Performance criteria of DNA fingerprinting methods for typing of Helicobacter pylori isolates: experimental results and meta-analysis

Abstract

Typing systems are used to discriminate between isolates of Helicobacter pylori for epidemiological and clinical purposes. Discriminatory power and typeability are important performance criteria of typing systems. Discriminatory power refers to the ability to differentiate among unrelated isolates; it is quantitatively expressed by the discriminatory index (DI). Typeability refers to the ability of the method to provide an unambiguous result for each isolate analyzed; it is quantitatively expressed by the percentage of typeable isolates. We evaluated the discriminatory power and the typeability of the most currently used DNA fingerprinting methods for the typing of H. pylori isolates: ribotyping, PCR-based restriction fragment length polymorphism (PCR-RFLP) analysis, and random amplified polymorphism DNA (RAPD) analysis. Forty epidemiologically unrelated clinical isolates were selected to constitute a test population adapted to the evaluation of these performance criteria. A meta-analysis of typeability and discriminatory power was conducted retrospectively with raw data from published studies in which ribotyping, PCR-RFLP, RAPD, repetitive extragenic palindromic DNA sequence-based PCR (REP-PCR), or pulsed-field gel electrophoresis (PFGE) was used. Experimental results and the meta-analysis demonstrated the optimal typeability (100%) and the excellent discriminatory powers of PCR-based typing methods: RAPD analysis, DIs, 0.99 to 1; REP-PCR, DI, 0.99; and PCR-RFLP analysis, DIs, 0.70 to 0.97). Chromosome restriction-based typing methods (ribotyping and PFGE) are limited by a low typeability (12.5 to 75%) that strongly decreases their discriminatory powers: ribotyping, DI, 0.92; PFGE, DIs, 0.24 to 0.88. We do not recommend the use of ribotyping and PFGE for the typing of H. pylori isolates. We recommend the use of PCR-based methods.

FIG. 1

Ribopatterns (HaeIII digests) of DNAs from 12 H. pylori isolates (isolates 11-D, 94-60, 94-48, 94-55, 94-47, 93-37, 93-38, 92-4, 86-6, 85-B, V17, V3). Different problems with typeability are shown: DNA was not cleaved by HaeIII (lanes 6, 7, and 9), insufficient intensities of DNA bands (lanes 3 and 11), degradation of chromosomal DNA (lane 10), and incomplete digestion of chromosomal DNA (lane 12). Lanes M, molecular size marker Raoul 1 (bands of 48.5, 18.5, 14.9, 10.6, 9.0, 7.4, 5.6, 4.4, 4.0, 3.6, 2.9, 2.3, 1.8, 1.4, 1.2, 1, 0.9, 0.7, and 0.6 kbp, from top to bottom, respectively).

FIG. 2

PCR-RFLP patterns of the amplified ureC gene products from 12 H. pylori isolates (isolates 93-32, 93-33, 93-34, 93-35, 93-37, 93-38, 93-40, 93-41, 93-42, 94-47, 94-48, and 94-55) whose amplified DNAs were digested with Sau3A. Two unrelated isolates (isolates 93-34 and 93-35 [lanes 3 and 4, respectively]) yielded identical pattern. Lanes M, molecular size marker pBR328-BglI + pBR328-HinfI (bands of 2,176, 1,766, 1,230, 1,033, 653, 517, 453, 394, 298, 234, 220, and 154 bp, from top to bottom, respectively).

FIG. 3

RAPD patterns of the amplified DNAs of 12 H. pylori isolates (isolates 85-B, 86-G, 92-3, 92-4, 92-7, 92-8, 92-9, 92-10, 92-11, 92-16, 93-25, and 93-31) obtained by RAPD analysis with primer 1254. Two pairs of unrelated isolates (lanes 2 and 3 and lanes 6 to 10, respectively) yielded identical patterns. Lanes M, molecular size marker pBR328-BglI + pBR328-HinfI (bands of 2,176, 1,766, 1230, 1033, 653, 517, 453, 394, 298, 234, 220, and 154 bp, from top to bottom, respectively).