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Phylogenetic diversity of NO reductases, new tools for monitoring, and insights into NO production in natural and engineered environments

《环境科学与工程前沿（英文）》 2022年第16卷第10期 doi: 10.1007/s11783-022-1562-3

摘要：

● 548 representative nor genes were collected to create complete phylogenetic trees.

关键词： N₂O Greenhouse gas NO reductase NO dismutase Primer Crystal structure

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Design and use of group-specific primers and probes for real-time quantitative PCR

Juntaek LIM, Seung Gu SHIN, Seungyong LEE, Seokhwan HWANG

《环境科学与工程前沿（英文）》 2011年第5卷第1期页码 28-39 doi: 10.1007/s11783-011-0302-x

摘要： Real-time quantitative polymerase chain reaction (qPCR) has gained popularity as a technique to detect and quantify a specific group of target microorganisms from various environmental samples including soil, water, sediments, and sludge. Although qPCR is a very useful technique for nucleic acid quantification, accurately quantifying the target microbial group strongly depends on the quality of the primer and probe used. Many aspects of conducting qPCR assays have become increasingly routine and automated; however, one of the most important aspects, designing and selecting primer and probe sets, is often a somewhat arcane process. In many cases, failed or non-specific amplification can be attributed to improperly designed primer-probe sets. This paper is intended to provide guidelines and general principles for designing group-specific primers and probes for qPCR assays. We demonstrate the effectiveness of these guidelines by reviewing the use of qPCR to study anaerobic processes and biologic nutrient removal processes. qPCR assays using group-specific primers and probes designed with this method, have been used to successfully quantify 16S ribosomal Ribonucleic Acid (16S rRNA) gene copy numbers from target methanogenic and ammonia- oxidizing bacteria in various laboratory- and full-scale biologic processes. Researchers with a good command of primer and probe design can use qPCR as a valuable tool to study biodiversity and to develop more efficient control strategies for biologic processes.

关键词： absolute quantification design guideline primer probe real-time quantitative polymerase chain reaction (qPCR)

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检测GI和GII族人源诺如病毒双重RT-qPCR的优化设计 Article

刘丹蕾, 张子蕾, 吴清平, 田鹏, 耿浩然, 徐婷, 王大鹏

《工程（英文）》 2020年第6卷第4期页码 442-448 doi: 10.1016/j.eng.2019.08.018

摘要：

人源诺如病毒（human norovirus, HuNoV）是重要的食源性病毒之一，能够引起全世界范围人类的非细菌性急性胃肠炎。由于HuNoV的体外培养体系尚不成熟，无法用于常规的病毒分离和检测，因此目前对HuNoV的检测依赖于分子方法，如逆转录聚合酶链反应（reverse transcription polymerase
chain reaction, RT-PCR）和逆转录实时定量聚合酶链反应（reverse transcription quantitative real-time polymerase chain reaction, RT-qPCR）。21世纪初期设计的引物和探针依然被广泛地用于RTqPCR检测体系。HuNoV基因组具有变异度高的特点，导致设计的引物和（或）探针无法有效地与已进化的新病毒核酸进行匹配，从而随着时间的推移效率降低。为了提高HuNoV检出效率，本研究基于2010年后GenBank公布的病毒序列，分析设计了一套HuNoV检测引物和探针，并优化了一种新的双重RT-qPCR(new duplex RT-qPCR, ND-RT-qPCR)检测体系。以体外转录获得的长链病毒RNA为模板，ND-RT-qPCR可对低至一个基因组的GI和GII族HuNoV进行有效检测。以23份HuNoV临床样本为对象，评估了ND-RT-qPCR和常用RT-qPCR(Kageyama RT-qPCR)的检测性能。结果显示：ND-RT-qPCR检出所有GI族样本（5/5），而Kageyama RT-qPCR只检出两个样本（2/5）。ND-RT-qPCR检出18个GII族样本（18/18），而Kageyama RT-qPCR漏检1个样本。另外，ND-RTqPCR的灵敏度显著高于Kageyama RT-qPCR（前者Cq值低于后者Cq值）。因此，ND-RT-qPCR有助于提高HuNoV检出率，是目前该病毒检测良好的选择。

关键词：人源诺如病毒 RT-qPCR 优化设计引物探针检测