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From the Divisions of Emerging and Transfusion Transmitted Diseases
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and Hematology,
Office of Blood Research and Review, and the Division of Viral Products,
Office of Vaccines Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In a potential contamination event, the identity of the pathogen will not be known with certainty. Simultaneously screening for multiple bioterror agents would be desirable. A number of multiplex real-time polymerase chain reaction (PCR) assays have been tested that perform highly sensitive and rapid detection of conventional pathogens in blood.2, 3, 4 However, in multiplexing PCR, as the number of primer pairs in a reaction is increased there is a trend to generate more nonspecific PCR products due to inappropriate primer pairing.5, 6 The nonspecific PCR products make identification of the diagnostic PCR product by gel electrophoresis difficult to impossible.7 Techniques such as Southern blotting or DNA sequencing to identify the multiplex PCR products are mostly considered too time consuming for screening purposes.
An additional technology with even greater potential to screen for a multitude of pathogens simultaneously is the DNA microarray. A DNA microarray is a miniature device with many unique oligonucleotide probes printed on a solid surface. These oligonucleotide probes would be able to simultaneously discriminate, by hybridization, many specific PCR products amplified from pathogens using a small amount of sample. The microarray platform has the potential to screen blood for bioterror pathogens. Microarray-based diagnostic techniques already have proven effective in matrices other than blood.6, 7, 8
In our study, a microarray was developed for detecting three bioterror pathogens and a human sequence for internal positive control. Sensitivity and specificity of the multiplex PCR microarray assay for detection of three bacterial agents in blood was evaluated by spiking a known amount of pathogens into whole blood. In addition, the performance of the assay was evaluated by screening blinded blood samples from mice infected with B. anthracis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and probe sequences in Table 2
.
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and Figure 2
). The parasite probes were included as a negative control and because one of the genera in the family, Leishmania, has been a long-standing research organism in our laboratory, presenting a need to monitor its contamination. Two replicate copies of an oligonucleotide for the human internal control (Table 3
and Figure 2
) were included and the whole set of probes printed within a 2 x 10 grid (total area, 1 mm x 4.95 mm) to form a subarray. Each subarray was printed eight times on a CSS-100 sylilated slide (CEL Associates, Inc., Pearland, TX) by a microspotting pin (Telechem, Inc., Sunnyvale, CA) with a PIXSYS5500 contact microspotting robotic system (Cartesian Technologies, Inc., Irvine, CA). Each oligonucleotide probe solution was a mixture that contained a 10 µmol/L concentration of quality control oligo (QCprobe, Table 2
),6
and a 100 µmol/L concentration of the pathogen oligo in 50% dimethyl sulfoxide. In addition, spots for marking the array position on the slide were made by using 1x microspotting solution (Telechem). The printed slides were dried for 10 minutes at 80°C and soaked in freshly prepared 0.25% sodium borohydride solution for 10 minutes at room temperature. The slides were washed with 0.2% sodium dodecyl sulfate in water for 5 minutes once and washed with distilled water for 5 minutes three times to eliminate unbound oligonucleotides. A sample-printed slide was hybridized with Cy5-QC-complement oligo (Table 2
and Figure 2A
) to confirm the quality of the print.
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8 to 10 hours. The culture was monitored at OD600 and stopped when it reached 0.15 to 0.2 (1 x 108 cells per ml.) indicating early to mid-log phase. Aliquots of 0.5 ml were frozen at –80°C for future use. Aliquots were thawed and quantitated by serial dilution and plate count.
Pathogen Spiking into Whole Blood and Total DNA Isolation
A liquid culture or spore suspension of each pathogen at known concentration was used to make a series of 10-fold dilutions in phosphate-buffered saline and 10 µl of each dilution was spiked into 200 µl of research use whole blood, which was obtained from the Department of Transfusion Medicine, National Institutes of Health, Bethesda, MD. Total DNA was prepared from the spiked blood by using a QIAamp DNA blood minikit (Qiagen Inc., Valencia, CA) according to the DNA blood kit’s protocol. The final eluted volume was 100 µl in Qiagen’s Buffer AE.
