Novel Genus-Specific PCR-Based Assays for Rapid Identification of Neisseria Species and Neisseria meningitidis

Abstract

This study presents the development of polymerase chain reaction (PCR)-based tests for the identification and detection of Neisseria species and Neisseria meningitidis. Currently, isolating and identifying these pathogens using conventional biochemical methods require 48-72 h. To improve speed and accuracy in diagnosing Neisseria infections, simple PCR-based tests that are specific for the genus Neisseria and the species Neisseria meningitidis have been developed. The genus-specific and species-specific DNA sequences were chosen by selecting and analyzing available database sequences. Neisseria-specific and Neisseria meningitidis-specific primer pairs were derived from the genes asd (coding for the aspartate beta-semialdehyde dehydrogenase) and ctrA (coding for a conserved outer membrane protein), respectively. Both the Neisseria-specific and Neisseria meningitidis-specific PCR assays were specific (they amplified only DNA from the target genus or species, out of 84 bacterial species tested). In addition, the Neisseria-specific assay amplified DNA from 321 of 322 strains tested representing 13 species of Neisseria, while the Neisseria meningitidis-specific assay amplified DNA from all 256 strains tested representing nine serogroups of Neisseria meningitidis. These PCR assays, which can be combined in multiplex, have been adapted to ensure that they are simple and can be performed within approximately 90 min. The tests provide new diagnostic tools for identifying Neisseria infections.

An analysis of the biofilms adhered to framework alloys using in vitro denture plaque models

“…Qualitative PCR was carried out according to the previous studies [28][29][30][31][32][33][34][35][36][37][38][39][40] using DNA templates extracted from the biofilms on five alloys, resin and the individual saliva samples (N=5). The primers used were for the genera of Actinomyces 28) , Burkholderia 29) , Fusobacterium 30) , Haemophilus 31) , Mycoplasma 32) , Neisseria 33) , Peptostreptococcus 34) , Pseudomonas 35) , Streptococcus 36) , Staphylococcus 37) , and Candida 38) and for the species of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Treponema denticola, Tannerella forsythia 39) and S. mutans 40) .…”

“…The primers used were for the genera of Actinomyces 28) , Burkholderia 29) , Fusobacterium 30) , Haemophilus 31) , Mycoplasma 32) , Neisseria 33) , Peptostreptococcus 34) , Pseudomonas 35) , Streptococcus 36) , Staphylococcus 37) , and Candida 38) and for the species of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Treponema denticola, Tannerella forsythia 39) and S. mutans 40) .…”

Neutrophilic bacterial meningitis: pathology and etiologic diagnosis of fatal cases

“…To evaluate the quality of DNA extract, each sample was tested for the amplification of the house-keeping genes human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and b-globin. 20,21 The DNA extracts were evaluated by conventional PCR assays for S. pneumoniae, 13,20 N. meningitidis, 14,22 Streptococcus species, and by paneubacterial PCR 23 using the High-Fidelity PCR kit (Roche Diagnostics, Indianapolis, IN). Primer sequences, target genes, PCR annealing temperatures, and amplification product sizes of conventional PCR assays are summarized in Table 2.…”

“…In addition, DNA extracts were also tested by staphylococcal real-time multiplex PCR assay targeting the Panton-Valentine leukocidin gene and toxic shock syndrome toxin 1 gene. PCR assays were performed using the previously published primers and probes 22,23 and the QuantiTect Multiplex PCR Kit (Qiagen) on the Stratagene Mx3000P QPCR system.…”

Direct and Rapid Identification and Genogrouping of Meningococci andporAAmplification by LightCycler PCR

“…Detection of nucleic acids of the 16S rRNA gene by PCR has been used for diagnosis of meningococcal disease (2,15,16,19,25). Recently, PCR assays have also focused on the conserved regulatory gene, crgA (28), as well as the ctrA gene (7,13,17,24), a target involved in the transport of the capsular polysaccharide (10).…”