DE19944168A1 - Helicobacter and Wolinella species-specific detection and identification using new oligonucleotide primers directed to 16S rRNA consensus sequences - Google Patents

Abstract

Detection and identification of Helicobacter and Wolinella species through use of oligonucleotide primers directed to 16S rRNA consensus sequences at positions corresponding to 540-584 nucleotides (nt) (sequence (I)) and 1173-1206 nt (sequence (II)) is new. All sequences are fully defined in the specification. A method for species-specific detection and species identification of bacteria of the species Helicobacter and Wolinella comprising treating a DNA or RNA sample to be investigated so that only a special gene fragment of the 16S rRNA is amplified, when the sample contains bacteria of the genus Helicobacter or Wolinella. At least one novel oligonucleotide primer of about 18 bp is used for the method, which are localized to consensus regions A and B of 16S rRNA corresponding to positions 540-584 nt (I) or 1173-1206 nt (II) of sequences fully defined in the specification. Amplification is carried out by using the chosen oligonucleotide primers fixed to the nucleotide sequence by a known molecular biology method, for example PCR. In the event of a positive detection, the resulting amplification product can be sequenced by a known DNA sequencing technique using the described oligonucleotide primers or internal primers as sequencing primers and identification of the Helicobacter species in question from the resulting nucleotide sequence by sequence comparison.

Description

Bacteria of the genus Helicobacter are Gram negative, spiral to curved Stick bacteria with flagella. Gene sequence analyzes show one Relationship between the genera Helicobacter, Wolinella, Campylobacter and Arcobacter. Together these genera form the epsilon Subdivision of Proteobacteria, also known as the RNA superfamily VI.

Today more than 30 different Helicobacter species are known. One and only previously known representative of the genus Wolinella is Wolinella succinogenes. All the time new representatives of the genus Helicobacter are rediscovered. The best so far researched Helicobacter species is Helicobacter pylori: This bacterium is found in People on the onset of chronic gastritis, stomach ulcers and Stomach tumors involved. In addition to Helicobacter pylori, others can Helicobacter species occur in humans. This is how Helicobacter settles Heilmannii as well as Helicobacter pylori the stomach and can be similar Cause diseases like Helicobacter pylori. More in humans Helicobacter species described colonize other localizations of the body. To date, only the pathogenic potential of these Helicobacter species exists very little data. Helicobacter cinaedi, Helicobacter fennelliae and Helicobacter rappiini have been detected in the gut and are associated with diarrhea Connected. In HIV-infected patients, blood cultures or Joint punctate Helicobacter strain Mainz, Helicobacter westermeadii and Helicobacter cinaedi isolated.

Low host specificity has been shown for some of the Helicobacter species. So some Helicobacter species are capable of different host organisms settle. Several of the known species are found in both humans and found in animals. Other Helicobacter species have so far only been found in animals found where they have been associated with various diseases become. The spectrum ranges from miscarriages to tumor development to inflammatory bowel disease and pancreatitis.

Helicobacter infections are often difficult to detect. In principle exist cultural, microscopic and molecular biological detection methods, which are used for Screening for Helicobacter infections can be used. Cultural Detection methods are due to low sensitivity, disruptive physiological flora or slow growth, or impossibility of cultivation  certain Helicobacter species are of limited use. Microscopic Techniques for the detection of Helicobacter can only be used to a limited extent. Especially The low sensitivity and specificity, as well as the high, have a disadvantageous effect Workload. The molecular biological described so far Detection methods are essentially based on the detection of specific ones bacterial nucleic acid sequences. Here is the quality of the examination in particular on the properties of the special DNA or RNA probes dependent on the implementation of the respective detection method (e.g. Northern or Southern blot, polymerase chain reaction (PCR), ligase chain reaction, etc.) can be used. The properties of these oligonucleotide probes will decisively influenced by the selection of the nucleic acid sequence. Suitable Probes that are unspecific enough around several organisms of one genus to be able to prove and at the same time are specific enough to be present genetically similar organisms are unable to provide false positive evidence however difficult to develop.

The object of the invention is to provide new oligonucleotide samples for genus-specific Detection and identification of Helicobacter and Wolinella species. According to the invention the object is achieved as indicated in the claims.

By locating the new oligonucleotide samples in two newly discovered Homology areas (see Tables 1 and 2) are these oligonucleotide samples particularly suitable for the genus-specific detection of Helicobacter species. These two new areas of homology show within the genus Helicobacter almost total homology, but indicate the sequences of bacteria related genera significant sequence differences, causing cross reactions be avoided. By using these oligonucleotides also as Following a positive detection, the sequencing primer can be the present Helicobacter species identified by sequencing the amplification products become.

A variation of the method described in claim 1 is in Claim 2 described. The identification of the present takes place here Helicobacter species by using a restriction fragment length Polymorphism (RFLP). That generated with the help of the described oligonucleotides Amplification product is processed by analyzing the band pattern can be differentiated between individual Helicobacter species.  

Exemplary embodiment for claim 1

1. DNA preparation: The total DNA is isolated from clinical samples. This happens through the use of commercially available kits (e.g. QlAmp Tissue Kit, QIAGEN GmbH, Hilden, Germany) according to the Manufacturer instructions are done.

2. Specific detection of Helicobacter DNA using genus-specific PCR: 1 µg of the DNA obtained in step 1 is used in a PCR with a total volume of 100 µl. The salt concentrations correspond to a standard PCR buffer with 1.5 mM MgCl ₂ . The nucleotide concentration is 200 µM for each of the four dNTP's. 50 pmoles each of the primers Seq1 [5'-CGCGTGGAGGATGAAGG-3 '] and Seq2 [5'-CGTGCAGCACCTGTTTTC-3'] are used. A thermostable polymerase is added after the batch has been heated to -950C. 35 cycles with denaturation at 95 ° C. for 30 seconds, annealing at 58 ° C. for 30 seconds and elongation at 72 ° C. for 60 seconds are carried out. Finally, there is a final elongation at 72 ° C for 10 min.

