A total of 39 bacterial isolates including 16 isolates of P. aeruginosa, 8 isolates of Pseudomonas spp. and 15 isolates of other bacteria were used in the present study (Table I). The 16S rRNA gene amplifications were performed to verify the bacterial identification as described in a previous report (23). The origins and sources of all 40 bacterial isolates tested were indicated in Table I. All bacterial isolates were grown for overnight at 37°C in trypticase soy agar (TSA). A single colony from TSA was picked and inoculated into trypticase soy broth (TSB) at 37°C for overnight.

DNA was extracted from bacteria cultured in TSB by using QIAamp DNA mini kit (Qiagen, Inc., Valencia, CA, USA) according to the manufacture's manual. The extracted DNA was stored at −20°C until use and an isolate of P. aeruginosa (PA07) ATCC was used for the assay of optimization and sensitivity testing with pure culture.

The DNA extracted from bacterial samples was used as a template for PCR amplification. The PCR amplification of ecfX gene was performed as described in previous reports (3). Briefly, the PCR was carried out with primers ECF 1 (ATGGATGAGCGCTTCCGT) and ECF 2 (TCATCCTTCGCCTCCCTG) for 35 cycles, each of which consisted of denaturation at 94°C for 45 sec, annealing at 58°C for 45 sec and extension at 72°C for 30 sec. The PCR amplicon size was 528 bp. PCR products were separated by electrophoresis on 1% (w/v) agarose gel and visualized using a gel documentation system (Dynamica GelView Master).

A set of four primers consisting of 2 outer primers (F3 and B3) and 2 inner primers (FIP and BIP) recognized six distinct regions on ecfX gene (GenBank, accession no. DQ 996559.1) of P. aeruginosa was designed using PrimerExplorer ver. 4 (http://primerexplorer.jp/elamp4.0.0/index.html). The FITC-labeled oligonucleotide probe was synthesized and labeled with a thiol group at 5′-end (Bio Basic Inc., Markham, Ontario, Canada). The sequences of primers and probe are shown in Table II.

In order to prevent the carryover contamination of LAMP product, the UDG-LAMP reactions were performed as described by a previous study with some modification. The dTTP in a standard dNTP mix was partially replaced with dUTP and UDG was used to degrade uracil-labeled LAMP amplicons (22).

The UDG-LAMP assay was carried out in a total of 25 µl reaction mixture consisted of 40 pmol each of the inner primers (FIP and BIP), 5 pmol each of the outer primers (F3 and B3), 1.4 mM each of dATP, dGTP, dCTP and dUTP: dTTP (different ratios at 100% dUTP; 80% dUTP + 20% dTTP; 60% dUTP + 40% dTTP; 40% dUTP + 60% dTTP; 20% dUTP + 80% dTTP; and 100% dTTP) (New England Biolabs, Inc., Ipswich, MA, USA), 5.0 mM of MgSO₄, 0.8 M Betaine (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), 8 U of Bst 2.0 DNA polymerase (New England Biolabs), 5 U of UDG (New England Biolabs), 1X of supplied buffer and DNA template. The reaction mixture was incubated at 37°C for 5 min. and then further incubated at 60, 63 and 65°C for 60 min to determine the optimal temperature. To establish the optimal time, the UDG-LAMP reaction was carried out at pre-determined temperature of 65°C for 30, 45, 60, 75 and 90 min and heated at 80°C for 10 min to terminate the reaction. The products were analyzed by 2% agarose gel electrophoresis and visualized using a gel documentation system (Dynamica GelView Master).

The AuNPs probe was prepared according to a previous report (21). Briefly, 4 ml of colloidal AuNPs with diameter of 10 nm (Sigma-Aldrich; Merck KGaA) was added to 2.5 nmol of the thiol-probe and incubated with shaking at 150 rpm, 45°C for 16 h. The following solutions consisted of 4 µl of 10% SDS, 400 µl of 100 mM phosphate buffer, pH 7.5 (0.02 M NaH₂PO₄·H₂O, 0.08 M Na₂HPO₄·7 H₂O) and 200 µl of 2 M NaCl were added to the mixture and incubated at 45°C, 150 rpm shaking for 48 h. The AuNPs-probe was precipitated with centrifugation at 20,000 × g, 4°C for 30 min. The pellet was washed twice with 700 µl of solution containing 10 mM phosphate buffer, 100 mM NaCl, and 0.01% (w/v) SDS. Finally, the pellet was resuspended in 700 µl of 10 mM phosphate buffer, monitored for absorbance at 525 nm in the range of 0.3–0.4, stored at 4°C and protected from light.

