Identification of upper respiratory tract pathogens using electrochemical detection on an oligonucleotide microarray

Abstract

Bacterial and viral upper respiratory infections (URI) produce highly variable clinical symptoms that cannot be used to identify the etiologic agent. Proper treatment, however, depends on correct identification of the pathogen involved as antibiotics provide little or no benefit with viral infections. Here we describe a rapid and sensitive genotyping assay and microarray for URI identification using standard amplification and hybridization techniques, with electrochemical detection (ECD) on a semiconductor-based oligonucleotide microarray. The assay was developed to detect four bacterial pathogens (Bordetella pertussis, Streptococcus pyogenes, Chlamydia pneumoniae and Mycoplasma pneumoniae) and 9 viral pathogens (adenovirus 4, coronavirus OC43, 229E and HK, influenza A and B, parainfluenza types 1, 2, and 3 and respiratory syncytial virus. This new platform forms the basis for a fully automated diagnostics system that is very flexible and can be customized to suit different or additional pathogens. Multiple probes on a flexible platform allow one to test probes empirically and then select highly reactive probes for further iterative evaluation. Because ECD uses an enzymatic reaction to create electrical signals that can be read directly from the array, there is no need for image analysis or for expensive and delicate optical scanning equipment. We show assay sensitivity and specificity that are excellent for a multiplexed format.

Figure 1. Strategy for amplification and labeling of pathogen-specific target.

The one-tube RT-PCR reaction includes three stages: A) Reverse transcription reaction to produce first strand cDNA for viral targets (vRNA); B) Low temperature (56°C), exponential amplification of target and C) High temperature (68°C), linear amplification to product single-strand, biotin-labeled target. Target is labeled by incorporation of biotin-14-dCTP during amplification or by the use of biotinylated reverse primers.

Figure 2. Comparison of assay sensitivity with target labeled by biotin-incorporation and biotinylated primers.

B. pertussis (B.pert, arrow) genomic DNA at 15 ng/ul was diluted to 1∶1,000 (top panels) and 1∶10,000 (bottom panels) and labeled either by biotin-incorporation (left panels) or with biotinylated reverse primers (right panels). The average of type-specific probe group signals in picoamps is shown at the left and identity of pathogen type-specific probe groups is shown below panels. The horizontal bar indicates the assay cutoff, which was determined by the mean value of negative control probes plus 3 standard deviations of the mean.

Figure 3. Pseudoimage of raw data from scanned chips illustrating Mycoplasma pneumoniae (A) and coronavirus 229E (B) detection.

The left panels (Probe Map) show the position of all pathogen-specific probes and include 3 sub-type probes for coronavirus and parainfluenza virus. Geometric shapes indicated by arrows are areas on the microarray dedicated to array quality control probes. Positive control target was included only with M. pneumoniae (A). Coronavirus probes are divided into three sections on the array: 1) Type 1, 2) Type 2, and 3) Type 3.

Figure 4. URI chip validation studies showing average RNA genome (A) and DNA genome (B) target signal intensity for the 10 upper respiratory pathogens studied.

The left panels (GENOTYPE) show the average genotype-specific probe signal in picoamps that is used to determine a genome identity. The right panels (PROBES) illustrate the average probe signal intensities of 12 replicates for each probe and also illustrate signal specificity for both positive and negative probes. Pathogens are ADV (adenovirus 4), B.p. (Bordetella pertussis), C.p. (Chlamydia pneumoniae), CV (coronavirus), InfA (influenza A), InfB (influenza B), M.p. (Mycoplasma pneumoniae), PIV (parainfluenza virus), RSV (respiratory syncytial virus), and S.p. (Streptococcus pyogenes) at nucleic acid concentrations described in Table 2.

Figure 5. Comparison of assay sensitivity with primer pools 1A, 1B, and 1C.

Mycoplasma pneumoniae (M.p.) DNA, at 1 ng/ul, was serially diluted and amplified with primer pools 1A (3 M.p. amplicons), 1B (2 M.p. amplicons), and 1C (1 M.p. amplicon). Panels in (A) illustrate the sensitivity obtained with the 3 pools at M.p. DNA dilutions of 1∶1,000 (left) and 1∶10,000 (right). Average signal intensities in picoamps for each set of amplicon probes (vertical bars) are shown at the left. The horizontal bar within graphs indicates the assay cutoff (mean 3±SDM). The PCR amplicons analyzed for each pathogen are listed in panel (B) and the three M.p. amplicons are indicated by thick underline.

Figure 6. Results of sensitivity studies for Mycoplasma pneumoniae (A); coronavirus 229E (B); influenza B (C); and PIV 1, and RSV (D).

Vertical bars indicate the mean signal intensities in picoamps for positive genotypes (above cutoff bar) and negative genotypes (below cutoff bar). Horizontal bars indicate the assay cutoff, which is based on the mean of the negative control probes, plus 3 standard deviations of the mean. Relative probe electrochemical signal intensities in picoamps are shown to the left and probe identities are shown below. The identity of the pathogen tested and the dilution factor are shown on each graph. Beginning target nucleic acid concentrations are shown in Table 2.

Figure 7. Example of sensitivity and specificity tests for multiple-organism assays.

Ten genotypes of interest (adenovirus 4, B. pertussis, C. pneumoniae, coronavirus 229E, influenza A, influenza B, M. pneumoniae, parainfluenza Type 1, respiratory syncytial virus, and S. pyogenes) were simultaneous amplified in two PCR reactions (5 DNA and 5 RNA targets) that were combined and hybridized on the same chip. Assays were run with pathogen nucleic acid dilutions of: (A) 1 to 20 (1∶20), (B) 1 to 200 (1∶200), and (C) 1 to 2,000 (1∶2000) that were based on the sample concentrations listed in Table 2. Vertical bars indicate average genotype probe signals in picoamps and horizontal bars indicate the assay cutoff (mean of negative control probes plus three standard deviations of the mean). Asterisks indicate the 10 target genomes that were amplified.

Figure 8. Hybridization and detection of viral and bacterial targets on microarrays.

Biotinylated, single-strand target is hybridized to complementary probes on the microarray (A, B and C) and labeled by the addition of streptavidin (SA)-horseradish peroxidase (HRP) (for electrochemical detection) or streptavidin-Cy3/Cy5 (for fluorescent detection).