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Abstracts of Oak Ridge Posters |
(1 Keck Graduate Institute of Applied Life Sciences, Claremont, CA; 2 ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT; 3 Department of Pathology, University of Utah, Salt Lake City, UT 84108;
aaddress correspondence to this author at: Keck Graduate Institute of Applied Life Sciences, Claremont, CA 91711; fax 909-607-9826, e-mail aniemz{at}kgi.edu)
There is a need for rapid nucleic acid amplification and detection technologies suitable for point-of-care settings. PCR-based molecular diagnostics, portable thermocyclers, and rapid microfluidic techniques are emerging technologies (1), but the use of isothermal nucleic acid amplification would make less demanding instrumentation feasible. Most isothermal methods reported to date(2)(3)(4)(5)(6)(7) require reaction times >30 min. In this study, we describe very rapid isothermal DNA amplification through EXPAR (exponential amplification reaction)(8)(9) coupled with visual colorimetric detection facilitated by aggregation of DNA-functionalized gold nanospheres(10)(11)(12) applied to a genomic sequence derived from herpes simplex virus (HSV)1.
Rapid detection of HSV infections is important in HIV-positive and immunocompromised individuals (13)(14), pregnant women, and newborns(15). PCR-based assays are superior to viral cultures and immunoassays for the diagnosis of HSV infections from cerebrospinal fluid(16) and genital, oral, and topical swabs(17)(18)(19)(20). New rapid, low-cost molecular diagnostic tools can enable broad-based testing of at-risk populations, providing effective patient treatment and preventing further disease spread.
EXPAR (8)(9) isothermally amplifies a short oligonucleotide trigger 106- to 109-fold in <10 min. EXPAR occurs at 55 °C, permitting activity and stability of the polymerase and nicking endonuclease involved in the reaction(9). Trigger X primes an amplification template containing 2 complimentary X' sequences and enables generation of the nicking enzyme recognition and cleavage site (see Fig. S1 in the Data Supplement that accompanies the online version of this Abstracts of Oak Ridge Posters at http://www.clinchem.org/content/vol53/issue11). Trigger extension and single-strand nicking create another trigger oligonucleotide, which dissociates from the amplification template. Through repeating cycles of elongating and nicking, the trigger is linearly amplified. Newly formed triggers prime other amplification templates, causing exponential amplification. The progression of EXPAR can be monitored in real time using SYBR Green. As an indirect measure for trigger amplification, the sigmoidal increase in fluorescence intensity reflects conversion of template into the partially double-stranded trigger-producing form, (see Fig. S2 in the online Data Supplement). The time at which each amplification curve reaches its inflection point is linearly correlated to the logarithm of the starting trigger concentration (see Fig. S3 in the online Data Supplement), similar to the correlation between cycle threshold and target DNA concentration in real-time PCR.
Despite the positive attributes of EXPAR, the detection limit of this method is currently high because of nonspecific background amplification. Investigations are ongoing to determine the nature of this amplification observed in the absence of trigger, and our data suggest that unconventional DNA synthesis may be involved (unpublished data). The timing of background amplification depends on the template sequence and mastermix composition. For suitable EXPAR templates, trigger concentrations of 10 fmol/L (1.8 x 105 copies in a 30-µL reaction volume) can be reproducibly differentiated from background, using as a metric a 
10% difference between the inflection points of the real-time fluorescence curves for the trigger-containing (positive, P) and no trigger–containing (negative, N) samples [(N-P)/N in %]. This value equals approximately 60-s absolute difference in the inflection points of the real-time fluorescence curves. The timing of background amplification has a typical intraassay imprecision (CV) value of approximately 2%, and an interassay CV of approximately 10%. This interassay variability does not negatively affect the relative separation of trigger and no-trigger–containing samples within each experiment. We are optimizing EXPAR, and have occasionally been able to differentiate much lower trigger concentrations from background (1 amol/L, 18 copies in 30 µL; see Fig. S4 in the online Data Supplement). Although such low limits of detection are not routinely attainable, we are optimistic that through systematic assay optimization it will be possible to perform EXPAR in a robust manner with PCR-like sensitivity.
To facilitate visual detection, we have developed a 2-stage EXPAR amplification reaction, coupled to aggregation of DNA-functionalized gold nanospheres through a bridging reporter sequence (9). DNA nanosphere aggregation causes a red-to-blue color change attributable to a shift in plasmon resonance(10)(11)(12). We previously reported that 2-stage EXPAR coupled to DNA-nanosphere aggregation enables detection of 100 fmol/L trigger oligonucleotide in <10 min. Through continuing optimization, we can now reproducibly detect 10 fmol/L trigger (60 000 copies in 10 µL) with amplification times of 3.5 or 4 min. We have occasionally achieved low copy number detection limits (1 amol/L, 6 copies in 10 µL; see Fig. S5 in the online Data Supplement), but variability in assay performance exists at starting trigger concentrations <10 fmol/L.
