Bacterial strains and DNA extraction
Nine toxigenic and 31 nontoxigenic Corynebacterium strains were used in the study. The toxigenic strains included 5 C. diphtheriae clinical isolates, one C. ulcerans clinical isolate, and reference C. diphtheriae strains such as PW8, NCTC 10648, and NCTC 3984. The nontoxigenic strains included 30 C. diphtheriae clinical isolates and the reference C. diphtheriae strain NCTC 10356. Additionally, other bacterial species that might be present in the respiratory tract, such as Streptococcus salivarius, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus epidermidis, Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus, Corynebacterium pseudodiphtheriticum, Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae, were included in the study.
DNA extraction was performed for 24 h bacterial cultures on the medium appropriate for the bacterial species by using DNeazy Blood and Tissue Kit (Qiagen, Germany) according to the manufacturer’s instructions.
Loop-Mediated Isothermal Amplification (LAMP)
LAMP primer sets for the detection of tox gene coding diphtheria toxin and dtxR gene coding global regulator were designed by using LAMP designer software PrimerExplorer V4 (https://primerexplorer.jp/e/). Each LAMP primer set included two outer (F3, B3), two inner (FIB, BIP), and two loop primers (LF, LB). The sequences of the oligonucleotide primer sets used in the study are presented in Table 1. For the detection of amplified products using the lateral flow dipsticks, the FIB and BIP primers were labeled with biotin and fluorescein isothiocyanate (FITC), respectively, at the 5′ end. For the colorimetric detection of amplified products, unmodified primers were used. Modified primers were obtained from Metabion (Germany) and unmodified primers were obtained from Genomed (Poland). LAMP was carried out in a final reaction volume of 25 µl. The concentration of primers in the reaction mixture was optimized for each target individually. Finally, the reaction mixture for both targets contained 0.8 µM of FIB and BIP primers each, 0.2 µM of F3 and B3 primers each, 0.4 µM of LF and LB primers each, 1× reaction buffer containing 20 mM Tris-HCl, 50 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Tween 20 (New England Biolabs, USA), 0.2 mM dNTP (Sigma-Aldrich, USA), 0.2 M betaine (Sigma-Aldrich, USA), 8 units Bst 2.0 DNA Polymerase (New England Biolabs, USA), and 2 µl sample DNA. The reaction was optimized at the temperature ranging from 62°C to 70°C and conducted for 60 min. During the optimization step, the results of the reaction were analyzed using agarose gel electrophoresis.
Detection of product amplification with the lateral flow dipsticks
Milenia HybriDetect dipsticks (Milenia Biotec, Germany) were used for the detection of the amplified products labeled with biotin and FITC. Ten microliters of the reaction mixture were pipetted directly on the sample application area on the dipstick. Then, the dipstick was placed with the same application area into 100 µl of HybriDetect assay buffer and incubated for 5–15 min in an upright position. The results were regarded as positive when two bands were visible (a control band and a test band) or as negative when only a control band was visible.
Colorimetric detection of amplified products
For the colorimetric detection of amplified products, 5 indicators were used comparatively: neutral red, phenol red, hydroxynaphthol blue (HNB), calcein and QuantiFluor. Neutral red and phenol red are pH indicators. They are added to the pre-reaction solution. The progress of LAMP reaction is related to lowering of the solution pH, which can be observed directly as color change of faint orange to pink (neutral red) or red to yellow (phenol red) . Hydroxynaphthol blue and calcein are metal ion indicators. They are also added to the pre-reaction solution. When Mg2+ ion concentration decreases in the progress of LAMP reaction, the color change of the indicators can be observed directly . The color shift is violet to blue for HNB and orange to fluorescent green for calcein. QuantiFluor is a DNA intercalating dye. It is added to the solution after the reaction is completed. When the LAMP reaction is positive, a color change of orange to fluorescent yellow is observed under ambient light condition.
Neutral red (Sigma-Aldrich, USA) and phenol red (Sigma-Aldrich, USA) were dissolved in deionized water or 1 M NaOH, respectively, at 50 mM to prepare a stock solution and diluted to 2.5 mM. For the optimization of the concentration of the indicators in the reaction solution, the following final concentrations were tested: 0.2, 0.15, 0.1, 0.05, and 0.01 mM. HNB was dissolved in deionized water at 50mM to prepare a stock solution. Then, the solution was diluted and tested in the LAMP reaction at the following final concentrations: 1, 0.5, 0.32, 0.25, 0.16, 0.125, 0.08, and 0.04 mM. The calcein stock solution consisted of 0.5 mM calcein (Novazym, Poland) and 10 mM MnCl2 (Sigma-Aldrich, USA) in deionized water. To select an optimal concentration, the following volumes of the stock solution were added to the reaction solution: 0.25, 0.5, 1, 1.5, and 2 µl. The amount of QuantiFluor (Promega, Germany) in the post-reaction solution was optimized by the addition of the following volumes of the dye: 2, 1, and 0.5 µl.
Determination of specificity, sensitivity, detection limit, and minimal reaction time
Specificity and sensitivity of the LAMP were investigated using abovementioned bacterial species that can be present in respiratory tracts. The sensitivity was calculated as follows: A/(A + C) × 100%, and the specificity was calculated as follows: D/(B + D) × 100%, where A is the number of true positive results, B is the number of false-positive results, C is the number of false-negative results, and D is the number of true negative results.
The limit of detection was investigated using 10-fold serial dilutions of the total genomic DNA.
To determine required minimal LAMP reaction time, we examined the results of the reactions for tox and dtxR markers after 10, 20, 30, 40, 50, and 60 min of incubation using 10-fold serial dilutions of the total genomic DNA as a reaction template.