随着人们越来越关注B组链球菌(GBS)感染及其对围产期妊娠的不利影响,包括感染,早产,新生儿败血症,和脑膜炎,迫切需要在所有怀孕阶段推广GBS筛查。这项研究的目的是建立一个独立于设备的,快,敏感,视觉GBS检测方法。利用重组酶聚合酶等温扩增(RPA)的特点,nfo核酸酶裂解碱基类似物(四氢呋喃,THF)现场,以及横向流动色谱条(LFS)的可视化读取的优势,开发了一种GBS检测方法。这种方法集中在由cfb基因编码的Christie-Atkins-Munch-Petersen因子的保守区域,对GBS特异的毒力基因。两个正向引物,两个生物素标记的反向引物,设计了一种异硫氰酸荧光素(FITC)标记和C3间隔区阻断的探针。该研究涉及优化引物对和探针组合,确定最佳反应温度和时间,评估特异性,分析检测限,并对87例围产期孕妇阴道拭子进行了检测。结果表明,GBS-RPA-LFS的视觉检测方法,使用cfb-F1/R2/P1引物探针,可以在39°C至42°C的温度下在15分钟内检测到GBS。此外,该方法仅特异性扩增GBS,不会与乳酸菌等病原体发生交叉反应,卷曲乳杆菌,白色念珠菌,单核细胞增生李斯特菌,小肠结肠炎耶尔森氏菌,肺炎克雷伯菌,阴沟肠杆菌,Freundii柠檬酸杆菌,溶藻弧菌,副溶血性弧菌,鼠伤寒沙门氏菌,金黄色葡萄球菌,铜绿假单胞菌,或者阴道毛滴虫.它可以检测到每个反应最少100个拷贝。在临床98个孕妇阴道拭子样本中,GBS-RPA-LFS法与TaqMan实时荧光定量法的符合率为95.92%。总之,本研究成功建立了RPA和LFS联合GBS原位检测平台,反应时间短,高灵敏度,高特异性,便携性,和设备独立性,为临床GBS筛查提供了可行的策略。
With growing concerns about Group B streptococcal (GBS) infections and their adverse effects on perinatal pregnancies, including infection, premature delivery, neonatal septicemia, and meningitis, it is urgent to promote GBS screening at all pregnancy stages. The purpose of this study is to establish a device-independent, fast, sensitive, and visual GBS detection method. Taking advantage of the characteristics of the recombinase polymerase isothermal amplification (RPA), the activity of the nfo nuclease cleavage base analog (tetrahydrofuran, THF) site, and the advantages of visual reading of the lateral flow chromatography strip (LFS), a GBS detection method was developed. This method focused on the conservative region of the Christie-Atkins-Munch-Petersen factor encoded by the cfb gene, a virulence gene specific to GBS. Two forward primers, two biotin-labeled reverse primers, and one fluorescein isothiocyanate (FITC)-labeled and C3spacer-blocked probe were designed. The study involved optimizing the primer pair and probe combination, determining the optimal reaction temperature and time, evaluating specificity, analyzing detection limits, and testing the method on 87 vaginal swabs from perinatal pregnant women. The results showed that the visual detection method of GBS-RPA-LFS, using the cfb-F1/R2/P1 primer probe, could detect GBS within 15 min at the temperature ranging from 39°C to 42°C. Furthermore, the method specifically amplified only GBS, without cross-reacting with pathogens like Lactobacillus iners, Lactobacillus crispatus, Candida albicans, Listeria monocytogenes, Yersinia enterocolitica, Klebsiella Pneumoniae, Enterobacter cloacae, Citrobacter freundii, Vibrio alginolyticus, Vibrio parahaemolyticus, Salmonella typhimurium, Staphylococcus aureus, Pseudomonas aeruginosa, or Trichomonas vaginalis. It could detect a minimum of 100 copies per reaction. In clinical 98 samples of vaginal swabs from pregnant women, the agreement rate between the GBS-RPA-LFS method and TaqMan real-time fluorescence quantification method was 95.92%. In conclusion, this study successfully established a combined RPA and LFS GBS in situ detection platform, with short reaction time, high sensitivity, high specificity, portability, and device independence, providing a feasible strategy for clinical GBS screening.