Direct TAMRA-dUTP labeling of M. tuberculosis genes using loop-mediated isothermal amplification (LAMP)

Abstract

Fluorescent molecule-based direct labeling of amplified DNA is a sensitive method employed across diverse DNA detection and diagnostics systems. However, using pre-labeled primers only allows for the attachment of a single fluorophore to each DNA strand and any modifications of the system are less flexible, requiring new sets of primers. As an alternative, direct labeling of amplified products with modified nucleotides is available, but still poorly characterized. To address these limitations, we sought a direct and adaptable approach to label amplicons produced through Loop-mediated isothermal amplification (LAMP), using labeled nucleotides (dUTPs) rather than primers. The focus of this study was the development and examination of a direct labeling technique of specific genes, including those associated with drug resistance in Mycobacterium tuberculosis. We used 5-(3-Aminoallyl)-2'-deoxyuridine-5'triphosphate, tagged with 5/6-TAMRA (TAMRA-dUTP) for labeling LAMP amplicons during the amplification process and characterized amplification and incorporation efficiency. The optimal TAMRA-dUTP concentration was first determined based on amplification efficiency (0.5% to total dNTPs). Higher concentrations of modified nucleotides reduced or completely inhibited the amplification yield. Target size also showed to be determinant to the success of amplification, as longer sequences showed lower amplification rates, thus less TAMRA incorporated amplicons. Finally, we were able to successfully amplify all four M. tuberculosis target genes using LAMP and TAMRA-modified dUTPs.

Figure 1

Determination of appropriate TAMRA-dUTP amount. LAMP reactions were performed for 20 min (65°C), using the 16S rRNA gene in M. tuberculosis DNA strain as target. (a: left) SYBR Green based rtLAMP curves for amplification of 1.00E + 05 copies/µl DNA target per reaction, with various ratios of TAMRA-dUTP (0.5–4%). (NTC: no template control, unlabeled), 0% TAMRA-dUTP: positive control, unlabeled). (a: right) Onset-values and corresponding Onset-value shifts performing amplification with unlabeled and x% TAMRA-dUTP-labeled nucleotides. (b) Gel electrophoresis and (c) graphical visualization of fluorescence signals with purified amplicons after LAMP reaction with different TAMRA-dUTP ratios compared to the unlabeled reaction (NTC no template control).

Figure 2

Detection of four different M. tuberculosis gene sequences: IS6110, 16S rRNA, katG, and pncA. LAMP reactions were performed for 30 min (65 °C), using 1.00E+05 copies/µl DNA target per reaction, and 0.5% TAMRA-dUTP. (a) Graphical visualization of fluorescence signals and (b) gel electrophoresis with purified amplicons after LAMP reaction with TAMRA-dUTP.

Figure 3

Detection of TAMRA-dUTP in LAMP amplicons. LAMP reactions were performed for 30 min (65 °C), using 1.00E+05 copies/µl DNA target per reaction, and 0.5% TAMRA-dUTP. Incorporation of fluorescent-labeled nucleotides was analyzed by fluorescence spectroscopy. (a) Calibration curve for calculation of incorporated TAMRA-dUTP in amplified target DNA samples. (b) SYBR Green based graphical representation illustrating the quantity of amplified DNA targets, comparing reactions containing TAMRA-dUTP (w/) to reactions without the inclusion of TAMRA-dUTP (w/o). (c) Graphical visualization of TAMRA intensity signals within target amplicons at a concentration of 100 ng/µl, along with the proportion of thymidine (dT) associated with each amplified DNA fragment.

Figure 4

LOD determination for the 16S rRNA gene. LAMP reactions were performed for 30 min (65 °C), using a serial dilution of 10 to 1.00E+05 copies/µl target genomic DNA per reaction, and 0.5% TAMRA-dUTP. Fluorescence intensities were measured of purified amplicons after LAMP reactions with TAMRA-dUTP. (NTC no template control; Buffer: 1 × Isothermal buffer).