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Abstracts of Oak Ridge Posters |
(1 Abacus Diagnostica Ltd., Turku, Finland; 2 Department of Biotechnology, University of Turku, Turku, Finland;
aaddress correspondence to this author at: Abacus Diagnostica Ltd., Tykistökatu 4D, FIN-20520 Turku, Finland; fax 358-20-7188381, e-mail jussi.nurmi{at}abacusdiagnostica.com)
Nucleic acid diagnostic techniques are increasingly used for bacterial detection (1). However, because of a lack of automation, DNA testing has thus far been mainly performed by laboratory personnel with a high level of expertise in molecular biology. Recently, homogeneous detection technologies(2) have greatly decreased the risk of cross-contamination. To further improve the reproducibility of PCR, manufacturers have introduced universal PCR master mixes containing all reagents at predetermined concentrations. Although these ready-to-use mixes have facilitated PCR setup, they still require cold storage and careful liquid handling for consistent performance. More recently, the automation of all assay steps including sample preparation has become feasible, and fully integrated, high-performance nucleic acid analyzers(3) have become commercially available. However, the high cost of analyzers and assays still limits their routine use.
To facilitate the everyday use of PCR, we have developed a new instrument platform, GenomEraTM, which combines rapid thermal cycling (4); low-cost plastic reaction vessels(4); a homogeneous, dual-label assay technology based on end-point time-resolved fluorescence (TRF) detection; software with an intuitive user interface; and ready-to-use dry-reagent reagent sets(5). The GenomEra assay chips are made of polypropylene and metal foil, allowing optimal optical characteristics and a high speed of thermal transfer. The foil acts as a mirror to enhance the intensity of the long-lifetime fluorescence measured from the closed reaction vessels. Thermal cycling in the GenomEra instrument is based on a conveyor that transfers the reaction chips cyclically between thermal blocks maintained at constant temperatures. Combined with the assay chips that are laminated with metal foil, the rate of temperature change inside the reaction chamber can be highly accelerated compared to conventional 1-block instruments. In addition to the denaturation, annealing/extension, and measurement blocks, "hot" and "cold" blocks set to more extreme temperatures are used to further increase the speed of temperature change. The automated platform is intended for rapid, sensitive, small-scale analysis of any nucleic acid that can be detected by PCR. The target-specific reagents are predispensed and dried inside the assay vessels, and after the user has added the preprocessed sample (various sample preparation methods can readily be used), the analysis is performed automatically starting from the selection of an applicable temperature profile and ending with the reporting of unambiguous qualitative results.
We developed a homogeneous GenomEra PCR assay for Borrelia burgdorferi sensu lato, a species complex of tick-transmitted spirochetes causing Lyme borreliosis (6). The end-point detection of PCR amplification is based on competitive hybridization(7)(8). In the dual-label assay, terbium (Tb) is used to detect amplification of the target sequence, and europium (Eu) the amplification of the internal amplification control (IAC), which minimizes the possibility of false negative results. All oligonucleotides were obtained from Thermo Fisher Scientific. The Borrelia-specific primer sequences were as described by Schwaiger et al.(9), and the label- and quencher-probe sequences were 5'-aminolink C6-TGCTACAACCTCATCTGTCATTG-3' and 5'-CAATGACAGATGAGGTTGTAGCADabcyl-3', respectively. For the IAC, the primers were as described by Nurmi et al.(10), and the label- and quencher-probe sequences were 5'-aminolink C6-CCTTCTGAGGGTGATTGCG-3' and 5'-GCGAATCACCCTCAGAAGG-Dabcyl-3', respectively. The amino-modified label probes for the target and the IAC amplification products were labeled with intrinsically fluorescent GenomEra Tb [{2,2',2'',2\#821;lsqb;-{[2-(4-isothiocyanatophenyl)ethylimino]bis(methylene) bis{4-[2-methoxy-4-(carboxymethoxy)phenyl]pyridine-6,2-diyl}bis(methylenenitrilo)}tetrakis(acetato)}Tb(III)] and GenomEra Eu(11) chelates, respectively, and HPLC-purified by the labeling service of Abacus Diagnostica. The target cells (Borrelia garinii and Borrelia afzelii) in 1 x PBS (137 mmol/L NaCl, 2.7 mmol/L KCl, 4.3 mmol/L Na2HPO4 · 7 H2O, 1.4 mmol/L KH2PO4, pH 7.2) were provided by the Department of Biotechnology, University of Turku, Finland. The concentration of 2 x 106 cells/mL was determined using a Bürker counting chamber, and the cells were stored frozen in small aliquots at –20 °C until use. Before PCR, the cells were diluted in molecular biology grade water (Eppendorf).
The PCR reagents (Abacus Diagnostica) calculated for a 30-µL reaction (buffer containing 10 mmol/L Tris and 50 mmol/L KCl, pH 8.3, 5.5 mmol/L MgCl2, 0.32 mmol/L deoxynucleotide triphosphates, 1 g/L BSA), target-specific (0.5 µmol/L primers, 12 nmol/L label probe, 30 nmol/L quencher probe) and IAC-specific (0.15 µmol/L primers, 10 nmol/L label probe, 100 nmol/L quencher probe) oligonucleotides, and 10 000 copies of the IAC were dispensed onto the plastic reaction chips. The heat-stable polymerase in a buffer containing 1 g/L BSA was added in a separate drop, and the reagents were dried under vacuum. The chips were then laminated with aluminum foil, barcoded, and stored with desiccant in sealed aluminum foil bags at room temperature until use. According to our stability studies, the dry-reagent assay chips are stable at room temperature for 4 months.
