Meningitis Lab Manual PCR Detection and Characterization. Species specific real time PCR assays. PCR detection of N. H. influenzae, and S. The following assays have been developed and validated to be used on DNA extracted from clinical specimens typically, blood and CSF and bacterial isolates. N. meningitidis. Two genes can be targeted in N. A and sod. C. The capsule transport to cell surface gene, ctr. Bacterial Polysaccharides Pdf Reader' title='Bacterial Polysaccharides Pdf Reader' />A, is highly conserved among isolates responsible for invasive meningococcal infections and has been used in both real time and conventional PCR to detect N. It is a gene within the capsule locus Figure 2. Our experience has been that naturopathic approaches to irritable bowel syndrome IBS tend to be highly successful. Often, uncovering and removing hidden food. Safety Assessment of Microbial Polysaccharide Gums as Used in Cosmetics Status Final Report for public distribution Release Date October 5, 2012. Lipopolysaccharide Endotoxinhost defense antibacterial peptides interactions Role in bacterial resistance and prevention of sepsis. Yosef Rosenfeld. Target gene assays for detection and characterization of bacterial meningitis etiologies. Over the past several years, conventional and realtime PCR assays have been. However, since at least 1. A 1. 0, 1. 4, 4. The new england journal of medicine n engl j med 36216 nejm. Advances in the Development of Vaccines. This is based on New Zealand Qualification Authority Unit 8024 entitled Demonstrate knowledge of bacterial structure. To view a power point on this unit follow this. Bacterial Polysaccharides Pdf Editor' title='Bacterial Polysaccharides Pdf Editor' />PCR assay to detect all meningococci, regardless of encapsulation status, was recently developed and validated 1. This assay targets the Cu, Zn superoxide dismutase gene, sod. C, which is not genetically linked to the capsule locus. The sod. C assay detects encapsulated meningococci, but it is also useful for detecting nongroupable meningococci that do not contain an intact ctr. A, as will be recovered during carriage studies. For this reason, it is recommended that sod. Bacterial Polysaccharides Pdf To DocBacterial Polysaccharides Pdf MergeC be used for detection of N. C and ctr. A primers and probes are listed in Table 2. H. influenzae. The protein D encoding gene, hpd, encodes protein D, a highly conserved, surface exposed lipoprotein that is present in all encapsulated and non encapsulated H. The conserved nature of this gene and its presence in all strains of H. H. influenzae species specific real time PCR assay. The recently developed and validated hpd real time PCR assay is capable of detecting all six serotypes a f and nontypeable Hi. NT H. influenzae with high sensitivity and specificity 6. Real time PCR assays targeting bex. A were developed and distributed because bex. A is present in all six serotypes of H. However, though sensitive for detection of Hib, it is less sensitive for Hia, Hic, and Hid, and does not detect Hie, Hif, or Hi. NT and should no longer be used. The primers and probes for the hpd assay are listed in Table 2. S. pneumoniae. Both conventional and real time PCR assays have been developed for the detection of S. A, and pneumococcal surface adhesion psa. A genes 8. However, false positive results with ply based PCR have been reported when applied to upper respiratory tract specimens. A suggested explanation for these false positives is the detection of non pneumococcal alpha hemolytic streptococci 3. Streptococcus mitis group and Streptococcus oralis which sometimes contain a ply gene 6. The PCR detection assay for S. A is recommended because it is highly conserved within the species and it has been shown that this assay bestseparates S. S. mitis, S. oralis, and S. Serial Power Dvd 5.0 on this page. The real time PCR assay lyt. A primers and probes that have been found to be extremely reliable for detection of S. Table 2. Due to recombination events that occur between pneumococci and closely related streptococci, there will probably be rare false positives or false negatives for virtually any real time assay for pneumococcal identification. Serogroupserotype