Detection and typing of Helicobacter pylori cagA/vacA genes by radioactive, one-step polymerase chain reaction in stool samples from children.
The detection and molecular typing of Helicobacter pylori virulence genes in human stool specimens by polymerase chain reaction (PCR) require an adequate amount of bacterial DNA and an appropriately adjusted PCR protocol. DNA was isolated from stool samples of 39 H. pylori-infected and nine uninfected Colombian children using the QIAamp Kit following the manufacturer's instructions but with modifications. DNA templates were amplified for the vacA s and m regions and for the cagA gene by PCR using radioactively labeled (32P) primers. The modifications in the standard Qiagen protocol of stool DNA extraction increased the final concentration of eluted total stool DNA 4.7 times (117 +/- 17 versus 22 +/- 3 ng/microl; P < 0.0001). Nevertheless, its amplification by regular PCR programs (30-40 cycles) did not generate visible signals because of the very low ratio of H. pylori DNA to other DNA. PCR for 80 cycles successfully amplified vacA in 36/39 samples (sensitivity, 92.3%) and cagA fragments in 21/39 (53.8%) fecal DNA samples. Both s and m vacA regions were amplified in 33/36 (91.7%) DNA samples. The s1m1 genotype was the most commonly isolated variant, accounting for 17/36 or 47.2% of positive samples. The s2m2 genotype was ascertained to be frequent also (14/36 or 38.9%). Almost all (94.1%) s1m1 genotypes were cagA positive. The majority of s2m2 genotypes (78.6%) were not associated with the cagA gene. Neither cagA nor vacA fragments were amplified from DNA isolates of H. pylori-uninfected children nor from DNA isolated from six gastrointestinal bacterial strains (specificity, 100%). The data suggest that the proposed modified technique of DNA extraction and PCR assay of stool samples may be an effective and reliable noninvasive tool for the detection and typing of H. pylori cagA/vacA virulence genes in infected individuals.
Sicinschi
Liviu A
LA
Department of Pathology, Louisiana State University Health Sciences Center, 1901 Perdido Street, New Orleans, LA 70112-1393, USA. lsicinschi@hotmail.com
Correa
Pelayo
P
Bravo
Luis E
LE
Schneider
Barbara G
BG
D000942:Antigens, Bacterial; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification; D015373:Bacterial Typing Techniques / Q000379:methods* / Q000706:statistics & numerical data; D001483:Base Sequence; D002648:Child; D002675:Child, Preschool; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis; D005243:Feces / Q000382:microbiology*; D005798:Genes, Bacterial; D016480:Helicobacter pylori / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D007223:Infant; D010761:Phosphorus Radioisotopes; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D013045:Species Specificity; D014774:Virulence
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D010761:Phosphorus Radioisotopes; C074689:VacA protein, Helicobacter pylori; C080866:cagA protein, Helicobacter pylori