suggest that the Yellow and the carboxylated Yellow Green beads have the best combination of features for this RNA assay with the 2100 Bioanalyzer readout. 1446

dx.doi.org/10.1021/ac103109v |Anal. Chem. 2011, 83, 1443–1447

Analytical Chemistry Our results also showed that there are major variations in the level of signals in assays performed with the same beads and in the same conditions. Similar variations were observed for nonspecific binding. Moreover, different beads had different spectral bleed-throughs in the red channel. Variability of Assay Signal. The variability of the assay signal was the most important limitation on the minimum detectable amount of analyte. We observed two types of variability: day-today and variability observed between samples produced by exactly the same procedures and measured simultaneously under the same conditions. The former type can be managed by handling the assays and the control samples together on the same day. The latter type was observed mainly in assays and control samples, with a variation between three samples of up to 80.7% around the average. For beads and probe-bead complexes, the maximum variation was 7.7%. The variability was higher when RNA was present, suggesting that these differences may arise during the hybridization of RNA and probes. They may be caused partly by variations in amounts of reagents during the hybridization stage. We note that in the hybridization protocol, some amounts of fluid were close to or even smaller than 0.5 μL, the limit of our fluid handling capability. Therefore, we attribute the observed signal variability to bead aggregation, discussed previously, in combination with fluid handling issues during the hybridization procedure.

’ CONCLUSIONS We demonstrated a highly specific bead assay targeting the organism E. coli K12, with the assay signal up to 24 times higher than the nonspecific binding and with practically no detectable binding to rRNA of the control organism, B. subtilis. The assays proved to be very robust, resulting in a successful proof of binding in the vast majority of attempts. We reported results of optimization of the amount of beads and RNA. A higher than recommended amount of beads produces readouts of multiple beads at a time, and this artificially raises the value of the observed signal. We also evaluated a method of limiting the nonspecific binding, by incubation with Tween. The minimum detectable amount of RNA was 125 ng, 16 times less than reported in ref 1. Sample-to-sample variability was the main factor determining the limit of detection. It was attributed to a combination of limited precision of fluid handling and aggregation of beads, especially for assay samples with beads conjugated to sample RNA which increases signal averages and creates sample-to-sample fluctuations. Furthermore, we also compared four types of commercially available beads, noting large differences between the assay signals they are able to produce; these are due to very different surface functionalization. Our investigations were conducted using kits available in the market for RNA extraction, RNA labeling, binding of probes to beads, and hybridization of RNA and probes. The relevant protocols are relatively time-consuming, especially the extraction of good quality RNA with RIN values between 8.2 and 10. The assay time could be reduced significantly by using rapid extraction kits currently on the market. However, tests will be required to confirm adequate RNA quality.

ARTICLE

’ ACKNOWLEDGMENT The authors thank Peter Bergquist for scientific advice and critical eading of the manuscript, Russell Connally and Ian Paulsen (Macquarie University) for their scientific advice, and Noosha Ehya (Macquarie University) for technical support. The authors acknowledge funding by Agilent Life Sciences. ’ REFERENCES (1) Ikeda, M.; Yamaguchi, N.; Tani, K.; Nasu, M. J. Microbiol. Methods 2006, 67, 241–247. (2) Dunbar, S. A. Clin. Chim. Acta 2006, 363, 71–82. (3) Nolan, J. P.; Sklar, L. A. Trends Biotechnol. 2002, 20, 9–12. (4) Verpoorte, E. Lab Chip 2003, 3, 60N–68N. (5) Kellar, K. L.; Iannone, M. A. Exp. Hematol. 2002, 30, 1227–1237. (6) Carson, R. T.; Vignali, D. A. J. Immunol. Methods 1999, 227, 41–52. (7) Kettman, J. R.; Davies, T.; Chandler, D.; Oliver, K. G.; Fulton, R. J. Cytometry 1998, 33, 234–243. (8) Staats, H. F.; Kirwan, S. M.; Whisnant, C. C.; Stephenson, J. L.; Wagener, D. K.; Majumder, P. P. Clin. Vaccine Immunol. 2010, 17, 412– 419. (9) Dunbar, S. A.; Vander Zee, C. A.; Oliver, K. G.; Karem, K. L.; Jacobson, J. W. J. Microbiol. Methods 2003, 53, 245–252. (10) Diaz, M. R.; Fell, J. W. J. Clin. Microbiol. 2004, 42, 3696–3706. (11) Smith, P. L.; WalkerPeach, C. R.; Fulton, R. J.; DuBois, D. B. Clin. Chem. 1998, 44, 2054–2056. (12) Amann, R. I.; Binder, B. J.; Olson, R. J.; Chisholm, S. W.; Devereux, R.; Stahl, D. A. Appl. Environ. Microbiol. 1990, 56, 1919–1925. (13) Neidhardt, F. C.; Umbarger, E. In Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd ed.; Neidhardt, F. C., Curtiss, R., III, Ingraham, J. L., Lin, E. C. C., Low, K. B., Reznikiff, W. S., Riley, M., Schaechter, M., Umbarger, H. E., Eds.; AMS Press: Washington, DC, 1996; Vol. 1, p 2822. (14) Houseley, J.; Tollervey, D. Cell 2009, 136, 763–776. (15) Assmus, B.; Hutzler, P.; Kirchhof, G.; Amann, R.; Lawrence, J. R.; Hartmann, A. Appl. Environ. Microbiol. 1995, 61, 1013–1019. (16) Fulton, R. J.; McDade, R. L.; Smith, P. L.; Kienker, L. J.; Kettman, J. R., Jr. Clin. Chem. 1997, 43, 1749–1756. (17) Schroeder, A.; Mueller, O.; Stocker, S.; Salowsky, R.; Leiber, M.; Gassmann, M.; Lightfoot, S.; Menzel, W.; Granzow, M.; Ragg, T. BMC Mol. Biol. 2006, 7, 3. (18) Joachimsthal, E. L.; Ivanov, V.; Tay, S. T. L.; Tay, J. H. World J. Microbiol. Biotechnol. 2003, 19, 527–533. (19) Stender, H.; Broomer, A. J.; Oliveira, K.; Perry-O’Keefe, H.; Hyldig-Nielsen, J. J.; Sage, A.; Coull, J. Appl. Environ. Microbiol. 2001, 67, 142–147. (20) Sakamoto, C.; Yamaguchi, N.; Nasu, M. Appl. Environ. Microbiol. 2005, 71, 1117–1121. (21) Ikeda, M.; Yamaguchi, N.; Nasu, M. J. Health Sci. 2009, 55, 851– 856. (22) Nitsche, R. Application note from Agilent Technologies, 2002. (23) Jenkins, S. H.; Heineman, W. R.; Halsall, H. B. Anal. Biochem. 1988, 168, 292–299. (24) Maggio, E. T. Enzyme-Immunoassay; CRC Press: Boca Raton, FL, 1980.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected] 1447

dx.doi.org/10.1021/ac103109v |Anal. Chem. 2011, 83, 1443–1447