Simple and rapid detection of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans by loop-mediated isothermal amplification assay

Background: Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans are two main causative agents associated with periodontitis, an inflammatory reaction of tissues around the teeth. The aim of this study was to develop and evaluate the loop-mediated isothermal amplification (LAMP) assay for simple and rapid detection of P. gingivalis and A. actinomycetemcomitans. Methods: A total of ten subgingival plaque and saliva samples were evaluated to detect the presence of both bacteria by LAMP and PCR assays. Two sets of six primers each were designed to amplify pepO and dam gene. The LAMP assay was carried out using a Loopamp DNA amplification kit in 25 μl volumes. The reaction mixture was incubated at 65oC for 60 minutes and terminated at 80oC for 5 minutes in heating block. The amplification reactions were visualized using naked eye detection and by agarose gel electrophoresis. The sensitivity of the LAMP assay was investigated ranging from 10 μg to 100fg of P. gingivalis(ATCC 33327) and A. actinomycetemcomitans (ATCC 33384). Results: The lowest detection limit of both LAMP and PCR methods were 1 ng and 10 ng of DNA, respectively. When crude template of subgingival plaques were used, P. gingivalisand A. actinomycetemcomitans were tested80% (8/10) and 60% (6/10) positive respectively through LAMP detection. Whereas by PCR, P. gingivaliswas tested 40% (4/10) positive and no significant detection rate for A. actinomycetemcomitans. When a crude template of saliva was used, P. gingivalisand A. actinomycetemcomitans were tested 70% (7/10) and 30% (3/10) positive respectively through LAMP detection. Whereas, when using PCR, there was no significant detection rate for P. gingivalisand A. actinomycetemcomitans. Conclusion: The LAMP assay using a crude template offers greater advantage as it is simple, rapid and cost-effective to detect periodontal pathogens.


Introduction
Periodontitis is an inflammation that occur in the gum area of the mouth which lead to loss of the tooth.It is statistically proven (p<0.001) that Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans are two main putative microorganisms responsible to this issue. 1,2,3 s both of the bacteria are anaerobic organisms, there are longer time taken in term of culturing and growing these bacteria by convectional culture methods.These bacteria particularly take 48 to 72 hours to grow in an anaerobic environment consisted of 80% nitrogen, 10% carbon dioxide and 10% hydrogen.Moreover, in-vitro culture of the bacteria is laborious in term of preparation the agar in the petri dish.To overcome all the shortcoming by culture methods, researcher are now using molecular techniques for detection the bacterial pathogens.To date, there are diverse molecular procedures have been developedlike polymerase chain reaction (PCR), qPCR, FISH and hybridization assay for the detection of microbial pathogens. 4,5 mong all, conventional PCR are by a far the most acclaimed and widely used technique.However, there are disadvantages on PCR such like the requirement for a costly thermocyclar and the needed of post enhancement investigation.These downsideshas limit the application of PCR in lowresources setting. 6oop-mediated isothermal amplification (LAMP) is a molecular technique that has been developed by Notomi et al (2000) to amplify a few copies of DNA with high sensitivity and specificity under isothermal conditions in a hour or less. 7Over conventional PCR and qPCR, LAMP has been reported to be simple and cheaper because water bath or heating block can be used as amplification tool instead of thermocycler.As a consequence, LAMP method for detecting pathogens has been described in many previous studies including bacteria and virus.In addition, the amplification product of LAMP are easily accessible by direct visualization of the whiteprecipateformation. Positive reaction of LAMP will produce large amount of white magnesium pyrophosphate due to interaction of pyrophosphate ions with magnesium ions. 8Therefore, this precludes the need for post amplification analysis and hence, decreases cost and manpower. 1,7,9 Tre are also a few studies have been carried out to identify periodontal pathogens by utilizing LAMP. 1,9,10 I addition to that, the aim of this study was to evaluate the LAMP method for simple and rapid detection of P. gingivalis and A. actinomycetemcomitants in clinical samples of subgingival plaque and saliva from periodontallyinfected patients.This study employed a simplified template preparation technique without the DNA extraction step and extended the use of the LAMP technique to the crude template of subgingival plaque and saliva.

Bacterial strains and culture conditions
All bacterial strains used in this study were obtained from the American Type Culture Collection (ATCC) as listed in Table 1.P. gingivalis(ATCC 33327) and A. actinomycetemcomitans (ATCC 33384) were used as reference strains.The P. gingivalis strain was grown on supplemented tryptic soy broth as reported previously. 12A. actinomycetemcomitans was grown anaerobically at 37°C in Todd-Hewiit broth (Difco Laboratories, Detroit, MI) and supplemented with 1.0% yeast extract (Difco Laboratories, Detroit, MI).Other oral bacteria were grown on blood agar (Oxoid, Nepean, CA).The growth of bacteria was observed through microscopy.

