Quantification of Frankia in soils using SYBR Green based qPCR.
A SYBR Green based qPCR method was developed for the quantification of clusters 1 and 3 of the actinomycete Frankia in soils. Primer nifHr158 was designed to be used as reverse primer in combination with forward primer nifHf1 specifically amplifying a 191-bp fragment of the nifH gene of these Frankia. The primer combination was tested for specificity on selected pure cultures, and by comparative sequence analyses of randomly selected clones of a clone library generated with these primers from soil DNA extracts. After adjustments of DNA extraction conditions, and the determination of extraction efficiencies used for sample normalization, copy numbers of nifH genes representing Frankia of clusters 1 and 3 were quantified in different mineral soils, resulting in cell density estimates for these Frankia of up to 10(6) cells [g soil {dry weight}](-1) depending on the soil. Despite indications that the nifH gene is not a perfect target for the quantification of Frankia, the qPCR method described here provides a new tool for the quantification and thus a more complete examination of the ecology of Frankia in soils.
Texas State University, Department of Biology, San Marcos, TX 78666, USA.
Suvidha
S
Samant
Qiong
Q
Sha
Anita
A
Iyer
Priti
P
Dhabekar
Dittmar
D
Hahn
D058491:Bacterial Load / Q000379:methods*; D052160:Benzothiazoles; D017931:DNA Primers / Q000235:genetics; D003959:Diamines; D040161:Frankia / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D009930:Organic Chemicals / Q000378:metabolism; D010088:Oxidoreductases / Q000235:genetics; D011804:Quinolines; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D012988:Soil Microbiology; D013194:Staining and Labeling / Q000379:methods
D052160:Benzothiazoles; D017931:DNA Primers; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; D010088:Oxidoreductases; C029130:nitrogenase reductase