Rapid combined characterization of microorganism and host genotypes using a single technology.
BACKGROUND
Genetic information is becoming increasingly important in diagnosis and prognosis of infectious diseases. In this study we investigated the possibility of using a single technology, the Pyrosequencing trade mark technology (Biotage AB, Uppsala, Sweden), to gather several kinds of important genetic information from the human pathogen Helicobacter pylori, as well as from the carrier of the H. pylori infection.


METHODS
DNA from 87 clinical isolates of H. pylori, 50 isolates from H. pylori-infected transgenic mice and nine gastric biopsies from H. pylori-infected patients was analyzed for targets in the 16S rRNA, 23S rRNA and cytotoxin associated gene A (cagA) genes to determine species identity, clarithromycin susceptibility and virulence level, respectively. In addition, three single nucleotide polymorphisms in the human interleukin-1B (IL-1B) gene, reported to affect the risk of developing gastric cancer, were analyzed in the gastric biopsy samples.


RESULTS
All DNA targets were processed and analyzed in parallel, enabling convenient genetic characterization of both pathogen and host. All genotypes were easily and accurately assigned. In the 16S rRNA analysis, 99.83% of the bases were correctly called.


CONCLUSIONS
We conclude that genetic analysis using Pyrosequencing trade mark technology was nonlaborious, and gave highly accurate data for different kinds of target. We therefore believe that this technology has the potential to complement or in the future substitute the time-consuming traditional microbial identification and typing methods, as well as enabling rapid typing of relevant host genetic markers.
Hjalmarsson
Sandra
S
Department of Medical Sciences, Clinical Bacteriology, Uppsala University, Uppsala, Sweden.
Alderborn
Anders
A
Fock
Caroline
C
Muldin
Ingrid
I
Kling
Helene
H
Uhlén
Mathias
M
Engstrand
Lars
L
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000942:Antigens, Bacterial / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics; D001706:Biopsy; D017291:Clarithromycin / Q000494:pharmacology; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification; D004275:DNA, Ribosomal / Q000737:chemistry / Q000302:isolation & purification; D024881:Drug Resistance, Bacterial / Q000235:genetics; D005753:Gastric Mucosa / Q000737:chemistry; D005798:Genes, Bacterial; D020459:Genes, rRNA; D044404:Genomic Islands / Q000235:genetics; D005838:Genotype; D016481:Helicobacter Infections / Q000235:genetics* / Q000382:microbiology; D016480:Helicobacter pylori / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D007375:Interleukin-1 / Q000235:genetics*; D051379:Mice; D008822:Mice, Transgenic; D017354:Point Mutation; D020641:Polymorphism, Single Nucleotide; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012338:RNA, Ribosomal, 23S / Q000235:genetics; D017422:Sequence Analysis, DNA; D014774:Virulence / Q000235:genetics
D000900:Anti-Bacterial Agents; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D007375:Interleukin-1; D012336:RNA, Ribosomal, 16S; D012338:RNA, Ribosomal, 23S; C080866:cagA protein, Helicobacter pylori; D017291:Clarithromycin