Rapid pathogen detection using a microchip PCR array instrument

¹ Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, 7000 East Ave., Livermore, CA 94551.

² Naval Medical Research Institute, 8901 Wisconsin Ave., Bethesda, MD 20814.
^a Address correspondence to this author at: LLNL, BBRP, P.O. Box 808, L-452, Livermore, CA 94551. Fax 925-422-2282; e-mail belgrader1{at}llnl.gov.

	Abstract

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An array of PCR microchips for rapid, parallel testing of samples for pathogenic microbes is described. The instrument, called the Advanced Nucleic Acid Analyzer (ANAA), utilizes 10 silicon reaction chambers with thin-film resistive heaters and solid-state optics. Features of the system include efficient heating and real-time monitoring, low power requirements for battery operation, and no moving parts for reliability and ruggedness. We analyzed cultures of Erwinia herbicola vegetative cells, Bacillus subtilis spores, and MS2 virions, which simulated pathogenic microbes such as Yersinia pestis, Bacillus anthracis spores, and Venezuelan equine encephalitis, respectively. Detection of microbes was achieved in as little as 16 min with detection limits of 10⁵–10⁷ organisms/L (10²–10⁴ organisms/mL).

	Introduction

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Emerging and re-emerging infectious diseases contribute to ~25% of physician visits in the United States and are the major cause of death worldwide (1). Changing ecological, environmental, and human demographical factors and ongoing evolution of microorganisms are causing an increase in the number of reported ailments caused by pathogenic agents (2)(3). Moreover, bioterrorism has become an issue of serious concern, especially because policies and plans to prevent and counter a potential incident are not well established (4). Improving the readiness of healthcare and emergency service providers to respond to microbial threats is critical for the proper implementation of effective surveillance, treatment, and control measures. However, this will only happen if new tools that enable first responders and clinicians to rapidly detect infectious agents are made available.

PCR-based (5) tests for detecting microorganisms are increasingly being implemented in clinical laboratories (6). These tests offer high sensitivity and specificity but have been relatively slow compared with immunoassays. However, recent innovations in PCR chemistry and thermal cycling technology now enable DNA testing to be performed in a matter of minutes instead of hours. Fluorogenic PCR assays (7)(8)(9)(10)(11) eliminate the necessity of post-PCR analysis, which typically involves subjecting the PCR product to enzymatic treatment, hybridization capture, and/or electrophoretic separation. Using two-temperature PCR reduces the complexity of the thermal cycling profile and increases the speed and efficiency of the reaction (12)(13). Advanced spectrofluorometric thermal cyclers with extremely fast heating properties make rapid fluorogenic PCR and real-time monitoring possible (11)(14)(15).

Previously, a Miniature Analytical Thermal Cycling Instrument (MATCI) for performing rapid fluorogenic TaqMan assays (7)(8) was reported (14)(16). This instrument contained a single silicon reaction chamber with thin-film heaters and integrated solid-state optics, enabling battery-powered operation, efficient and rapid heating of the reaction chamber, and real-time data analysis. The complete instrument, including a laptop computer and a battery power supply, was fitted in a small suitcase for easy transport and operation in virtually any location. The potential for using the MATCI for rapid environmental, clinical, and forensic testing was demonstrated by analyzing samples of human, bacterial, and viral DNA (14)(16)(17).

The MATCI allowed only sequential sample analysis. Therefore, the next logical step in microchip PCR development was to build a system consisting of an array of 10 silicon reaction chambers for multiple sample capability. Here we demonstrate our first prototype microchip PCR array system, called the Advanced Nucleic Acid Analyzer (ANAA).

	Materials and Methods

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samples and assay conditions
Bacillus subtilis spores and Erwinia herbicola vegetative cells were provided by Dugway Proving Ground (Dugway, Utah). PCR amplification and fluorescence detection was accomplished using the ANAA or the ABI Prism 7700 thermal cycler (Perkin-Elmer) in a volume of 25 µL or 50 µL, respectively. Reaction assays for Bacillus and Erwinia consisted of 10x TaqMan Buffer (Perkin-Elmer), 0.4 mmol/L each PCR primer and probe, 5 mmol/L MgCl₂, 0.2 mmol/L each dNTP, 50 000 U/L (0.05 U/µL) of AmpliTaq polymerase (Perkin-Elmer), and 5 µL of sample for the ANAA or 10 µL of sample for the ABI 7700. Each probe contained 6-carboxy fluorescein and 6-carboxytetramethylrhodamine fluorescent dyes attached at the 5' and 3' ends, respectively. MS2 assays were prepared using the EZ rTth RNA PCR Kit (Perkin-Elmer), which couples reverse-transcription with TaqMan analysis. Thermal cycling was accomplished on the ANAA at 95 °C for 30 s, followed by 50 cycles of 94 °C for 4 s and 60 °C for 19 s (Figs. 1 , 2 , and 4 ) or 30 s (Fig. 3 ). Thermal cycling was performed on the ABI Prism 7700 using the manufacturer's recommended settings of 95 °C for 60 s, followed by 50 cycles of 95 °C for 15 s and 60 °C for 60 s.

