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Journal of Clinical Microbiology, August 2000, p. 3010-3015, Vol. 38, No. 8
0095-1137/00/$04.00+0

Differentiation of Pathogenic Bartonella Species by Infrequent Restriction Site PCR

Scott A. Handley and Russell L. Regnery*

Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, 30333

Received 21 December 1999/Returned for modification 3 April 2000/Accepted 18 May 2000

Infrequent restriction site PCR (IRS-PCR) is a recently described DNA fingerprinting technique based on selective amplification of restriction endonuclease-cleaved fragments. Bartonella isolates associated with human disease and related nonhuman isolates were analyzed by IRS-PCR genomic fingerprinting. Preparation of DNA templates began with double digestion using three different restriction endonuclease combinations. Combinations included the frequently cutting endonuclease HhaI in conjunction with an infrequently cutting endonuclease, EagI, SmaI, or XbaI. Digestion was followed by ligation of oligonucleotide adapters designed with ends complementary to the restriction endonuclease sites. Amplification of fragments flanked with an EagI, SmaI, or XbaI site in combination with an HhaI site produced a series of different-sized amplicons resolvable into patterns by polyacrylamide gel electrophoresis (PAGE). The pattern complexity was varied by the addition of selective nucleotides to the 3' ends of the EagI-, SmaI-, or XbaI-specific primers. Amplicons were also generated with fluorescently labeled primers and were subsequently resolved and detected by capillary electrophoresis. Analysis by traditional slab PAGE and capillary electrophoresis provided suitable resolution of patterns produced with the enzyme combinations EagI-HhaI and SmaI-HhaI. However, the combination of XbaI-HhaI produced too many fragments for sufficient resolution by traditional PAGE, thus requiring the better resolving properties of capillary electrophoresis. Due to the flexibility in modulating the pattern complexity and electrophoresis methods, these techniques allow for a high level of experimental optimization. The results provide evidence of the discriminatory power, ease of use, and flexibility of the IRS-PCR method as it applies to the identification of human-pathogenic Bartonella species.

* Corresponding author. Mailing address: Center for Disease Control and Prevention, 1600 Clifton Rd., Mail Stop G-13, Atlanta, GA 30333. Phone: (404) 639-1075. Fax: (404) 639-4436. E-mail: rur1{at}cdc.gov.

Journal of Clinical Microbiology, August 2000, p. 3010-3015, Vol. 38, No. 8
0095-1137/00/$04.00+0


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