Progress 07/01/21 to 12/31/22

Outputs
Target Audience: Nothing Reported Changes/Problems:We encountered several challenges that resulted in modifications to our initial approach. First, we had initially planned to develop assays for multiple targets including stx1 and stx2 genes as well as the uidA point mutation specific to O157 E. coli. While efforts to detect the stx1b gene were successful we were unable to develop an assay that was suitably selective for the uidA target. Another challenge was developing a test that could be performed in under an hour. The initial electrochemical assay that was developed required not only a 2+ hour thermocycling program but also a multi-step process involving the capture and detection of ligated primers using an immunoassay style approach. By switching to FRET-based detection, we were able to replace the time-intensive electrochemical detection process with a fluorescent-based method that could be performed immediately after completion of the thermocycling step. These modifications allowed for sample-to-answer turnaround time in approximately 90 minutes. We also encountered sensitivity challenges using amplification reactions mediated only by SPAAC ligation reactions. We improved the sensitivity by three orders of magnitude by coupling fluorescent based SPAAC ligation with PCR amplification. This modified approach, called PCR-Enhanced SPAAC Ligation (PESL), allows for high sensitivity detection (~6.0x104 DNA copies/mL) of stx1b target DNA. We found that the use of PCR amplification could be performed without any additional cell lysis or DNA purification steps even when directly testing live E. coli cells. After a short PCR amplification step, SPAAC-enabled primers could be added directly to PCR product for further amplification and detection. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Designed SPAAC ligation primer sets and positive control target DNA sequences for two targets gene sequences specific to O157 E. coli. Designed a 200-nucleotide (NT) single-stranded positive control target DNA sequence that is representative of the stx1b gene sequence present in O157 E. coli. Three primer sets were designed to target the stx1b gene target sequence. Designed a 200-nucleotide (NT) single-stranded positive control target DNA sequence that is representative of the uidA- gene sequence present in E. coli O157:H7. Three primer sets were designed to target the uidA- gene target sequence. Demonstrated SPAAC ligation-mediated target amplification for st1xb and uidA- positive controls. Three primer sets were screened for the ability to amplify the stx1b positive control and the optimal primer set was downselected for further development. Three primer sets were screened for the ability to amplify the uidA- positive control. Electrochemical detection of amplified stx1b target DNA sequence was shown to be sensitive to at least 0.1 pM of target DNA sequence. The selected primer sequence was shown to be both specific to and selective for the stx1b target sequence DNA. Developed a FRET-Enhanced SPAAC Ligation method to improve assay speed and efficiency. Proof of concept of the FRET-Enabled SPAAC ligation was demonstrated using FRET-enabled primers that were not specific to E. coli targets Primer sets downselected for stx1b or uidA- detection were redesigned for FRET-enabled SPAAC ligation and purchased. A methodology for PCR-Enhanced SPAAC Ligation (PESL) using FRET-enabled primers was developed to improve assay sensitivity. Proof of concept of the PCR-Enhanced SPAAC ligation method using FRET-enabled SPAAC primers was demonstrated using non-E. coli primer sets and targets. PCR primers for PCR-Enhanced SPAAC ligation of E. coli stx1b and uidA- targets were designed and purchased. Using the PESL method, we were able to achieve the following milestones: Detection of the stx1b target sequence at a sensitivity of 440 copies/mL purified genomic DNA from O157 E. coli confirmed to have the stx1b target sequence. Selective detection of 440 copies/mL genomic O157 E. coli DNA in the presence of 1000-fold excess non-stx1b K12 E. coli genomic DNA. Detection O157 E. coli bacteria confirmed to have the stx1b gene at a dilution of 1000 cells/mL prior to filter concentration and 100 cells/mL after filter concentration.