View larger version (111K): [in this window] [in a new window]   Figure 1. The ANAA. The 10 silicon-based reaction chambers are arranged in two columns on the right side of the instrument.

View larger version (18K): [in this window] [in a new window]   Figure 2. Real-time detection of E. herbicola cells. A sample of 50 µL of Erwinia at 108 CFU/L (105 CFU/mL) was added to 200 µL of PCR master mix. Aliquots of 25 µL were placed in each chamber and subjected to rapid TaqMan analyses. The detection profile for each chamber is represented by a distinct curve. RQ is the 530/590 nm emission ratio normalized to the emission ratio for the 10th cycle.

View larger version (13K): [in this window] [in a new window]   Figure 4. Simultaneous analysis of Bacillus spores, Erwinia cells, and MS2 virions on the ANAA. A 5-µL aliquot of each sample was analyzed. The MS2 reaction was loaded on the instrument first to accomplish a 15-min incubation at 60 °C for reverse transcription. At the completion of the incubation, the thermal cycling phase commenced, and the Bacillus and Erwinia reactions were loaded. The panels indicate the concentration of the tested sample and represent the limits of detection. Samples at lower concentrations did not produce positive signals. Each primer and probe set is specific for the respective target microbe and does not cross-react with the other microbes (data not shown).

View larger version (31K): [in this window] [in a new window]   Figure 3. Quantitative analysis of B. subtilis spores. Spores were lysed by subjecting them to 2 min of ultrasonics in the presence of glass beads. Five and ten micoliters of spores at concentrations of 107, 108, and 109/L (104, 105, and 106/mL) were subjected to TaqMan analysis on the ANAA (top) and the ABI Prism 7700 (bottom), respectively. For the ANAA, chambers 3, 6, 8, and 10 were used. Rn is the normalized emission at 530 nm; NTC, no-template control.

the anaa
The ANAA consisted of 10 reaction modules, with each module containing a silicon reaction chamber with thin-film resistive heaters and an optical window, a light-emitting diode with a 500-nm bandpass filter as the excitation source, and two photodiodes with bandpass filters centered at 530 nm and 590 nm to detect 6-carboxy fluorescein and 6-carboxytetramethylrhodamine emissions, respectively. Fabrication of the chambers has been described previously (18). The instrument included a Macintosh Powerbook laptop computer running Igor software (Wavemetrics) to control thermal cycling, to provide real-time display of all the reactions, and to automatically call a positive via a red indicator and an audible signal. Assays were accomplished by filling plastic, disposable polypropylene reaction tubes with 25 µL of PCR mixture overlayed with 4 µL of mineral oil and sliding the tubes in the reaction chambers.

	Results

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The ANAA was built to demonstrate simultaneous control of an array of silicon PCR reaction chambers with continuous real-time monitoring (Fig. 1 ). This required considerable redesigning of hardware and software originally developed for MATCI to accommodate the order of magnitude increase in capability. The basic element of the system was the reaction module. Each module harbored a silicon PCR microchip, a thermistor to interface the thermal controller with the microchip, and a complete set of optics for fluorogenic detection. The modules were arranged in two rows, with five modules per row. Performance of the instrument based on sensitivity, signal-to-noise ratio, and speed of analysis was substantially improved compared with the MATCI.

PCR microchips produced by our fabrication process exhibit slight variations in thermal characteristics. This variability did not cause problems for the MATCI, in which a single PCR routine controlled one chip. A calibration program was written that corrected for chip-to-chip differences and ensured that the thermal cycling profile remained consistent when the chip was replaced. However, the ANAA introduced a new level of complexity because the PCR routine had to simultaneously control a parallel array of 10 chips. The calibration program could not accommodate adjusting for variability within an array. Therefore, fabricated chips were carefully tested, compared, and matched until 10 chips were identified that shared similar heating and cooling profiles.