Bacterial DNA extraction
Reference bacterial strains from lyophilised stocks were cultured anaerobically on blood agar plates for a span of 5-7 days at 37ºC.Bacterial DNA from reference strains were extracted using a commercial DNeasy Blood and Tissue Kit (Qiagen, Inc., Valencia, CA, USA) according to manufacturer's instructions as follow: The pellet was briefly re-suspended in 180 µl of phosphate-buffered saline (PBS) [0.12M NaCl, 0.01M Na 2 HPO 4 , 5 mM KH 2 PO 4 , pH 7.5].Then, 20 µl of Protease K and 200 µl of Buffer AL were added to the sample andwas incubated at 56°C for 10 minutes.Following lysis, 100% ethanol was added to the samples, and the samples were transferred to spin columns which bind DNA.Wash buffers (AW1 and AW2) were used to remove unbound substances.DNA was eluted using 150 µl of AE buffer and was used as a template for LAMP and PCR assays.The DNA concentration was determined by measuring the A 260 , and its quality was estimated using the A 260 / A 280 ratio.

Study population
Ten periodontitis patients who attended Dental Clinic at Hospital UniversitiSains Malaysia, KubangKerian, Kelantan, Malaysia were selected in this study.All patients showed clinical signs of periodontitis, presented with periodontal pocket depth equal or exceeding 4 mm with radiographic evidence of alveolar loss.The selected patients were those who did not take any antibiotics for the past 3 months prior to the sample collection.

Sample collection
Subgingival plaque and saliva samples were collected from each patient after obtaining their informed consent.Ethical approval was obtained from the Research Ethics Committee (Human), UniversitiSains Malaysia (USM) (USMKK/PPP/ JEPeM[259.4.(5.5)].Briefly, by using sterile Gracey curette, subgingival plaque were obtained by from the periodontal pocket at the deepest part.The plaque was collected by vertical stroke of curette and was suspended into 200 µl PBS.For saliva collection, were made a request to hold their head marginally forward and expectorate all amassed salivainto sterile collection tubes.The samples were transferred on ice and sent to the laboratory for processing.

Preparation of crude template from clinical specimens
The subgingival plaque and saliva samples were homogenized with a glass rod and 200 µl aliquot of each sample was centrifuged at 10,000 X g for 5 minutes.Two µl of the supernatant was used as a template for PCR and LAMP.

PCR assay
The crude templates were subjected to singleplex PCR targeting pepO (P.gingivalis) and dam (A.actinomycetemcomitans) genes.Conventional PCR was performed using forward outer primer (F3) and backward outer primer (B3) for each gene (Table 2).The PCR mixture (25 μL) consisted of 0.2 mM of each deoxyribonucleoside triphosphate, 10 mMTris-HCl buffer (pH 8.0), 100 mMKCl, 25 mM MgCl 2 , 2.5 U of Ex Taq DNA polymerase (TaKaRa Bio Inc., Otsu, Japan), 0.4 μM of each primer, and 2 μL of template DNA.The PCR was performed as follow: The mixture was subjected to initial denaturation at 94°C for 5 min followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 58°C for 30 s, and extension at 72°C for 1 min, followed by a final extension at 72°C for 10 min in a T100 Thermal Cycler (Bio-Rad Inc., Hercules, CA).The products were visualized by resolution on a 2% agarose gel followed by staining with SYBRSafe (Invitrogen, CA, USA.).

Primer design for LAMP
The primers for P. gingivalis (ATCC 33327) and A. actinomycetemcomitans (ATCC 33384) were designed from the pepO and dam genes, respectively.A set of six primers for each gene amplified for LAMP were designed using the Primer Explorer software, version 2.0 (Fujitsu Co., Ltd., Tokyo, Japan)(https:// primerexplorer.jp/e/).The primers comprise of forward inner primer (FIP), backward inner primer (BIP), forward outer primer (F3) and backward outer primer (B3).Two additional primers (forward loop primer, FLP and backward loop primer, BLP) were used to increase amplification efficacy.The sequences of each primer are listed in Table 2.The specificities of the designed primers were initially confirmed using BLAST in the National Center for Biotechnology Information server (http://www.ncbi.nlm.nih.gov/).Validations of the designed primers were performed using different species of Porphyromonas and Aggregatibacteras well as other oral bacteria to confirm their specificities.

Analysis of LAMP products
The LAMP reaction was evaluated through visual inspection based on turbidity of insoluble magnesium pyrophosphate, a byproduct of DNA synthesis produced in proportion to the amount of amplified DNA.LAMP amplicons in the reaction mixture were detected by naked eye on addition of 1.0 µl of 1/10-diluted original SYBR Green I (Molecular Probes, Eugene, USA) to the mixture and observing othe color changes.For further confirmation, the amplicons were also analyzed by running 2% (w/v) agarose gel electrophoresis and stained with SYBR Safe (1 mg/ml) and assessed photographically under ultraviolet light (302 nm).

Sensitivity and specificity of LAMP
To determine the detection limit, both LAMP and PCR assays were carried out using 10-fold serial dilutions of P. gingivalis and A. actinomycetemcomitans extracted DNA.To evaluate the species specificity of the LAMP, other oral bacteria strains were tested (Table 1).

