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Representational Difference Analysis (RDA) for Detection of Genetic Elements Associated with Increased Incidence of Serogroup C Neisseria meningitidis Infection

Kostelnik, Leah M. (2006) Representational Difference Analysis (RDA) for Detection of Genetic Elements Associated with Increased Incidence of Serogroup C Neisseria meningitidis Infection. Master's Thesis, University of Pittsburgh. (Unpublished)

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Previous studies have demonstrated that the increased incidence of invasive disease caused by serogroup C Neisseria meningitidis in the United States during the 1990s was attributed primarily to strains belonging to the ST11 clonal complex. Subcapsular genotyping of a subset of isolates from Maryland identified distinct "early" and "late" clones defined by antigenic shift at the FetA outer membrane protein. Representational difference analysis (RDA) was used to identify additional genetic differences that may have contributed to the emergence of the late clone. A collection of serogroup C isolates representative of the early and late clone was subjected to pulsed field gel electrophoresis (PFGE) to determine genetic relatedness among the isolates and to identify a candidate tester/driver pair for RDA. RsaI-digested tester genomic DNA (late clone) was ligated to specific adaptors followed by two rounds of subtractive hybridization with RsaI-digested driver genomic DNA (early clone). PCR amplification of subtracted tester DNA with adaptor specific primers generated at least three late clone-specific bands that were absent from the early clone. These products were cloned and sequenced and confirmed by Southern blotting with tester and driver digoxigenin-labeled genomic DNA probes to be tester specific. A BLAST search of late clone-specific sequences identified homology to either IS1301 or pJS-B plasmid N. meningitidis sequences. PCR with primers specific to either IS1301 or pJS-B plasmid sequences amplified these elements from late clone isolates but not from early clone isolates. Thus, RDA successfully identified two unique genetic elements present in an emergent N. meningitidis serogroup C ST-11 clone that had undergone antigenic shift at FetA. Further investigation is required to determine the potential role of these elements in clonal emergence and N. meningitidis pathogenesis. The public health significance of this project stems from increased incidence of meningococcal disease being a major concern: morbidity and mortality increase, outbreaks produce panic and disruption in communities, public health agencies must respond for control and prevention, and mass immunization and antibiotic prophylaxis are often required.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Kostelnik, Leah
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairHarrison, Lee Hlharriso@edc.pitt.eduLHARRISO
Committee MemberMartinson, Jeremyjmartins@pitt.eduJMARTINS
Committee MemberChang, Yuanyc70@pitt.eduYC70
Date: 7 June 2006
Date Type: Completion
Defense Date: 7 April 2006
Approval Date: 7 June 2006
Submission Date: 12 April 2006
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Institution: University of Pittsburgh
Schools and Programs: School of Public Health > Infectious Diseases and Microbiology
Degree: MS - Master of Science
Thesis Type: Master's Thesis
Refereed: Yes
Uncontrolled Keywords: representational difference analysis; antigenic shift; Neisseria meningitidis
Other ID:, etd-04122006-165816
Date Deposited: 10 Nov 2011 19:36
Last Modified: 15 Nov 2016 13:39


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