Impact of prehybridization PCR amplification on microarray detection of nitrifying bacteria in wastewater treatment plant samples

Slil Siripong, John J. Kelly, David A. Stahl, Bruce Rittmann

Research output: Contribution to journalArticle

20 Scopus citations

Abstract

A gel-based microarray that included a set of 26 oligonucleotide probes targeting all nitrifying bacteria at varying levels of specificity suggested the presence of targeted microorganisms when hybridized to RNA isolated from a wastewater treatment plant, but could not discriminate between perfectly matched and mismatched sequences due in part to low signal intensity. To enhance sensitivity and improve discrimination, polymerase chain reaction was used to selectively amplify the 16S rRNA genes of specific nitrifier groups. RNA transcribed from these DNA templates was hybridized to the microarray and thermal dissociation analysis was used to characterize the specificity of hybridization. Amplification with Nitrospira-specific primers resulted in the selective amplification of this target group, confirmed by both a significant increase in signal intensity and a melting profile identical to the reference RNA. In contrast, Nitrobacter was not detected in the environmental samples with probe Nbac1000 despite pre-amplification with Nitrobacter-specific primers, indicating the absence of strains containing this Nitrobacter-specific sequence. Pre-amplification using primers specific for β-Proteobacterial ammonia-oxidizing bacteria resulted in a significant increase in signal intensity for probe Nso190, but melting profiles for probe Nso190 showed a slight deviation between amplified RNA and the reference microorganism, suggesting that the amplification products contained some sequences that varied by a single nucleotide difference in the probe target region.

Original languageEnglish (US)
Pages (from-to)1564-1574
Number of pages11
JournalEnvironmental microbiology
Volume8
Issue number9
DOIs
StatePublished - Sep 1 2006

ASJC Scopus subject areas

  • Microbiology
  • Ecology, Evolution, Behavior and Systematics

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