Evaluation of LAMP with clinical samples
For further evaluation of the LAMP assay, a total of 10 crude templates of subgingival plaque and saliva samples from periodontitis patients were tested using method as above.

DNA sequencing
To confirm the presence of P. gingivalis and A. actinomycetemcomitans, the purified product of positive sample was sent for DNA sequencing using P.ging_F3 and P.ging_B3, A.act_F3 and A.act_B3 primers targeted on pepOand dam genes, respectively.For sequence analysis, the NCBI blast website was used (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

Statistical analysis
Statistical analysis was performed using SPSS version 22.0 software (IBM, Chicago, IL, USA).Descriptive data were expressed as frequencies and percentages for categorical variables, and means and standard deviations for continuous variables.Differences between categorical variables were evaluated using Fisher's Exact tests.

Optimization of LAMP reaction
The LAMP reaction was carried out using P. gingivalis and A. actinomycetemcomitans DNA as templates to determine the optimal temperature and reaction time.After addition of 1.0 µl of diluted SYBR Green I to the reaction tube, positive amplification products turned green, while the negative reaction remained orange.The results were considered valid if turbidity was present in the positive control and absent in the negative control.The analysis on 2.0% agarose gel indicated successful amplification by LAMP which showed a ladder-like pattern at 50, 55, 60 and 65 °C (Figure 1).There was no reaction at 70°C and 75°C.The color intensity (identified through naked-eye detection) as well as amplification pattern and band intensity (identified through agarose gel electrophoresis) was similar at 50, 55, 60 and 65°C.With respect to reaction time, the conditions of the LAMP assay were optimised for duration of 20-100 mins at 65°C.The amplification products could be clearly detected as early as 20 mins (Figure 2).Thus, for optimal reaction condition at 65°C, the reaction time of 30 min was chosen to ensure positive detection of lower template concentrations.intensity of the reaction and electrophoresis band.As indicated by Notomiet al. 7 specificity increased at higher temperatures and the electrophoresis bands will clearly well-formed at optimum conditions.Therefore, the temperature of 65°C was pickedas double-stranded DNA is at dynamic equilibrium around 65°C to start amplification.Bst polymerase used in this LAMP reaction is good at strand displacement, but at 70°C, the enzyme was heat-inactivated and consequently no response occurred. 15In this study, positive amplification can be detected as early as 20 mins.Combining these two factors (temperature & amplification time), the optimal reaction conditions of 65°C and 30 minutes of amplification were chosen to ensure positive detection of lower template concentrations.Since the reaction occurs under isothermal conditions, there is no time loss during thermal change. 16n the present study, the detection limit for both P. gingivalis and A. actinomycetemcomitans-LAMP was 1 ng of DNA and 10 ng of DNA for PCR.The detection limit of LAMP was 10-fold more sensitive than the conventional PCR.When tested using crude template of subgingival plaque and saliva, LAMP demonstrated higher sensitivity when compared with PCR.The LAMP had ended up being less influenced by various inhibitors when compared with the convectional PCR.In agreement with previous work, LAMP reaction was said to be tolerant and not affected with the other inhibitor or biological substances incorporated into the assays.In contrast, the use of crude samples of saliva and subgingival plaque that contain inhibitor or substances canhinder PCR reactions. 13The trail of crude template demonstrated a promising outcomes in LAMP.However, in PCR, extraction and purification steps of DNA was suggested to be applied to diminish the amount of inhibitory substances in the sample. 17Among all listed periodontal bacteria, P. gingivalis ranked the highest in prevalence followed by A. actinomycetemcomitans.Our results are as per Göhleret al. 4 which showed that P. gingivalis are high in periodontally infected patients.In the present study, the clinical samples were collected fromchronic periodontitis patients.As in chronic periodontitis, P. gingivalis bacteria is dominating, compared to A. actinomycetemcomitans which highly detected in subgingival plaque of patients with aggressive periodontitis. 18Moreover, there are positive and negative interactions in the microbial community inside the mouth.Ready et al. 19 demonstrated that P. gingivalis was able to antagonise the ability of other bacterial species including A. actinomycetemcomitans.Compared to P. gingivalis,A.actinomycetemcomitans had to reach the critical threshold to be significantly associated with disease and it is suspected that since our sample did not achieve the threshold point, they were low in detection.Conversely, the mere presence of P. gingivalis was significantly associated with periodontitis. 3 In the present study, the prevalence of P. gingivalisand A. actinomycetemcomitans was higher in subgingival plaque as compared to that of in saliva.The presence of P. gingivalisand A. actinomycetemcomitans in the clinical sample was further affirmed by sequencing.These outcomes are in concurrence with previous findings, which reported that P. gingivalis was higher in subgingival plaque (53.5%) as compared to that of in saliva (23.5%). 20,21

Conclusion
This study validates the application of LAMP assays for detection of P. gingivalis and A. actinomycetemcomitans in the crude template of subgingival plaque and saliva samples from periodontitis patients.In conclusion, the LAMP assay using a crude template will be of great advantage as it is simple, rapid and cost-effective to detect periodontal pathogens as compared to other detection assays.