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

ABSTRACT:

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.

SUBMITTER: Lai YYL 

PROVIDER: S-EPMC11341660 | biostudies-literature | 2024 Aug

REPOSITORIES: biostudies-literature