A real-time qPCR assay for the detection of the nifH gene of Methanobrevibacter smithii, a potential indicator of sewage pollution.

AIMS: To develop a quantitative, real-time PCR assay to detect the nifH gene of Methanobrevibacter smithii. Methanobrevibacter smithii is a methanogenic archaea found in the intestinal tract of humans that may be a useful indicator of sewage pollution in water. METHODS AND RESULTS: Quantification standards were prepared from Meth. smithii genomic DNA dilutions, and a standard curve was used to quantify the target gene and calculate estimated genome equivalency units. A competitive internal positive control was designed and incorporated into the assay to assess inhibition in environmental extracts. Testing the assay against a panel of 23 closely related methanogen species demonstrated specificity of the assay for Meth. smithii. A set of 36 blind water samples was then used as a field test of the assay. The internal control identified varying levels of inhibition in 29 of 36 (81%) samples, and the Meth. smithii target was detected in all water samples with known sewage input. CONCLUSIONS: The quantitative PCR assay developed in this study is a sensitive and rapid method for the detection of the Meth. smithii nifH gene that includes an internal control to assess inhibition. Further research is required both to better evaluate host specificity of this assay and the correlation with human health risks. SIGNIFICANCE AND IMPACT OF THE STUDY: This research is the first description of the development of a rapid and sensitive quantitative assay for a methanogenic archaeal indicator of sewage pollution.

Moving beyond GK-12.

The National Science Foundation GK-12 program has made more than 300 awards to universities, supported thousands of graduate student trainees, and impacted thousands of K-12 students and teachers. The goals of the current study were to determine the number of sustained GK-12 programs that follow the original GK-12 structure of placing graduate students into classrooms and to propose models for universities with current funding or universities interested in starting a program. Results from surveys, literature reviews, and Internet searches of programs funded between 1999 and 2008 indicated that 19 of 188 funded sites had sustained in-classroom programs. Three distinct models emerged from an analysis of these programs: a full-stipend model, in which graduate fellows worked with partner teachers in a K-12 classroom for 2 d/wk; a supplemental stipend model in which fellows worked with teachers for 1 d/wk; and a service-learning model, in which in-classroom activity was integrated into university academic coursework. Based on these results, potential models for sustainability and replication are suggested, including establishment of formal collaborations between sustained GK-12 programs and universities interested in starting in-classroom programs; development of a new Teaching Experience for Fellows program; and integration of supplemental fellow stipends into grant broader-impact sections.

Detection of the nifH gene of Methanobrevibacter smithii: a potential tool to identify sewage pollution in recreational waters.

AIMS: The goal of this study was to develop and test the efficacy of a PCR assay for the environmental detection of the nifH gene of Methanobrevibacter smithii, a methanogen found in human faeces and sewage. METHODS AND RESULTS: PCR primers for the nifH gene of M. smithii were designed, tested and used to detect the presence or absence of this organism in faecal and environmental samples. Specificity analysis showed that the Mnif primers amplified products only in M. smithii pure culture strains (100%), human faeces (29%), human sewage samples (93%) and sewage-contaminated water samples (100%). No amplification was observed when primers were tested against 43 bacterial stock cultures, 204 animal faecal samples, 548 environmental bacterial isolates and water samples from a bovine waste lagoon and adjacent polluted creek. Sequencing of PCR products from sewers demonstrated that a 222-bp product was the nifH gene of M. smithii. The minimal amount of total DNA required for the detection of M. smithii was 10 ng for human faeces, 10 ng for faecally contaminated water and 5 ng for sewage. Recreational water seeded with M. smithii established a lower detection limit of 13 cells ml(-1). CONCLUSIONS: The Mnif assay developed during this investigation showed successful detection of M. smithii in individual human faecal samples, sewage and sewage-contaminated water but not in uncontaminated marine water or bovine-contaminated waters. The Mnif assay appears to be a potentially useful method to detect sewage-polluted coastal waters. SIGNIFICANCE AND IMPACT OF THE STUDY: This study was the first to utilize methanogens as an indicator of sewage pollution. Mnif PCR detection of M. smithii was shown to be a rapid, inexpensive and reliable test for determining the presence or absence of sewage pollution in coastal recreational waters.