Method for detecting
The present invention provides a PCR based method for detecting chronic gastritis causing bacterium, namely Helicobacter pylori , in a stool sample. The present invention is based on the use of oligonucleotide primers and probes specific to H. pylori 23S rRNA gene.
1. A method of detecting Helicobacter pylori DNA in a stool sample, the method comprising:
a) performing a nested PCR reaction comprising DNA isolated from a stool sample as a template and a first and second oligonucleotide primer set specific for amplifying H. Pylori -specific target sequences in an H. pylori 23S rRNA gene in an amplification reaction,
wherein the first oligonucleotide primer set comprises an oligonucleotide primer comprising the nucleotide sequence as set forth in SEQ ID NO: 1 or consisting of at least 10 contiguous nucleotides present in the nucleotide sequence as set forth in SEQ ID NO: 1 and an oligonucleotide primer comprising the nucleotide sequence as set forth in SEQ ID NO: 2 or consisting of at least 10 contiguous nucleotides present in the nucleotide sequence as set forth in SEQ ID NO: 2,
wherein the second oligonucleotide primer set comprises an oligonucleotide primer comprising the nucleotide sequence as set forth in SEQ ID NO: 3 or consisting of at least 10 contiguous nucleotides present in the nucleotide sequence as set forth in SEQ ID NO: 3 and an oligonucleotide primer comprising the nucleotide sequence as set forth in SEQ ID NO: 4 or consisting of at least 10 contiguous nucleotides present in the nucleotide sequence as set forth in SEQ ID NO: 4,
wherein the H. pylori -specific target sequence of the first oligonucleotide primer set is a nucleotide region of an H. pylori 23S rRNA gene corresponding to positions 1937-2793 of SEQ ID NO: 13 and at least part of said nucleotide region is amplified in the PCR reaction by the first oligonucleotide primer set, and
wherein the H. Pylori -specific target sequence of the second oligonucleotide primer set is a nucleotide region of an H. pylori 23S rRNA gene corresponding to positions 2482-2624 of SEQ ID NO: 13 and at least part of said nucleotide region is amplified in the PCR reaction by the second oligonucleotide primer set; and
b) detecting H. pylori DNA in said stool sample when the H. pylori -specific target sequences are amplified.
2. The method according to claim 1 , wherein the mutations in H. pylori 23S rRNA gene associated with clarithromycin resistance in H. pylori are further detected by a first probe consisting of at least 10 contiguous nucleotides present in a nucleotide sequence as set forth in SEQ ID NO: 7 and a second probe consisting of at least 10 contiguous nucleotides present in a nucleotide sequence as set forth in SEQ ID NO: 8 or wherein said first probe comprises the nucleotide sequence as set forth in SEQ ID NO: 7 and said second probe comprises the nucleotide sequence as set forth in SEQ ID NO: 8.
3. The method according to claim 1 or 2 , wherein the detection of H. pylori DNA in the stool sample when the H. pylori -specific target sequences are amplified is performed using a DNA chip, gel electrophoresis, a radiation measurement, a fluorescence measurement, or a phosphorescence measurement.
4. The method according to claim 1 , wherein steps a) and b) are performed in a single vessel.
5. The method according to claim 1 , wherein said method is performed as a real-time PCR assay.