A unique amplification chemistry was developed that allows highly specific isothermal detection of bacteria, and presumably eukaryotic cells, at sub-single-cell sensitivities. This is based on the ability to detect rRNA (ribosomal RNA) rather than DNA, as is typical. The difference is that rRNA is present in metabolizing cells at high copy number compared to the cognate DNA coding sequence. This formulation has the additional characteristics of simplified sample preparation, isothermal incubation conditions, and long-term stability in ambient conditions.
While detection of bacteria and other organisms by amplification of genomic DNA is routinely achieved using PCR (polymerase chain reaction), there are many applications that require sensitivity below the limit of detection for PCR. The target for water testing on the International Space Station, for example, requires that individual pathogens can be detected at down to 10 cells per liter. Even with filter concentration, this detection level is essentially impossible by PCR or any other method that relies on amplification of DNA targets. Another limitation of PCR on the ISS is the weight and power consumption of a thermocycled amplification and the requirement for elaborate sample preparation regimens.
The detection method of this invention is isothermal and therefore does not require the complex instrumentation associated with PCR. The isothermal amplification method, LAMP, is not novel; however, the unique capacity of the chemistry to detect RNA targets that are several logs more abundant than the genomic DNA that is typically targeted by PCR or other isothermal methods, is novel. This amplification advantage is due to the unique reverse transcriptase and thermostability properties of the OmniAmp Polymerase, a novel enzyme. These rRNA targets are highly divergent even among closely related species, allowing a high degree of discrimination. The stability of the enzyme allows dry formulation that is highly stable in ambient conditions. The formulation also proved highly tolerant of unextracted and otherwise unprocessed bacterial samples.