Department of Hygienic Inspection, School of Public Health, Jilin University, 1163 Xinmin Street, Changchun 130021, China.
Changchun Customs Technology Center, 4448 Freedom Road, Changchun, China.
FEMS Microbiol Lett. 2023 Jan 17;370. doi: 10.1093/femsle/fnad080.
In the surveillance of outbreaks of Clostridioides difficile infection, the rapid detection and diagnosis of C. difficile remain a major challenge. Polymerase spiral reaction (PSR) is a nucleic acid amplification technique that uses mixed primers and the strand displacement activity of Bst DNA polymerase to achieve a pair of primers and a single enzyme in an isothermal environment. The primer design is simple, the reaction is efficient, and a color indicator can be used to visualize the result. In this study, we developed a rapid and visually interpretable PSR to detect C. difficile by analyzing artificially contaminated feces samples and clinical isolates from patient feces samples. We designed two pairs of primers for a PSR that specifically targeted the conserved tcdB gene of C. difficile. The amplification results were visualized with the chromogenic dye hydroxynaphthol blue. The entire process was accomplished in 50 min at 64°C, with high specificity. The limit of detection of C. difficile with PSR was 150 fg/μl genomic DNA or 2 × 10 CFU/ml in artificially contaminated feces samples. With this method, we analyzed four clinical isolates and also compared the PSR with an isolation-and-culture detection method, polymerase chain reaction, and the Sanger sequencing. The four clinical isolates were found positive for tcdB, which confirmed the high specificity of the primers. The positive rates of tcdB in toxigenic C. difficile detected with PSR, PCR, and Sanger sequencing were 100%. The proportions of toxin types in these clinical C. difficile strains were 50% tcdA+tcdB+CDT- and 50% tcdA+tcdB+CDT+. The assay described should extend our understanding of the incidence of C. difficile. This may allow the rapid diagnosis and screening of C. difficile-related disease outbreaks in the field.
在艰难梭菌感染暴发的监测中，快速检测和诊断艰难梭菌仍然是一个主要挑战。聚合酶螺旋反应（PSR）是一种核酸扩增技术，它使用混合引物和 Bst DNA 聚合酶的链置换活性，在等温环境中实现一对引物和单个酶。引物设计简单，反应效率高，并且可以使用显色指示剂来可视化结果。在这项研究中，我们通过分析人工污染粪便样本和来自患者粪便样本的临床分离株，开发了一种快速且可直观解释的 PSR 来检测艰难梭菌。我们设计了两对针对艰难梭菌保守 tcdB 基因的 PSR 引物。扩增结果用显色染料羟萘酚蓝进行可视化。整个过程在 64°C 下 50 分钟内完成，具有高度特异性。PSR 检测艰难梭菌的检测限为 150 fg/μl 基因组 DNA 或人工污染粪便样本中的 2×10 CFU/ml。使用该方法，我们分析了四个临床分离株，并将 PSR 与分离培养检测方法、聚合酶链反应和 Sanger 测序进行了比较。四个临床分离株的 tcdB 均为阳性，证实了引物的高特异性。PSR、PCR 和 Sanger 测序检测的产毒艰难梭菌 tcdB 阳性率均为 100%。这些临床艰难梭菌菌株的毒素类型比例为 50% tcdA+tcdB+CDT-和 50% tcdA+tcdB+CDT+。该检测方法应扩展我们对艰难梭菌发病率的认识。这可能允许在现场快速诊断和筛查艰难梭菌相关疾病暴发。
