Comparison of urease gene primer sequences for PCR-based amplification assays in identifying the gastric pathogen Helicobacter pylori.
Different genomic DNA samples and primer sequences were evaluated in urease (ure) gene-based PCR assays for rapid identification of Helicobacter pylori. Purified DNA and heated (boiled) cell lysates of bacterial cultures from gastric biopsies were tested with three primer sets for unique internal ureA, ureA+B and ureC sequences. The heated-lysates of H. pylori were quick to prepare but more frequently gave unexpected variable or negative PCR results than assays performed on purified DNA, which were highly specific and reproducible for all three primer sets. Results indicated that sensitivity of the assay was linked to the size of the amplified target region rather than any particular strain feature, with the small 294 bp ureC product providing more accurate assays with heated-lysates of H. pylori. We strongly recommend that negative results in any PCR assay should be checked on purified DNA to exclude the possibility of a false-negative result.
Romero Lopez
C
C
National Collection of Type Cultures, Central Public Health Laboratory, London, UK.
Owen
R J
RJ
Banatvala
N
N
Abdi
Y
Y
Hardie
J M
JM
Davies
G R
GR
Feldman
R
R
D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D015139:Blotting, Southern; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics*; D005188:False Negative Reactions; D005798:Genes, Bacterial; D016480:Helicobacter pylori / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification; D006358:Hot Temperature; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D013698:Templates, Genetic; D014510:Urease / Q000235:genetics*
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D014510:Urease