# The Loop-Mediated Isothermal Amplification for the Rapid Detection of Porphromonas Gingivalis
## 4 Discussion
gingivalis_ from the material secured for isolation by TE buffer. Therefore, the results presented in this work confirm the significant usefulness of LAMP for an easy and quick microbiological diagnostic of periodontitis. The high sensitivity and specificity of the isothermal method in conjunction with the easy preparation of the analysis sample thanks to the Difast buffer allows for skipping the bacterial DNA extraction. This makes the LAMP method can be a serious alternative for qPCR reaction on an outpatient basis. [PERSON] et al. compared LAMP to PCR in detection of _F. nucleatum_. The presented results suggest that LAMP has a 10-fold higher sensitivity than conventional PCR when used for the detection of this particular pathogen [25]. The specificity and sensitivity of the LAMP, PCR, and qPCR methods conducted using samples of patients from whom the bacterial DNA was extracted, secured with TE buffer (buffer Tris 10 mM pH 8.0, 1 mM EDTA) or Difast buffer, was also compared. The TE buffer is a very durable medium, securing the bacterial DNA thanks to an EDTA that deactivates the nucleases by chelating the divalent ions, whereas the Tris buffer present in the solution allows for sustaining the stable form of DNA. On the other hand, the Difast buffer allows for streamlining the process of acquiring the DNA in a much shorter detection time. This buffer has been used before among others, such as Pieczul and Wasowska, in order to extract the DNA genome of the _Zymosetporia tritici_ fungus in a 15 min procedure [27]. In comparison to the method based on using the Difast buffer, the standard DNA isolation procedure lasts for around 60-70 min. Due to the presence of solid particles, the Difast buffer requires continuous stirring during the acquisition process of the examined material. Not following the procedure can negatively influence the effectiveness of the DNA isolation process. The comparison of LAMP, PCR, and qPCR techniques used in the analysis of the samples secured by TE and Difast buffers showed a statistically significant difference in detecting _P. gingivalis_ in favour of the TE buffer. Moreover, not following the procedure of using the Difast buffer strictly can lead to disturbance of the bacterial DNA isolation process. This is associated with an insufficient amount of the bacterial DNA, which may cause a false-negative reaction result. _P. gingivalis_ is the main microbiological etiological factor of the parietal periodontitis [28]. A quantitative real-time analysis of _P. gingivalis_ using the LAMP method with a fluorescent dye SYBR Green I with a linearity of 102-106 cells was conducted by [PERSON] et al. Using this technique, the scientists obtained almost identical results to those of qPCR. However, the comparison of both methods showed LAMP to be superior in terms of reaction speed [23]. A study of 58 patients showed that _T. forythia_ was detected in 48 samples (82.75%) when using a PCR test on a DNA template isolated from material secured with TE buffer. Moreover, the advancement stage showed an upward trend as periodontitis progressed. The studies conducted by [PERSON] et al. confirmed that _T. forythia_ contributes to the disease's progression by activating many mechanisms such as the gelatine and collagen type I and III degradation process with the activity of serine protease [29]. The processes of periodontium destruction and progression of periodontitis include the activity of Gram-negative bacteria _Fusobacterium_. These pathogens have developed the ability to aggregate with early and late colonisers such as _P. gingivalis_. The presented results of a study conducted on a group of 35 patients tested with using the LAMP method with the DNA isolated with the use of Difast buffer showed that _F. nucleatum_ was detected in only 5 samples (14.29%). An additional interesting aspect of the study is the fact that all of the patients (5 people) were active smokers. Therefore, the results are consistent with previous observations of [PERSON] et al.