News & Events

When conducting experiments or detections using ERA (Enzymatic Recombinase Amplification) technology, the occurrence of false positive results in no-template control (NTC) experiments is mainly caused by contamination, primer design issues, and non-specific amplification of the reaction system.  1. Contamination of Environment and Reagents  False positives caused by contamination are similar to those in PCR, and the handling methods are basically the same. Here, we focus on aerosol contamination and its treatment.   Aerosols are generated by friction between air and liquid surfaces. Opening tubes, shaking reaction tubes, and repeated pipetting with contaminated pipettes can all form aerosols, leading to cross-contamination. It is estimated that a single aerosol particle can contain 48,000 copies; therefore, special attention should be paid to contamination caused by aerosols.   The key to the treatment method lies in the experimental space and the surfaces of related instruments:   - Fumigation disinfection of the space can effectively remove nucleic acid contamination sources.   - Disinfect the surfaces of objects in the working area with disinfectant or 75% ethanol.   - Use new amplification reagents (such as solubilizers, activators, etc.).   Repeated cleaning may be required to completely eliminate contamination.  2. False Positives Caused by Primer-Probe Mismatch  Such false positives are generally caused by mismatches between primers, between primers and probes, or within primers themselves.   Verification methods for such false positives:   1) Remove any one primer. If false positives still occur, it indicates that the primers and probes used in the reaction have mismatches. The optimal solution is to redesign the primers or probes.   2) If there is no suitable position for redesign, the dosage of each primer and probe can be adjusted to minimize false positives. The specific dosage should be determined through gradient experiments, with a recommended adjustment range of 10%-20%. Priority should be given to testing dosage reduction.  3. False Positives Caused by Non-Specific Amplification  Isothermal amplification technologies rely on related enzymes to achieve nucleic acid amplification reactions, and non-specific amplification may occasionally occur in such technologies. To reduce or eliminate non-specific amplification, strict design of primers and probes is required on the one hand, and appropriate adjustments to the reaction system based on specific primers and probes are necessary on the other hand.   Common adjustment schemes:   1) Adjust reaction temperature and time: The product manual provides recommended reaction temperatures and times, but differences may arise due to variations in design and projects. Therefore, the reaction temperature and time can be adjusted according to specific primers and probes. The recommended reaction temperature range is 37℃-42℃, and the reaction time range is 10min-30min.   2) Adjust the dosage of solubilizers and activators: The dosages of solubilizers and activators listed in the reaction system of the product manual are general recommendations. During specific experiments, the dosages can be appropriately adjusted to improve detection sensitivity or control false positives. (Activators can be adjusted within ±25% of the normal dosage; solubilizers can be adjusted within ±10% of the normal dosage.)   3) Operational proficiency and loading time: Due to the characteristics of ERA technology, the amplification reaction starts immediately after the system is prepared. Faster loading speed, appropriate mixing intensity and time, suitable centrifugation time, and accurate pipetting all affect the final experimental results. Details have been specified in the manual; please refer to it.