Quantification of Frankia in soils using SYBR Green based qPCR

Samant, Suvidha; Sha, Qiong; Iyer, Anita K.; Dhabekar, Priti; Hahn, Dittmar

doi:10.1016/j.syapm.2011.12.004

Cited by 23 publications

(26 citation statements)

References 51 publications

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“…CA). Extraction efficiencies were determined by qPCR quantification of added DNA of the nitrogen-fixing symbiont Frankia (strain Ag45/Mut15) before and after purification [35], and used to normalize quantitative analyses of salmonellae [36,37]. Detection of salmonellae by qPCR was based on an established protocol for end-point PCR using primers 139 (5′-GTG AAA TTA TCG CCA CGT TCG GGC AA) and 141 (5′-TCA TCG CAC CGT CAA AGG AAC C) [38] to amplify a 284-bp fragment of the invA gene that encodes a protein of a type III secretion system, essential for the invasion of epithelial cells by salmonellae [39,40].…”

Section: Quantification Of Salmonellae In Biofilms and Water By Qpcrmentioning

confidence: 99%

Quantifying Salmonella Population Dynamics in Water and Biofilms

et al. 2012

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Members of the bacterial genus Salmonella are recognized worldwide as major zoonotic pathogens often found to persist in non-enteric environments including heterogeneous aquatic biofilms. In this study, Salmonella isolates that had been detected repeatedly over time in aquatic biofilms at different sites in Spring Lake, San Marcos, Texas, were identified as serovars Give, Thompson, Newport and -:z10:z39. Pathogenicity results from feeding studies with the nematode Caenorhabditis elegans as host confirmed that these strains were pathogenic, with Salmonella-fed C. elegans dying faster (mean survival time between 3 and 4 days) than controls, i.e., Escherichia coli-fed C. elegans (mean survival time of 9.5 days). Cells of these isolates inoculated into water at a density of up to 10(6) ml(-1) water declined numerically by 3 orders of magnitude within 2 days, reaching the detection limit of our quantitative polymerase chain reaction (qPCR)-based quantification technique (i.e., 10(3) cells ml(-1)). Similar patterns were obtained for cells in heterogeneous aquatic biofilms developed on tiles and originally free of Salmonella that were kept in the inoculated water. Cell numbers increased during the first days to more than 10(7) cells cm(-2), and then declined over time. Ten-fold higher cell numbers of Salmonella inoculated into water or into biofilm resulted in similar patterns of population dynamics, though cells in biofilms remained detectable with numbers around 10(4) cells cm(-2) after 4 weeks. Independent of detectability by qPCR, samples of all treatments harbored viable salmonellae that resembled the inoculated isolates after 4 weeks of incubation. These results demonstrate that pathogenic salmonellae were isolated from heterogeneous aquatic biofilms and that they could persist and stay viable in such biofilms in high numbers for some time.

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Section: Quantification Of Salmonellae In Biofilms and Water By Qpcrmentioning

confidence: 99%

Quantifying Salmonella Population Dynamics in Water and Biofilms

et al. 2012

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“…While these results indicate a similar abundance of frankiae in soils vegetated with birch and alder, any correlation of nodulation units with cell numbers is highly biased because a nodule can theoretically be induced by a single spore, a hyphal fragment, or a colony [18]. We recently developed a SYBR Green-based quantitative PCR (qPCR) that used either nifH or 23S rRNA gene sequences as target in DNA extracts from soil samples that allowed us to quantify nitrogen-fixing members of the genus Frankia directly in soil samples [19,20]. Quantification results in different mineral soils from temperate regions using both targets were comparable, with cell density estimates for frankiae of up to more than 10 6 cells [g soil {dry wt.}]…”

Section: Introductionmentioning

confidence: 99%

Abundance and Relative Distribution of Frankia Host Infection Groups Under Actinorhizal Alnus glutinosa and Non-actinorhizal Betula nigra Trees

et al. 2015

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Quantitative polymerase chain reaction (qPCR) was used to assess the abundance and relative distribution of host infection groups of the root-nodule forming, nitrogen-fixing actinomycete Frankia in four soils with similar physicochemical characteristics, two of which were vegetated with a host plant, Alnus glutinosa, and two with a non-host plant, Betula nigra. Analyses of DAPI-stained cells at three locations, i.e., at a distance of less than 1 m (near stem), 2.5 m (middle crown), and 3-5 m (crown edge) from the stems of both tree species revealed no statistically significant differences in abundance. Frankiae generally accounted for 0.01 to 0.04 % of these cells, with values between 4 and 36 × 10(5) cells (g soil)(-1). In three out of four soils, abundance of frankiae was significantly higher at locations "near stem" and/or "middle crown" compared to "crown edge," while numbers at these locations were not different in the fourth soil. Frankiae of the Alnus host infection group were dominant in all samples accounting for about 75 % and more of the cells, with no obvious differences with distance to stem. In three of the soils, all of these cells were represented by strain Ag45/Mut15. In the fourth soil that was vegetated with older A. glutinosa trees, about half of these cells belonged to a different subgroup represented by strain ArI3. In all soils, the remaining cells belonged to the Elaeagnus host infection group represented by strain EAN1pec. Casuarina-infective frankiae were not found. Abundance and relative distribution of Frankia host infection groups were similar in soils under the host plant A. glutinosa and the non-host plant B. nigra. Results did thus not reveal any specific effects of plant species on soil Frankia populations.

