Specific and sensitive, ready-to-use universal fungi detection by visual color using ITS1 loop-mediated isothermal amplification combined hydroxynaphthol blue

Microbial strains, culture and crude DNA extraction

Microorganisms used in this study included 30 fungal isolates (22 species in phyla Zygomycota, Ascomycota and Basidiomycota) and 17 bacterial species (in classes Bacilli, Antinobacteria, Chlamydiae, Gammaproteobateria and Betaproteobacteria) (Table S1). All fungi were cultured in potato dextrose agar (20 g dextrose, 200 g potatoes infusion form, and 15 g agar, per 1 L water). Bacteria were cultured in blood agar (trypticase soy agar enriched with 5% human blood). Crude DNA extraction was performed by resuspending isolate colonies of fungi (or bacteria) in 20 μL of Tris-EDTA buffer (10 mM Tris Base and 1 mM EDTA, in pH 8.0), boiling at 95 ^∘C for 15 min (fungi) or 10 min (bacteria), and placed on ice prior to centrifugation at 11,772 × g for 10 min. The supernatant was used as DNA template. Quality and concentration of crude DNA extracts were determined by A260/A280 and A260 nanodrop spectrophotometer, respectively.

LAMP primer design for universal fungi detection

Nucleotide sequences of ITS1 gene from >40,000 fungal species covering all four phyla of kingdom fungi were downloaded from GenBank (http://www.ncbi.nlm.nih.gov/genbank/), and multiple sequence alignments were performed using ClustalW (http://www.megasoftware.net/). Six degenerate primers were manually designed to locate on eight conserved regions on ITS1 and flank variable region among fungal species (https://primerexplorer.jp/e/) (Table 1). The specificity and coverage of each primer was checked using BLASTN (Camacho et al., 2009).

ITS1 LAMP-HNB

LAMP-HNB reaction (15 μ L) comprised 0.2 μM each of primers F3 and B3, 1.6 μ M each of FIP (F1c/F2) and BIP (B1c/B2), 1.4 μ M each of LF and LB, 1.4 mM dNTP (SibEnzyme Ltd., Novosibirsk, Russia), 1 M betaine (Sigma–Aldrich, St. Louis, MO, USA), 6 mM MgSO4, 8 U Bst DNA polymerase (large fragment) (New England Biolabs Inc., Beverly, MA, USA), 1 × ThermoPol^TM Reaction Buffer (New England Biolabs Inc.), 133μ M HNB (Sigma-Aldrich, St. Louis, MO, USA), and 100 ng DNA (unless specified). The reaction conditions (shortest incubation time with highest sensitivity and correct specificity) were optimized using temperatures of 55, 58, 60, and 63 ^oC, and incubation periods of 20, 25, 30, 40 and 45 min. A. flavus MSCU0850 was used as template for finding optimized isothermal condition and a limit of detection of ITS1 LAMP-HNB. Result was analyzed by naked eyes from violet (negative, ∼550–575 nm) to sky blue (positive, ∼650 nm). On 1.75% agarose gel electrophoresis at 100V (GE), the LAMP products appeared as intercalating bands of various base pair (bp) sizes.

ITS1 and 18S rDNA PCR-GE

PCR reaction (25 μL) comprised 0.3 μM each of forward and reverse primers (Table 1: ITS1_F3 and ITS1_B3 for ITS1, and Euk1A and Euk516A for 18S rRNA gene) (Wang et al., 2014), 12.5 μL EmeraldAmp^® GT PCR Master Mix (TakaRa Bio Inc., Shiga, Japan), and 100 ng DNA (unless specified). The thermal cycling condition was 94 ^∘C 4 min followed by 35 cycles of 94 ^∘C 45 s, 50 ^∘C 1 min and 72 ^∘C 1.5 min, and final extension at 72 ^∘C 10 min. The PCR product was analyzed by GE.

Comparison of specificity and sensitivity (detection limit) among ITS1 LAMP-HNB, ITS1 PCR-GE and 18S rDNA PCR-GE