specific real time PCR assays. The capsule gene loci of both N. H. influenzae have areas that are both unique and conserved within each serogroup N. H. influenzae thus providing gene targets for the development or real time PCR assays designed to identify each specific serogroup or serotype. N. meningitidis. N. Major disease causing serogroups include A, B, C, Y, and W1. A produces a poly 1 6 linked N acetylmannosamine 6 phosphate capsule 3. Outbreaks caused by serogroup X meningococci, which express poly 1 4 linked N acetylglucosamine 1 phosphate capsule 6 have also been reported 2, 2. Serogroup D is no longer recognized as a serogroup of N. As illustrated in Figure 2, the genetic organization of the capsule locus is conserved among the serogroups. The capsule expression genes are located in four operons one that encodes capsule biosynthesis called syn or sia genes, depending on which nomenclature system is used and three that encode the capsule transport to the cell surface proteins ctr. The gene products of the ctr operon share high similarity with the ATP dependent transporters of the ABC family 1. X 5. 6. Sensitive real time PCR assays targeting ctr. A, which is the first gene in the capsule transport operon, have been developed for detection of all encapsulated and some non encapsulated nongroupable N. C gene as a target has been developed. The genetic differences among the capsule biosynthesis operons of meningococcal serogroups have facilitated the development of real time PCR assays targeting serogroup specific genes for capsule biosynthesis to determine the capsule genotype of a meningococcal isolate 3. The gene sia for sialic acid biosynthesis 1. B syn. D, C syn. E, Y syn. F and W1. G. The sac. B gene is targeted for serogroup A and the xcb. A gene, which most likely encodes the capsule polymerase, is targeted for serogroup X 2, 3. These target genes are depicted within the structure of the capsule gene complex for serogroups A, B, C, Y, W1. X Figure 2. The most current adapted primer and probe sequences for the serogrouping real time PCR assays are listed in Table 3, though, periodically, the primers and probes are adapted as new information regarding probe chemistries and allelic variations become available. There have been several different systems in place for naming the genes for meningococcal capsule biosynthesis. Some groups have called this operon syn for capsule biosynthesis 2. E. coli K1 genes for N acetylneuraminic acidbiosynthesis 2. While the sia. D genes of serogroups B, C, W1. Y were initially thought to be alleles, more extensive sequencing analysis demonstrated that this is not so. The syn. D and syn. E genes of serogroups B and C, respectively, are alleles and encode capsular polysaccharide polymerases that catalyze different linkages of sialic acid monomers 28 linkage for serogroup B and 29 linkage for serogroup C. However, the Y and W1. B and C genes and differ in nucleotide sequence 1. In addition, the polymerases for serogroups Y and W1. Thus, the capsular polysaccharide polymerase genes of serogroups Y and W1. To continue to call all of these polymerase genes sia. D would be a misnomer. For these reasons and for simplicity, this text will use the syn. ABCDEFG nomenclature listed above and in Table I. Table 1. Serogroup capsule type and gene targets for genotyping real time PCR assays. Sero group. Capsule type. Gene Target Name. Alternate Gene Names. Ref. A16 N acetyl D mannosamine 1 phosphatesac. B 3. 1B28 N acetylneuraminic acidsyn. Dsia. Dsia. D of Bsia. DB5, 9, 2. 3, 4. C29 N acetylneuraminic acidsyn. Esia. D of Csia. DC5, 9, 5. W1. 35. 6 D Gal14 N acetylneuraminic acid26syn. Gsia. D of W1. 35sia. DW4, 9, 1. 4, 3. X14 N acetyl D glucosamine 1 phosphatexcb. B 2, 6Y6 D Glc14 N acetylneuraminic acid26syn. Fsia. D of Ysia. DY4, 9, 1. For serogroups B, C, Y and W1. The fourth gene product is a polymerase that catalyzes the formation of polymers with the serogroup specific linkage. In serogroups B and C, the products of a four gene operon syn. ABC plus the polysialyltransferase gene syn. D Nmen B or syn. E Nmen C are responsible for biosynthesis of the sialic acid also known as N acetylneuraminic acid, Neu. NAc, or NANA homopolymer.