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To develop and evaluate a novel multiplex PCR assay that enables definition of Helicobacter pylori vacA allelic type in a single reaction.Application of the one-step system to DNA extracts from 22 cultures of known vacA genotype demonstrated that it was highly accurate. Analysis of 15 matched gastric biopsy/culture pairs generated exactly correlating genotype profiles. vacA genotypes were determined from an additional 62/70 gastric biopsies from dyspeptic patients of known H. pylori positive status by the one-step assay, compared with 63/70 by the original two-reaction test. Types s1/m1, s1/m2 and s2/m2 were identified in 51·9%, 31·2% and 16·9% of biopsies, respectively.The multiplex PCR system developed enables rapid one-step vacA genotyping that is accurate, easy to interpret and more economical than the alternative multiple-reaction tests. Application of this system to gastric biopsies from patients in South-east England demonstrated that s1/m1 was the most common genotype, while s1/m2 and s2/m2 were less prevalent.This simple one-step system can be applied direct to antral gastric biopsies without the need for culture, thereby facilitating rapid surveillance of vacA genotype in relation to geographical location and disease status.