Multiplex PCR Amplification
The three different pathogen primer pairs and an internal control primer pair (Table 1)
were combined in the standard PCR mixture (25 µl), which contained 1.5 U of TaqDNA polymerase with 1x TaqMaster reagent, 1x Eppendorf PCR buffer, 2.0 mmol/L Mg(OAc)2, 0.6x Applied Biosystems (Roche, Indianapolis, IN) PCR buffer (300 mmol/L KCl, 60 mmol/L Tris-HCl, pH 8.3), 200 µmol/L concentration of each dNTP (Invitrogen, Carlsbad, CA), 320 nmol/L concentration of each pathogen forward and reverse primers, 160 nmol/L each internal control primer, and 5 µl of DNA template. Thermocycling was preformed using a T gradient 96 PCR machine (Whatman Biometra, Goettingen, Germany) following the program of an initial 94°C for 4 minutes, and 35 cycles of 94°C for 30 seconds, 58°C for 30 seconds, 72°C for 40 seconds, finally 72°C for 10 minutes. A negative control tube containing water in place of the DNA template was used to monitor for contamination. An internal control primer pair was used that targeted the 18S human ribosomal RNA gene to monitor the performance of PCR, labeling, and hybridization. PCR products were evaluated by electrophoresis on a 1.5% agarose gel stained with ethidium bromide.
Nested PCR Amplification
Each of the three pathogen-nested primer pairs was combined with the internal control primer pair in three separate nested PCR reactions. The total PCR volume of each was 40 µl. The PCR mixture contained 2.0 U TaqDNA polymerase, 1x Eppendorf PCR buffer, 2.0 mmol/L Mg(OAc)2, 0.6x Applied Biosystems (Roche) PCR buffer (300 mmol/L KCl, 60 mmol/L Tris-HCl, pH 8.3), 200 µmol/L dNTP, 300 nmol/L of the pathogen forward and reverse primers, and 100 nmol/L internal control primers. The primary PCR products were diluted 1 to 10. Three µl of the diluted primary PCR products were added to each of the three nested PCR tubes. The thermocycle conditions were the same as the first PCR and also negative controls were performed. The synthesis of PCR products was confirmed by gel electrophoresis. The three tubes of nested PCR were pooled and purified using a MinElute PCR purification kit (Qiagen, Inc.) following the manufacturer’s instructions with 10 µl as the final elution volume.
Primer Extension Thermocycling (PET) for Synthesis of Amino Allyl-Labeled Single-Strand DNA (ssDNA)
The purified pooled nested PCR products were used as DNA templates in primer extension reactions for incorporation of amino allyl-dUTP. The reaction contained one primer for each of the three pathogens and the human control (PET primers listed in Table 1
), chosen to generate single DNA strands that were complementary to the probe on the microarray for each amplicon. The PET mixture (50 µl) contained 3.0 U of TaqDNA polymerase, 1x Taq Master, 1x Eppendorf PCR buffer, 0.6x Applied Biosystems (Roche) PCR buffer (300 mmol/L KCl, 60 mmol/L Tris-HCl, pH 8.3), 2.0 mmol/L Mg(OAc)2, 200 µmol/L, dGTP, dCTP, dATP, 40 µmol/L dTTP, 40 µmol/L amino allyl-dUTP (AA-dUTP) (Sigma Aldrich, St. Louis, MO) with 320 nmol/L of each primer and 10 µl of the purified PCR product. The cycling program was initially 95°C for 1 minute, then 35 cycles (94°C for 30 seconds, 58°C for 30 seconds, and 72°C for 30 seconds) and finally, 72°C for 10 minutes. The PET product was purified using the MinElute PCR purification kit. The final eluted solution was dried in a Speed Vac (ThermoSavant, Holbrook, NY) for 15 minutes.
Fluorescent Dye Coupling to the Amino Allyl Groups Incorporated into ssDNA
The dried ssDNA was resuspended with 5 µl of 0.2 mol/L NaHCO3, coupling buffer, and 5 µl of Cy3 or Cy5 monofunctional dye solution [the dry contents of one tube, Q15108 (Amersham Biosciences, NJ), was resuspended in 62 µl of dimethyl sulfoxide] was added, mixed, and incubated in a dark box for 1 hour at room temperature. Subsequently the fluorescent ssDNA was purified using the MinElute PCR purification kit. The final eluted solution was dried in Speed Vac for 10 minutes.
Hybridization
The dried fluorescent DNA was resuspended in 5 µl of distilled water and mixed with 5 µl of a 2x hybridization buffer containing 10x Denhardt’s solution, 12x standard saline citrate, and 0.2% Tween 20 in distilled water. The sample was denatured at 96°C for 1 minute and placed in ice. Seven µl of the hybridization solution was placed on two subarrays and covered with a plastic coverslip (5.5 mm x 20 mm) (PGC Scientifics, Frederick, MD). Hybridization was conducted at 55°C for 30 minutes in a hybridization chamber (Telechem Inc.) immersed in a water bath. After incubation, the slide was washed with a solution of 2x standard saline citrate and 0.1% sodium dodecyl sulfate for 10 minutes. Next, it was washed with 2x standard saline citrate solution for 10 minutes. Finally, it was washed with 0.2x standard saline citrate solution for 10 minutes. All three washes were done at room temperature. Subsequently, the slide was dried by a centrifugation at 1000 rpm for 5 minutes.
