Helicobacter pylori detection in human biopsies: a competitive PCR assay with internal control reveals false results.
A polymerase chain reaction assay (PCR) for the diagnosis of Helicobacter pylori in human gastric biopsies was developed. To prevent false-negative results while performing PCR on human tissues, an internal control is necessary. Primer set ACT1-ACT2 which specifically amplifies a 542-bp fragment of the 16S rRNA gene of H. pylori was used. dUTP and hot-start were used to prevent false-positives from carryover of previous products and avoid non-specific extension products. A competitive internal control DNA fragment was constructed to detect the presence of inhibitors. Biopsies from 101 unselected patients with gastric symptoms were tested. PCR results were compared with results from microscopy of histological sections and conventional culturing for H. pylori. Forty-two percent of the biopsies were found to contain compounds inhibiting the PCR. The addition of the internal control assures the performance of the PCR assay and is an important quality control parameter.
Thoreson
A C
AC
Department of Gastrointestinal Infections, Statens Serum Institut, Copenhagen, Denmark.
Borre
M
M
Andersen
L P
LP
Jørgensen
F
F
Kiilerich
S
S
Scheibel
J
J
Rath
J
J
Krogfelt
K A
KA
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001706:Biopsy; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis; D004275:DNA, Ribosomal; D005189:False Positive Reactions; D005260:Female; D005773:Gastroscopy; D016481:Helicobacter Infections / Q000382:microbiology*; D016480:Helicobacter pylori / Q000302:isolation & purification*; D006801:Humans; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods*; D011706:Pyloric Antrum / Q000382:microbiology* / Q000473:pathology; D011786:Quality Control; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity
D017931:DNA Primers; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S