The specificities of the assays were determined with representative fungi (positive) and bacteria (negative) isolates (listed in Table S1). Additional negative controls included crude extracted DNA from human (Homo sapiens) (Somboonna et al., 2018; study was approved by the Institutional Review Board of Buddhachinaraj Phitsanulok Hospital (101/54) and the Ethics Committee for Research in Human Subjects of the Department of Disease Control, Bangkok (FWA00013622)), pork (Sus scrofa), chicken (Gallus gallus) and fish (Pangasius hypophthalmus) meats from local supermarket in Bangkok, Thailand. Crude DNA extraction from additional negative controls was performed by resuspending a sample in 20 μL of Tris-EDTA buffer, boiling at 95 ^∘C for 15 min, and placed on ice prior to centrifugation at 11,772 × g for 10 min. The supernatant was used as DNA template. The detection limits were carried out using a series of 10-fold dilutions of ITS1 and 18S rDNA of A. flavus MSCU0850 in the range of 9.6 ×10 ⁶ to 0.96 (equivalent to 4.6 pg to 0.46 ag) and 9.71 ×10 ⁶ to 0.97 copy numbers (equivalent to 5.9 pg to 0.59 ag), respectively. The results of LAMP-HNB were monitored via naked eyes and confirmed by UV-vis spectra measurement and GE. The performance of ITS1 LAMP-HNB was compared in parallel with ITS1 PCR-GE (using primers corresponding to the same nucleotide region, which were ITS1_F3 and ITS1_B3 in Table 1) and 18S rDNA PCR-GE. Noted that because LAMP for universal fungi had none reported, this assay compared with 18S rDNA PCR-GE from established protocols (Wang et al., 2014). Further, the assay performance of the ITS1 LAMP-HNB was validated using real dry food samples (five samples each of peanuts, chili and garlics from local markets) with and without artificial microbial contamination, and a healthy human blood sample in EDTA buffer (Ethical Committee of the Faculty of Medicine, Chulalongkorn University, E.C.NO.654/60) with artificial microbial contamination, and was compared with PCR-GE. Each microbial isolate was grown in pure culture, then centrifuged and 1 pg microbial isolate pellet was used for artificial microbial contamination. For crude DNA extraction, 0.3 g food sample (or 0.1–0.3 mL blood sample) was mashed and transferred to an empty one mL eppendorf tube, and the sample was irradiated in a microwave oven at ≥ 800 W 5 min, along a presence of water-containing beaker during the irradiation to avoid the microwave damage. Next, 300 mL TE (10 mM Tris-HCl pH 7.5, 1 mM EDTA) was added to resuspend, vortexed, centrifuged at 11772 × g 1 min, and the supernatant was used as template for LAMP-HNB and PCR-GE (Ferreira, Silva & Panek, 1996).

Effect of preserving additive on shelf-life of ready-to-use ITS1 LAMP-HNB reagents

Choices of preserving additives and concentrations (3% and 5% glycerol, and 3% and 5% polyvinyl alcohol (PVA)) were added to the whole ITS1 LAMP reagents including Bst DNA polymerase (representing “ready-to-use LAMP reagents” owning to their containment of every ingredient except HNB and DNA template of interest), and the ready-to-use reagents were stored at 30, 50 and 80 ^∘C for a period of 2-30 days. In brief, the methods of identifying shelf-life of the ready-to-use LAMP reagents with each preserving additive were followed established protocols (Labuza, 1984; Khalid et al., 2011). A fluorescent DNA dye, Novel Juice (BIO-HELIX, Keelung, Taiwan), was used to track the level of LAMP reaction activity (the amplified product) at each time series of storage. The florescent intensity was analyzed by ImageJ software (https://imagej.nih.gov/ij/download.html), and was proportional to the amount of the LAMP products. Subsequently, the reduction in the fluorescent intensity could define a percentage of LAMP activity remaining. Three independent replicates were performed and measured the percent remaining activity per time point to obtain the average and standard deviations. The remaining percentages of the LAMP activity against storage times (days) for each choice of preserving additives and concentrations were plotted, and derived a regressive equation with storage days and remaining % LAMP activities as X and Y variables, respectively, along the goodness-of-fit index (R²).

Primer design and optimal isothermal condition for ITS1

LAMP-HNB

Regions for 6 LAMP primer sequences were designed based on conserved regions among fungal species following ITS1 sequence alignment analysis. Figure S1 displayed minor nucleotide variation among these conserved primer regions, and degenerate primers (Table 1) were thereby designed to cover all possible sequences of fungal species (Iserte et al., 2013). The ITS1 region between 18S and 5.8S rRNA genes is a specific target for universal fungi, and the variable region in ITS1 between our designed primers ITS1_F1 and ITS1_BIP is rather specific to allow identification of fungi at a species level (Iwen, Hinrichs & Rupp, 2002; Blaalid et al., 2013; Yang et al., 2018).

Following a range of reaction temperatures and time durations, the optimal isothermal condition for our LAMP-HNB reaction (shortest incubation time with highest sensitivity and correct specificity, Fig. S2) was found at 58 ^∘C and 40 min, using crude fungal DNA lysate from simple boiling at 95 ^∘C for 15 min (details in Materials and Methods) as a template.

