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Using pyrosequencing and quantitative PCR to analyze microbial communities

Husen ZHANG

《环境科学与工程前沿（英文）》 2011年第5卷第1期页码 21-27 doi: 10.1007/s11783-011-0303-9

摘要： New high-throughput technologies continue to emerge for studying complex microbial communities. In particular, massively parallel pyrosequencing enables very high numbers of sequences, providing a more complete view of community structures and a more accurate inference of the functions than has been possible just a few years ago. In parallel, quantitative real-time polymerase chain reaction (QPCR) allows quantitative monitoring of specific community members over time, space, or different environmental conditions. In this review, the principles of these two methods and their complementary applications in studying microbial ecology in bioenvironmental systems are discussed. The parallel sequencing of amplicon libraries and using barcodes to differentiate multiple samples in a pyrosequencing run are explained. The best procedures and chemistries for QPCR amplifications are also described and advantages of applying automation to increase accuracy are addressed. Three examples in which pyrosequencing and QPCR were used together to define and quantify members of microbial communities are provided: in the human large intestine, in a methanogenic digester whose sludge was made more bioavailable by a high-voltage pretreatment, and on the biofilm anode of a microbial electrolytic cell. The key findings in these systems and how both methods were used in concert to achieve those findings are highlighted.

关键词： polymerase chain reaction (PCR) microbial communities pyrosequencing gut microbial fuel cell sludge

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Interaction and independence on methane oxidation of landfill cover soil among three impact factors: water, oxygen and ammonium

Pinjing HE, Na YANG, Wenjuan FANG, Fan Lü, Liming SHAO

《环境科学与工程前沿（英文）》 2011年第5卷第2期页码 175-185 doi: 10.1007/s11783-011-0320-8

摘要： To understand the influence patterns and interactions of three important environmental factors, i.e. soil water content, oxygen concentration, and ammonium addition, on methane oxidation, the soils from landfill cover layers were incubated under full factorial parameter settings. In addition to the methane oxidation rate, the quantities and community structures of methanotrophs were analyzed to determine the methane oxidation capacity of the soils. Canonical correspondence analysis was utilized to distinguish the important impact factors. Water content was found to be the most important factor influencing the methane oxidation rate and Type II methanotrophs, and the optimum value was 15% (w/w), which induced methane oxidation rates 10- and 6- times greater than those observed at 5% (w/w) and 20% (w/w), respectively. Ambient oxygen conditions were more suitable for methane oxidation than 3% oxygen. The addition of of ammonium induced different effects on methane oxidation capacity when conducted at low or high water content. With regard to the methanotrophs, Type II was sensitive to the changes of water content, while Type I was influenced by oxygen content. Furthermore, the methanotrophic acidophile, , was detected in soils with a pH of 4.9, which extended their known living environments.

关键词： quantitative polymerase chain reaction (PCR) denaturing gradient gel electrophoresis (DGGE) principal component analysis (PCA) canonical correspondence analysis (CCA)

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低成本PCR微反应器阵列的温度测量与控制技术

李现明,张玉林,李建田

《中国工程科学》 2007年第9卷第7期页码 76-81

摘要：

设计了一种用于聚合酶链式反应的低成本微反应器，其内部无微加热器、微传感器。针对这种低成本的微反应器，提出了一种温度测量与控制方案，其特点是将各反应器温度的集中控制与分散控制有机结合，能够同时完成上百只微反应器快速、准确的温度循环。重点阐述了温度传感器负荷效应的提取与补偿、动态误差补偿、微反应器温度间接测量误差评定、微反应器温度串级控制策略。

关键词：微反应器测量控制聚合酶链式反应

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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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基于抗原和聚合酶链式反应的组合策略对模拟社区内遏制COVID-19传播效果的定量分析 Article

黄蔷如, 孙艳侠, 贾萌萌, 张婷, 陈方圆, 姜明月, 王晴, 冯录召, 杨维中

《工程（英文）》 2023年第28卷第9期页码 237-245 doi: 10.1016/j.eng.2023.01.004

摘要：研究结果表明抗原检测及其组合策略比聚合酶链式反应（polymerase chain reaction, PCR）相关策略更高效，更有助于降低医院床位和重症监护病房床位需求。较短的结果返回时间是抗原检测策略的主要优势所在，也是优化PCR策略的关键因素。优化调整检测结果返回时间后，低频次的检测策略的效果可以媲美每日连续检测策略的效果。

