In some instances, differentiation between species may be problematic when using 16S rRNA sequences due to similarity. In such instances, 23S rRNA may be a better alternative. The global standard library of rRNA sequences is constantly becoming larger and continuously being updated, and thus the possibility of a random hybridization event between a specifically-designed probe (based on complete and current data from a range of test organisms) and an undesired/unknown target organism cannot be easily dismissed. On the contrary, it is plausible that there exist microorganisms, yet to be identified, which are phylogenetically members of a probe target group, but have partial or near-perfect target sites, usually applies when designing group-specific probes.
Probably the greatest practical limitation to this technique is the lack of available automation.