Bioscience Method 2024, Vol.15, No.1, 1-8 http://bioscipublisher.com/index.php/bm 4 Figure 3 Evaluation of the sensitivity of the RPA-Badh2-E7 Note: M: DL2000 Marker; P1: Shen CR1 (Fragrant Rice); P2: Shen Hui 26 (Non-fragrant Rice); The template concentration of the first P1 and P2 from left to right was 10 ng/μL, and the template concentration was diluted 10 times from the second. 1.3 Optimization of RPA amplification reaction temperature and time In order to further improve the efficiency of Badh2-E7 genotype detection, we optimized the RPA amplification reaction conditions. Firstly, the amplification effect of RPA amplification reaction was compared under different temperature conditions, and it was found that specific DNA bands could be amplified within the temperature range of 25 °C~45 °C (Figure 4A). Therefore, we used the recommended amplification temperature of 39 °C in the product manual of the RPA isothermal amplification kit (TABAS03KIT, TwistDx) as the amplification temperature for subsequent experiments. Then, we compared the amplification effects of different RPA reaction times and found that clear DNA bands could be obtained after 5 minutes of amplification at 39 °C (Figure 4B). When using conventional PCR amplification, it often takes about 2 hours to complete the amplification reaction (Cheng et al., 2018). This result indicates that compared to conventional PCR techniques, RPA amplification can significantly shorten the amplification reaction time and does not require the use of expensive instruments such as PCR machines, greatly improving the efficiency and convenience of badh2-E7 detection. Figure 4 Optimize the temperature and time of RPA amplification reaction (A: Comparison of RPA amplification effect under different temperature conditions; B: Comparison of RPA amplification effect under different reaction times.) Note: M: DL2000 Marker; P1: Shen CR1; P2: Shenhui 26 1.4 Application of RPA-badh2-E7 method for detecting badh2-E7 Using the RPA-badh2-E7 method created, 36 core restoring lines of hybrid japonica rice and 1 hybrid japonica rice Shenyou R1 (Shanghai Shendao 2022002) from the Rice Center Resource Library of Shanghai Academy of Agricultural Sciences were identified. Five restoring lines containing the aroma gene Badh2-E7 were screened, including 'Shenfan 24', 'Shenfan 30', 'Shenfan 33', 'Shenfan 43', and 'ShenCR1' (Figure 5). Banerjee et al. (2023) designed the Badh2-E7 allele detection marker using RPA technology, which cannot distinguish between homozygous and heterozygous Badh2-E7 genotypes. In this study, we tested the hybrid japonica rice 'Shenyou R1' (‘Shen23A’בShen CR1’), it can be found that two bands can be amplified in heterozygous plants, indicating that the RPA-badh2-E7 designed in this study is a co dominant marker (Figure 5).
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