Abstract：
OBJECTIVE: This article aims to establish a rapid visual method for the detection of Streptococcus pyogenes (GAS) based on recombinase polymerase amplification (RPA) and lateral flow strip (LFS).
METHODS: Utilizing speB of GAS as a template, RPA primers were designed, and basic RPA reactions were performed. To reduce the formation of primer dimers, base mismatch was introduced into primers. The probe was designed according to the forward primer, and the RPA-LFS system was established. According to the color results of the reaction system, the optimum reaction temperature and time were determined. Thirteen common clinical standard strains and 14 clinical samples of GAS were used to detect the selectivity of this method. The detection limit of this method was detected by using tenfold gradient dilution of GAS genome as template. One hundred fifty-six clinical samples were collected and compared with qPCR method and culture method. Kappa index and clinical application evaluation of the RPA-LFS were carried out.
RESULTS: The enhanced RPA-LFS method demonstrates the ability to complete the amplification process within 6 min at 33 °C. This method exhibits a high analytic sensitivity, with the lowest detection limit of 0.908 ng, and does not exhibit cross-reaction with other pathogenic bacteria.
CONCLUSIONS: The utilization of RPA and LFS allows for efficient and rapid testing of GAS, thereby serving as a valuable method for point-of-care testing.
METHODS: Utilizing speB of GAS as a template, RPA primers were designed, and basic RPA reactions were performed. To reduce the formation of primer dimers, base mismatch was introduced into primers. The probe was designed according to the forward primer, and the RPA-LFS system was established. According to the color results of the reaction system, the optimum reaction temperature and time were determined. Thirteen common clinical standard strains and 14 clinical samples of GAS were used to detect the selectivity of this method. The detection limit of this method was detected by using tenfold gradient dilution of GAS genome as template. One hundred fifty-six clinical samples were collected and compared with qPCR method and culture method. Kappa index and clinical application evaluation of the RPA-LFS were carried out.
RESULTS: The enhanced RPA-LFS method demonstrates the ability to complete the amplification process within 6 min at 33 °C. This method exhibits a high analytic sensitivity, with the lowest detection limit of 0.908 ng, and does not exhibit cross-reaction with other pathogenic bacteria.
CONCLUSIONS: The utilization of RPA and LFS allows for efficient and rapid testing of GAS, thereby serving as a valuable method for point-of-care testing.
摘要：
目的：本文旨在建立一种基于重组酶聚合酶扩增（RPA）和侧向流条（LFS）的化脓性链球菌（GAS）快速可视化检测方法。
方法：利用GAS的speB作为模板，设计了RPA引物,进行碱性RPA反应。为了减少引物二聚体的形成，将碱基错配引入引物。根据正向引物设计探针，并建立了RPA-LFS系统。根据反应体系的颜色结果，确定了最佳反应温度和时间。采用13株常见临床标准菌株和14例GAS临床样品检测该方法的选择性。以10倍梯度稀释GAS基因组为模板检测该方法的检出限。收集了一百五十六个临床样品，并与qPCR方法和培养方法进行了比较。对RPA-LFS进行Kappa指数和临床应用评价。
结果：增强的RPA-LFS方法证明了在33°C下在6分钟内完成扩增过程的能力。该方法具有很高的分析灵敏度，最低检测限为0.908ng，与其他致病菌不发生交叉反应。
结论：使用RPA和LFS可以高效快速地检测气体，从而作为一种有价值的即时检测方法。
方法：利用GAS的speB作为模板，设计了RPA引物,进行碱性RPA反应。为了减少引物二聚体的形成，将碱基错配引入引物。根据正向引物设计探针，并建立了RPA-LFS系统。根据反应体系的颜色结果，确定了最佳反应温度和时间。采用13株常见临床标准菌株和14例GAS临床样品检测该方法的选择性。以10倍梯度稀释GAS基因组为模板检测该方法的检出限。收集了一百五十六个临床样品，并与qPCR方法和培养方法进行了比较。对RPA-LFS进行Kappa指数和临床应用评价。
结果：增强的RPA-LFS方法证明了在33°C下在6分钟内完成扩增过程的能力。该方法具有很高的分析灵敏度，最低检测限为0.908ng，与其他致病菌不发生交叉反应。
结论：使用RPA和LFS可以高效快速地检测气体，从而作为一种有价值的即时检测方法。
