Multiplex-Touchdown PCR Assay for the Detection and Genotyping of Helicobacter pylori from Artificially Contaminated Sheep Milk
Abstract:Helicobacter pylori (Hp) is an organism commonly present worldwide in the human population, sometimes causing serious illnesses such as duodenal and gastric ulcers, adenocarcinoma of the stomach, and low-grade B-cell mucosa-associated lymphoid tissue lymphoma of the stomach. This article describes a multiplex-touchdown PCR method for the identification and genotyping (vacA—s1/m1, s1/m2, and s2/m2—and cagA genes) of Hp directly from sheep milk artificially contaminated with Hp strains from human gastric biopsies and with Hp ATCC 43504. The strains from humans carried s1/m2 cagA + and s2/m2 cagA − allelic combinations, while the ATCC strains carried an s1/m1 cagA + allelic combination. The technique showed a sensitivity of 15 CFU/ml for species identification and of 1,500 CFU/ml for the detection of genes encoding for VacA and CagA. It has proven to be specific and rapid, and the authors suggest that it be used as a rapid screening method to ensure that sheep milk is uncontaminated with this organism.
Document Type: Research Article
Affiliations: 1: Department of Health and Animal Welfare–Faculty of Veterinary Medicine, Str. Prov. per Casamassima Km 3, 70010 Valenzano (Ba), Italy 2: Istituto Zooprofilattico Sperimentale della Puglia e Basilicata, V. Chiancolla n. 1, Putignano (Bari), Italy 3: Istituto Zooprofilattico Sperimentale della Sardegna, V. F. lli Kennedy, 08100, Nuoro, Italy