艰难梭菌感染粪便PCR的微量样本制备
Microscale sample preparation for PCR of C. difficile infected stool.
作者信息
Gillers Sara, Atkinson Christopher D, Bartoo Aaron C, Mahalanabis Madhumita, Boylan Michael O, Schwartz John H, Klapperich Catherine, Singh Satish K
机构信息
Department of Medicine, Section of Gastroenterology, Boston University School of Medicine, Suite 504, 650 Albany Street, Boston, MA 02118, United States.
出版信息
J Microbiol Methods. 2009 Aug;78(2):203-7. doi: 10.1016/j.mimet.2009.05.020. Epub 2009 Jun 6.
Abstract
In this paper, we describe the design of a microfluidic sample preparation chip for human stool samples infected with Clostridium difficile. We established a polymerase chain reaction able to distinguish C. difficile in the presence of several other organisms found in the normal intestinal flora. A protocol for on-chip extraction of nucleic acids from clinical samples is described that can detect target DNA down to 5.0x10(-3) ng of template. The assay and sample preparation chip were then validated using known positive and known negative clinical samples. The work presented has potential applications in both the developed and developing world.
摘要
在本文中，我们描述了一种用于感染艰难梭菌的人类粪便样本的微流控样品制备芯片的设计。我们建立了一种聚合酶链反应，能够在正常肠道菌群中发现的几种其他生物体存在的情况下区分艰难梭菌。本文描述了一种从临床样本中进行芯片上核酸提取的方案，该方案能够检测低至5.0×10⁻³纳克模板的目标DNA。然后使用已知的阳性和已知的阴性临床样本对该检测方法和样品制备芯片进行了验证。所展示的这项工作在发达国家和发展中国家都有潜在的应用。
相似文献
1
Microscale sample preparation for PCR of C. difficile infected stool.
J Microbiol Methods. 2009 Aug;78(2):203-7. doi: 10.1016/j.mimet.2009.05.020. Epub 2009 Jun 6.
2
Rapid detection of toxigenic Clostridium difficile in fecal samples by magnetic immuno PCR assay.
J Clin Microbiol. 1994 Jul;32(7):1629-33. doi: 10.1128/jcm.32.7.1629-1633.1994.
3
Rapid detection of Clostridium difficile in feces by real-time PCR.
J Clin Microbiol. 2003 Feb;41(2):730-4. doi: 10.1128/JCM.41.2.730-734.2003.
4
Evaluation of a new commercial TaqMan PCR assay for direct detection of the clostridium difficile toxin B gene in clinical stool specimens.
J Clin Microbiol. 2009 Dec;47(12):3846-50. doi: 10.1128/JCM.01490-09. Epub 2009 Oct 21.
5
Rapid detection assay of toxigenic Clostridioides difficile through PathOC RightGene, a novel high-speed polymerase chain reaction device.
Diagn Microbiol Infect Dis. 2021 Feb;99(2):115247. doi: 10.1016/j.diagmicrobio.2020.115247. Epub 2020 Oct 16.
6
Specific detection of toxigenic strains of Clostridium difficile in stool specimens.
J Clin Microbiol. 1993 Mar;31(3):507-11. doi: 10.1128/jcm.31.3.507-511.1993.
7
Comparison of a commercial real-time PCR assay for tcdB detection to a cell culture cytotoxicity assay and toxigenic culture for direct detection of toxin-producing Clostridium difficile in clinical samples.
J Clin Microbiol. 2009 Feb;47(2):373-8. doi: 10.1128/JCM.01613-08. Epub 2008 Dec 10.
8
Clostridium difficile testing in the clinical laboratory by use of multiple testing algorithms.
J Clin Microbiol. 2010 Mar;48(3):889-93. doi: 10.1128/JCM.01801-09. Epub 2010 Jan 13.
9
Evaluation of tcdB real-time PCR in a three-step diagnostic algorithm for detection of toxigenic Clostridium difficile.
J Clin Microbiol. 2010 Jan;48(1):124-30. doi: 10.1128/JCM.00734-09. Epub 2009 Nov 18.
