Q1：What are the differences between RPA and RAA?
● RPA and RAA are both recombinase-based isothermal amplification. ● RPA: Recombinase polymerase amplification. RAA: Recombinase aided amplification.
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Q2：How to select the right RPA kit？What is the difference of RPA kits?
● To select the right one, you need to know your requirements and understand the features and differences of the RPA kits. - RPA Basic kit: mainly used to amplify DNA — meaning it makes millions to billions of copies of a specific DNA segment at isothermal temperature. You can observe the amplification product using agarose gel. - RPA Exo kit: It adds fluorescent probe (Exo probe) in the RPA basic kit to improve the specificity. You can read fluorescent value using Q-PCR. - RPA Nfo kit: It adds lateral flow probe (Nfo probe) in the RPA basic kit to improve the specificity. You can read the result using a test strip. ● Experience sharing: RPA Exo kit gives quantitative results. It is suitable for primer and probe optimization. With the most effective primer set and probe in Exo kit, you can easily get effective primer set and probe for Nfo kit.
Q3：How to design primer and probe for RPA?
EZassay website offers free online software for primer and probe design. (www.ezassay.com) ●First, paste the target DNA sequence in FASTA format into the dialog box. Due to server computational limitations, the target sequence should not be too long. Generally, 80-500 bp is sufficient. ●The web output shows different combinations of forward and reverse primers. Keeping the forward primer constant, multiple reverse primers can be selected, and vice versa. Each combination represents a different pairing. ●Download the Excel table and select 5-10 forward primers and 5-10 reverse primers from top to bottom for the first round of screening to identify the optimal combination. *Note: The best primers must be determined through experimental screening. The software currently cannot predict this accurately. Please be aware of this.
Q4：Can I use PCR primers for RPA kit?
Yes. However, due to different amplification mechanisms, PCR-validated primers may not perform optimally in RPA. Primer specificity and sensitivity are crucial for RPA. It is recommended to design primers using RPA online software and then screen them through experiment to select the best primer pairs.
Q5：Why does the DNA gel result of RPA amplification show non-specific bands?
We have to admit that non-specific products in isothermal amplification are more common than in classical PCR. This is due to the inherent mechanism of the technique. But don’t be worry. We can optimize it through the following solutions: ● Primer optimization： RPA primers usually need to go through three or more rounds of screening. In the first round, maximize the differences in the amplification region. In the second round, select the best primer pairs from the first round. Then, for the forward and reverse primers, shift them left or right by 1 to 3 nucleotides respectively for further screening and optimization. ● Primer Dimer Formation: Primers may form dimers, which can produce non-specific bands. Adjusting the primer concentration or redesigning the primers can help solve this problem. ● Temperature: The reaction temperature may affect the specificity of amplification. RPA typically works best at 39-41°C. If the temperature is too low, it may increase non-specific amplification. The temperature of dry bath may be not accurate. Adjusting the temperature to the optimal range can improve specificity. ● Inspired by the design principles of TaqMan probes in PCR, RPA can significantly enhance detection specificity by introducing Exo probes, Nfo probes, or CRISPR-crRNA.
Q6：How to Interpret Results for products of RPA basic kit? Is DNA purification necessary?
● The amplification products can be visualized by running a DNA agarose gel. ● Before electrophoresis, it is recommended to terminate the reaction by heating at 65°C for 10 minutes. ● Optional step: Purify the products before gel electrophoresis for cleaner bands. Product purification can be performed using column-based, magnetic bead, or other methods. Refer to the purification kit manual for specific procedures. * Note: Products of RPA-Exo kit cannot be analyzed by gel electrophoresis because the exonuclease degrades the products.
Q7：Why do the negative control group show signals?
● If the negative control group initially had no signal but later shows a signal, or if the signal is very strong, there is high possibility of carry-over or aerosol contamination. It is recommended to use new reagents and conduct the experiment in a different environment. Suggestion for sample addition order: Add the negative control first, followed by the sample, and finally the positive control. ● If the negative control shows a signal in the first experiment, it is generally due to poor specificity of the primers/probes, leading to non-specific amplification. It is recommended to redesign and optimize the primers and probes. ● High probe concentration can also cause non-specific cleavage, resulting in increased background signals. (In this case, the signal usually increases slowly.) ● Upon receipt, it is suggested to aliquot and store the reagents. For example, divide them into 3 to 5 tubes to avoid repeated use of the same tube, which can cause cross-contamination. EZassay team offers “Contamination-proof” RPA kit by adding UDG enzyme. It is very helpful for IVD product development. Please contact your distributor or EZassay team for further enquiry.
Q8：What is the sensitivity of RPA isothermal amplification? What is the concentration of the positive control provided in the kit?
● The sensitivity of RPA kits can generally reach a minimum of 10~100 copies per reaction. ● The concentration of the positive control in the kit is approximately 10^5 copies/μl.
Q9：Why is there no signal for positive control group?
If PCR thermal cycler is used, make sure the heated lid function is turned off. You may pre-select the "heated lid off" option and run the program to allow the lid to cool. For specific temperature control, consult the manufacturer based on the instrument model. Generally, the heated lid of PCR/qPCR instruments heats up to 105°C immediately after powering on. Even if the heated lid is turned off, if it remains at 105°C, wait until it cools to an appropriate temperature (e.g., below 50°C) before placing reaction tubes inside. Otherwise, heat conduction from the lid to the tube bottom may partially or completely inactivate the enzyme.
Q10：Positive control works well. But why is there no signal for sample group?
● There may be template degradation or low concentration. ● Make sure thoroughly mix. After adding template, mix well by inverting or flicking the tube several times, followed by a quick spin. Repeat this process three times. ● The primer may not be suitable. ● Add the Starter last. It is suggested to apply the Starter to the tube cap or wall (avoid direct addition to the template or reagents), then mix well and then quick spin.