Multiple analyses of different regions of the 16S rRNA gene increase the diagnostic efficiency of PCR-DGGE technique for Helicobacter spp.

Abstract number: 1134_02_46

Abu Al-Soud W., Ouis I.-S., Ljungh Å., Wadström T.

Objectives:

Since the first identification of Helicobacter pylori by Warren and Marshal in 1983, members of the genus Helicobacter increased to more than 26 species, which have been detected in human and animals. The construction of species specific PCR assays is difficult due to the close relatedness between different Helicobacter species. The PCR-DGGE technique was developed for the identification of Helicobacter colonization. The aim of this study was to investigate the effect of multiple regions of the 16 rRNA gene on the diagnostic efficiency PCR-DGGE.

Methods:

DNA was extracted from 40 Helicobacter strains, which represent 20 Helicobacter species. Amplification of ~1.2 kb of helicobacter 16S rDNA, which contains V3 and V6-7 regions, was done using previously published Helicobacter genus specific primers C97 and C05. The PCR product can be used as a template for two Helicobacter genus PCR assays, the v6-7 regions 16S rDNA was amplified using the primers 1FC254 and 2RC686, the v-3 region was amplified using the primers BSF917 and BSR1114 published at (http://rrna.uia.ac.be/primers). DGGE analysis of the V3 and V6-7 regions was performed on 9% polyacrylamide gels containing urea and formamide gradient from 20–40% or 15–30%, respectively. Electrophoresis was performed in a DCode electrophoresis unit (BioRad) at constant voltage (200 V) at 60°C for 4 hours.

Results:

DGGE analysis of two regions of Helicobacter 16SrRNA gene showed mobility patterns that allowed discrimination of most Helicobacter species except those which are closely related such as H. pullorum–H. pametensis, H. ganmani–H. rodentium and H. bizozeroni–H. felis–H. salmonis.

Conclusions:

Helicobacter species are widely distributed in the gastrointestinal tract of mammals, birds and other animals. The PCR-DGGE technique has proven to be an easy, inexpensive and efficient tool for the identification of Helicobacter species and for the detection of colonisation by more than one Helicobacter species, without the need for species-specific PCR assays. In addition, the diagnostic efficiency of this technique was increased by analysis of multiple regions of the 16S rRNA gene.