To be useful for clinical diagnostics, this assay must be coupled with trigger generation from a genomic target DNA sequence. One approach for trigger generation, called fingerprinting, is based on adjacent nicking enzyme recognition sites within genomic DNA (Fig. 1A
). When these sites are oriented head-to-head, both strands are cut by the nicking enzyme, and the genomic DNA dissociates, creating templates for 2 complementary triggers that are linearly amplified through consecutive cycles of polymerase extension and single-strand nicking, (analogous to linear amplification within EXPAR; see Fig. S1 in the online Data Supplement). Fingerprinting can be performed isothermally at the same temperature and in the same mastermix as EXPAR, without additional reagents. Differential HSV diagnosis requires distinction from other pathogens and from human genomic DNA. On the basis of in silico sequence analysis, HSV1 fingerprinting produces 14 trigger oligonucleotides without overlap with the predicted trigger sequences of the other human herpes viruses (see Table S1 in the online Data Supplement), other potentially interfering pathogens, or of human genomic DNA.
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In this proof-of-principle study, we targeted a 28-mer fingerprinting site within the open reading frame of the nuclear protein UL3 (Human herpesvirus 1) gene UL3 containing 2 GAGTC sites oriented head-to-head. We have performed fingerprinting with a preliminary model system, a pUC19 vector with a 273-bp HSV1-derived insert that contains the targeted UL3 fingerprinting site (Table 1
, sequence 1) and enables generation of the complementary trigger oligonucleotides HSV1-1a and HSV1-1b (Table 1
, sequences 2 and 3). Of these 2 triggers, we have selected HSV1-1a for subsequent assay design. This vector model system (termed HSV1-vector1), which can be obtained inexpensively in pure form and at high concentrations, serves as a practical intermediate step in establishing the assay. The same reaction applies in principle to trigger generation from HSV1 genomic DNA.
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Through negative ion mode liquid chromatography/electrospray ionization TOF mass spectrometry, we verified that HSV1-1a is generated via fingerprinting in the presence of 1 nmol/L HSV1-vector1. No product peaks were observed in the absence of the vector, a finding that corroborates the specificity of the reaction (Fig. 1B
). Because fingerprinting involves linear amplification only, nanomolar starting concentrations of vector are required without coupling to EXPAR. To improve this detection limit, we coupled fingerprinting with EXPAR by including the appropriate X'-X' template in the reaction mixture (Table 1
sequence 4, Fig. 1C
). According to the SYBR Green fluorescence data (Fig. 1D
), fingerprinting coupled with single-stage EXPAR can differentiate 1 pmol/L HSV1-vector1 from nonspecific background amplification, with marginal differentiation at the 100 fmol/L level, a 103- to 104-fold lower detection limit than fingerprinting alone.
We coupled trigger generation and amplification with visual colorimetric detection (Table 1
sequence 6, Fig. 1E
) by including the vector and both templates (X'-X' and X'-Y', Table 1
sequences 4 and 5) in the reaction mixture. After amplification at 55 °C, the reaction was quenched by adding a DNA nanosphere detection reagent, which contains 2 sets of gold nanospheres bearing the 5' and 3' probes, respectively (Table 1
, sequences 7 and 8). During incubation at room temperature for 2 min, reporter Y generated through 2-stage EXPAR hybridizes to and aggregates the DNA nanospheres. After being spotted on a C18 reverse-phase silica thin-layer chromatography plate, aggregated nanospheres yield a blue spot (positive) and monodisperse nanospheres yield a red spot (negative). We are currently able to detect 1 pmol/L HSV1-vector1 (6 x 106 copies in a 10 µL-reaction volume) after 4-min amplification at 55 °C (Fig. 1F
) with good intra- and interassay reproducibility and 4-min amplification time at 55 °C. These results are based on 3 different experiments conducted on 3 different days, each in duplicate. Longer amplification times produce nonspecific background as displayed by the "No Vector" negative control. The 2 additional controls labeled positive and negative signify that DNA nanospheres are stable under the reaction conditions and aggregate in the appropriate manner.
In conclusion, we have demonstrated detection of an HSV1-derived genomic sequence through a simple, rapid molecular diagnostic assay that can be performed in 
10 min and requires only a heating block and minimal consumption of reagents and supplies, characteristics that make this method suitable for point-of-care and limited resource settings. We are performing systematic assay optimization to ensure sensitivity, reproducibility, and robustness required for clinical diagnostic applications, and are investigating the cause of nonspecific background amplification. At a minimum, we are targeting a limit of detection appropriate for diagnosis of HSV from swab samples of herpetic lesions, reported to contain on average 8 x 104 virus particles/mL lysis buffer (19). The proof-of-principle results presented were obtained using a plasmid vector model system with HSV1 insert. We are in the process of establishing the reaction with DNA isolated from clinical samples, and expanding the assay to HSV2. Fingerprinting is the simplest approach for trigger generation from genomic DNA, but has limitations. We are therefore also exploring alternative probe-based trigger generation reactions that can interrogate arbitrary genomic DNA or RNA sequences.
Acknowledgments
Grant/funding support: Funding for this project was provided by National Institutes of Health-National Institute of Allergy and Infectious Disease award 1R21AI064804, by the Keck Graduate Institute, and by ARUP Laboratories.
Financial disclosures: None declared.
Acknowledgments: We thank Megan Buechel and Bruce Irvine for their work in investigating the performance of EXPAR.
References
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