■快速准确地检测副溶血性弧菌,这是一种与海鲜污染有关的主要病原体,需要有效对抗食源性疾病和伤口感染。toxR基因在副溶血弧菌中相对保守,并且主要以显著程度的特异性参与毒力基因的表达和调节。这项研究的目的是开发一种快速、简单,临床和非专业实验室环境中副溶血性弧菌的恒温检测方法。
■在这项研究中,使用特异性引物和CRISPRRNA靶向toxR基因,构建将重组酶聚合酶扩增(RPA)与CRISPR-Cas13a相结合的反应系统.采用自制十二烷基硫酸钠(SDS)核酸快速提取试剂提取样品的全基因组DNA,并通过侧流试纸(LFD)对检测结果进行视觉解释。
■RPA-CRISPR/Cas13a-LFD方法的特异性使用副溶血性弧菌菌株ATCC-17802和其他六种非溶血性弧菌物种进行了验证。结果证明了100%的特异性。此外,副溶血性弧菌的基因组DNA被连续稀释和分析,此方法的最低检测限为1拷贝/微升,高于TaqMan-qPCR方法(102拷贝/μL)。所建立的方法成功地应用于检测野生型副溶血性弧菌,结果与TaqMan-qPCR和MALDI-TOFMS质谱鉴定结果一致。最后,将建立的RPA-CRISPR/Cas13a-LFD方法应用于副溶血性弧菌感染小鼠的全血标本,该方法对副溶血性弧菌的检出率与常规PCR方法一致。
■在这项研究中,我们描述了一种RPA-CRISPR/Cas13a检测方法,该方法专门针对toxR基因,并提供了简单性等优点,快速性,高特异性,和视觉解释。该方法是在非专业实验室环境中迅速检测副溶血弧菌的有价值的工具。
UNASSIGNED: Swift and accurate detection of Vibrio parahaemolyticus, which is a prominent causative pathogen associated with seafood contamination, is required to effectively combat foodborne disease and wound infections. The toxR gene is relatively conserved within V. parahaemolyticus and is primarily involved in the expression and regulation of virulence genes with a notable degree of specificity. The aim of this study was to develop a rapid, simple, and constant temperature detection method for V. parahaemolyticus in clinical and nonspecialized laboratory settings.
UNASSIGNED: In this study, specific primers and CRISPR RNA were used to target the toxR gene to construct a reaction system that combines recombinase polymerase amplification (
RPA) with CRISPR‒Cas13a. The whole-genome DNA of the sample was extracted by self-prepared sodium dodecyl sulphate (SDS) nucleic acid rapid extraction reagent, and visual interpretation of the detection results was performed by lateral flow dipsticks (LFDs).
UNASSIGNED: The specificity of the
RPA-CRISPR/Cas13a-LFD method was validated using V. parahaemolyticus strain ATCC-17802 and six other non-parahaemolytic Vibrio species. The results demonstrated a specificity of 100%. Additionally, the genomic DNA of V. parahaemolyticus was serially diluted and analysed, with a minimum detectable limit of 1 copy/µL for this method, which was greater than that of the TaqMan-qPCR method (102 copies/µL). The established methods were successfully applied to detect wild-type V. parahaemolyticus, yielding results consistent with those of TaqMan-qPCR and MALDI-TOF MS mass spectrometry identification. Finally, the established
RPA-CRISPR/Cas13a-LFD method was applied to whole blood specimens from mice infected with V. parahaemolyticus, and the detection rate of V. parahaemolyticus by this method was consistent with that of the conventional PCR method.
UNASSIGNED: In this study, we describe an
RPA-CRISPR/Cas13a detection method that specifically targets the toxR gene and offers advantages such as simplicity, rapidity, high specificity, and visual interpretation. This method serves as a valuable tool for the prompt detection of V. parahaemolyticus in nonspecialized laboratory settings.