Comparison of pre-labelled primers and nucleotides as DNA labelling method for lateral flow detection of Legionella pneumophila amplicons

Abstract

Labelling of nucleic acid amplicons during polymerase chain reaction (PCR) or isothermal techniques is possible by using both labelled primers and labelled nucleotides. While the former is the widely used method, the latter can offer significant advantages in terms of signal enhancement and improving the detection limit of an assay. Advantages and disadvantages of both methods depend on different factors, including amplification method, detection method and amplicon length. In this study, both methods for labelling PCR products for lateral flow assay (LFA) analysis (LFA-PCR) were analysed and compared. It was shown that labelling by means of nucleotides results in an increase in label incorporation rates. Nonetheless, this advantage is negated by the need for post-processing and competitive interactions. In the end, it was possible to achieve a detection limit of 3 cell equivalents for the detection of the Legionella-DNA used here via primer labelling. Labelling via nucleotides required genomic DNA of at least 3000 cell equivalents as starting material as well as an increased personnel and experimental effort.

Figure 1

Illustration of the amplification product labelling and subsequent detection on an antibody-based lateral flow assay. (A) Preparation of dual-labelled PCR product using FITC- and biotin-conjugated primers. (B) Labelling of the PCR product by incorporation of pre-labelled nucleotides (e.g. fluorescein and biotin conjugated dUTP). (C) Detection of the dual labelled primer-derived amplicon via lateral flow assay as a general example for the LFA detection of dual-labelled amplification products. Detection is achieved by the binding of anti-FITC antibodies to the fragment labelled with FITC and the subsequent binding of the biotin via biotin ligands on the LFA test line. Gold nanoparticles (Au) attached to the anti-FITC antibody are used to make the nucleic acids visible.

Figure 2

Gel electrophoresis after amplification of various gene regions from Legionella pneumophila and subsequent LFA detection of the mip149 amplicon. (A) Amplification products of different genes and sequence lengths are shown both with and without labelled primers (F− unlabelled forward primer, F+ forward primer labelled with FITC, B− unlabelled reverse primer, B+ reverse primer labelled with biotin, NTC no template control, M molecular weight marker, the numbers of the fragment names correspond to the fragment lengths to be expected). (B) Detection of the mip149 amplicon after labelling with FITC and biotin primers. To determine the detection limit of the test strips, the original PCR product (uppermost test strip) was diluted and analysed as a concentration series (V1 to V6) by LFA.

Figure 3

Gel electrophoresis after amplification of the gene fragment mip149 from Legionella pneumophila with fluorescein- or biotin-conjugated nucleotides. For labelling, different concentrations (2–20 µM) of the labelled dUTPs were used in the PCR reaction (Fluo-dUTP fluorescein-conjugated nucleotides, Bio-dUTP biotin-conjugated nucleotides, NTC no template control for each primer combination, M molecular weight marker).

Figure 4

LFA-comparison of the different labelling methods. The labelling of the amplicons was performed both exclusively with primers or with nucleotides and in combination of the two methods. The rows show the concentration of the labelled nucleotides used (c [µM]) for amplification of the different fragment lengths (amplicon). The minimum amount of PCR product [pg] and the resulting amount of substance of the fragments of different lengths (number of amplicons) that was necessary to be able to recognise a signal on the LFA with the naked eye were determined. The images of the LFA strips were digitally processed in terms of contrast, exposure and sharpness in such a way as to make the weak test strip visible on the digital images (see “Methods” section).

Figure 5

Detection limit of the LFA-PCR assay with three different labelling variations. Shown are the LFA strips for the detection of a 25 cycle PCR with different initial concentrations of genomic DNA during amplification. The samples which were amplified with FITC-forward primer/Bio-reverse primer were added to the test strips directly after PCR. All other samples of different labelling methods were purified with magnetic beads before LFA analysis. The images of the LFA strips (NTC to 1.2E−03 ng genomic DNA) were digitally processed in terms of contrast, exposure and sharpness in such a way as to make the weak test line visible on the digital images (see “Methods” section). NTC no template control.