Application Overview
Ensuring the accuracy and reliability of reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assays is vital, especially in point-of-care settings for screening and surveillance of viral RNA infections. One significant vulnerability of RT-LAMP, due to its high sensitivity, is the risk of carry-over DNA contamination, which can lead to false positives.
ArcticZymes Cod UNG is engineered to mitigate this issue by efficiently eliminating carry-over amplicons in subsequent RT-LAMP runs, thereby restoring assay sensitivity. This is particularly crucial in diagnostic and research labs where high specificity and sensitivity are paramount
Application Overview
Ensuring the accuracy and reliability of reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assays is vital, especially in point-of-care settings for screening and surveillance of viral RNA infections. One significant vulnerability of RT-LAMP, due to its high sensitivity, is the risk of carry-over DNA contamination, which can lead to false positives.
ArcticZymes Cod UNG is engineered to mitigate this issue by efficiently eliminating carry-over amplicons in subsequent RT-LAMP runs, thereby restoring assay sensitivity. This is particularly crucial in diagnostic and research labs where high specificity and sensitivity are paramount
The Challenges These Enzymes Address
Carry-over contamination in RT-LAMP assays can significantly impact the level of detection (LOD), especially when dealing with samples that have limited RNA. This contamination can lead to false positives and reduced assay sensitivity, undermining the reliability of the results. The challenge lies in efficiently eliminating these contaminating amplicons without affecting the assay's performance.
This vulnerability occurs during synthesis, when new product is being made and a conventional UNG has no natural point in the reaction where it is switched off.
LAMP & RT-LAMP: one enzyme, every stage
THE PROBLEM These ENZYME SOLVES
Carry-over contamination in RT-LAMP assays can significantly impact the level of detection (LOD), especially when dealing with samples that have limited RNA. This contamination can lead to false positives and reduced assay sensitivity, undermining the reliability of the results. The challenge lies in efficiently eliminating these contaminating amplicons without affecting the assay's performance.
This vulnerability occurs during synthesis, when new product is being made and a conventional UNG has no natural point in the reaction where it is switched off.
LAMP & RT-LAMP: one enzyme, every stage
THE PROBLEM These ENZYMes SOLVE
Carry-over contamination in RT-LAMP assays can significantly impact the level of detection (LOD), especially when dealing with samples that have limited RNA. This contamination can lead to false positives and reduced assay sensitivity, undermining the reliability of the results. The challenge lies in efficiently eliminating these contaminating amplicons without affecting the assay's performance.
This vulnerability occurs during synthesis, when new product is being made and a conventional UNG has no natural point in the reaction where it is switched off.
LAMP & RT-LAMP: one enzyme, every stage
The Solution
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.
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.
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 1 U 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 1 U of Cod UNG, restoring assay sensitivity even in extreme cases.
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 1 U 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.
Contamination control in RT-LAMP
Cod UNG is ideal for contamination control in RT-LAMP.
One unit of Cod UNG per 30 µL reaction is sufficient for removing even high concentrations of carry-over contamination.
- Ensure that you use dNTP mixes containing dUTP in your experiments.
- Check that the RT-LAMP reaction is compatible with dUTP by running side-by-side reactions containing different ratios of dUTP to dUTP (100% dUTP, 90% dUTP, 80% dUTP and 0% dUTP).
- Add 1 U Cod UNG directly to your 30 µL RT-LAMP reaction.
- Prepare the reaction mix on ice.
- Analyse your RNA at 65°C, no preincubation is necessary.
This protocol is a recommended starting point for evaluation and optimization, not the only or necessarily optimal approach for every workflow. Reaction conditions may need adjustment depending on your specific assay, sample type, and equipment.
For more information, please refer to Protocol.
Application Background
The dUTP/UDG carry-over prevention system, introduced by Longo and colleagues in 1990, is the standard approach across PCR-based assays. RT-LAMP's constant, single-temperature format removes any point in the workflow where a conventional UNG would reliably switch off — an even more complete loss of that safety net than one-step RT-qPCR faces. Cod UNG's low-temperature activity and loss of activity before significant new product has formed were developed to fit this isothermal, closed-tube format, extending carry-over control to point-of-care and surveillance use.
Manufacturing you can trust
Cod UNG has been supplied to diagnostic kit manufacturers for over two decades. Manufactured under ISO 13485, it offers the consistent quality and supply that regulated workflows depend on — contamination control that doesn't compromise sensitivity or performance, enabling robust assay development. Further details on manufacturing, specifications, and supply are available on the Cod UNG page.
Among different UNGs, Cod-UNG was selected for this study (Carry-over control in POC RT-LAMP) because this enzyme was active at a wider range of temperatures ranging from 20°C to 40°C, and the activity was lost at above 42°C.
Quyen et al., Frontiers in Cellular and Infection Microbiology, 2022