Differentiation of pathogenic Bartonella species by infrequent restriction site PCR

Abstract

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.

FIG. 1

Schematic of IRS-PCR analysis. (Step 1) Double digestion using EagI and HhaI. (Step 2) Adapter ligation. (Step 3) Primary denaturation followed by EagI primer binding. (Step 4) If the selective primer extension is a cytosine, then Taq polymerase reads through and initial extension occurs. If X ≠ C, extension does not occur. (Step 5) Initial extension produces complementary binding sequence for oligonucleotide AH1 (from HhaI adapter).

FIG. 2

Duplicate IRS-PCR patterns produced by amplification of restricted-ligated SmaI-HhaI DNAs using primer PSmaI-C. The first and last lanes in each gel are 100-bp ladders. (A) Second and third lanes, B. henselae Houston-1; fourth and fifth lanes, B. henselae Marseilles; sixth and seventh lanes, B. quintana Fuller; eighth and ninth lanes, B. quintana OK90-268. (B) Second and third lanes, B. bacilliformis KC583; fourth and fifth lanes, B. elizabethae; sixth and seventh lanes, B. clarridgeiae; eighth and ninth lanes, B. grahamii. (C) Second and third lanes, B. vinsonii subsp. vinsonii; fourth and fifth lanes, B. vinsonii subsp. arupensis; sixth and seventh lanes, B. vinsonii subsp. berkhoffii; eighth and ninth lanes, B. weissii.

FIG. 3

Duplicate IRS-PCR patterns produced by amplification of restricted-ligated EagI-HhaI DNAs using primer PEagI-C. Lane assignments are the same as for Fig. 2.

FIG. 4

Examples of IRS-PCR electropherograms generated by analysis of fluorescent IRS-PCR products with capillary electrophoresis. Patterns were generated using fluorescent primer PX-C. Patterns were subsequently normalized and sized against a common internal standard.

FIG. 5

Effects of selective nucleotide addition on IRS-PCR patterns of B. henselae Houston-1. Patterns were generated by amplification of EagI-HhaI restricted-ligated fragments by using various forward primers in combination with primer AH-1. First lane, 100-bp ladder; second lane, primer EagI-A; third lane, PEagI-T; fourth lane, PEagI-G; fifth lane, PEagI-C.