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Development of Multi-Locus Variable Number Tandem Repeat Analysis for Outbreak Detection of Neisseria meningitidis

Price, Alicia Anne (2006) Development of Multi-Locus Variable Number Tandem Repeat Analysis for Outbreak Detection of Neisseria meningitidis. Master's Thesis, University of Pittsburgh. (Unpublished)

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Neisseria meningitidis is a major cause of septicemia and meningitis worldwide. Traditional typing methods like pulsed-field gel electrophoresis (PFGE) for identifying outbreaks are subjective and time consuming. Multi-locus variable number tandem repeats analysis (MLVA) is an objective typing method amenable to automation that has been used to type other bacterial pathogens. This report describes the development of MLVA for outbreak detection of N. meningitidis. Tandem Repeats Finder software was used to identify variable number tandem repeats (VNTRs) from 3 sequenced N. meningitidis genomes. PCR amplification of identified VNTRs was performed on DNA from 7 serogroup representative isolates. PCR products were sequenced and repeats were manually counted. VNTR loci identified by this screen were evaluated on a collection of 46 outbreak and sporadic serogroup C isolates. Alleles at each locus were concatenated to define the MLVA type for each isolate. Minimum spanning tree (MST) analysis was performed to determine the genetic relationships among the isolates. The genetic distance was defined as the summed tandem repeat difference (STRD) between isolates MLVA types. Outbreak clusters were defined by a STRD less than or equal to 3. These data was compared to PFGE data to determine the utility of MLVA for outbreak detection. Twenty-one VNTR loci with variable copy numbers among the sequenced genomes were identified that met the established criteria of short repeat length and consensus sequence > 85%. Seven VNTR loci were reliably amplified among the 7 serogroups tested. These loci had repeat lengths between 4 and 20 nucleotides and exhibited between 10 and 26 alleles among 61 isolates belonging to 7 different serogroups. MST analysis with 7 loci differentiated serogroups, discriminated sporadic isolates and identified 7 out of 8 serogroup C outbreaks. In summary, MLVA with 5 VNTR loci distinguished N. meningitidis isolates from 7 different serogroups and sporadic isolates within each serogroup. In addition, MLVA identified 88% of PFGE-defined serogroup C outbreaks. Further investigation of these and other outbreak-associated isolates is necessary to define the optimal combination of VNTR loci and to evaluate MST analysis criteria in order to determine the utility of MLVA for N. meningitidis outbreak detection.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Price, Alicia
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairHarrison, Lee Hlharriso@pitt.eduLHARRISO
Committee MemberMcEllistrem, Catherineschmiede@pitt.eduSCHMIEDE
Committee MemberMartinson, Jeremyjmartins@pitt.eduJMARTINS
Committee MemberKhan, Saleemkhan@pitt.eduKHAN
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: Multi-Locus Variable Number Tandem Repeat Analysis; Neisseria meningitidis
Other ID:, etd-04122006-174415
Date Deposited: 10 Nov 2011 19:36
Last Modified: 15 Nov 2016 13:39


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