Multiplex-touchdown PCR assay for the detection and genotyping of Helicobacter pylori from artificially contaminated sheep milk.
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, sl/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 sl/m2 cagA+ and s2/m2 cagA allelic combinations, while the ATCC strains carried an sl/ml 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.
Quaglia
N C
NC
Department of Health and Animal Welfare-Faculty of Veterinary Medicine, Str. Prov. per Casamassima Km 3, 70010 Valenzano (Ba), Italy.
Normanno
G
G
Dambrosio
A
A
Celano
G V
GV
Parisi
A
A
Firinu
A
A
Buonavoglia
C
C
D000818:Animals; D001483:Base Sequence; D015169:Colony Count, Microbial; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis*; D005506:Food Contamination / Q000032:analysis*; D005516:Food Microbiology; D005838:Genotype; D016480:Helicobacter pylori / Q000235:genetics* / Q000302:isolation & purification*; D008892:Milk / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012756:Sheep
D017931:DNA Primers; D004269:DNA, Bacterial