关键词： COVID-19 抗原检测 PCR核酸检测检测策略

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Seasonal microbial community shift in a saline sewage treatment plant

Qingmei YAN, Xuxiang ZHANG, Tong ZHANG, Herbert H P FANG

《环境科学与工程前沿（英文）》 2011年第5卷第1期页码 40-47 doi: 10.1007/s11783-011-0304-8

摘要： Activated sludge was monthly sampled from a saline sewage treatment plant of Hong Kong (China) during June 2007 to May 2008 to analyze the microbial community shift along with environmental variations using denaturing gradient gel electrophoresis of polymerase chain reaction amplified 16S rDNA fragments. Environmental changes resulted into a seasonal microbial community shift characterized by alterations in species number and abundance in the sewage treatment plant. Correspondence analysis and cluster analysis on community structure profile showed that the 12 monthly samples fell into four groups, which is in accordance with season changing in Hong Kong. Canonical correspondence analysis revealed that and posed more significant effects on community structure than total phosphorus and total nitrogen, respectively. Compared with sludge retention time, influent total phosphorus had an inverse effect on the community structure shift, and chemical oxygen demand and showed a similar effects. Results of this study may contribute to the development of new knowledge involving the microbial community shift in sewage treatment plants.

关键词： sewage treatment plants polymerase chain reaction (PCR) denaturing gradient gel electrophoresis (DGGE) canonical correspondence analysis correspondence analysis

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A pooling strategy of a PCR-based assay to detect Angiostrongylus cantonensis in snail intermediate host

Fu-Rong WEI MS, Shan LV PhD, He-Xiang LIU, Ling HU, Yi ZHANG MS,

《医学前沿（英文）》 2010年第4卷第2期页码 204-207 doi: 10.1007/s11684-010-0043-4

摘要： Pooling field specimens could reduce the number of assay and thus increase the efficiency in detecting and screening pathogen infections by polymerase chain reaction (PCR)-based assay. We investigated a pooling strategy in diagnosis of in Two settings of specimens were prepared, divided into portions and detected by multiplex PCR. Specimens A was 0.4490 g positive lung tissue of 28 larval nodes from four snails mixed with 1.310€g negative lung tissue from six snails and divided into 32 portions. Specimens B was 0.5448 g positive lung tissue with 26 larval nodes from two snails mixed with 1.092 g negative lung tissue from seven snails and divided into 48 portions. Repeated sampling was performed and sample size-accumulated positive rate curves were drawn. According to the sample size-accumulated positive rate curves, the appropriate sample size of the two specimens were 18 and 15, respectively, which is 0.36–0.58 to the total sample size. These test characteristics and the relevant factors to the sample size would need to be determined in much larger studies and more appropriately in field populations. The result indicates that the number of larval node is not the most important, nor the only factor to the sample size. It also implies the feasibility to detect in by pooling strategies.

关键词： Angiostrongylus cantonensis Pomacea canaliculata multiplex polymerase chain reaction diagnosis pooling

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Cultivation of aerobic granular sludge in a conventional, continuous flow, completely mixed activated sludge system

Xi CHEN,Linjiang YUAN,Wenjuan LU,Yuyou LI,Pei LIU,Kun NIE

《环境科学与工程前沿（英文）》 2015年第9卷第2期页码 324-333 doi: 10.1007/s11783-014-0627-3

摘要： Aerobic granules were formed in a conventional, continuous flow, completely mixed activated sludge system (CMAS). The reactor was inoculated with seed sludge containing few filaments and fed with synthetic municipal wastewater. The settling time of the sludge and the average dissolved oxygen (DO) of the reactor were 2 h and 4.2 mg·L , respectively. The reactor was agitated by a stirrer, with a speed of 250 r·min , to ensure good mixing．The granular sludge had good settleability, and the sludge volume index (SVI) was between 50 and 90 mL·g . The laser particle analyzer showed the diameter of the granules to be between 0.18 and 1.25 mm. A scanning electron microscope (SEM) investigation revealed the predominance of sphere-like and rod-like bacteria, and only few filaments grew in the granules. The microbial community structure of the granules was also analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). Sequencing analysis indicated the dominant species were α, β, and γ- , , and . The data from the study suggested that aerobic granules could form, if provided with sufficient number of filaments and high shear force. It was also observed that a high height-to-diameter ratio of the reactor and short settling time were not essential for the formation of aerobic granular sludge.