Microarray Scanning and Analyzing
The processed slides were scanned on a GenePix 4000B array scanner, and fluorescent images were analyzed by GenePix Pro 5.0 software (Axon Instruments, Union City, CA). The mean fluorescent signal intensities minus background were calculated and recorded for each spot with signal value that was more than two times the background value. Each detection experiment resulted in an image with two arrays. The average of the four internal control mean spot intensities contained in these arrays was calculated. Each pathogen had six spots total on the two arrays. The average of the pathogen mean spot intensities was divided by the average of the mean intensities of the four internal control spots because the positive control signal was the most consistent measure of the performance of the assay. Using this ratio as the measure of a pathogen signal normalized to the internal control eliminated the variations in signal intensity from assay to assay due to variables other than the quantity of the pathogen. This value, termed the relative pathogen signal (RPS), is expressed by the formula:
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Testing Unknown B. tularensis-Infected Mouse Blood Samples
The performance of the multiplex assay was tested on the blood obtained from mice infected with B. anthracis (Sterne). The mice were inoculated intraperitoneally with a target dose of 2 to 2.5 x 107 spores of bacteria essentially as described previously.12
Blood samples were collected in the presence of anti-coagulant from these infected mice at different time points after challenge. Samples from noninoculated mice were included in the analysis as negative controls. Each mouse blood sample (5 to 10 µl) was brought up to 200 µl with human whole blood and then DNA was extracted from the sample with the DNA blood mini kit (Qiagen Inc.). The extracted DNA (5 µl) was mixed into the first multiplex PCR mixture using the amplifying conditions described above. All subsequent assay procedures were as above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. In addition, an internal control amplicon and appropriate primers from human 18S rRNA genes (Table 1)
were selected and added into this PCR system for evaluating the efficiency of both multiplex PCR and microarray hybridization. To test the ability of the selected primer pairs to detect pathogens, bacteria cells were grown in laboratory culture and spiked into fresh blood in a series of 10-fold dilutions for each pathogen. DNA was extracted from the spiked pathogen blood samples and from a sample of blood alone as a negative control to assess the potential for false-positives due to contamination during PCR amplification or cross-hybridization to the microarray.
The first multiplex PCR contained three pathogen primer pairs, a primer pair for the internal control and DNA extracted from one of the spiked blood samples. Each pathogen could generate a PCR fragment of a unique size in this primary multiplex PCR reaction: 150 bp for B. anthracis, 407 bp for F. tularensis, 399 bp for Y. pestis, and 141 bp for the human rRNA gene. At this stage, the multiplex PCR sensitivity ranged from 500 to 50,000 CFU/ml based on visualizing an ethidium bromide-stained band on an agarose gel. An example, shown in Figure 1A
, contains PCR products resulting from multiplex amplification of serial dilutions in blood of Y. pseudotuberculosis from 5000 CFU/ml to 5 CFU/ml. The 141-bp human rRNA gene band is visible with all samples, as expected, except lane 1 which is the no template control. The 399-bp band for Y. pseudotuberculosis is only visible with the 5000 CFU/ml sample (lane 5). The 50-bp band in all lanes may be due to some primer dimer formation and the faint 500-bp band was a nonspecific product of some of the eight primers in this reaction, which do not amplify in the nested reaction described below. Attempts were made to detect the PCR products from the lower dilutions by fluorescent labeling and hybridization to the microarray, but this approach did not reach sufficient sensitivity (data not shown). To improve the sensitivity of the assay, a secondary, nested PCR reaction was added.
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were designed following the criteria described above to generate a secondary PCR product of smaller size using the primary PCR product as template. The secondary reactions generated new unique fragments: 144 bp for B. anthracis., 265 bp for F. tularensis, 396 bp for Y. pestis, and 136 bp for the human internal control. To overcome the interference that is increased in a nested, multiplex reaction, the secondary reactions were separated into a set of three reactions for each primary reaction. Each tube contained a small amount of the primary PCR products and two primer pairs, one of the three pathogen primer pairs and the internal control pair. The secondary PCR achieved a sensitivity of 50 CFU/ml for some pathogens by gel electrophoresis. Figure 1B
shows one example of a gel containing the PCR products from sets of three nested PCR reactions using PCR products from reactions shown in Figure 1A
as templates. Although detection of Y. pseudotuberculosis at 50 CFU/ml was possible in this example, identifying the PCR products from all pathogens consistently on agarose gels was not practical due to low yield at times and size similarity of many of the fragments, such as the 144-bp B. anthracis band and the 136-bp internal control band. Therefore PCR products were identified by hybridization to a microarray. To achieve multiplex detection using a microarray, fluorescent labeled, single-strand copies of the PCR products were made in an indirect PET reaction and subsequent chemical coupling reaction attaching either a Cy5 or Cy3 fluorophore to the amino groups incorporated into the DNA.
