Department of Infectious Diseases, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China.
Ningbo Health Gene Technologies Co., Ltd., Ningbo, China.
J Clin Lab Anal. 2022 Oct;36(10):e24686. doi: 10.1002/jcla.24686. Epub 2022 Aug 31.
In recent years, the incidence of bloodstream infections (BSI) has increased, the composition of pathogenic bacteria has changed, and drug resistance among bacteria has gradually increased due to the widespread use of interventional techniques, broad-spectrum antibacterial drugs, hormones, and immunosuppressive agents. Here, we have developed a multiplex PCR assay kit for the detection of pathogens (14 Gram-negative bacteria, 15 Gram-positive bacteria, and 4 fungi) in whole blood from patients with BSI using five-color fluorescent multiplex PCR followed by capillary electrophoresis. Our assay exhibits a diagnosis of higher quality and an improved detection rate for common pathogens.
A local genome DNA database of 33 pathogenic bacteria was constructed. Next, "Exhaustive" primer search of the full coding sequence of the reference genomes of these bacteria was performed. Panels with minimal interactions between primers and amplicons were selected by random sampling and testing by a recursive algorithm. Primers and Mg concentrations and PCR reaction procedures were optimized to maximize the detection efficacy.
The LOD of the kit was determined as 100 copies/μl. Using clinical samples, results generated by this kit and regular blood culture method were found to be 95.08% consistent. Additionally, six pathogens which were unidentifiable by blood culture were successfully detected by this kit.
Our study provided a bioinformatics approach to the challenge of primer design in multiplex PCR, and combined with optimized wet lab practice, a multiplex PCR-based assay kit for BSI with higher sensitivity and accuracy than blood culture was produced.
近年来，由于介入技术、广谱抗菌药物、激素和免疫抑制剂的广泛应用，血流感染（BSI）的发病率增加，病原菌构成发生变化，细菌耐药性逐渐增加。在这里，我们使用五色荧光多重 PCR 结合毛细管电泳，开发了一种用于检测 BSI 患者全血中病原体（14 种革兰氏阴性菌、15 种革兰氏阳性菌和 4 种真菌）的多重 PCR 检测试剂盒。我们的检测方法具有更高的质量和改进的常见病原体检测率。
构建了 33 种致病细菌的局部基因组 DNA 数据库。然后，对这些细菌的参考基因组的全长编码序列进行“穷尽”引物搜索。通过随机抽样和递归算法测试选择引物和扩增子之间最小相互作用的面板。优化引物和 Mg 浓度以及 PCR 反应程序以最大限度地提高检测效果。
试剂盒的 LOD 确定为 100 拷贝/μl。使用临床样本，发现该试剂盒和常规血培养方法生成的结果一致性为 95.08%。此外，该试剂盒成功检测到血培养无法识别的六种病原体。
我们的研究为多重 PCR 中的引物设计挑战提供了一种生物信息学方法，并结合优化的湿实验室实践，产生了一种比血培养更敏感和准确的基于多重 PCR 的 BSI 检测试剂盒。