关键词： aerobic granular sludge completely mixed activated sludge system (CMAS) continuous flow shear force filamentous bacteria polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE)

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基于探针图的并行型图顶点着色DNA计算模型 Article

许进, 强小利, 张凯, 张成, 杨静

《工程（英文）》 2018年第4卷第1期页码 61-77 doi: 10.1016/j.eng.2018.02.011

摘要：其主要贡献有：①通过如下三种方法来克服解空间指数爆炸问题：顶点颜色集的确定方法；子图分解方法以及子图中的顶点优化排序方法；②设计了一种并行型聚合酶链反应（PCR）操作技术，应用这种技术一次可以对图中多条边来删除非解本文以一个3-着色的61 个顶点的图为例，实验表明，99% 的非可行解在构建初始解空间时就被删除，并利用DNA 自组装和并行PCR 方法，通过识别、拼接以及组装等技术得到解。

关键词： DNA计算图顶点着色问题聚合酶链反应（PCR）

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Erratum to: Using pyrosequencing and quantitative PCR to analyze microbial communities

Husen ZHANG

《环境科学与工程前沿（英文）》 2011年第5卷第3期页码 488-488 doi: 10.1007/s11783-011-0342-2

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involved in nitrogen cycling in wastewater treatment plants using DNA microarray and most probable number-PCR

Junqin PANG, Masami MATSUDA, Masashi KURODA, Daisuke INOUE, Kazunari SEI, Kei NISHIDA, Michihiko IKE

《环境科学与工程前沿（英文）》 2016年第10卷第4期 doi: 10.1007/s11783-016-0846-x

摘要： Nitrogen-cycling microbial communities in municipal WWTPs were characterized. Numbers of , and genes were quantified by MPN-PCR. Diversity of whole nitrogen-cycling communities was analyzed with DNA microarray. CAS process retained diverse nitrogen cycling populations. Specific, limited populations may be dominated in nitrogen removal processes. To improve nitrogen removal performance of wastewater treatment plants (WWTPs), it is essential to understand the behavior of nitrogen cycling communities, which comprise various microorganisms. This study characterized the quantity and diversity of nitrogen cycling genes in various processes of municipal WWTPs by employing two molecular-based methods:most probable number-polymerase chain reaction (MPN-PCR) and DNA microarray. MPN-PCR analysis revealed that gene quantities were not statistically different among processes, suggesting that conventional activated sludge processes (CAS) are similar to nitrogen removal processes in their ability to retain an adequate population of nitrogen cycling microorganisms. Furthermore, most processes in the WWTPs that were researched shared a pattern:the and the bacterial genes were more abundant than the and archaeal genes, respectively. DNA microarray analysis revealed that several kinds of nitrification and denitrification genes were detected in both CAS and anaerobic-oxic processes (AO), whereas limited genes were detected in nitrogen removal processes. Results of this study suggest that CAS maintains a diverse community of nitrogen cycling microorganisms; moreover, the microbial communities in nitrogen removal processes may be specific.

关键词： DNA microarray analysis Nitrogen cycling functional genes Most probable number-polymerase chain reaction (MPN-PCR) Wastewater treatment plants (WWTPs)

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一种通过检测特定粪便微生物群来评估人体肠道微生物稳态的方法 Article

Zhongwen Wu, Xiaxia Pan, Yin Yuan, Pengcheng Lou, Lorina Gordejeva, Shuo Ni, Xiaofei Zhu, Bowen Liu, Lingyun Wu, Lanjuan Li, Bo Li

