發(fā)布者：抗性基因網(wǎng) 時間：2023-06-14 瀏覽量：2306
摘要
? ? ? 由于人畜共患病原體正在威脅全球公眾健康，它們攜帶的毒力因子基因（VFG）是環(huán)境中潛在風險的基礎。然而，由于缺乏高效可靠的量化工具，在環(huán)境中分析VFG仍處于初級階段。在這里，我們開發(fā)了一種新的高通量qPCR（HT-qPCR）芯片，稱為VFG芯片，以全面量化環(huán)境中靶向VFG的豐度。120對引物共靶向了來自肺炎克雷伯菌、鮑曼不動桿菌、大腸桿菌和腸炎沙門氏菌等四種細菌病原體的96個VFG，這些引物參與編碼毒素、粘附、分泌系統(tǒng)、免疫逃避/侵襲和鐵攝取等五種毒力因子。VFG芯片的特異性在計算和實驗上都得到了驗證，擴增子測序和熔解曲線分析的高度一致性證明了其強大的能力。VFG芯片也顯示出高靈敏度（通過質(zhì)粒系列稀釋測試）和平均97.7%的擴增效率。我們成功地將VFG芯片應用于廢水處理系統(tǒng)中VFG的分布，共檢測到69個VFG?？偟膩碚f，VFG芯片為全面量化環(huán)境中的VFG提供了一個強大的工具，從而為評估環(huán)境中人畜共患病原體的健康風險提供了新的信息。
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