Detection of Actinobacteria cultivated from environmental samples reveals bias in universal primers

Abstract

Aims: The aims of this study were to develop media to cultivate actinomycetes, screen the resulting isolates with Actinobacteria‐specific primers, and examine the efficacy of detection of the actinobacterial isolates with universal primers. Methods and Results: Soil‐extract medium was developed for a terrestrial bluff environment. Recovered isolates were subjected to polymerase chain reaction (PCR) with taxon‐specific primers to identify Actinobacteria. Universal bacterial primers 24f and 1492r (modified and original versions) were used to amplify the 16S rRNA gene from the putative Actinobacteria. While both reverse primers failed to provide amplification products from 20% to 50% of the isolates, the 1492r primer detected Actinobacteria more effectively than 1492r‐mod. The region of the gene containing the annealing site for the 1492r primers from 15 isolates that failed to amplify showed no differences in nucleotide sequence to the original 1492r primer. Conclusions: Universal 16S rRNA gene primers are not capable of amplifying this gene from all bacteria within an environmental sample. Some Actinobacteria may share 100% sequence similarity to universal primers but remain undetected. Significance and Impact of the Study: These findings are important for studies of particular taxa in environmental samples where reactions utilizing universal primers may not reveal the extent of their presence and diversity.

Empirical Testing of 16S rRNA Gene PCR Primer Pairs Reveals Variance in Target Specificity and Efficacy Not Suggested by In Silico Analysis

“…This strategy made it challenging to identify two specific, opposing primers that would generate a readily detectable PCR product, and we therefore paired a target-specific forward primer with the same general reverse primer used in the original cloning exercise. (4,10,12) have shown that so-called "universal" primer sets are incapable of efficiently amplifying all targets, even those from pure cultures and with perfect sequence matches, casting doubts on the universality of such primers. Further, other groups have suggested that 16S rRNA gene group-specific primers can produce upwards of 25% nontarget amplification, making them unsuitable for qPCR (10).…”

“…(4,10,12) have shown that so-called "universal" primer sets are incapable of efficiently amplifying all targets, even those from pure cultures and with perfect sequence matches, casting doubts on the universality of such primers. Further, other groups have suggested that 16S rRNA gene group-specific primers can produce upwards of 25% nontarget amplification, making them unsuitable for qPCR (10). It has also been suggested that reliance on the slowly evolving 16S rRNA gene makes it difficult to recognize recent events in the evolutionary history of a species, such as those associated with incipient speciation (24), which might also be a contributing factor to the high levels of nonspecific binding for some of the primer sets employed here.…”

Extracellular biosynthesis of silver nanoparticle using Streptomyces sp. 09 PBT 005 and its antibacterial and cytotoxic properties

“…The cyclic conditions were as follows: initial denaturation at 94°C for 3 min, 35 cycles of 94°C for 1 min, 54°C for 1 min, and 72°C for 2 min, and final extension of 10 min at 10 min and hold at 4°C. The PCR products were confirmed by 1 % agarose gel electrophoresis (Farris et al 2007). …”

PCR bias associated with conserved primer binding sites, used to determine genotype diversity within Citrus tristeza virus populations

“…Polyspecific primers are usually designed to amplify multiple templates present in samples, often including a variety of organisms (Farris and Olsen, 2007). It is critical to design polyspecific primers that are specific enough to target only the taxonomic groups of interest but at the same time not biasing the PCR reaction toward only certain components of a population (Anderson et al, 2003).…”