10
Detection of C. difficile toxin as a model assay for performing fully automated high-throughput RT-PCR on clinical stool samples.
J Microbiol Methods. 2020 May;172:105882. doi: 10.1016/j.mimet.2020.105882. Epub 2020 Feb 28.
引用本文的文献
1
Overcoming donor variability and risks associated with fecal microbiota transplants through bacteriophage-mediated treatments.
Microbiome. 2024 Jul 1;12(1):119. doi: 10.1186/s40168-024-01820-1.
2
An Aniline-Substituted Bile Salt Analog Protects both Mice and Hamsters from Multiple Clostridioides difficile Strains.
Antimicrob Agents Chemother. 2022 Jan 18;66(1):e0143521. doi: 10.1128/AAC.01435-21. Epub 2021 Nov 15.
3
On-chip stool liquefaction via acoustofluidics.
Lab Chip. 2019 Mar 13;19(6):941-947. doi: 10.1039/c8lc01310a.
4
Low concentration DNA extraction and recovery using a silica solid phase.
PLoS One. 2017 May 5;12(5):e0176848. doi: 10.1371/journal.pone.0176848. eCollection 2017.
5
Combining electrochemical sensors with miniaturized sample preparation for rapid detection in clinical samples.
Sensors (Basel). 2014 Dec 30;15(1):547-64. doi: 10.3390/s150100547.
6
Low cost extraction and isothermal amplification of DNA for infectious diarrhea diagnosis.
PLoS One. 2013;8(3):e60059. doi: 10.1371/journal.pone.0060059. Epub 2013 Mar 28.
7
An integrated disposable device for DNA extraction and helicase dependent amplification.
Biomed Microdevices. 2010 Apr;12(2):353-9. doi: 10.1007/s10544-009-9391-8.
本文引用的文献
1
Rapid lab-on-a-chip profiling of human gut bacteria.
J Microbiol Methods. 2008 Jan;72(1):82-90. doi: 10.1016/j.mimet.2007.10.011. Epub 2007 Nov 4.
2
Thermoplastic microfluidic device for on-chip purification of nucleic acids for disposable diagnostics.
Anal Chem. 2006 Feb 1;78(3):788-92. doi: 10.1021/ac051449j.
3
Toxin production by an emerging strain of Clostridium difficile associated with outbreaks of severe disease in North America and Europe.
Lancet. 2005;366(9491):1079-84. doi: 10.1016/S0140-6736(05)67420-X.
4
Environmental microbiology-on-a-chip and its future impacts.
Trends Biotechnol. 2005 Apr;23(4):174-9. doi: 10.1016/j.tibtech.2005.02.004.
5
Quantification of intestinal bacterial populations by real-time PCR with a universal primer set and minor groove binder probes: a global approach to the enteric flora.
J Clin Microbiol. 2004 Jun;42(6):2566-72. doi: 10.1128/JCM.42.6.2566-2572.2004.
6
Rapid detection of Clostridium difficile in feces by real-time PCR.
J Clin Microbiol. 2003 Feb;41(2):730-4. doi: 10.1128/JCM.41.2.730-734.2003.
7
Development and evaluation of a PCR method for detection of the Clostridium difficile toxin B gene in stool specimens.
J Clin Microbiol. 2002 Jun;40(6):2288-90. doi: 10.1128/JCM.40.6.2288-2290.2002.
8
Clinical practice. Antibiotic-associated diarrhea.
N Engl J Med. 2002 Jan 31;346(5):334-9. doi: 10.1056/NEJMcp011603.
9
Porous polymer monoliths: simple and efficient mixers prepared by direct polymerization in the channels of microfluidic chips.
Electrophoresis. 2001 Oct;22(18):3959-67. doi: 10.1002/1522-2683(200110)22:18<3959::AID-ELPS3959>3.0.CO;2-5.
10
Comparison of PCR and microscopy for detection of Cryptosporidium parvum in human fecal specimens: clinical trial.
J Clin Microbiol. 1998 Apr;36(4):995-8. doi: 10.1128/JCM.36.4.995-998.1998.
Zotero 插件