Twistamp liquid dna amplification kit

RPA reactions were performed using the TwistAmp^® Liquid DNA Amplification Kit (TwistDx Inc., Maidenhead, UK) according to the manufacturer’s instructions. The 50 µL reaction system contained 25 µL 2× reaction buffer, 5 µL 10× Basic e-mix, 2.5 µL 20× core mix, 2.4 µL of 10 µM forward primer, 2.4 µL of 10 µM reverse primer, and 9.2 µL of distilled water. A 2.5 µL volume of 280 mM magnesium acetate and 1 µL of template were added to the lid of the reaction tube. After a brief centrifugation, the reaction mixture was incubated for 30 min at 37°C. RPA amplification products were purified by a PCR cleaning kit (Shanghai Meiji Biotechnology Co., Ltd., Shanghai, China) and used for 2% agarose gel electrophoresis.

To screen the best forward and reverse primer pairs, RPA amplification was performed using the TwistAmp Liquid DNA amplification Kit (TwistDx Inc., Maidenhead, United Kingdom). Each 50 μL mixture contained 25 μL of 2× reaction buffer, 5 μL of 10× basic mix, 2.5 μL of 20× core mix, 2.1 μL of forward primers (10 μM), 2.1 μL of reverse primers (10 μM), 9.8 μL of ddH₂O, and 1 μL of the template genome. To ensure that all reaction systems reacted at the same time, 2.5 μL of 280 mM magnesium acetate was added to the PCR tube caps and transiently centrifuged into all reaction tubes. The reaction system was vortexed and immediately incubated at 37°C in a thermostat heater for 30 min. The amplification products were purified using the DNA Purification Kit (Tiangen Biochemical Technology Co., Ltd, Beijing, China) and detected using 1.5 percent agarose gel electrophoresis.

The forward and reverse primers were modified with fluorescein isothiocyanate (FITC) and biotin at the 5′ ends, respectively (General Biosystems, Co. Ltd., Anhui, China). RPA reactions were performed according to the manufacturer’s instructions of the TwistAmp^® Liquid DNA Amplification Kit (TwistDx, Inc., Maidenhead, United Kingdom). Five microliters of the amplification products were used for LFS (Ustar Biotech, Ltd., Hangzhou, China) detection. The amplification products were mixed with 95 μl of sample buffer (Ustar Biotech), and the stick of LFS was inserted into the mixture for 3 min and then for visual reading.

The RPA assay was performed in accordance with the manufacturer’s instructions of the TwistAmp^® Liquid DNA Amplification Kit (TwistDx Inc., Maidenhead, United Kingdom). Each RPA reaction contained 25 μl of 2 × reaction buffer, 5 μl of 10 × Basic e-mix, 2.5 μl of 20 × core mix, 2.1 μl of each primer (10 μM), 9.8 μl of distilled water, and 1 μl of the template. To initiate the reaction, 2.5 μl of magnesium acetate (280 mM) was added to the mixture. After protein-induced DNA bending and centrifugation, the mixture was immediately incubated for 30 min at 37°C. The amplification products were purified with the MonPure™ Gel and PCR Clean Kit (Monad Biotech Co., Ltd., Wuhan, China) and separated by electrophoresis on a 1.5% agarose gel.

To initially screen the forward and reverse primers, RPA amplification was performed with the TwistAmp Liquid DNA amplification kit (TwistDx Inc., Maidenhead, UK), according to the manufacturer’s instructions. Each 50 μL mixture contained 25 μL of 2 × Reaction buffer, 5 μL of 10 × Basic mix, 2.5 μL of 20 × Core mix, 2.1 μL of forward primers and 2.1 μL of reverse primer (both 10 μM), 1 μL of genomic DNA as template, and 9.8 μL of distilled water. To ensure that all the reaction systems reacted simultaneously, 2.5 μL of 280 mM magnesium acetate was added to the PCR tube cap, and was added to the reaction system simultaneously by transient centrifugation.
The reaction mixture was briefly centrifuged and then incubated in a heater at 37°C for 30 min. Reactions with distilled water as a template were used as negative controls. The amplification products were purified with a DNA purification kit (Tiangen, Beijing, China) and resolved with 1.5% agarose gel electrophoresis.

For initial screening of forward and reverse primer sets, RPA amplification was conducted using a TwistAmp Liquid DNA Amplification Kit (TwistDx, UK) according to the manufacturer’s instructions. Each 50 μL mixture contained 25 μL reaction buffer, 11.9 μL double-distilled H₂O (ddH₂O), 5 μL Basic e-mix, 2.5 μL core mix, 2.1 μL RPA forward and reverse primers (10 μM), and 1 μL genomic DNA. The reaction was initiated by adding 2.5 μL of 280 mM Mg(Ac)₂ to the mixture and immediately incubating at 37°C for 30 min. Amplification products were purified with Universal DNA Purification Kit (TIANGEN, Beijing) and were detected via 1.5% agarose gel electrophoresis.

RPA reactions were performed using the TwistAmp^® Liquid DNA Amplification Kit (TwistDx Inc., Maidenhead, UK) according to the manufacturer’s instructions. The 50 µL reaction system was prepared as follows: 25 µL of 2× reaction buffer, 5 µL of 10× Basic e-mix, 2.5 µL of 20× core mix, 2.4 µL of 10 µM forward primer, 2.4 µL of 10 µM reverse primer, and 9.2 µL of distilled water; 2.5 μL of 280 mM magnesium acetate and 1 μL of template were added to the lid of the reaction tube. After a short centrifugation, the reaction mixture was incubated at 37°C for 30 min. The RPA amplification products were purified using the PCR Cleanup Kit (Meiji Biotechnology, Shanghai, China) and electrophoresed on a 2% agarose gel.