Validating novel oligonucleotide primers targeting three classes of bacterial non-specific acid phosphatase genes in grassland soils

Abstract

Microbially driven mineralization of organic phosphorus forms is of particular importance in the soil environment, where it becomes available to plants as inorganic orthophosphates. In acidic soils, microbes produce non-specific acid phosphatases (NSAPs; E.C. 3.1.3.2) which act on the most common forms of organic P in the soil. Our understanding of phosphorus turnover in soils would greatly benefit from an improvement in research tools targeting these genes. Thus, in this study we developed two novel oligonucleotide PCR primer sets, that will enable researchers to target the present and active communities of bacteria with the genetic potential of acid phosphatase production. A total of three primer sets were validated to target the three classes of NSAPs. Utilizing Illumina MiSeq, amplicons from grassland pasture soils were sequenced. The resulting target specificity was high for all three groups; CAAP (97.2%), CBAP (99.5%), and CCAP (94.8%). Quantification of target genes by qPCR indicated measurable differences between classes, ranging from 5 log to 7.5 log for CAAP, 6 log to 8 log for CBAP, and 4 log to 5 log for CCAP. The validated primer sets were specific to the target genes and identified potential quantitative differences between the NSAP classes.