To establish the optimal salt concentration for induction of free probe aggregation, 5 µl of MgSO₄ was added to the mixture (the total volume of 15 µl) to achieve the final concentration of 5, 10, 20, 40, 100 and 200 mM. The positive reaction (red-purple color) and negative reaction (blue-gray or colorless) were observed and recorded by naked eyes and by UV-visible analysis (Thermo Fisher Scientific, Inc., Waltham, MA, USA).

To optimize the hybridization temperature for detection of UDG-LAMP products, 2.5 nmol of AuNPs-probe solution was added to the UDG-LAMP products in a 1:1 ratio at 57, 62 and 65°C for 5 min. The optimal hybridization time was investigated at 5, 10 and 20 min under pre-determined temperature (65°C).

The specificity of UDG-LAMP-AuNPs and PCR based on ecfX gene was performed to examine the 39 bacterial isolates including 16 isolates of P. aeruginosa, 8 isolates of Pseudomonas spp. and 15 isolates of other bacteria as shown in Table I. DNA templates isolated from bacterial cultures by QIAamp DNA mini kit (Qiagen) were used to evaluate the specificity test.

The sensitivity of the UDG-LAMP-AuNPs assay for the detection of P. aeruginosa in pure culture was performed according to a previous study (24) with some modifications using known amounts of P. aeruginosa (PA 07). Briefly, bacterial cells from a single colony on TSA were inoculated into 4 ml of trypticase soy broth (TSB; Difco) and incubated at 37°C for overnight. Approximately 40 µl of TSB culture was added to a new 4 ml of TSB and incubated at 37°C with shaking at 225 rpm to mid-log phase (OD 600 nm=0.5–0.6). Then, 10-fold serial dilutions of the cultures were prepared in phosphate-buffered saline solution (PBS).

To extract DNA from pure culture, 100 µl of each dilution was transferred into a new microcentrifuge tube and centrifuged at 20,000 × g for 5 min. Then, the supernatant was removed and the pellet was resuspended in 50 µl of 25 mM NaOH and subsequently heated at 95°C for 5 min. After neutralization with 4 µl of 1 M Tris-HCl buffer (pH 7.5), the suspension was centrifuged at 4°C, 20,000 × g for 5 min and used as a template (1 µl) with optimization condition for UDG-LAMP-AuNPs and PCR assays. The detection limit of UDG-LAMP-AuNPs detection methods was compared with that of PCR assay.

In parallel, to count the bacteria colony number, 100 µl of each dilution was spread on TSA in duplicate and incubated at 37°C for overnight. The bacterial colonies were counted at the dilution yielding 30–300 colony-forming units (CFUs) and then the CFU ml⁻¹ of bacterial suspension was calculated.

Contact lens samples (Pretty lens; VASSEN Co., Ltd., Pyeongtaek-si gyeonggido, Republic of Korea) were purchased at a store in Bangkok, Thailand. The contact lens samples were washed with 4 ml of sterile PBS. The DNA template was prepared as described below and tested with PCR specific to ecfX gene as indicated in the above section to confirm that they were negative for P. aeruginosa. The bacterial suspension and adherence of P. aeruginosa to contact lens was employed according to a previous report (25) with some modifications using known amounts of P. aeruginosa (PA 07). Briefly, a single bacterial colony on TSA was inoculated into a 5 ml of trypticase soy broth and incubated for overnight at 37°C. Then, bacterial cells were collected by centrifugation at 2,000 x g for 10 min and the pellet was washed with sterile PBS twice before resuspended in PBS. The absorbance at 660 nm was adjusted to the range of 0.1 which is ~10⁸ CFU ml⁻¹. Sterile contact lenses were transferred into 2 ml of 10⁸ CFU ml⁻¹ of PA07 and incubated at 37°C with shaking at 125 rpm for 24 h.

After 24 h, the contact lenses were removed aseptically and washed gently with PBS to remove loosely attached microorganisms before transferred to 4 ml sterile PBS and vortexed for 1 min to remove the adhered microorganisms. Then, 10-fold serial dilutions of the cultures were prepared in PBS. For DNA extraction, the 100 µl of each dilution was centrifuged at 20,000 × g for 5 min, the supernatant was removed and the pellet was resuspended in 50 µl of 25 mM NaOH and subsequently heated at 95°C for 5 min. After neutralization with 4 µl of 1 M Tris-HCl buffer (pH 7.5), the suspension was centrifuged at 4°C, 20,000 × g for 5 min and the supernatant was used as a template (1 µl) for UDG-LAMP-AuNPs and PCR assay. The sensitivity of UDG-LAMP-AuNPs was compared with that of PCR assay. In parallel, the bacterial colonies were counted as described above.