3. Detection of the amplified DNA: An aliquot of 10 ul of the PCT volume is separated with the aid of an agarose gel stained with ethidium bromide. After electrophoretic separation of the sample is the result under UV light analyzed. An approximately 630 bp PCR product is then visible when Helicobacter DNA was included in the sample.

4. The remaining 90 µl of the PCR volume can be used for species identification be used. By sequencing the nucleotide sequence of the approximately 630 bp large PCR product, which a portion of the 16S rRNA gene of the Helicobacter or Wolinella species, the Helicobacter species present - determined. Thereby for the Sequencing reaction commercially available kits used (e.g. ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit, PerkinElmer, Weiterstadt, Germany). The individual sequencing reactions are carried out in Manufacturer protocol defined amount of the PCR product used. As Sequencing primer serves Seq 1 when the sense sequence is sequenced should. For sequencing the antisense sequence is used as a sequencing primer Seq 2 used. The further steps of the sequencing reaction take place according to the manufacturer's recommendations.  

Embodiment for claim 2

According to the current state of research, the human gastric mucosa can be colonized by Helicobacter pylori or Helicobacter heilmannii. Around Differentiating between these two infections is as follows proceeded:

1. Human gastric mucosal biopsies obtained by endoscopy are, according to application example for claim 1, Steps 1-3 dealt with. This creates both in the case of a Helicobacter pylori infection as well as in the presence of a Helicobacter Heilmannii infection an approximately 630 bp amplification product.

2. This amplification product with the restriction enzyme Hhal (e.g. New England Biolabs, Schwalbach / Taunus, Germany) according to the Manufacturer recommendations digested. Helicobacter pylori results in three smaller fragments, with Helicobacter heilmannii the size of the Amplification product unchanged, since no Hha I interface within the Amplification product exists.

3. Stained by electrophoretic separation using ethidium bromide These fragments can be separated by agarose gels and visible under UV light be made. Based on the fragment sizes, it can be concluded whether Helicobacter pylori or Helicobacter heilmannii is present in the samples.  

Table 1

Homology region 1 (position 540-584) in which oligonucleotide 1 is located

The preferred for the genus-specific detection of Helicobacter species Oligonucleotide Seq1 used is shown in bold. In particular, differs the nucleotide sequence in the 3 'end of the oligonucleotide in front of the nucleotide sequence from related bacteria such as Arcobacter (A.), Campylobacter (C.) and Bacteroides (B.) Species. The base numbering is based on the counting method Helicobacter bilis, strain Hb2, GenBank Accession U18767 [Fox, J.G. et al. (1995) J. Clin. Microbiol. 33: 445-454].  

Table 2

Homology region 2 (position 1173-1206) in which oligonucleotide 2 is located

The preferred for genus-specific detection of Helicobacter species Oligonucleotide Seq2 used corresponds to the antisense strand of the bold sequence area shown. In particular, the Nucleotide sequence in the 3 'end of the oligonucleotide from the nucleotide sequence of related bacteria such as Arcobacter (A.), Campylobacter (C.) and Bacteroides (B.) Species. The base numbering is based on the counting method Helicobacter bilis, strain Hb2, GenBank Accession U18767 [Fox, J.G. et al. (1995) J. Clin. Microbiol. 33: 445-454].

Claims (2)

1. A method for the genus-specific detection and species identification of bacteria of the genera Helicobacter and Wolinella, characterized in that

• a) DNA or RNA samples to be examined are treated in such a way that a specific gene fragment of the 16S ribosomal RNA (16S rRNA) only replicates if bacteria of the genus Helicobacter or Wolinella are contained in the sample,
• b) this is achieved by using one or more novel oligonucleotide primers of approximately 18 base pairs (bp) in length for the method, which are characterized in that they are located in one of the two 16S rRNA consensus regions A, corresponding to positions 540 to 584, or B, are located according to items 1173-1206 as shown in Tables 1 and 2 of Appendix 1 and 2;
• c) the oligonucleotide primers designated with Seq1 [5'-CGCGTGGAGGATGAAGG-3 '] and Seq2 [5'-CGTGCAGCACCTGTTTTC-3'] or oligonucleotides derived therefrom are preferably used for the process, which are chosen such that they form the 16S rRNA gene , the RNA itself or its antisense sequence are sufficiently homologous to hybridize specifically to all known Helicobacter species, but at the same time differ as much as possible from the nucleotide sequences of related organisms in order to prevent cross-reactions,
• d) the amplification of the nucleotide sequence determined by the selected oligonucleotide primers is carried out in a manner known per se by molecular biological techniques, such as, for example, the polymerase chain reaction;
• e) in the event of a positive detection, the resulting amplification product will be sequenced by known DNA sequencing techniques, using the above-described oligonucleotide primers or internal primers as sequencing primers,
• f) on the basis of the resulting nucleotide sequence, the Helicobacter species present are identified by sequence comparison.

2. The method according to claim 1, characterized in that

• a) as described in claim 1a) to d), specific oligonucleotides are used to detect Helicobacter species by amplifying their 16S rRNA, the identification of which, however, takes place in that
• b) the amplification product is digested in a known manner by selected restriction enzymes in the sense of a restriction fragment length polymorphism (RFLP),
• c) the DNA fragments are separated by electrophoresis techniques,
• d) the resulting Helicobacter species is inferred from the resulting band pattern.