Detection of periodontal pathogenPorphyromonas gingivalisby loop-mediated isothermal amplification method

Abstract

A method for nucleic acid amplification, loop-mediated isothermal amplification (LAMP) was employed to develop a rapid and simple detection system for periodontal pathogen, Porphyromonas gingivalis. A set of six primers was designed by targeting the 16S ribosomal RNA gene. By the detection system, target DNA was amplified and visualized on agarose gel within 30 min under isothermal condition at 64 degrees C with a detection limit of 20 cells of P. gingivalis. Without gel electrophoresis, the LAMP amplicon was directly visualized in the reaction tube by addition of SYBR Green I for a naked-eye inspection. The LAMP reaction was also assessed by white turbidity of magnesium pyrophosphate (a by-product of LAMP) in the tube. Detection limits of these naked-eye inspections were 20 cells and 200 cells, respectively. Although false-positive DNA amplification was observed from more than 10(7) cells of Porphyromonas endodontalis, no amplification was observed in other five related oral pathogens. Further, quantitative detection of P. gingivalis was accomplished by a real-time monitoring of the LAMP reaction using SYBR Green I with linearity over a range of 10(2)-10(6) cells. The real-time LAMP was then applied to clinical samples of dental plaque and demonstrated almost identical results to the conventional real-time PCR with an advantage of rapidity. These findings indicate the potential usefulness of LAMP for detecting and quantifying P. gingivalis, especially in its rapidity and simplicity.

Efficient Identification of Clinically Relevant Candida Yeast Species by Use of an Assay Combining Panfungal Loop-Mediated Isothermal DNA Amplification with Hybridization to Species-Specific Oligonucleotide Probes

“…Compared to a standard PCR protocol, LAMP requires a single bath at a constant temperature instead of a thermal cycler. The performances of LAMP-and PCR-based diagnostic systems, including real-time technologies (23,40), have been extensively compared. In general, LAMP was found to be either similar or superior to PCR, and more specific (e.g., 8,13,15), but a few studies proved otherwise, such as those reported by Kato et al (18), who showed that although LAMP was 10-fold more sensitive than standard PCR for the detection of the Clostridium difficile toxin B gene (tcdB), an optimized nested-PCR assay performed much better than LAMP.…”

Use of a Combination of Brushing Technique and the Loop-Mediated Isothermal Amplification Method as a Novel, Rapid, and Safe System for Detection of Helicobacter pylori

“…H. pylori occurs beneath the mucus layer, predominantly in the antrum (34). Infection is generally confirmed by histology (21), culturing (13), and the rapid urease test (RUT) (23) performed on endoscopic gastric mucosal biopsy samples or by the urea breath test (14). This latter test is a noninvasive method that detects 13 CO 2 generated by H. pylori urease (5); because it is indirect, however, it does not allow the measurement of antibiotic susceptibility, a disadvantage given that the recent gradual increase in the number of antibiotic-resistant strains has mandated susceptibility testing before eradication therapy (9,25).…”

“…The principle of LAMP involves autocycling strand displacement DNA synthesis using a DNA polymerase with high strand displacement activity. Recent reports demonstrating the usefulness of LAMP in detecting various bacteria and viruses (10,16,17,21,27,28,32,35,37) suggested it may be useful for the rapid detection of H. pylori. The LAMP procedure is simple, and its selectivity is extremely high owing to its use of four primers that recognize six distinct regions of the target DNA.…”

Molecular diagnostic methods for detection of Thelohania contejeani (Microsporidia), the causative agent of porcelain disease in crayfish

“…Although the LAMP assay required only 4 primers, 2 outer and 2 inner, that recognise 6 different regions of the target sequence (Notomi et al 2000), addition of 2 loop primers was found to enhance and accelerate the reaction, and increase not only the sensitivity but also the specificity of the assay. The 6 primers recognised 8 distinct regions of the target DNA, which minimised the probability of false positive results (Nagamine et al 2002, Maeda et al 2005. Although the LAMP assay could detect T. contejeani DNA in as little as 25 min (Fig.…”