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本文引用的文献

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Sensitivity and performance characteristics of a direct PCR with stool samples in comparison to conventional techniques for diagnosis of Shigella and enteroinvasive Escherichia coli infection in children with acute diarrhoea in Calcutta, India.印度加尔各答急性腹泻儿童粪便样本直接PCR与传统技术相比诊断志贺菌和肠侵袭性大肠杆菌感染的敏感性及性能特征
J Med Microbiol. 2001 Aug;50(8):667-674. doi: 10.1099/0022-1317-50-8-667.
2
Contamination and sensitivity issues with a real-time universal 16S rRNA PCR.实时通用16S rRNA聚合酶链反应的污染和敏感性问题。
J Clin Microbiol. 2000 May;38(5):1747-52. doi: 10.1128/JCM.38.5.1747-1752.2000.
3
Rapid identification of bacteria in blood cultures by using fluorescently labeled oligonucleotide probes.利用荧光标记的寡核苷酸探针快速鉴定血培养中的细菌。
J Clin Microbiol. 2000 Feb;38(2):814-7. doi: 10.1128/JCM.38.2.814-817.2000.
4
Direct analysis of genes encoding 16S rRNA from complex communities reveals many novel molecular species within the human gut.对来自复杂群落的编码16S rRNA的基因进行直接分析，揭示了人类肠道内许多新的分子物种。
Appl Environ Microbiol. 1999 Nov;65(11):4799-807. doi: 10.1128/AEM.65.11.4799-4807.1999.
5
Analysis of the intestinal microflora: a renaissance.肠道微生物群分析：一场复兴。
Antonie Van Leeuwenhoek. 1999 Jul-Nov;76(1-4):265-78.
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Development and validation of an automated, microscopy-based method for enumeration of groups of intestinal bacteria.一种基于显微镜的自动化肠道细菌菌群计数方法的开发与验证
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7
Application of denaturant gradient gel electrophoresis for the analysis of the porcine gastrointestinal microbiota.变性梯度凝胶电泳在猪胃肠道微生物群分析中的应用。
J Microbiol Methods. 1999 Jun;36(3):167-79. doi: 10.1016/s0167-7012(99)00029-9.
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Quantification of 16S rRNAs in complex bacterial communities by multiple competitive reverse transcription-PCR in temperature gradient gel electrophoresis fingerprints.通过温度梯度凝胶电泳指纹图谱中的多重竞争性逆转录PCR对复杂细菌群落中的16S rRNA进行定量分析。
Appl Environ Microbiol. 1998 Nov;64(11):4581-7. doi: 10.1128/AEM.64.11.4581-4587.1998.
9
Temperature gradient gel electrophoresis analysis of 16S rRNA from human fecal samples reveals stable and host-specific communities of active bacteria.对人类粪便样本中的16S rRNA进行温度梯度凝胶电泳分析，揭示了活跃细菌的稳定且宿主特异性群落。
Appl Environ Microbiol. 1998 Oct;64(10):3854-9. doi: 10.1128/AEM.64.10.3854-3859.1998.
10
Variations of bacterial populations in human feces measured by fluorescent in situ hybridization with group-specific 16S rRNA-targeted oligonucleotide probes.使用针对特定菌群的16S rRNA靶向寡核苷酸探针通过荧光原位杂交技术检测人类粪便中细菌群体的变化。
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通过实时聚合酶链反应对附着于胃肠道黏膜的细菌进行定量分析。

Quantification of bacteria adherent to gastrointestinal mucosa by real-time PCR.

作者信息

Huijsdens Xander W, Linskens Ronald K, Mak Mariëtte, Meuwissen Stephan G M, Vandenbroucke-Grauls Christina M J E, Savelkoul Paul H M

机构信息

Department of Medical Microbiology, VU University Medical Center Amsterdam, Amsterdam, The Netherlands.

出版信息

J Clin Microbiol. 2002 Dec;40(12):4423-7. doi: 10.1128/JCM.40.12.4423-4427.2002.

DOI:10.1128/JCM.40.12.4423-4427.2002
PMID:12454130
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC154607/
Abstract

The use of real-time quantitative PCR (5' nuclease PCR assay) as a tool to study the gastrointestinal microflora that adheres to the colonic mucosa was evaluated. We developed primers and probes based on the 16S ribosomal DNA gene sequences for the detection of Escherichia coli and Bacteroides vulgatus. DNA was isolated from pure cultures and from gut biopsy specimens and quantified by the 5' nuclease PCR assay. The assay showed a very high sensitivity: as little as 1 CFU of E. coli and 9 CFU of B. vulgatus could be detected. The specificities of the primer-probe combinations were evaluated with samples that were spiked with the species most closely related to E. coli and B. vulgatus and with eight other gut microflora species. Mucosal samples spiked with known amounts of E. coli or B. vulgatus DNA showed no PCR inhibition. We conclude that the 5' nuclease PCR assay may be a useful alternative to conventional culture techniques to study the actual in vivo composition of a complex microbial community like the gut microflora.

摘要

评估了使用实时定量聚合酶链反应（5'核酸酶聚合酶链反应测定法）作为研究黏附于结肠黏膜的胃肠道微生物群的工具。我们基于16S核糖体DNA基因序列开发了引物和探针，用于检测大肠杆菌和普通拟杆菌。从纯培养物和肠道活检标本中分离DNA，并通过5'核酸酶聚合酶链反应测定法进行定量。该测定法显示出非常高的灵敏度：低至1个大肠杆菌菌落形成单位和9个普通拟杆菌菌落形成单位都能被检测到。用与大肠杆菌和普通拟杆菌关系最密切的物种以及其他八种肠道微生物群物种加标的样品评估了引物-探针组合的特异性。用已知量的大肠杆菌或普通拟杆菌DNA加标的黏膜样品未显示出聚合酶链反应抑制。我们得出结论，5'核酸酶聚合酶链反应测定法可能是一种有用的替代传统培养技术的方法，用于研究像肠道微生物群这样的复杂微生物群落的实际体内组成。