Publications

Progress 07/01/21 to 06/30/22

Outputs
Target Audience: Nothing Reported Changes/Problems:We encountered several challenges during this reporting period. The first problem was that we were unable to discriminate between GUD- and GUD+ targets. Although this might be an unrealistic target due to the presence of only a single point mutation, we were able to selectively and specifically detect the stx1b gene. We also had difficulty generating reliable electrochemical data. We have recently found that SPAAC ligation assays can be performed using FRET-enabled primers and we may employ this strategy for E. coli detection. This would obviate any issues with electrochemical detection and would also result in a significantly simpler and faster assay. Finally, we had significant issues with regards to reagent procurement and the acquisition of additional personnel. For example, a delay in the shipment O157 genomic DNA prevented us from being able to generate data for Task 3. The lack of personnel has also strained the ability of our scientists to meet the goals for this program in a timely fashion. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we will use the 20/18nts assay to test for the presence of the Stx1b gene in water samples spiked STEC genomic DNA purchased from ATCC (cat# 43895DQ). We will also test selectivity and specificity using genomic DNA from non-O157 E. coli (ATCC, cat#s MP-10 and 10798DQ). We will also report on a non-electrochemical based detection method that utilizes fluorescent resonance energy transfer (FRET). We believe that this method will reduce test time and improve assay sensitivity and reproducibility.

