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.
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.
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.
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.
Certain applications of ArcticZymes Technologies ASA products may require licenses from others. It is the expressed duty of any receiver of ArcticZymes Technologies ASA products to acquire such licenses, if necessary. In no event shall ArcticZymes Technologies ASA be liable for claims for any damages, whether direct, incidental, foreseeable, consequential, or special (including but not limited to loss of use, revenue, or profit), arising due to the violation of third parties Intellectual Property Rights by any receiver of ArcticZymes Technologies ASA products. ArcticZymes Technologies ASA products may be covered by pending or issued patents, designs, or design applications and/or trademarks or trademark applications or any other registered or unregistered Intellectual Property Right.