Identification of PCR Products by Microarray Hybridization
The labeled PET products were mixed in a hybridization solution and applied to two replicate arrays containing oligonucleotide probes for the three bioterror pathogens, a negative control pathogen and two replicate copies of the probe for the internal control (Table 3
and Figure 2
). After hybridization, an image of the washed slide was captured with a laser scanner. The fluorescent images revealed a hybridization pattern that correctly identified the spiked pathogen and showed strong signals on the two human rRNA gene internal control spots (Figure 2; B to D)
. To establish rigorous criteria for distinguishing a positive microarray result from a negative result, any spot on the microarray with signal above background was quantitated and a mean pixel intensity was calculated for each spot. The RPS was calculated as described in the Materials and Methods section.
To evaluate the analytical sensitivity and specificity of the multiplex PCR microarray assay for bioterror pathogens, cultured pathogens were spiked into normal donor whole blood as described above in 10-fold dilution series from 5000 to 5 CFU/ml for B. anthracis animal vaccine strain, F. tularensis live vaccine strain and Y. pseudotuberculosis. Each dilution series was prepared three times from independent cultures to test the consistency of the assay. Primary and secondary PCR reactions, labeling, hybridization, and image analysis were performed for each pathogen at each dilution as well as unspiked blood in three separate experiments on 3 separate days to show reproducibility. From the results of known positives and known negatives, a cutoff value of RPS = 0.3 allowed separation of almost all positives from negatives. However, some of the probes printed on the array were particularly cross-reactive and in a few cases resulted in an RPS > 0.3 for a pathogen not present in the sample. An example can be seen in Figure 2B
in which a visible signal appears on spot E1, the first probe for F. tularensis in the assay of a B. anthracis-spiked sample. Therefore, a second criterion for positive detection was applied that required all three probes for a pathogen to have a spot mean intensity greater than 8% of the average mean intensity of the internal control spots. When both criteria were met, all known positives could be discriminated from all known negatives (Table 4)
. All three pathogens were consistently detectable in this assay at a concentration of 50 CFU/ml. At 5 CFU/ml, the assay could detect bacteria only intermittently (Table 4)
. Applying the dual criteria for detection resulted in no false-positives. The specificity of the assay was also challenged with common bacteria that could be present in samples submitted for testing. DNA extracted from Bacillus cereus (1.9 µg or 3 x 108 genome equivalents) was spiked into the standard amount of human blood DNA and assayed as above resulting in strong signals for the internal control, but no positive RPS for any pathogen (data not shown). Similarly, DNA extracted from Escherichia coli did not result in a positive signal for any of the bioterror pathogens (data not shown).
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. Time 0 represented blood samples collected before injection (negative controls) and were all negative for B. anthracis. In three of the five mouse blood samples taken 36 hours after infection or later, B. anthracis organisms were detected, although all of the mice that were challenged eventually developed symptoms of anthrax and died from infection.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The development of this assay design involved optimization at many steps. Primer design relied heavily on computer-based methods, but ultimately trial and error still played a role, because some primer sequences failed to perform well and were replaced with others that were superior. Choice of primer sequence also involved optimizing amplicon length, a property that must balance efficiency of amplification (shorter length) with specificity of detection (sufficient length to accommodate multiple probes of up to 45 nucleotides). Reaction optimization involved choosing annealing temperatures, Mg++, and primer concentrations that were a compromise between all eight primers in the multiplex reactions. The addition of a second, nested PCR reaction to improve sensitivity increased the danger of contamination as reported by others.5 However, care in handling, separation of PCR setup and PCR product manipulation into different rooms, and monitoring each reaction with a no template control tube eliminated false-positives. Reaction optimization leading to increased sensitivity also occurred in the fluorescent-labeling step. Blending the single-strand primer extension method6 with indirect labeling resulted in greater sensitivity. Single-strand primer extension improved the hybridization efficiency over double-stranded thermocycle labeling because the extension products generated were all complementary to the probes on the array thereby avoiding competitive hybridization. Indirect labeling enhanced the detection sensitivity because the small amino-allyl group is more readily incorporated into DNA than a bulky molecule such as the Cy5-dUTP used in direct labeling. Thus, the direct labeling would likely cause early chain termination and incomplete elongation of the labeled single-strand product, with a corresponding loss of pathogen-specific fluorescent signal. The indirect labeling method may also contribute to specificity by reducing the amount of short, premature termination products that would be more likely to cross hybridize. The inclusion of a human internal control target sequence was fundamentally important to the performance of the assay. Previous multiplex pathogen detection PCR assays have demonstrated the importance of an internal control18, 19 to verify extraction of DNA from the tissue and performance of all of the components in the enzyme reaction. In our assay, the human rRNA gene target verifies the efficiency of the labeling and hybridization as well. Thus, absence of fluorescence signal at the four internal control spots forces a repetition of the assay and avoids false-negatives. Additionally, the internal control is a reference to normalize the quantitation of the fluorescent signal from the pathogen probes. This normalization provides the basis to set criteria for positive detection in the assay. A crucial step in optimizing the use of the internal control in the multiplex PCR was adjusting the primer concentration. The samples of whole blood have a constant and abundant quantity of human DNA, whereas the pathogen DNA becomes vanishingly rare at the limit of detection. Excess amounts of the human rRNA gene PCR product inhibited the amplification of the rare pathogen DNA. Therefore lower concentrations of the internal control primers permitted greater sensitivity of pathogen detection.