《工程（英文）》 2023年第29卷第10期页码 110-119 doi: 10.1016/j.eng.2023.03.007

摘要：

Research on microecology has been carried out with broad perspectives in recent decades, which has enabled a better understanding of the gut microbiota and its roles in human health and disease. It is of great significance to routinely acquire the status of the human gut microbiota; however, there is no method to evaluate the gut microbiome through small amounts of fecal microbes. In this study, we found ten predominant groups of gut bacteria that characterized the whole microbiome in the human gut from a largesample Chinese cohort, constructed a real-time quantitative polymerase chain reaction (qPCR) method and developed a set of analytical approaches to detect these ten groups of predominant gut bacterial species with great maneuverability, efficiency, and quantitative features. Reference ranges for the ten predominant gut bacterial groups were established, and we found that the concentration and pairwise ratios of the ten predominant gut bacterial groups varied with age, indicating gut microbial dysbiosis. By comparing the detection results of liver cirrhosis (LC) patients with those of healthy control subjects, differences were then analyzed, and a classification model for the two groups was built by machine learning. Among the six established classification models, the model established by using the random forest algorithm achieved the highest area under the curve (AUC) value and sensitivity for predicting LC. This research enables easy, rapid, stable, and reliable testing and evaluation of the balance of the gut microbiota in the human body, which may contribute to clinical work.

关键词： Gut microbiota Machine learning Microbial dysbiosis Quantitative polymerase chain reaction Chinese cohort

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Expression and clinical implication of PRL-1 and PRL-3 in transitional cell carcinoma of bladder

Bin HAO, Changwei LIU, Huixiang LI

《医学前沿（英文）》 2009年第3卷第2期页码 197-203 doi: 10.1007/s11684-009-0036-3

摘要： The mRNA and protein expression of phosphatase of regenerating liver 1 (PRL-1) and phosphatase of regenerating liver 3 (PRL-3) in transitional cell carcinoma of bladder (BTCC) and normal epithelia of bladder was investigated, and the relationship between the BTCC and pathological changes was clarified. The expression of PRL-1 and PRL-3 mRNA was detected by using reverse transcription polymerase chain reaction (RT-PCR) in 30 cases of BTCC and 10 cases of normal bladder, and the expression of PRL-1 and PRL-3 protein was checked by using immunohistochemistry in 30 cases of BTCC and 15 cases of normal bladder. The expression levels of PRL-1 and PRL-3 mRNA and protein were higher in BTCC than those in normal bladder epithelia ( <0.05). The increased expression of PRL-1 and PRL-3 mRNA and protein was detectable in deep invasion and metastasis of BTCC ( <0.05). There was no correlation between the expression of PRL-1 and PRL-3 and gender, age or recurrence of BTCC (all >0.05). A significantly positive correlation was found between PRL-1 and PRL-3 in BTCC ( <0.05). PRL-1 and PRL-3 are expressed consistently and may contribute to the growth, differentiation, invasion and metastasis of BTCC.

关键词： transitional cell carcinoma of bladder phosphatase of regenerating liver 1 phosphatase of regenerating liver 3 reverse transcription polymerase chain reaction immunohistochemistry

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A computational toolbox for molecular property prediction based on quantum mechanics and quantitative

《化学科学与工程前沿（英文）》 2022年第16卷第2期页码 152-167 doi: 10.1007/s11705-021-2060-z

摘要： Chemical industry is always seeking opportunities to efficiently and economically convert raw materials to commodity chemicals and higher value-added chemical-based products. The life cycles of chemical products involve the procedures of conceptual product designs, experimental investigations, sustainable manufactures through appropriate chemical processes and waste disposals. During these periods, one of the most important keys is the molecular property prediction models associating molecular structures with product properties. In this paper, a framework combining quantum mechanics and quantitative structure-property relationship is established for fast molecular property predictions, such as activity coefficient, and so forth. The workflow of framework consists of three steps. In the first step, a database is created for collections of basic molecular information; in the second step, quantum mechanics-based calculations are performed to predict quantum mechanics-based/derived molecular properties (pseudo experimental data), which are stored in a database and further provided for the developments of quantitative structure-property relationship methods for fast predictions of properties in the third step. The whole framework has been carried out within a molecular property prediction toolbox. Two case studies highlighting different aspects of the toolbox involving the predictions of heats of reaction and solid-liquid phase equilibriums are presented.

