Development and Comparison of In-house Line Probe Assay (LiPA) and SYBR Green Real-time PCR Regarding the Detection of Periodontal Pathogens

Periodontal disease, which can become a chronic condition, is an inflammatory disease that upsets the soft and hard structures supporting the teeth. The aim of the present study was to design and develop an in-house Line Probe Assay (LiPA), to detect putative periodontitis-related bacterial pathogens, and compare it with SYBR Green Real-time PCR. The LiPA method was launched using biotinylated 16s rRNA universal primers and specific probes for each of the five bacteria including Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Tannerella forsythia, Porphyromonas gingivalis and Treponema denticola. For this, optimized quantities of the primers and specific probes were dotted onto nylon membrane stripes in a defined pattern. Hybridization was performed between the probes and the single-stranded biotinylated PCR products. The stripes were developed via biotin-streptavidin reaction. Ultimately, the analytical and diagnostic sensitivity and specificity of the in-house LiPA was evaluated and compared with SYBR Green Real-time PCR. The detection limit of the LiPA was 2760 copies of targeted genes. In testing analytical specificity, only signals corresponding to the specific biotinylated products were produced. The calculated diagnostic sensitivity of the LiPA for the five bacterial targets ranged from 96.4 to 100%, whereas the diagnostic specificity was between 90.9 and 100%. Comparing the results, no noticeable difference (p=0.4795) was observed between the two methods. To screen periodontal pathogens, a simple, inexpensive and accurate method is desirable. The in-house LiPA, having advantages such as high specificity and sensitivity, and the ability to detect five major periodontal pathogens, offers the option of evaluating samples without the need for a post-PCR platform.