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“…Primer combinations for Sybr Green-based qPCR targeted nifH gene sequences (16) or 23S rRNA gene sequences (15) (Table 1). Sybr Green-based analyses were carried out in triplicate in a total volume of 10 l containing 5 l of SsoADV Sybr Green mix (Bio-Rad, Hercules, CA), 0.125 l of forward and reverse primers (100 nM each), and 1 l of DNA template using an initial denaturation at 95°C for 5 min and 40 cycles of denaturation at 95°C, annealing at 62, 64, or 66°C depending on the primer combination (Table 1), and extension at 72°C, each for 30 s (15, 17).…”

Section: Methodsmentioning

confidence: 99%

“…Information on the ecology of cluster 2 and 4 frankiae therefore is quite limited. We have recently developed Sybr Green-based quantitative PCR (qPCR) methods that used nifH or 23S rRNA genes as a target to quantify uncultured Frankia populations in different soils (15)(16)(17). nifH as a target only detected the combination of members of clusters 1 and 3 but not those of clusters 2 and 4, while 23S rRNA genes as targets covered all frankiae on the genus level, i.e., clusters 1, 2, 3, and 4 together.…”

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Sybr Green- and TaqMan-Based Quantitative PCR Approaches Allow Assessment of the Abundance and Relative Distribution of Frankia Clusters in Soils

Tekaya

Ganesan

Guerra

et al. 2017

Appl Environ Microbiol

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The nodule-forming actinobacterial genus Frankia can generally be divided into 4 taxonomic clusters, with clusters 1, 2, and 3 representing nitrogen-fixing strains of different host infection groups and cluster 4 representing atypical, generally non-nitrogen-fixing strains. Recently, quantitative PCR (qPCR)-based quantification methods have been developed for frankiae of clusters 1 and 3; however, similar approaches for clusters 2 and 4 were missing. We amended a database of partial 23S rRNA gene sequences of Frankia strains belonging to clusters 1 and 3 with sequences of frankiae representing clusters 2 and 4. The alignment allowed us to design primers and probes for the specific detection and quantification of these Frankia clusters by either Sybr Green-or TaqMan-based qPCR. Analyses of frankiae in different soils, all obtained from the same region in Illinois, USA, provided similar results, independent of the qPCR method applied, with abundance estimates of 10 ϫ 10 5 to 15 ϫ 10 5 cells (g soil) Ϫ1 depending on the soil. Diversity was higher in prairie soils (native, restored, and cultivated), with frankiae of all 4 clusters detected and those of cluster 4 dominating, while diversity in soils under Alnus glutinosa, a host plant for cluster 1 frankiae, or Betula nigra, a related nonhost plant, was restricted to cluster 1 and 3 frankiae and generally members of subgroup 1b were dominating. These results indicate that vegetation affects the basic composition of frankiae in soils, with higher diversity in prairie soils compared to much more restricted diversity under some host and nonhost trees. IMPORTANCE Root nodule formation by the actinobacterium Frankia is host plant specific and largely, but not exclusively, correlates with assignments of strains to specific clusters within the genus. Due to the lack of adequate detection and quantification tools, studies on Frankia have been limited to clusters 1 and 3 and generally excluded clusters 2 and 4. We have developed tools for the detection and quantification of clusters 2 and 4, which can now be used in combination with those developed for clusters 1 and 3 to retrieve information on the ecology of all clusters delineated within the genus Frankia. Our initial results indicate that vegetation affects the basic composition of frankiae in soils, with higher diversity in prairie soils compared to much more restricted diversity under some host and nonhost trees.

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Quantification of Frankia in soils using SYBR Green based qPCR

Cited by 23 publications

References 51 publications

Quantifying Salmonella Population Dynamics in Water and Biofilms

Quantifying Salmonella Population Dynamics in Water and Biofilms

Abundance and Relative Distribution of Frankia Host Infection Groups Under Actinorhizal Alnus glutinosa and Non-actinorhizal Betula nigra Trees

Sybr Green- and TaqMan-Based Quantitative PCR Approaches Allow Assessment of the Abundance and Relative Distribution of Frankia Clusters in Soils

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