Past research has demonstrated the substitution of dTTP for dUTP in the modification of amplified products. While this approach is still not widely adopted, a handful of studies have employed this method to label products of polymerase chain reaction (PCR) 25,26, rolling circle amplification (RCA) 16, recombinase polymerase amplification (RPA) 17,27, and loop-mediated isothermal amplification (LAMP) 7,9,14,15 techniques.
Previous studies have demonstrated the modification of amplified products through the utilization of labeled nucleotides. These investigations highlight that the efficiency of amplification and the incorporation effectiveness are dependent on factors such as the structural features, size, and concentration of the used labeled nucleotides 9,16.
In this study we showed the successful incorporation of 5-(3-Aminoallyl)-2'-deoxyuridine-5'triphosphate, labeled with 5/6-TAMRA (TAMRA-dUTP) in four different M. tuberculosis gene sequences directly during the LAMP reaction. To our knowledge, this is the first time TAMRA-dUTP has been used in direct labeling during the LAMP process.
Performing different detection methods such as rtLAMP, fluorescence spectroscopy and agarose gel electrophoresis, we were able to characterize TAMRA-dUTP incorporation into amplicons during LAMP reaction. Furthermore, the effect of the labeled nucleotides on the LAMP process and the ratio of incorporation were analysed.
Warmt et al. demonstrated a positive correlation between the fluorescence intensity of the amplicons in LAMP and the increasing concentration of labeled nucleotides. In that study, the quantities of Cy5-dUTP employed per LAMP reaction ranged from 4 µM (0.28%) to 200 µM (12.5%). Notably, employing 1.5% Cy5-dUTP introduced a 3.5-minute delay in the LAMP reaction. The study suggested an optimal ratio of 1–2% of fluorescence-labeled dUTP, striking a balance between fluorescence intensity and the speed of amplification.
Similarly, in this study, the best ratio of labeled nucleotides used in a LAMP reaction was determined to be 10 µM (0.5%) TAMRA-dUTP to 2 mM non-labeled nucleotides. A delay in the onset of the amplification reaction was observed to be an effect of using higher concentrations of labeled nucleotides. An Onset-value shift of more than 11.00 min from the non-labeled LAMP reaction to 1% amount of TAMRA-dUTP per reaction in samples was observed. While using 2% or higher concentrations was seen to inhibit LAMP reaction completely. Based on that, TAMRA-dUTP/dTTP substitution rate was assumed to be 1 per 200 dTTP when using 0.5% labeled dUTP during the LAMP reaction.
Comparable inhibitory effects were observed in rolling circle amplification (RCA) reactions with incorporation of (sulfo-Cy3-dUTP, sulfo-Cy5-dUTP, sulfo-Cy5.5-dUTP, BDP-FL-dUTP, and NH2-dUTP) labeled nucleotides. It was reported that reaction yields decreased with an increasing percentage of labeled nucleotides. Consequently, an optimal percentage of each labeled nucleotide had to be determined to avert the observed reduction in reaction efficiency 16.
Four different gene sequences in M. tuberculosis were successfully labeled using TAMRA-dUTP in LAMP. Amplification efficiency and incorporation rate were compared between these four DNA targets. It was observed that the total TAMRA fluorescence intensity depends highly on the length of the amplified fragment. The shortest amplicon (pncA, 162 bp) had more than double the fluorescence signal intensity compared to the longest amplified fragment (IS6110 ,273 bp). To further analyse the total incorporated TAMRA-dUTP in each amplification reaction of the target genes, a calibration curve was constructed from the measurement of the signal intensity of a serial dilution of the free labeled nucleotide. We observed that LAMP reactions of the shortest fragment had 40% more TAMRA-dUTP when compared to the longest fragment. Further results determined that the variation in TAMRA signals stemmed from differing amplification rates associated with the length of the target DNA product.
Similar results have been reported in the literature, evaluating the fragment length in recombinase polymerase amplification (RPA) reactions to fluorescent signal intensity on incorporated Cy5-labeled nucleotides 27. It was observed that the shortest RPA fragment (141 bp) gave a 20-fold higher fluorescence signal compared to the largest fragment (809 bp).
Furthermore, this study revealed that the incorporation rate of TAMRA-dUTP was relatively consistent regardless of the length of the amplified target gene. However, the length of the amplicon significantly influences amplification efficiency, correlating with the quantity of generated products. Amplification of the DNA target was seen to increase as the fragment length decreased. Consequently, this gives rise to varying quantities of labeled amplicons generated with distinct DNA targets. This was evident through the measurement of TAMRA signal intensities in target DNA products with equal concentrations. Notably, it was seen that there was a slight increase in the signal intensity of the 16S rRNA gene sequence amplicon (Fig. 3c), which could be related to the higher proportion of thymidine (dT) within the DNA sequence, compared to the other DNA targets.
Length of the amplified product can indeed impact both amplification efficiency and the quantity of amplicons produced (Holm et al., 2021; Shagin et al., 1999). The effectiveness of LAMP is influenced by the size of the target DNA due to the involvement of strand displacement DNA synthesis as a rate-limiting step in this method. It has been determined that optimal outcomes were achieved with DNA target size smaller than 200 base pairs, while DNA of more than 500 bp amplified, but very poorly 3.
Additionally, upon conducting a comparative analysis of amplification levels in LAMP reactions that incorporated labeled vs. unlabeled nucleotides, it became evident that the incorporation of TAMRA-dUTP led to an overall reduction in amplification efficiency. This reduction in LAMP products ranged from 9–17% when compared with unlabeled reactions. These results reinforce the observed impact of TAMRA-dUTP incorporation in diminishing the efficiency of amplification within LAMP reactions.
The assessment of the labeling LAMP reaction's sensitivity and efficacy at lower DNA copy numbers involved conducting a dilution series using M. tuberculosis DNA strain. The results indicated that the labeled amplification reaction remained functional even at a minimum of 10 DNA copies per reaction. This finding underscores that the utilized concentration of 10 µM TAMRA-dUTP was satisfactory for effective labeling of M. tuberculosis even at low copy numbers. Importantly, this concentration of labeled nucleotide did not impede the reaction's progression despite the lower amount of target DNA in the reaction.
This study showed the successful integration of the TAMRA fluorophore into four distinct target genes of M. tuberculosis. This incorporation was achieved through a direct labeling technique applied during the LAMP process. Moreover, the efficiency of both their amplification and TAMRA integration exhibited variability based on factors such as the length of the amplified product and the concentration of the labeled nucleotide implemented. Furthermore, it was established that TAMRA-dUTP exhibits a consistent incorporation rate throughout LAMP reactions. However, variations in the signal output align with the quantity of generated amplicons, a relationship directly influenced by the length of the DNA fragment.
In conclusion, employing labeled nucleotides for direct amplicon labeling during LAMP reactions provides adaptability and harmonizes with various detection methods like fluorescence spectrometry and microarray technology. This enables simultaneous analysis of multiple LAMP products, eliminating the need for an extensive array of labeled primers and probes, as well as the need for additional post-amplification labeling steps; thus, saving time and resources, and making it suitable for point-of-care settings where rapid and accurate results are essential. Nonetheless, when applying TAMRA-labeled nucleotides in LAMP reactions, certain factors warrant consideration. These factors encompass the effectiveness of the chosen primer set, the length of the target gene arranged for amplification, the elongation time, and the proportion of labeled nucleotides integrated per reaction.