The multiplex PCR microarray assay was able to detect B. anthracis, F. tularensis, and Y. pseudotuberculosis with a sensitivity of 50 CFU/ml of whole blood. This sensitivity was reproducible in replicate spiked samples. This limit of detection is only slightly higher than reports of single target PCR for select agents20
and is comparable to recently published multiplex assays.15
The limit of detection compares well to real-time multiplex PCR when samples are prepared from pathogen-spiked blood.2, 4
Our assay was highly specific because no false-positive detection occurred in 45 assays. At pathogen concentrations of 500 CFU/ml or more, this assay is a simple, qualitative assay. The microarray images appear as in Figure 2
, showing signal at the detected pathogen spots, the internal control spots, and no signal at all of the other spots. However, as the target DNA becomes rare at lower pathogen concentrations, false priming events are more likely to give rise to unwanted PCR products that can cross hybridize with probes on the array. This assay achieved a sensitivity of 50 CFU/ml while retaining high specificity by quantitating the scanned image. The software provided with all models of microarray scanners is equipped with tools that allow calculation of the mean signal intensity at each spot. Once this value is collected, simple manipulation on a calculator, or in any spreadsheet program for higher throughput, allows calculation of the RPS as described above. The high specificity of this assay is also due to the multiplicity of probes that are exposed to hybridization simultaneously. There are three unique probes printed on the array for each of the pathogen targets. Amplification of a target present in a sample results in hybridization to all three probes, while spurious amplification products do not hybridize evenly to all three probes preventing false-positive detection.
The multiplex PCR microarray assay was demonstrated capable of detecting a bioterror pathogen from peripheral blood of an infected organism. The majority of mouse blood samples collected 36 hours or more after B. anthracis infection tested positive. Although all of the mice after time 0 can be presumed infected because they all died of anthrax within 5 days, the level of bacteria in the blood at the time of collection is uncertain. Bacteremia occurs late in B. anthracis infection in humans,21 and it is not known at what hour after inoculation of mice it will rise more than 50 CFU/ml (the analytical sensitivity of this assay). Some loss of sensitivity may have occurred with the mouse samples due to the very low blood volume. Human samples tested in the analytical study were 200 µl in volume. The mouse blood samples were 5 to 10 µl representing at least a 20-fold lower sampling of pathogen DNA. Larger sample volumes or modification of the sample preparation procedure that would result in a higher concentration of infected sample DNA in the PCR reaction could improve the sensitivity. The results suggested a high preclinical specificity because none of the time 0 samples showed B. anthracis-positive and no positive signals were seen for the other pathogens detectable by this microarray.
In conclusion, we have demonstrated a proof of the concept of a DNA microarray for simultaneously screening blood for contamination with three bioterror bacteria. An analytical sensitivity of 50 CFU/ml was demonstrated with no false-positives. Effectiveness in screening infected blood was demonstrated with B. anthracis-infected mouse samples. Although considerable work remains in simplifying and consolidating the assay procedures, this work is the necessary first step toward application of the microarray to high-throughput blood screening.
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Acknowledgments |
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Footnotes |
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Supported by a competitive grant from the Counter Terrorism Special Needs Projects, Center for Biologics Evaluation and Research, Bethesda, MD, and Cooperative Research and Development Agreement, Hematech, Westport, CT.
Accepted for publication June 6, 2005.
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