Meeting the goal of providing point of care (POC) tests for molecular detection of pathogens in low resource settings places stringent demands on all aspects of the technology. OmniAmp DNA polymerase (Pol) is a thermostable viral enzyme that enables true POC use in clinics or in the field by overcoming important barriers to isothermal amplification. In this paper, we describe the multiple advantages of OmniAmp Pol as an isothermal amplification enzyme and provide examples of its use in loop-mediated isothermal amplification (LAMP) for pathogen detection. The inherent reverse transcriptase activity of OmniAmp Pol allows single enzyme detection of RNA targets in RT-LAMP. Common methods of nucleic acid amplification are highly susceptible to sample contaminants, necessitating elaborate nucleic acid purification protocols that are incompatible with POC or field use. OmniAmp Pol was found to be less inhibited by whole blood components typical in certain crude sample preparations. Moreover, the thermostability of the enzyme compared to alternative DNA polymerases (Bst) and reverse transcriptases allows pretreatment of complete reaction mixes immediately prior to amplification, which facilitates amplification of highly structured genome regions. Compared to Bst, OmniAmp Pol has a faster time to result, particularly with more dilute templates. Molecular diagnostics in field settings can be challenging due to the lack of refrigeration. The stability of OmniAmp Pol is compatible with a dry format that enables long term storage at ambient temperatures. A final requirement for field operability is compatibility with either commonly available instruments or, in other cases, a simple, inexpensive, portable detection mode requiring minimal training or power. Detection of amplification products is shown using lateral flow strips and analysis on a real-time PCR instrument. Results of this study show that OmniAmp Pol is ideally suited for low resource molecular detection of pathogens.

PCR has found immediate use in developmental DNA diagnostics procedures and in molecular cloning from genomic DNA. (1~ Current protocols for PCR amplification require the dispensing and mixing of aqueous solutions stored at subambient temperatures. In addition, dispensing of such aqueous reagents involving enzymes, buffers, and nucleotides is time consuming. It also may lead to pipetting errors as well as an increased probability of carry over contamination of pristine PCR mixtures. Having considered the need for rapid testing and automation, we have developed an alternate approach to stabilizing PCR reaction mixtures in carbohydrate polymers. This process leads to the formation of glassy matrices that provide room-temperature stability (referred to as stabilized): This paper describes the utility of stabilized PCR reagents and subsets of PCR reagents containing buffered Taq DNA polymerase and/or nucleotides for routine PCR applications. RESULTS AND DISCUSSIONThe PCR reagent mixture, containing 2.5 units of Taq DNA polymerase (Perkin Elmer Cetus), 0.2 mM each of dNTPs (Pharmacia), and I x standard PCR reaction buffer [10 mM Tris-HC1 (pH 8.3), 50 mM KC1, 1.5 mM MgC12, 0.001% gelatin (Perkin Elmer Cetus)], was mixed with an equal volume of 20% (wt/vol) carbohydrate polymer and processed to form a stable, glassy matrix. (2) The stabilized PCR mixtures were stored at room temperature for extended periods of time and tested in PCR. Figure 1 shows the amplification of a pBR322 DNA template utilizing PCR reagents prepared from aqueous solutions (stored at -20°C) and stabilized reagents (stored at room temperature for 11 months). The stabilized reagent mixture was rehydrated with 100 t~l of water containing the specific primers and DNA template. Equivalent amounts of PCR product were observed with fresh reagents and stabilized reagents ( Fig.