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Specific and sensitive detection of H. pylori in biological specimens by real-time RT-PCR and in situ hybridization.

Liu H, Rahman A, Semino-Mora C, Doi SQ, Dubois A - PLoS ONE (2008)

Bottom Line: This sub-domain was then used to design (1) a set of high quality RT-PCR primers and probe that encompassed a 76-bp sequence and included at least two mismatches with other Helicobacter sp. 16S rRNA; and (2) in situ hybridization antisense probes.The sensitivity and specificity of the approaches were then demonstrated by using gastric biopsy specimens from patients and rhesus monkeys.This H. pylori-specific region of the 16S rRNA sequence is highly conserved among most H. pylori strains and allows specific detection, identification, and quantification of this bacterium in biological specimens.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland, United States of America.

ABSTRACT
PCR detection of H. pylori in biological specimens is rendered difficult by the extensive polymorphism of H. pylori genes and the suppressed expression of some genes in many strains. The goal of the present study was to (1) define a domain of the 16S rRNA sequence that is both highly conserved among H. pylori strains and also specific to the species, and (2) to develop and validate specific and sensitive molecular methods for the detection of H. pylori. We used a combination of in silico and molecular approaches to achieve sensitive and specific detection of H. pylori in biologic media. We sequenced two isolates from patients living in different continents and demonstrated that a 546-bp domain of the H. pylori 16S rRNA sequence was conserved in those strains and in published sequences. Within this conserved sequence, we defined a 229-bp domain that is 100% homologous in most H. pylori strains available in GenBank and also is specific for H. pylori. This sub-domain was then used to design (1) a set of high quality RT-PCR primers and probe that encompassed a 76-bp sequence and included at least two mismatches with other Helicobacter sp. 16S rRNA; and (2) in situ hybridization antisense probes. The sensitivity and specificity of the approaches were then demonstrated by using gastric biopsy specimens from patients and rhesus monkeys. This H. pylori-specific region of the 16S rRNA sequence is highly conserved among most H. pylori strains and allows specific detection, identification, and quantification of this bacterium in biological specimens.

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Sequences of H. pylori 16S rRNA that are 100% homologous among USU-101, USU-102, J99, and 26695 H. pylori strains (A), and also do not match the 16S rRNA sequences of E. coli, S. bareilly, C. jejuni, and S. flexneri (B), nor the sequences of H. heilmannii (C), and encompass the set of primers and TaqMan probe (D).E shows the sequences of the two ISH probes used in the present study (546-bp from 187 to 732 of J99 16S rRNA sequence).
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pone-0002689-g002: Sequences of H. pylori 16S rRNA that are 100% homologous among USU-101, USU-102, J99, and 26695 H. pylori strains (A), and also do not match the 16S rRNA sequences of E. coli, S. bareilly, C. jejuni, and S. flexneri (B), nor the sequences of H. heilmannii (C), and encompass the set of primers and TaqMan probe (D).E shows the sequences of the two ISH probes used in the present study (546-bp from 187 to 732 of J99 16S rRNA sequence).

Mentions: To examine whether a particular domain of H. pylori 16S rRNA sequence was conserved among strains with markedly different fingerprints, the DNASTAR software was used to perform multi-alignment of the 16S rRNA sequences of the four strains described above. We discovered that a 546-bp nucleotide domain was 100% conserved among these five sequences (Figure 2A). To determine whether this domain was also conserved among various H. pylori strains, we performed a nucleotide BLAST of this sequence and observed that the sequence was 100% homologous to 49 H. pylori sequences published in GenBank to date.


Specific and sensitive detection of H. pylori in biological specimens by real-time RT-PCR and in situ hybridization.

Liu H, Rahman A, Semino-Mora C, Doi SQ, Dubois A - PLoS ONE (2008)

Sequences of H. pylori 16S rRNA that are 100% homologous among USU-101, USU-102, J99, and 26695 H. pylori strains (A), and also do not match the 16S rRNA sequences of E. coli, S. bareilly, C. jejuni, and S. flexneri (B), nor the sequences of H. heilmannii (C), and encompass the set of primers and TaqMan probe (D).E shows the sequences of the two ISH probes used in the present study (546-bp from 187 to 732 of J99 16S rRNA sequence).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2481290&req=5

pone-0002689-g002: Sequences of H. pylori 16S rRNA that are 100% homologous among USU-101, USU-102, J99, and 26695 H. pylori strains (A), and also do not match the 16S rRNA sequences of E. coli, S. bareilly, C. jejuni, and S. flexneri (B), nor the sequences of H. heilmannii (C), and encompass the set of primers and TaqMan probe (D).E shows the sequences of the two ISH probes used in the present study (546-bp from 187 to 732 of J99 16S rRNA sequence).
Mentions: To examine whether a particular domain of H. pylori 16S rRNA sequence was conserved among strains with markedly different fingerprints, the DNASTAR software was used to perform multi-alignment of the 16S rRNA sequences of the four strains described above. We discovered that a 546-bp nucleotide domain was 100% conserved among these five sequences (Figure 2A). To determine whether this domain was also conserved among various H. pylori strains, we performed a nucleotide BLAST of this sequence and observed that the sequence was 100% homologous to 49 H. pylori sequences published in GenBank to date.

Bottom Line: This sub-domain was then used to design (1) a set of high quality RT-PCR primers and probe that encompassed a 76-bp sequence and included at least two mismatches with other Helicobacter sp. 16S rRNA; and (2) in situ hybridization antisense probes.The sensitivity and specificity of the approaches were then demonstrated by using gastric biopsy specimens from patients and rhesus monkeys.This H. pylori-specific region of the 16S rRNA sequence is highly conserved among most H. pylori strains and allows specific detection, identification, and quantification of this bacterium in biological specimens.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland, United States of America.

ABSTRACT
PCR detection of H. pylori in biological specimens is rendered difficult by the extensive polymorphism of H. pylori genes and the suppressed expression of some genes in many strains. The goal of the present study was to (1) define a domain of the 16S rRNA sequence that is both highly conserved among H. pylori strains and also specific to the species, and (2) to develop and validate specific and sensitive molecular methods for the detection of H. pylori. We used a combination of in silico and molecular approaches to achieve sensitive and specific detection of H. pylori in biologic media. We sequenced two isolates from patients living in different continents and demonstrated that a 546-bp domain of the H. pylori 16S rRNA sequence was conserved in those strains and in published sequences. Within this conserved sequence, we defined a 229-bp domain that is 100% homologous in most H. pylori strains available in GenBank and also is specific for H. pylori. This sub-domain was then used to design (1) a set of high quality RT-PCR primers and probe that encompassed a 76-bp sequence and included at least two mismatches with other Helicobacter sp. 16S rRNA; and (2) in situ hybridization antisense probes. The sensitivity and specificity of the approaches were then demonstrated by using gastric biopsy specimens from patients and rhesus monkeys. This H. pylori-specific region of the 16S rRNA sequence is highly conserved among most H. pylori strains and allows specific detection, identification, and quantification of this bacterium in biological specimens.

Show MeSH