关键词： molecular property quantum mechanics quantitative structure-property relationship heat of reaction solid-liquid phase equilibrium

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Continuous reaction performances of benzene alkylation with long chain olefins catalyzed by ionic liquid

QIAO Congzhen, LI Chengyue

《化学科学与工程前沿（英文）》 2008年第2卷第1期页码 69-73 doi: 10.1007/s11705-008-0014-3

摘要： Based on a compulsive mixing-reacting-separating-recycling small experimental setup,the continuous reaction performances of benzene alkylation with long chain olefins catalyzed by [BMIM]Cl-AlCl ionic liquid were investigated. Three different situations including normal continuous operation mode (reagent materials), sidetrack feeding from different axial positions along the static mixing reactor (reagent materials) and normal continuous alkylation using industrial paraffin and olefins materials were examined. Even under the relatively hypecritical reaction conditions, the single pass conversion of pure 1-dodecene could reach to nearly 100.0%, and the selectivity of 2-phenyl isomer was higher than 37.7%. Although the positions along the reactor for sidetrack feeding were different, the 100.0% single pass conversion of 1-dodecene was also attained before the outlet of the reactor. The refined industrial olefins as raw material could meet with the requirements of continuous alkylation. The influences of impurities such as di-olefins and non-benzene aromatics on the catalytic activity and stability should be studied further.

关键词： stability different hypecritical compulsive mixing-reacting-separating-recycling non-benzene

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标题作者时间类型操作

Using pyrosequencing and quantitative PCR to analyze microbial communities

Husen ZHANG

期刊论文

Interaction and independence on methane oxidation of landfill cover soil among three impact factors: water, oxygen and ammonium

Pinjing HE, Na YANG, Wenjuan FANG, Fan Lü, Liming SHAO

期刊论文

低成本PCR微反应器阵列的温度测量与控制技术

李现明,张玉林,李建田

期刊论文

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

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

期刊论文

基于抗原和聚合酶链式反应的组合策略对模拟社区内遏制COVID-19传播效果的定量分析

黄蔷如, 孙艳侠, 贾萌萌, 张婷, 陈方圆, 姜明月, 王晴, 冯录召, 杨维中

期刊论文

Seasonal microbial community shift in a saline sewage treatment plant

Qingmei YAN, Xuxiang ZHANG, Tong ZHANG, Herbert H P FANG

期刊论文

A pooling strategy of a PCR-based assay to detect Angiostrongylus cantonensis in snail intermediate host

Fu-Rong WEI MS, Shan LV PhD, He-Xiang LIU, Ling HU, Yi ZHANG MS,

期刊论文

Cultivation of aerobic granular sludge in a conventional, continuous flow, completely mixed activated sludge system

Xi CHEN,Linjiang YUAN,Wenjuan LU,Yuyou LI,Pei LIU,Kun NIE

期刊论文

基于探针图的并行型图顶点着色DNA计算模型

许进, 强小利, 张凯, 张成, 杨静

期刊论文

Erratum to: Using pyrosequencing and quantitative PCR to analyze microbial communities

Husen ZHANG

期刊论文

involved in nitrogen cycling in wastewater treatment plants using DNA microarray and most probable number-PCR

Junqin PANG, Masami MATSUDA, Masashi KURODA, Daisuke INOUE, Kazunari SEI, Kei NISHIDA, Michihiko IKE

期刊论文

一种通过检测特定粪便微生物群来评估人体肠道微生物稳态的方法

Zhongwen Wu, Xiaxia Pan, Yin Yuan, Pengcheng Lou, Lorina Gordejeva, Shuo Ni, Xiaofei Zhu, Bowen Liu, Lingyun Wu, Lanjuan Li, Bo Li

期刊论文

Expression and clinical implication of PRL-1 and PRL-3 in transitional cell carcinoma of bladder

Bin HAO, Changwei LIU, Huixiang LI

期刊论文

A computational toolbox for molecular property prediction based on quantum mechanics and quantitative

期刊论文

Continuous reaction performances of benzene alkylation with long chain olefins catalyzed by ionic liquid

QIAO Congzhen, LI Chengyue

期刊论文

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