To start an assay, 30 µL of sample (Borrelia cells diluted in water) was dispensed in the chip. The chips were then transferred into the GenomEra nucleic acid analyzers 4 at a time, and the applicable temperature profile was automatically selected and initiated by the instrument based on the barcode on the analyte-specific chips. Inside the instrument, the chips were irreversibly sealed with heat and cycled between thermal blocks maintained at constant temperatures. After PCR (initial denaturation at 100 °C for 115 s, followed by 45 cycles of 27 °C for 1.7 s, 60 °C for 16 s, 108 °C for 5.5 s, and 100 °C for 3 s), all oligonucleotides present in the mixture were denatured (108 °C for 10 s, 100 °C for 60 s). The temperatures given above refer to the settings of the heat blocks, not to the temperatures inside the reaction vessels. Subsequently, TRF signals were measured twice using the built-in fluorometer. The result value was then calculated as the ratio of H x 100:D, in which H is the signal obtained from a hybridized probe (measured after 120 s at 50 °C) and D the signal obtained from a denatured probe (measured after the final denaturation step and 3.8 s at 50 °C) (12). The total turnaround time of the assay was 42 min.
The reproducibility of the system was studied by preparing a panel of 20 test samples (8 negative and 12 positive controls enriched with 20, 100, or 500 Borrelia cells) that was analyzed by 3 individual operators using 4 instruments; each operator running the samples on a different day (Table 1
). The results were automatically deduced by the software based on the cutoff values embedded in lot-specific barcodes. The results obtained from individual reaction vessels were not compared to any control samples in the same assay run. Typical of end-point PCR results, the CV was high, especially in samples enriched with target cells, because the end-point reading is not a quantitative measure of the initial amount of template in the sample. With the lot-specific cutoff values of 31 (mean result value of 20 negative controls + 4 SD) and 38 (mean result value of 20 negative controls + 7 SD) for the target and the IAC, respectively, result interpretation is straightforward, and a calculated result above the cutoff accurately indicates successful amplification of the sequence of interest (target, IAC, or both). For a positive result, a result value only above the target cutoff value is required, because the competition for reagents may suppress the amplification of the IAC. In this study, no operator effects were observed; each of the 3 users obtained an identical and 100% accurate set of qualitative results. The detection limit of the assay, corresponding to the cutoff value of 31, was 5 Borrelia cells per reaction. For simplicity, however, the numerical results are usually not visible to the operators; instead, only the qualitative conclusion of the assay run (i.e., target detected/not detected) is reported. If neither of the 2 sequences can be detected because of sample-derived PCR inhibition, the result is reported as "inconclusive".
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Increasingly sophisticated and automated nucleic acid detection methods are being developed at an advancing pace. Because all of the pioneering instruments are based on the real-time PCR methodology (2), the GenomEra platform can be viewed as an expedient alternative in settings in which quantitative answers are not required. The instrument is controlled by user-friendly software that guides the operator through assay initiation and, after completion of the assay, reports the results as simple answers instead of fluorescence readings or amplification graphs. All analytical results are automatically archived in a database that can be browsed at any time.
GenomEra is the only PCR instrument available to date that is capable of exploiting the excellent sensitivity of TRF in multiplex, homogeneous nucleic acid detection. Use of the lanthanide chelates of Eu and Tb allows elimination of the rapidly decaying background fluorescence originating from plastic ware, light scattering, and sample components. Furthermore, because of the sharp and nonoverlapping emission profiles of the lanthanides, TRF readily facilitates multiplex assays. Although the fluorescence reader of the instrument is specifically designed for assays that use TRF, the instrument can also be used to measure the more commonly used short-lifetime fluorophores, such as FAM and SYBR® Green. The TRF measurement mode leads to an up to 100-fold improved assay sensitivity compared to the latter label technology (10). Because the results are deduced by lot-specific cutoff values manufactured by the software, there is no need to run separate negative control samples in each batch of analysis or to perform real-time measurements. To eliminate variation originating from sample pretreatment, we chose not to use complex clinical sample matrices in the current study. Nevertheless, the assay can readily be combined with a range of specimen treatment methods, including filtration and immunomagnetic separation (data not shown).
In conclusion, we have developed an automated, homogeneous DNA-testing platform to simplify and speed up nucleic acid assays and to facilitate their routine use. The platform is specifically designed for users with minimal expertise in PCR chemistry. The platform has a high potential for adaptation to any application in nucleic acid diagnostics, with highly sensitive detection of target sequences in <45 min.
Acknowledgments
Grant/funding support: This study was partially financed by the Finnish Funding Agency for Technology and Innovation.
Financial disclosures: None declared.
Acknowledgments: We thank the Department of Biotechnology for collaboration in the development of fluorescent lanthanide labels, and for providing the Borrelia cells.
References
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