Thermostable DNA Polymerase from a Viral Metagenome Is a Potent RT-PCR Enzyme

Viral metagenomic libraries are a promising but previously untapped source of new reagent enzymes. Deep sequencing and functional screening of viral metagenomic DNA from a near-boiling thermal pool identified clones expressing thermostable DNA polymerase (Pol) activity. Among these, 3173 Pol demonstrated both high thermostability and innate reverse transcriptase (RT) activity. We describe the biochemistry of 3173 Pol and report its use in single-enzyme reverse transcription PCR (RT-PCR). Wild-type 3173 Pol contains a proofreading 3′-5′ exonuclease domain that confers high fidelity in PCR. An easier-to-use exonuclease-deficient derivative was incorporated into a PyroScript RT-PCR master mix and compared to one-enzyme (Tth) and two-enzyme (MMLV RT/Taq) RT-PCR systems for quantitative detection of MS2 RNA, influenza A RNA, and mRNA targets. Specificity and sensitivity of 3173 Pol-based RT-PCR were higher than Tth Pol and comparable to three common two-enzyme systems. The performance and simplified set-up make this enzyme a potential alternative for research and molecular diagnostics

A novel thermostable polymerase for RNA and DNA Loop-mediated isothermal amplification (LAMP)

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 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

Two step RT-PCR comparing 3173 Pol and MMLV RT.

<p><b>A.</b> MS2 viral RNA and <b>B., C.</b> total human liver RNA were reverse transcribed using either 3173 Pol or MMLV RT and then PCR amplified using Taq Polymerase. <b>Target amplicons</b>: <b>A.</b> MS2 RNA phage 77 bp amplicon, 2% gel, <b>B.</b> Human beta-actin 144 bp amplicon, 2% gel, <b>C.</b> Human beta-actin 821 bp amplicon, 1% gel. <b>Lanes</b>: <b>1</b>,<b>11</b>: oligo dT primer; <b>2–4</b>,<b>12–14</b>: Gene specific primers; <b>5</b>,<b>15</b>: random hexamers; <b>6</b>,<b>16</b>: random nonamers; <b>7</b>,<b>17</b>: No primer plus RT; <b>8</b>: No RT enzyme; <b>9</b>: PCR No Target Control; <b>10</b>: Molecular Weight Marker (MW), 100 bp (50 bp lowest) for Panels A, B and 1000 bp (300, 500, 700 lowest) for Panel C. Correct PCR product size indicated by black triangle.</p

Biochemical characterization of 3173 Pol.

<p>The thermal profile of the 3173 Pol was determined by assay at the indicated temperatures. Activity relative to maximal (77°C) is shown.</p

RT-PCR conditions.

a<p>Not including thermal melt. NA is not applicable.</p

RT-PCR detection of human transcript RNAs.

<p>Beta-actin, beta2-microglobulin and cyclophilin target sequences of the indicated sizes were amplified from human liver total RNA using the primers described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038371#pone-0038371-t001" target="_blank">Table 1</a>. Shown are products of two step reactions where either MMLV RT or 3173 Pol were used for first strand cDNA synthesis, as indicated. Taq Pol was used for PCR. Products were resolved on a 1% agarose gel.</p

Polymerase assay for detection of expression clones containing thermostable Pol activity from a boiling hot spring metagenomic library.

<p>Clones judged by sequence to encode complete <i>pol</i> genes were cultivated and thermostable proteins extracted as described in the Methods. Extension of a 37 nucleotide (nt) ROX-labeled primer <b>(peak 1)</b> on a 41 nt template oligonucleotide by a polymerase results in a shift from 37 to 41 nt <b>(peak 2)</b>. If a single nucleotide non-templated extension occurs as seen with Taq Pol, a peak at 42 nt results <b>(peak 3)</b>. Degradation of the ROX-labeled substrate by 3′ to 5′ exonuclease activity results in peaks of less than 37 nt <b>(peak 4)</b>. nt: size of standard markers in nucleotides.</p

Primers and Other Oligonucleotides.

*<p>ROX = Carboxy-X-rhodamine.</p>#<p>CF560 = CalFluor 560.</p

Single-enzyme, one step RT-PCR amplification of MS2 phage RNA using 3173 Pol.

<p>MS2 RNA was amplified by 40 cycles of RT-PCR using the primers shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038371#pone-0038371-t001" target="_blank">Table 1</a> and 3173 Pol. <b>A.</b> Products from 89 to 362 bp in length were amplified using one-step single-enzyme RT-PCR cycling conditions: 15 sec @ 94°C, (10 s @ 94°C, 30 s @ 72°C)*40. Products were resolved by 2% agarose gel electrophoresis. <b>B.</b> The MS2 RNA was diluted from 10¹ to 10⁷-fold and amplified using a primer pair corresponding to the 160 bp fragment in Panel A. Real-time PCR fluorescence in RFU (relative fluorescence units) vs. PCR cycles. <b>C.</b> Post-amplification thermal melt in -dRFU/dTemperature vs. Temperature (°C). Light blue region indicates melt curves for specific products. <b>D.</b> Standard curve PCR Cycle threshold vs. log₁₀ RNA copy number in triplicate with linear least squares best fit line.</p

Comparison of 3173 Pol (PyroScript) RT-PCR mix with two enzyme RT-PCR systems in detection of MS2 and influenza A.

<p>Ten-fold serial dilutions of an MS2, an influenza A RNA preparation and a water only control (NTC) were amplified by one-step RT-PCR reagent mixes (PyroScript, qScript (Quanta), Transcriptor (Roche), and SuperScript (Invitrogen), as indicated. <b>A.</b> MS2 detection. Left panel: 2% agarose gel, each group of four wells are 10⁻⁴, 10⁻⁶, 10⁻⁷-fold target dilutions and NTC, MW is 100 bp DNA ladder (50 bp smallest band). Right Panel: RT-qPCR analysis of 10⁻³, 10⁻⁴, 10⁻⁵, and 10⁻⁶-fold target dilutions. <b>B.</b> Influenza A RNA detection. Left panel: 4–20% gradient polyacrylamide gel, each group of three wells are 10⁻⁶, 10⁻⁷-fold target dilutions and NTC, MW is 25 bp DNA ladder (50 bp smallest band). Right Panel: RT-qPCR analysis of 10⁻³, 10⁻⁴, 10⁻⁵, and 10⁻⁶-fold target dilutions.</p