Removal of carry-over contamination in RT-LAMP

Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) has become the alternative nucleic acid test to the gold-standard RT-PCR due to its flexibility and simplicity for screening and surveillance of viral RNA infections at point-of-care. Nevertheless, because of its high sensitivity and productivity, RT-LAMP assays can be vulnerable to carry-over DNA contamination leading to false positives.

ArcticZymes Cod UNG is being widely used to avoid PCR carry-over contamination.  The same strategy can also be utilized to effectively remove carry-over contamination and restore sensitivity in RT-LAMP.

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IsoPol BST+ efficiently incorporates dUTP in RT-LAMP

Utilizing Cod UNG in contamination control requires the exchange of dTTP with dUTP in the RT-LAMP assay to ensure efficient clearance of potential amplicons in later runs.

To verify efficient incorporation of dUTP in RT-LAMP, IsoPol BST+ was used in combination with a commercially available AMV-RT. Figure 1 demonstrates that the RT-LAMP reaction is highly compatible with both 90 and 100% dUTPs in the reaction.

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Figure 1. IsoPol BST+ is highly compatible with dUTP in RT-LAMP. Viral and Human RNA was amplified with two different primer sets each.  Performance when using 90% and 100% dUTP (as a replacement to dTTP) was compared to a control without dUTP. Only small variations were observed after replacing dTTP with dUTP for all combinations tested. TTR: Time to results.

Cod UNG efficiently removes carry-over contamination in RT-LAMP with minimal effect on the level of detection (LOD)

To mimic carry-over contamination, diluted amplicons with dUTP were used as spike in RT-LAMP. In Figure 2 it is shown that detection of large amounts of RNA is not affected by the spike (blue and green).  However, it is affecting samples with limited RNA as the spike represents a large enough ratio of the amplified material to reduce the TTR (light green).  Also, the positive NTC is detrimental to the level of detection of the assay (grey). Addition of Cod UNG eliminated the contaminating amplicons, resulting in flat no template control (NTC) thus restoring assay sensitivity.

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Figure 2. Cod UNG restores assay sensitivity. RT-LAMP using MS2 RNA dilution series spiked with amplicons to mimic a carry-over contamination as template. The contamination amplifies and destroys the assay sensitivity (dotted lines). Adding 1U Cod UNG to the reaction eliminates contamination and restores assay sensitivity (solid lines).
The addition of 1 U of Cod UNG can remove large amounts of carry-over contamination. Figure 3 shows that amplicons introduced to the reaction at levels causing a TTR at less than 10 minutes is completely removed by 1U of Cod UNG, restoring assay sensitivity even in extreme cases.
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Figure 3. Cod UNG removes high concentrations of carry-over contamination.  An RT-LAMP using 0.001 ng RNA as template was added dUTP-containing amplicons to a level causing the NTC signal to exceed that obtained from the template alone. Adding 1U of Cod UNG to the material removed the contaminating amplicons to non-detectable levels in the NTC (A), while restoring the accuracy and sensitivity of the sample containing 0.001 ng RNA (B).

Cod UNG can be added to the reaction mix with other components in a single closed tube reaction without additional preincubation steps to effectively eliminate false positive results from carry-over contaminants and restore assay sensitivity.

For more information, please refer to Protocol.

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