Evaluation of a PCR primer based on the isocitrate dehydrogenase gene for detection of Helicobacter pylori in feces.
In order to improve detection and identification of Helicobacter pylori in highly contaminated samples, we evaluated new specific primers based on the DNA base sequence within the isocitrate dehydrogenase (icd) gene to amplify a 1,200-bp DNA segment. The specificity of the icd primer was tested against DNA derived from various bacteria, including 7 Helicobacter species and a panel of 1 gram-variable, 2 gram-positive, and 16 gram-negative bacteria, as well as DNA from houseflies and feces from H. pylori-negative patients. The primers permitted the detection of all clinical H. pylori isolates tested, but no reactions were observed with negative controls. Several procedures for DNA extraction from feces were evaluated using PCR with icd primers. The lower limits of detection of H. pylori DNA from two different sources containing the same number of H. pylori organisms, a pure culture and feces spiked with H. pylori, were established for each extraction method tested. The results were 8.0 x 10(3) CFU/ml for cultures of pure H. pylori, and 8.0 x 10(6) CFU/ml for H. pylori from feces, using the phenol-chloroform method; 8.0 x 10(2) and 7.0 x 10(3) CFU/ml, respectively, for a glass matrix and chaotropic solution protocol; 8.0 x 10(2) and 7.0 x 10(3) CFU/ml, respectively, for the QIAamp tissue kit; and 5.0 x 10(2) and 5.0 x 10(3) CFU/ml, respectively, for the XTRAX DNA extraction kit. We conclude that the use of the icd gene as a primer for PCR represents a specific and sensitive assay for detection of H. pylori in highly contaminated samples.
Argyros
F C
FC
Department of Biology, Northeastern University, Boston, Massachusetts, USA.
Ghosh
M
M
Huang
L
L
Masubuchi
N
N
Cave
D R
DR
Grübel
P
P
D000818:Animals; D017931:DNA Primers; D004381:Duodenal Ulcer / Q000382:microbiology; D005243:Feces / Q000382:microbiology*; D006090:Gram-Negative Bacteria / Q000235:genetics; D006094:Gram-Positive Bacteria / Q000235:genetics; D016481:Helicobacter Infections / Q000175:diagnosis*; D016480:Helicobacter pylori / Q000235:genetics / Q000302:isolation & purification*; D006793:Houseflies / Q000235:genetics / Q000382:microbiology; D006801:Humans; D007521:Isocitrate Dehydrogenase / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D017931:DNA Primers; D007521:Isocitrate Dehydrogenase