A group-specific PCR assay for the detection of Helicobacteraceae in human gut.
BACKGROUND
Enterohepatic Helicobacter species are emerging pathogens, which are increasingly isolated from humans with enteric diseases. Nevertheless, current methods to detect Helicobacteraceae in the human gut have significant limitations.


METHODS
Based on 16S-rRNA gene alignments and computer aided primer analysis a set of group-specific PCR primers was designed. The evaluation of the PCR assay was performed using 36 ATCC reference strains and intestinal biopsies from 10 patients with defined gastric Helicobacter pylori status. The amplification products derived from clinical samples were cloned and subsequently analyzed by DNA sequencing. Sensitivity of the PCR-assay was determined by spiking previously negative tested samples with decreasing amounts of Helicobacter DNA.


RESULTS
The analysis of the ATCC reference strains revealed amplification products in all 14 Helicobacter strains and Wolinella succinogenes, 21 other microorganisms representing negative controls did not produce PCR fragments. Four out of the 10 patient-derived samples were positive. Three of them represented H. pylori-derived DNA confirming the gastric H. pylori infection in these patients. In the fourth patient, who was suffering from Crohn's disease, H. pullorum was identified. The sensitivity of the PCR assay was 0.1 pg of Helicobacter-derived DNA representing about 40 bacteria.


CONCLUSIONS
The novel PCR assay described here is an important new tool in rapid and sensitive assessment for the presence of Helicobacteraceae in human gut.
Bohr
Ulrich R M
UR
Department of Gastroenterology, Hepatology and Infectious Diseases, Otto-von-Guericke-University, Magdeburg, Germany.
Primus
Anett
A
Zagoura
Alexandra
A
Glasbrenner
Bernd
B
Wex
Thomas
T
Malfertheiner
Peter
P
D017931:DNA Primers; D004275:DNA, Ribosomal / Q000032:analysis; D016998:Helicobacter / Q000235:genetics / Q000302:isolation & purification*; D016481:Helicobacter Infections / Q000382:microbiology*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D013270:Stomach / Q000382:microbiology*
D017931:DNA Primers; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S