Establishment And Application Of RPA-LFS Rapid Detection Methods For Porphyromonas Gingivalis

ObjectiveIn this study,we will establish a field rapid detection method of P.gingivalis based on recombinant enzyme polymerase amplification（RPA）and lateral flow test strip（LFS）technique,and the established RPA-LFS detection method will be evaluated in terms of specificity,sensitivity and feasibility.MethodsRPA is a new nucleic acid amplification method,which can be combined with LFS visualization method coated with colloidal gold,which has been developed for rapid detection of P.gingivalis.We use the 16 Sr RNA sequence of P.gingivalis standard strain（ATCC33277）as the target of RPA analysis to design primers and probes,and introduce specific base mismatches between primers and probe sequences to eliminate the false positive signal caused by primer-probe dimer,and finally select a group of specific primer probe combinations to amplify the target sequence.At the temperature of 25℃-48℃,reaction 5-40 min was used to optimize the reaction conditions to determine the best reaction temperature and time.20 clinical isolates of P.gingivalis and 20 strains of other common pathogenic bacteria were detected by RPA-LFS to verify the specificity of this method.The sensitivity of RPA-LFS method was detected by 10 times gradient diluted P.gingivalis genomic DNA,and the lowest detection limit of RPA-LFS method was determined by Probit probability regression analysis.Human genome DNA was added to detect whether the sensitivity of the system can resist the interference of human genome.Finally,RPA-LFS method and PCR method were applied to detect subgingival plaque samples from 130 patients with chronic periodontitis to verify the feasibility of RPA-LFS method.ResultsIn this study,we used the 16 S r RNA gene sequence of P.gingivalis standard strain（ATCC 33277）as template to design and screen primers and probes.After base mismatch,we obtained a set of best primer-probe combination #2F/m R/m P2.Finally,we established a set of RPA-LFS standard detection method for P.gingivalis.Under the optimized conditions,the RPA reaction can be amplified only by 25 min at the constant temperature of 37℃,and the amplification products can be detected intuitively by LFS without special equipment.The established RPA-LFS method can specifically and accurately detect 20 clinical isolates of P.gingivalis and distinguish P.gingivalis from other pathogens.This method has high sensitivity,and this sensitivity is not affected by human genome DNA,and the lowest detection limit is 9.27 CFU/μL.In the detection of subgingival plaque samples from patients with chronic periodontitis,118 cases were positive and 12 cases were negative by RPA-LFS.The results were consistent with those of conventional PCR,and the coincidence rate is 100%.However,compared with conventional PCR,the reaction time of RPA-LFS method is shorter.ConclusionIn this study,a rapid,specific,sensitive and accurate method for field detection of P.gingivalis was established based on RPA-LFS technology.This method helps to simplify the tedious work flow of the previous detection methods of P.gingivalis,and does not rely on laboratory equipment,which may not only meet the needs of chair-side diagnosis,but also achieve large-scale on-the-spot detection,and which has important guiding significance for the early diagnosis and clinical intervention of oral diseases and related systemic diseases.