qPCR assay optimisation for a clinical study comparing oral health risk in Rett syndrome

Purpose This study aimed to validate qPCR assays for specific microbiota, for use on dental plaque samples stored on Whatman FTA cards to compare relative oral health risk in Rett syndrome. Methods Supragingival dental plaque samples were collected, using a sterile swab, (COPAN FLOQswab™) swabbed onto Whatman FTA™ cards. DNA extraction was performed using a modified Powersoil™ protocol. Where published assays were unsuitable, species-specific qPCR assays for caries-associated, gingivitis-associated and oral-health-associated bacteria were designed using multiple sequence alignment, Primer3Plus and PrimerQuest. Assays were run using absolute quantification. Limit of detection (LOD) and limit of quantification (LOQ) were calculated, and PCR products verified by Sanger sequencing. Results Most assays allowed detection using real-time qPCR with high specificity on samples collected on FTA cards. Several assays showed low or even single gene copy numbers on the test samples. Conclusion Assays were optimised for detection and evaluation of oral health risk in dental plaque samples stored on FTA cards when cold storage is not feasible, except for F. nucleatum. Several assays showed gene copy numbers less than the LOQ or outside the range of the standard curve, so there is merit in optimising these assays using digital droplet PCR. Supplementary Information The online version contains supplementary material available at 10.1007/s40368-024-00912-8.


Figure 1b Figure 2 .
Figure 1bPerformed at 55°C annealing temperature on 3 October 2023.Non-precious samples were taken from broth culture which contained the respective species F nucleatum subspp.fusiforme, P gingivalis and S. mutans.Results showed a single band for S. mutans, S. wiggsiae, P. gingivalis and F. nucleatum subspp.fusiforme.NEB= New England Biolabs; NTC= no template control

Figure 3 .Figure 4 .
Figure 3. MSA For C Durum Sanger reverse complement for the reverse reading sequencing results (including SNPs) superimposed on Primer3Plus output of amplicon.The entire target amplicon sequence for C durum is shown, with forward primer (purple), probe (black), reverse primer (yellow) and multiple sequence alignment result for the amplicon and the Sanger sequencing product (blue highlight) highlight denotes an aligned sequence, with exception of single nucleotide polymorphism (shown in yellow highlight)

Figure 5 .
Figure 5. MSA For R dentocariosa Sanger reverse complement for the reverse reading sequencing results superimposed on Primer3Plus output of amplicon.

Figure 7 .
Figure 7. MSA For F. nucleatum subspp.fusiforme Sanger reverse complement for the reverse reading sequencing results superimposed on Primer3Plus output of amplicon.

Figure 8 .
Figure 8. MSA for P.gingivalis Sanger forward reading sequencing results (including SNPs) superimposed on Primer3Plus output of amplicon.
Figure 17.MSA For P.gingivalis Sanger reverse complement for the reverse reading sequencing results superimposed on Primer3Plus output of amplicon.The entire target amplicon sequence for P. gingivalis is shown, with forward primer (purple), probe (black), reverse primer (yellow) and multiple sequence alignment result for the amplicon and the Sanger sequencing product (blue highlight) Figure 19.MSA for S. mutans Sanger forward reading sequencing results (including SNPs) superimposed on Primer3Plus output of amplicon.The entire target amplicon sequence for S. mutans is shown, with forward primer (purple), probe (black), reverse primer (yellow) and multiple sequence alignment result for the amplicon and the Sanger sequencing product (blue highlight)

Figure 21 .
Figure 21.MSA for S. mutans Sanger reverse complement for the reverse reading sequencing results superimposed on Primer3Plus output of amplicon.The entire target amplicon sequence for S. mutans is shown, with forward primer (purple), probe (black), reverse primer (yellow) and multiple sequence alignment result for the amplicon and the Sanger sequencing product (blue highlight)

Figure 23 .
Figure 23.MSA for S. wiggsiae Sanger forward reading sequencing results (including SNPs) superimposed on Primer3Plus output of amplicon.The entire target amplicon sequence for C durum is shown, with forward primer (purple), probe (black), reverse primer (yellow) and multiple sequence alignment result for the amplicon and the Sanger sequencing product (blue highlight)

Figure 25 .
Figure 25.MSA for S. wiggsiae Sanger reverse complement for the reverse reading sequencing results superimposed on Primer3Plus output of amplicon.
Supplement 2 Table 2 DNA extraction results from DNA extraction