Method for determining Helicobacter pylori

An in vitro method for determining Helicobacter pylori (Hp) in a fecal sample isolated from an individual and for determining the resistance of said Helicobacter pylori to clarithromycin, said method comprising the steps of:

• I. obtaining a fecal sample isolated from the individual;
• II. purifying or isolating the DNA from said sample;
• III. amplifying SEQ ID NO:
• 1 or SEQ ID NO:
• 2 of the 23S rRNA gene of Helicobacter pylori comprising at least one mutation site responsible for the resistance to clarithromycin, wherein said at least one mutation site is/are selected from the group consisting of A2143G, A2142C and A2142G, said amplification being performed by real-time PCR using a pair of primers that have the sequences of SEQ ID NO:
• 5 and SEQ ID NO:
• 6 and two oligonucleotide probes, where the pair of primers hybridizes in the DNA region straddling said mutation site and where each oligonucleotide probe is marked with a distinguishing marker, and wherein one oligonucleotide probe is complementary and specific to SEQ ID NO:
• 1 or SEQ ID NO:
• 2 of the wild-type allele of the 23S rRNA gene and the other oligonucleotide probe is complementary and specific to SEQ ID NO:
• 1 or SEQ ID NO:
• 2 of the mutated allele of the 23S rRNA gene responsible for clarithromycin resistance;
• and
• IV. quantifying the amplified wild-type and/or mutated DNA by measuring the levels of the distinguishing markers with which said oligonucleotide probes are marked, where the presence of only the marker with which the probe complementary and specific to SEQ ID NO:
• 1 or SEQ ID NO:
• 2 of the wild-type allele is marked defines a normal homozygous genotype and thus the presence of said Hp which is not resistant to clarithromycin;
• the presence of only the marker with which the probe complementary and specific to SEQ ID NO:
• 1 or SEQ ID NO:
• 2 of the mutated allele of the 23S rRNA gene is marked defines a mutated homozygous genotype and hence the presence of said Hp resistant to clarithromycin;
• and the presence of both markers defines a heterozygous genotype with a wild-type allele and a mutated allele of the 23S rRNA gene, and thus the presence of said Hp resistant to clarithromycin.