The results were analysed using the descriptive statistics tests of SPSS version 23.0 software (IBM Corp., Armonk, NY, USA). Data are presented as the mean ± standard deviation of three independent experiments.

To determine the optimal conditions for UDG-LAMP assay, various temperatures for the UDG-LAMP assay were conducted at 60, 63 and 65°C for 60 min. The ladder-like pattern characteristic of LAMP reaction was only observed at 65°C tested temperature; therefore, the temperature at 65°C was chosen for the subsequent UDG-LAMP assays (Fig. 1A).

To determine the optimal time for UDG-LAMP assay, the reaction was conducted at 65°C for 30, 45, 60, 75 and 90 min. The LAMP amplicons could be clearly observed at 60, 75 and 90 min but no amplification was detected at 30 and 45 min. Therefore, the reaction time at 60 min was chosen as optimal time for the UDG-LAMP assay to minimize the reaction time (Fig. 1B).

The LAMP reaction using Bst 2.0 DNA polymerase was well progressed using dUTP in place of dTTP; however, the concentration of dUTP affected the visible of ladder-like pattern characteristic on agarose gel. When dTTP was totally replaced with dUTP, the UDG-LAMP reaction produced no ladder-like pattern (Fig. 2, lane 1). Therefore, the ratio between dUTP to dTTP was varied from 80% dUTP + 20% dTTP; 60% dUTP + 40% dTTP; 40% dUTP + 60% dTTP; 20% dUTP + 80% dTTP; and 100% dTTP. The ladder-like pattern was observed at the ratios of 60% dUTP + 40% dTTP; 40% dUTP + 60% dTTP; 20% dUTP + 80% dTTP; and 100% dTTP (Fig. 2). Therefore, the ratio of 40% dUTP to 60% dTTP was chosen for the subsequent UDG-LAMP assays to study the specificity and sensitivity.

To study the effect of salt concentration (MgSO₄), the UDG-LAMP product were hybridized to AuNPs probe under various concentrations of MgSO₄ including 5, 10, 20, 40, 100 and 200 mM. The results showed that 40 mM MgSO₄ yielded the clear difference between positive sample (red-purple) and negative control (blue-gray or colorless) (Fig. 3A and B). These results were corresponded to LAMP reactions followed by UV-visible analysis (Fig. 3C and D) and the absorbance at 525 nm clearly confirmed that results (Fig. 3E). Therefore, 40 mM MgSO₄ was used for all subsequent assays.

To determine the optimal temperature and time for hybridization between AuNPs probe and UDG-LAMP product, the hybridization was conducted at 57, 62 and 65°C for 5, 10, and 20 min at each temperature. The color reactions demonstrated similar results in all ranges of the tested temperature and time (Fig. 4A). However, the UV-visible analysis revealed that the hybridization at 65°C for 5 min yielded the highest absorbance at 525 nm (Fig. 4D). Therefore, the hybridization performed at the temperature of 65°C for 5 min was chosen for the subsequent reactions.

The specific testing demonstrated that UDG-LAMP-AuNPs and PCR specific to ecfX gene revealed positive results to all tested isolates of P. aeruginosa. While 8 other Pseudomonas species and all non-Pseudomonas bacteria yielded negative results (Table I).

Detection limit of UDG-LAMP-AuNPs and PCR assays with pure culture. To determine the sensitivity of UDG-LAMP-AuNPs and PCR assays for detection of P. aeruginosa (PA07), the stock solution of bacterial culture (1.6×10⁸ CFU ml⁻¹) was diluted in 10-fold serial dilution and DNA extracted from each dilution was used in the UDG-LAMP-AuNPs and PCR assays. The UDG-LAMP and UDG-LAMP-AuNPs assay exhibited the positive result at 1.6×10³ CFU ml⁻¹ or equivalent to ~3 CFU per reaction (Fig. 5A and C), whereas, the PCR assay showed the detection limit at 1.6×10⁴ CFU ml-1 or equivalent to 30 CFU per reaction (Fig. 5B).

The detection limit of UDG-LAMP-AuNPs and PCR assays for PA07 spiked into contact lenses was examined. After removal of loosely attached bacteria, the determined number of bacteria in washed solution was 1.1×10⁶ CFU ml⁻¹. After 10-fold serial dilutions of washed solution was performed, the total DNA extracted from each dilution was used to investigate the detection limit. The results showed the sensitivity of UDG-LAMP and UDG-LAMP-AuNPs at 1.1×10³ CFU ml⁻¹ or equivalent to 2 CFU per reaction (Fig. 6A and C). Whereas, the PCR assay showed detection limit at 1.1×10⁴ CFU ml⁻¹ or equivalent to 20 CFU per reaction (Fig. 6B).