Automated Method Based in VNTR Analysis for Rickettsiae Genotyping LILIANA VITORINO, a,b RITA DE SOUSA, b FATIMA BACELLAR, b AND L ´ IBIA Z ´ E-Z ´ E a,b a Universidade de Lisboa, Faculdade de Ciˆ encias, Centro de Gen´ etica e Biologia Molecular and Instituto de Ciˆ encia Aplicada e Tecnologia, Edif´ ıcio ICAT, Campus da FCUL, Campo Grande, 1749–016 Lisboa, Portugal b CEVDI, Instituto Nacional de Sa ´ ude Dr. Ricardo Jorge, 1649-016 Lisboa, Portugal ABSTRACT: A genetic locus named Rc-65, which is 5 ′ adjacent to gene dksA and 3 ′ adjacent to xerC gene, has previously been demonstrated to contain a VNTR with high discriminatory power in several rickettsial strains and thus, potentially useful for genetically similar strains identi- fication. In this work, we present an automated molecular identification method based on capillary electrophoresis separation of VNTRs am- plicons. The resulting electropherograms were in agreement with the sequence data obtained in a previous work. The presented genotyping method is fast and suitable for full automation, being a powerful tool for epidemiological surveillance in a large number of samples and enables the detection co-infected samples. The combination of other VNTR loci should improve the discriminatory capacity of this typing system, provid- ing greater resolution and contributing to a more accurate VNTR-based assay. To our knowledge, this is the first automated assay for rickettsial strains identification. KEYWORDS: Mediterranean spotted fever; Rickettsiae; variable number tandem repeats INTRODUCTION Tandem repeats (TR) (over 10 nt [nucleotides]) that represent a single lo- cus and show interindividual length polymorphisms are referred to as variable number tandem repeats (VNTRs). The VNTR analysis is a polymerase chain reaction (PCR)-based approach used to genotype several bacteria species, be- cause it is highly discriminatory and reproducible. 1 A VNTR assay for rick- ettsiae typing was recently described by Fournier et al. 2,3 and Vitorino et al. 4 Address for correspondence: L´ ıbia Z´ e-Z´ e, CEVDI, Instituto Nacional de Saud´ e Dr. Ricardo Jorge, INSA, Edif´ ıco LEMES, Av. Padre Cruz, 1649–016 Lisboa, Portugal. Voice: 00351-217-508-126; fax: 00351-217-508-121. e-mail: libia.zeze@insa.min-saude.pt Ann. N.Y. Acad. Sci. 1078: 582–586 (2006). C  2006 New York Academy of Sciences. doi: 10.1196/annals.1374.116 582