title: Development of a multiplex PCR assay for detection and genogrouping of Neisseria meningitidis.
abstract: Neisseria meningitidis is a leading pathogen of epidemic bacterial meningitis and fulminant sepsis worldwide. Twelve different N. meningitidis serogroups have been identified to date based on antigenic differences in the capsular polysaccharide. However, more than 90% of human cases of N. meningitidis meningitis are the result of infection with just five serogroups, A, B, C, W135, and Y. Efficient methods of detection and genogrouping of N. meningitidis isolates are needed, therefore, in order to monitor prevalent serogroups as a means of disease control and prevention. The capsular gene complex regions have been sequenced from only seven out of the 12 serogroups. In this study, the capsular gene complexes of the remaining five serogroups were sequenced and analyzed. Primers were designed that were specific for N. meningitidis species and for the 12 individual serogroups, and a multiplex PCR assay using these specific primers was developed for N. meningitidis detection and genogrouping. The assay was tested using 15 reference strains covering all 12 serogroups, 143 clinical isolates, and 21 strains from closely related species or from species that cause meningitis. The assay could detect N. meningitidis serogroups and was shown to be specific, with a detection sensitivity of 1 ng of genomic DNA (equivalent to ∼4 × 10(5) genomes) or 3 × 10(5) CFU/ml in noncultured mock cerebrospinal fluid (CSF) specimens. This study, therefore, describes for the first time the development of a molecular protocol for the detection of all N. meningitidis serogroups. This multiplex PCR-based assay may have use for the clinical diagnosis and epidemiological surveillance of N. meningitidis.
authors.affiliation: TEDA School of Biological Sciences and Biotechnology, Nankai University, TEDA, Tianjin, China.
authors.forename: Hongfei
authors.initials: H
authors.lastname: Zhu
authors.forename: Quan
authors.initials: Q
authors.lastname: Wang
authors.forename: Liuqing
authors.initials: L
authors.lastname: Wen
authors.forename: Jianguo
authors.initials: J
authors.lastname: Xu
authors.forename: Zhujun
authors.initials: Z
authors.lastname: Shao
authors.forename: Min
authors.initials: M
authors.lastname: Chen
authors.forename: Mingliang
authors.initials: M
authors.lastname: Chen
authors.forename: Peter R
authors.initials: PR
authors.lastname: Reeves
authors.forename: Boyang
authors.initials: B
authors.lastname: Cao
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
mesh_terms: D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D008589:Meningococcal Infections / Q000175:diagnosis* / Q000382:microbiology*; D017720:Molecular Epidemiology / Q000379:methods; D058889:Molecular Typing / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009345:Neisseria meningitidis / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial
references.citation: J Med Microbiol. 2005 May;54(Pt 5):463-466
references.pmid: 15824424
references.citation: Infect Immun. 2005 Mar;73(3):1491-505
references.pmid: 15731047
references.citation: J Clin Microbiol. 2001 Apr;39(4):1553-8
references.pmid: 11283086
references.citation: Mol Microbiol. 2009 Feb;71(4):960-71
references.pmid: 19170877
references.citation: J Infect Dis. 2003 May 15;187(10):1616-28
references.pmid: 12721942
references.citation: J Bacteriol. 1996 Jul;178(14):4052-9
references.pmid: 8763931
references.citation: N Engl J Med. 2001 May 3;344(18):1378-88
references.pmid: 11333996
references.citation: J Clin Microbiol. 2006 Mar;44(3):1127-31
references.pmid: 16517911
references.citation: Infect Immun. 2007 Jul;75(7):3594-603
references.pmid: 17470547
references.citation: Nucleic Acids Res. 2001 Jan 1;29(1):22-8
references.pmid: 11125040
references.citation: Proc Natl Acad Sci U S A. 2002 Apr 30;99(9):6103-7
references.pmid: 11983903
references.citation: Proc Natl Acad Sci U S A. 1997 Jan 7;94(1):271-6
references.pmid: 8990198
references.citation: Annu Rev Microbiol. 1996;50:285-315
references.pmid: 8905082
references.citation: Bioinformatics. 2000 Oct;16(10):944-5
references.pmid: 11120685
references.citation: J Clin Microbiol. 2004 Jan;42(1):320-8
references.pmid: 14715772
references.citation: J Clin Microbiol. 2005 Jan;43(1):144-9
references.pmid: 15634963
references.citation: Proc Natl Acad Sci U S A. 2004 Jul 6;101(27):10229-34
references.pmid: 15218099
references.citation: Mol Microbiol. 1994 Mar;11(5):885-96
references.pmid: 8022265
references.citation: Infection. 1989 Sep-Oct;17(5):309-10
references.pmid: 2513276
references.citation: Nucleic Acids Res. 2002 Jan 1;30(1):276-80
references.pmid: 11752314
references.citation: Plasmid. 2005 Nov;54(3):191-218
references.pmid: 16024078
references.citation: Mol Gen Genet. 1997 Dec;257(1):28-34
references.pmid: 9439566
references.citation: Am J Dis Child. 1980 Dec;134(12):1173
references.pmid: 6778202
references.citation: EMBO J. 1996 Jan 2;15(1):192-8
references.pmid: 8598202
references.citation: Nucleic Acids Res. 1997 Sep 1;25(17):3389-402
references.pmid: 9254694
references.citation: Proc Natl Acad Sci U S A. 2008 Mar 4;105(9):3473-8
references.pmid: 18305155
references.citation: Cell. 2006 Feb 24;124(4):703-14
references.pmid: 16497582
references.citation: J Clin Microbiol. 2002 Jan;40(1):75-9
references.pmid: 11773095
references.citation: Mol Biotechnol. 1996 Jun;5(3):233-41
references.pmid: 8837029
references.citation: J Clin Microbiol. 1984 Feb;19(2):187-90
references.pmid: 6365957
references.citation: J Bacteriol. 1996 Jun;178(11):3342-5
references.pmid: 8655518
references.citation: PLoS One. 2011 May 05;6(5):e19361
references.pmid: 21573213
references.citation: Am J Clin Pathol. 2004 Nov;122(5):754-64
references.pmid: 15491972
references.citation: FEMS Immunol Med Microbiol. 2008 Jul;53(2):178-82
references.pmid: 18623625
references.citation: FEMS Microbiol Lett. 1998 Feb 15;159(2):209-14
references.pmid: 9503614