Impacts
What was accomplished under these goals? Summary of Accomplishments: Two E. coli O157-specific target DNA sequences were identified: Stx1b and GUD-. Three primer sets were designed for click chemistry-mediated amplification of each target DNA sequence. Each primer set was screened for sensitivity, selectivity, and specificity. The Stx1b-specific primer set "20/18nts" demonstrated sensitivity of 0.1 pM (3x106 copies) and was selective for Stx1b in the presence of 10,000-fold excess non-target DNA. Template-specific ligation was demonstrated for 20/18nts primers using gel electrophoresis. Electrochemical detection was demonstrated for 20/18nts primers. The goal of this program is to develop rapid sensor for the detection of pathogenic E. coli O157, also known as shiga toxin-producing E. coli (STEC), in irrigation water sources. We proposed an inexpensive assay that has comparable sensitivity to PCR that can be performed quickly without the need for lab-based sample analysis. To achieve this, we developed an assay that utilizes strain-promoted alkyne-azide cycloaddition (SPAAC) click chemistry to amplify target sequences of nucleic acid (NA). The assay has two major steps, target amplification and detection. Target amplification is performed by combining a set of labeled oligos, referred to as "primers", with target sequence in a reaction buffer. Each primer set consists of four separate primers (labeled as P1, P2, P3, and P4) each modified with a unique set of functional groups to promote chemical ligation in the presence of a target DNA sequence. P1 and P2 are complementary to the target sequence and bind adjacent to each other in the presence of target. P1 is labeled with cyclooctyne (OCT) on the 5' end and P2 is labeled with azide on the 3' end. When annealed to the target NA sequence, azide and OCT are chemically ligated via SPAAC reactions. Ligated P1-P2 then serves as a template to ligate P3-P4. In order to facilitate this process, thermocycling is employed to promote denaturing and annealing, which serves to colocalize unbound primer and to dissociate ligated primer from target. This process drives exponential amplification of target sequence which can then be detected optically or electrochemically. Successive rounds of thermocycling promote amplification of ligated sequences. At the end of the thermocycling process ligated primers anneal to form a single double-stranded primer set containing all four primers. Once this duplex has formed, it can be captured with streptavidin via the 3' biotin tag on primer 4 and detected with horseradish peroxidase (HRP)-labeled antibody against the 3' dinitrophenol (DNP) tag on primer 1. Next, we designed two synthetic target oligos that were representative of sequences that are unique to shiga toxin-producing E. coli O157 strains. The first target we selected was the stx1b gene, which codes for the shiga toxin type 1 B subunit. We identified a 200 nucleotide (NT) sequence ranging from position 2639964 to 2640163 in E. coli O157:H7 strain 611 (Sequence ID: CP038428.1) that was used to synthesize synthetic single strand (ss) target DNA. Using this sequence, we created three unique primer sets that are designed to detect different regions within this target. The naming of each primer set is based on the number of nucleotides in each complementary primer pair. Primer sets "28/24nts", "22/26nts", and "20/18nts" were created for the detection of Stx1b. The second selected target was a highly conserved point mutation in the uidA gene that is present in O157 strains. This gene codes for β-glucuronidase (GUD) and is inactive in most O157 strains due to the point mutation. We selected a 200-NT positive control sequence ranging from position 2034761 to 2034960 also in strain 611, designated "GUD-". As a negative control, we designed an identical sequence that differs only by a mismatch at position 72 from a guanine to a thymine called "GUD+". This sequence is present in many non-O157 E. coli strains. We also developed three primer sets designed to detect the GUD- sequence by annealing to the region of the target where the mismatch occurs. These primer sets were called "17/11nts", 18/12nts", and "16nts". Primer sets specific for either Stx1b or GUD- targets were evaluated for sensitivity using replicate samples containing target DNA titrated from 1000 pM to 0.1 pM, including unspiked negative controls. Each primer set was screened for optimal annealing temperatures. The sensitivity of each primer set was evaluated using a preliminary assay cut point calculated using the formula 'mean negative control (NC) result + 3X NC standard deviation. Of the three Stx1b-specific primers, 20/18nts outperformed 28/24nts and 22/26nts. This primer set was associated with high sensitivity (0.1 pM or ~3X106 copies) and linear association with target concentration. We down selected this primer set for selectivity and specificity testing. By contrast, each of the GUD- primer sets performed nearly identically, although 17/11nts and 18/12nts had better sensitivity than 16nts (10 pM vs. 100 pM, respectively). We evaluated selectivity and specificity of 20/18nts using Stx1b target oligo that was prepared alone or in the presence of 100 nM of non-target oligo. In this case, we used a 124-NT oligo designed to represent a region of the mip gene found in Legionella pneumophila. Specificity was evaluated by testing a dilution series of NC mip oligo in the absence of stx1b. This experiment showed that 20/18nts is selective for and specific to Stx1b. Mip NC oligo did not generate signal at any concentration and Stx1b oligo prepared with 100 nM mip performed nearly identically to Stx1b tested alone. Non-target sequences have virtually no impact on 20/18nts assay performance. Unlike Stx1b, all the GUD- primers demonstrated equivalent ability to detect GUD-. We therefore decided to perform selectivity and specificity testing for all three primer sets. GUD+, which is identical to GUD- with the exception of a single point mutation, was used for selectivity and specificity testing in place of mip. We also tested selectivity using carrier DNA at 100 µg/mL. The three GUD- primer sets detected GUD- in the presence of carrier DNA but not in the presence of 100 nM GUD+. When GUD+ was tested alone, the results were similar to GUD- alone, suggesting that these primer sets are incapable of distinguishing the target based on a single mismatch. We also characterized primer ligation using gel electrophoresis. Stx1b-specific 20/18nts primers were prepared either alone, in the presence of Stx1b target, or non-target mip oligo from L. pneumophila and then tested with or without thermocycling. Unlike the previously described SPAAC assay which uses 25 nM of each primer, gel experiments require a significantly higher primer concentration for visualization. Therefore, each primer was tested at 2 µM and target and non-target oligos were tested at 0.2 µM. Non-thermocycled and thermocycled samples were then loaded onto a 4% agarose sodium borate gel and run at 120V for ~2hrs. GeneRuler Ultra Low Range DNA Ladder was used to demonstrate product size and the gel was stained using SybrSafe gel stain. Non-thermocycled primers prepared alone, with Stx1b, or non-target mip produced ~25-NT bands, demonstrating that no primer ligation occurred in the absence of thermocycling. Overall, the gel analysis showed that the presence of template significantly increased ligation efficiency relative to non-templated samples. We collected electrochemical data for the best 20/18nts samples from the temperature screen experiment for the 83.4°C denaturing and 47.9°C annealing temperature conditions. NC, 0.1 pM, 1 pM, and 10 pM samples were tested in duplicate by adding 50 µL of each sample to screen-printed carbon electrodes followed by chronoamperometry measurements. Signal >NC was observed at each target concentration.

Publications