Specificity and detection limit of ITS1 LAMP-HNB

The specificity of ITS1 LAMP-HNB was validated using 7 representative fungal species and 21 non-fungi species (Fig. 1A). Additional specificity assay on 23 fungal isolates with clinically relevance in human or foodborne diseases were also demonstrated (Fig. S3, e.g., Epidermophyton floccosum, Trichophyton rubrum, Cryptococcus neoformans, Microsporum gypseum, Flusarium sp., Phialophora verrucosa, Penicillium marneffei (also known as Talaromyces marneffei) and Candida albicans). Together, all 30 clinically relevant fungal isolates (accounting for 22 fungal species) were properly tested positive using ITS1 LAMP-HNB. The proper color and amplicon results were confirmed by UV-vis spectra (Fig. 1B) and GE (Fig. 1C), respectively. It is important to note that the positive color all had a ratio of A650/A580 >1.00, while the ratio <1.00 corresponded to negative results (Goto et al., 2009; Choopara et al., 2017). The PCR targeting ITS1 and 18S rRNA gene showed identical results (Figs. 2A and 2B), heightening the reliability of our ITS1 LAMP-HNB reaction protocols (compatible to PCR assays) and primers (compatible to other universal eukaryotic primers Euk1A and Euk516A for 18S rDNA) (Wang et al., 2014).

For determination of the detection limit of ITS1 LAMP-HNB, the minimum concentration that could be detected by naked eyes (Fig. 3A) consistent with UV-vis spectra (Fig. 3B) and GE (Fig. 3C), was found to be as low as 9.6 copies (equivalent to 4.6 ag) of ITS1. This limit of detection was compatible with ITS1 and 18S rDNA PCR-GE (Figs. 2C and 2D). The results of our ITS1 LAMP-HNBs were consistent, and thus the data from independent triplicates were able to generate the confident linear regression (goodness-of-fit measurement, R² = 0.98) for quantitative detection of a template with an unknown concentration from the ratio of A650/A580 by UV-vis spectra (Fig. 3D: Y = 0.448X + 0.9846, where Y is A650/A580 and X is log₁₀ copies).

Comparative performances between ITS1 LAMP-HNB and PCR-GE assays on dry food and clinical blood samples detection

The feasibility of the point-of-care ITS1 LAMP-HNB assay was evaluated, starting from 6-min crude DNA lysis to LAMP-HNB and visual color readout, in comparison to ITS1 and 18S rDNA PCR-GE assays. The reasons we compared with PCR-GE were that we could use our ITS1 LAMP primers (Table 1, ITS1_F3 and ITS1_B3) for ITS1 PCR, allowing parallel comparison with no bias on primer region selection, and that the standard methods for nucleotide amplification and amplicon visualization are PCR followed by GE. A total of 15 real dry food samples that might contain fungi contamination (peanuts, chili and garlics) were tested. Three independent experiments were performed. Based on the ITS1 PCR-GE as the standard assay (also the 18S rDNA PCR-GE), our ITS1 LAMP-HNB assay showed identical findings (Fig. 4). Hence, triplicate analyses of the 15 dry food samples by the ITS1 LAMP-HNB assay showed 100% sensitivity (7/7 positives) and specificity (8/8 negatives). Moreover, artificially contaminated dry food samples were performed to confirm the reliability of our ITS1 LAMP-HNB assay by adding A. flavus MSCU0850 culture to autoclaved (sterile) peanut, chili and garlic; meanwhile adding sterile water in replace of A. flavus MSCU0850 to autoclaved dry food sample (negative contamination) showed negative result (Figs. S4A and S4B). Our ITS1 LAMP-HNB was also evaluated its testing reliability on a clinical blood sample from a healthy (no infection) subject, without (as negative control) and with artificially microbial contamination by A. flavus MSCU0850 and A. flavus MSCU0850 mixed E. coli (positive tests) and E. coli (negative test) (Figs. S4C and S4D). Noted the visual color change of HNB may sometimes be unclear because of low presence of fungi or a color bias from an original sample, e.g., in Fig. 4A and Fig. S4, the crude extracted DNA from food samples had creamy white or reddish color.

Preserving additive to extend shelf life of ready-to-use ITS1 LAMP-HNB

Shelf life extension of our ready-to-use ITS1 LAMP-HNB reagents was attempted by adding 3% or 5%, of glycerol or PVA. After an addition of each preserving additive, the same detection limit (4.6 ag) was validated. Figures 5A-5D demonstrated that the higher concentration (5%) and the type of the preserving additive (PVA, instead of glycerol) both affected the shelf life of the ready-to-use ITS1 LAMP reagents. The shelf life at 90% remaining LAMP activity under 30 ^∘C storage using 3% glycerol, 5% glycerol, 3% PVA or 5% PVA as a preserving additive, was found to be 13.08, 13.11, 13.853 and 14.038 days, respectively. The ready-to-use ITS1 LAMP reagents with 3–5% glycerol and 3–5% PVA showed ≥ 50% remaining activity until 40.80–41.07 and 48.16–48.94 days, respectively (Table S2). The derived equations all had >96% goodness-of-fit, which meant that smaller than 5% variation to the estimate was only possible.