Three types of samples prepared from cultures were analyzed on the ANAA: E. herbicola vegetative cells, B. subtilis spores, and MS2 virions, which simulated pathogenic microbes such as Yersinia pestis (plague), Bacillus anthracis spores (anthrax), and Venezuelan equine encephalitis, respectively. TaqMan assays for each of the simulation organisms were developed and validated using the ABI Prism 7700, a commercial instrument. The most important criteria for the ANAA was to achieve equivalent amplification and detection efficiencies among the array of 10 chambers. Fig. 2 displays real-time monitoring of a replicate sample of E. herbicola cells analyzed using all 10 reaction chambers. The 10 detection profiles were very similar to one another, with chambers 4 and 9 exhibiting slightly stronger and weaker signals, respectively. The threshold cycles, the cycles where detectable signal was first observed, spanned 24–26 cycles, and the range of signal amplitudes after 50 cycles was relatively narrow (1.75–1.98). These results confirm that the modules shared similar heating, cooling, and fluorescence detection properties. In addition, rapid time of detection was demonstrated because cell lysis, PCR, and detection was completed by 16 min.

Quantitative TaqMan analysis of 10-fold serial dilutions of a sample of Bacillus spores was performed using the ANAA and the ABI 7700. Sensitivity was improved by lysing the spores by a 2-min ultrasonic procedure before analysis (Nasarabadi et al., manuscript submitted). Chambers 3, 6, 8, and 10 of the ANAA, which share nearly identical detection profiles as shown in Fig. 1 , were used for these tests. As expected, a direct correlation of spore concentration and threshold cycle was observed for both instruments (Fig. 3 ). Threshold signals were obtained 3–4 times faster with the ANAA as a consequence of faster heating rates, shorter step times, and higher reaction efficiency. This higher efficiency is particularly evident for the sample at 10 spores/L (10 spores/mL), exhibiting a threshold cycle of 21 for the ANAA and 25 for the ABI 7700. Furthermore, positive signals produced on the ANAA were viewed in real-time (18–26 min), whereas the ABI 7700 required the run to be completed (120 min) before the signal profiles could be visualized.

The multiple reaction chamber platform adds the utility of performing assays tailored for different microbes to be run simultaneously. Samples of Bacillus spores, Erwinia cells, and MS2 virions were analyzed at the same time. Thermal cycling conditions (1.6 cycles/min) were identical for the three simulation organisms, except that the MS2 reaction was loaded first to allow an incubation at 60 °C for 15 min to perform reverse transcription. Signal profiles representing the minimum detection limits are shown in Fig. 4 . Samples with concentrations below 10/L (10/mL) for Erwinia cells, 10/L (10/mL) for Bacillus spores, and 10/L (10/mL) for MS2 virions were not detected. These results demonstrate the potential to subject an unknown sample to a panel of different assays, in which each assay could rapidly detect a specific pathogen and/or identify markers for drug resistance or virulency.

	Discussion

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The samples in this study were used because they have been adopted by the Department of Defense as standards for simulating pathogenic agents released in a biological incident. Because the most effective way to release an agent is in the form of an aerosol, nasal swabs would be a primary sample source for determining whether individuals have been exposed. These swabs could potentially be analyzed by PCR with minimal required sample preparation. However, the testing of blood or tissues for infectious agents necessitates a reduction in sample complexity and/or purification of the microbial DNA to detect low copy numbers of microbial DNA in a high background of human genomic DNA and to remove potential inhibitors of PCR such as heme. Our previous reports on the single microchip PCR instrument, the MATCI, utilized conventional sample preparation methods to detect hantavirus in rodent blood (14) and orthopoxvirus in Vero cells (16). However, because these methods are relatively slow, expensive, and difficult to package into a small, autonomous, fluidic system, new concepts must be explored. Dielectrophoresis is one promising approach and has recently been demonstrated in a microchip format to separate Escherichia coli, Micrococcus lysodeikticus, and Staphylococcus epidermidis cells from a human whole blood sample in as little as 4 min (19).

The ANAA is still a prototype instrument with major improvements in progress. The utilization of an array of microchip reaction chambers with dedicated optical detectors offers the distinct advantages of efficient heating and real-time monitoring for rapid analyses, low power consumption for battery operation, and ruggedness because there are no moving or motorized components. Other potential advantages include independent control of each reaction chamber to accommodate different reaction parameters and a further reduction in size. Current work has focused on developing a microfluidic sample preparation module to interface with the instrument.

	Acknowledgments

 
We gratefully acknowledge funding from the Central Measurement and Signature Intelligence Office and the US Department of Energy Office of Non-Proliferation and National Security. This work was performed under the auspices of the US Department of Energy by Lawrence Livermore National Laboratory under contract no. W-7405-Eng-48.

	References

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