Development and clinical application of a rapid, visually interpretable polymerase spiral reaction for tcdB gene of Clostridioides difficile in fecal cultures
Development and clinical application of a rapid, visually interpretable polymerase spiral reaction for tcdB gene of Clostridioides difficile in fecal cultures
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DOI：
10.1093/femsle/fnad080
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发表时间：
2023-01-17
影响因子：
2.1
通讯作者：
Song，Xiuling
中科院分区：
文献类型：
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作者：
Yin，Caihong;Song，Zhanyun;Song，Xiuling
在艰难梭菌感染暴发疫情监测中，快速检测和诊断艰难梭菌是关键。difficile仍然是一个重大挑战。聚合酶螺旋反应（Polymerase spiral reaction，PSR）是一种利用混合引物和BstDNA聚合酶的链置换活性，在等温环境中实现一对引物和一种酶的核酸扩增技术。引物设计简单，反应有效，并且可以使用颜色指示剂来可视化结果。在这项研究中，我们开发了一个快速和直观的PSR检测C。通过分析人工污染的粪便样本和来自患者粪便样本的临床分离株，我们设计了两对特异性靶向C.很难扩增结果用羟基萘酚蓝显色。整个过程在64°C下在50分钟内完成，具有高特异性。C.人工污染的粪便样品中，PSR的最低检出浓度为150 fg/μl基因组DNA或2 × 10 - 3CFU/ml。用这种方法，我们分析了四个临床分离株，并比较了PSR的分离和培养检测方法，聚合酶链反应，和桑格测序。4株临床分离株的tcdB均为阳性，证实了引物的高度特异性。tcd在大肠杆菌中的阳性率为100%。PSR、PCR和桑格测序法检测的阳性率为100%。结果表明，临床上C. tcdA+tcdB+CDT−和tcdA+tcdB+CDT+的比例分别为50%和50%。所描述的测定法应扩展我们对C.很难这可能有助于快速诊断和筛查C。与疾病有关的疾病爆发。
In the surveillance of outbreaks ofClostridioides difficileinfection, the rapid detection and diagnosis ofC. difficileremain a major challenge. Polymerase spiral reaction (PSR) is a nucleic acid amplification technique that uses mixed primers and the strand displacement activity ofBstDNA 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 detectC. difficileby 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 conservedtcdBgene ofC. 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 ofC. difficilewith 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 fortcdB, which confirmed the high specificity of the primers. The positive rates oftcdBin toxigenicC. difficiledetected with PSR, PCR, and Sanger sequencing were 100%. The proportions of toxin types in these clinicalC. difficilestrains were 50% tcdA+tcdB+CDT−and 50% tcdA+tcdB+CDT+. The assay described should extend our understanding of the incidence ofC. difficile. This may allow the rapid diagnosis and screening ofC. difficile-related disease outbreaks in the field.