摘要
      由于人畜共患病原体正在威胁全球公众健康，它们携带的毒力因子基因（VFG）是环境中潜在风险的基础。然而，由于缺乏高效可靠的量化工具，在环境中分析VFG仍处于初级阶段。在这里，我们开发了一种新的高通量qPCR（HT-qPCR）芯片，称为VFG芯片，以全面量化环境中靶向VFG的丰度。120对引物共靶向了来自肺炎克雷伯菌、鲍曼不动杆菌、大肠杆菌和肠炎沙门氏菌等四种细菌病原体的96个VFG，这些引物参与编码毒素、粘附、分泌系统、免疫逃避/侵袭和铁摄取等五种毒力因子。VFG芯片的特异性在计算和实验上都得到了验证，扩增子测序和熔解曲线分析的高度一致性证明了其强大的能力。VFG芯片也显示出高灵敏度（通过质粒系列稀释测试）和平均97.7%的扩增效率。我们成功地将VFG芯片应用于废水处理系统中VFG的分布，共检测到69个VFG。总的来说，VFG芯片为全面量化环境中的VFG提供了一个强大的工具，从而为评估环境中人畜共患病原体的健康风险提供了新的信息。
Abstract
As zoonotic pathogens are threatening public health globally, the virulence factor genes (VFGs) they carry underlie latent risk in the environment. However, profiling VFGs in the environment is still in its infancy due to lack of efficient and reliable quantification tools. Here, we developed a novel high-throughput qPCR (HT-qPCR) chip, termed as VFG-Chip, to comprehensively quantify the abundances of targeted VFGs in the environment. A total of 96 VFGs from four bacterial pathogens including Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, and Salmonella enterica were targeted by 120 primer pairs, which were involved in encoding five types of virulence factors (VFs) like toxin, adherence, secretion system, immune evasion/invasion, and iron uptake. The specificity of VFG-Chip was both verified computationally and experimentally, with high identity of amplicon sequencing and melting curves analysis proving its robust capability. The VFG-Chip also displayed high sensitivity (by plasmid serial dilution test) and amplification efficiency averaging 97.7%. We successfully applied the VFG-Chip to profile the distribution of VFGs along a wastewater treatment system with 69 VFGs detected in total. Overall, the VFG-Chip provides a robust tool for comprehensively quantifying VFGs in the environment, and thus provides novel information in assessing the health risks of zoonotic pathogens in the environment.

https://www.sciencedirect.com/science/article/pii/S016041202300034X