2.13-2.15收获：rep-pcr

细菌耐药

这周本来想去Molecular diagnosis lab去看一看的。结果那边的实验室说让我等一等。在等的过程中，我到处看看，跟着老师做了一次rep-PCR。一块小小的芯片，16孔，要加一些试剂和对照，每次实验可以做10个样品，经过扩增后，上机1小时就可以出结果了。我也做过PFGE，相对而言，要比PFGE的操作简单不少。

我等会接着查一下相关的文献，大家有做过这个实验的吗？可以一起讨论一下。

樵夫

去年做过一些，rep-PCR的速度确实很快，灵敏度也很高，而且有专用的软件分析结果，可以跟以前的结果进行直接比对，但是感觉提纯DNA比较麻烦一点，特别是阳性球菌，如MRSA，经常达不到要求的浓度，对推荐的提纯方法进行了改良，效果还是不明显。
可以详细介绍一下他们的提取方法不？

蓝鱼o_0

你下次来可以跟我们这的小谭讨论。他技术能力蛮强的。尤其体现在遗传鉴定(PCR-BASED GENOTYPING)
我经常去讨教。

细菌耐药

樵夫 发表于 2012-2-16 12:54

                               
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去年做过一些，rep-PCR的速度确实很快，灵敏度也很高，而且有专用的软件分析结果，可以跟以前的结果进行直接 ...

UCLA这边就是用DNA提取试剂盒来提取的，不过有一点我觉得可能是比较重要的。在扩增前对所有标本进行DNA浓度的。再根据浓度的不同来设计扩增时的DNA量的。

细菌耐药

蓝鱼o_0 发表于 2012-2-16 13:02

                               
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你下次来可以跟我们这的小谭讨论。他技术能力蛮强的。尤其体现在遗传鉴定(PCR-BASED GENOTYPING)
我经常去 ...

OK，回国后一定当面去请教，谢谢。

细菌耐药

Braz J Infect Dis. 2003 Feb;7(1):32-43. Epub 2003 Dec 2.
Molecular techniques for MRSA typing: current issues and perspectives.
Trindade PA, McCulloch JA, Oliveira GA, Mamizuka EM.
SourcePharmaceutical Science School, São Paulo University, São Paulo, Brazil.

Abstract
Staphylococcus aureus has long been recognised as an important pathogen in human disease. Serious staphylococcal infections can frequently occur in inpatients and may lead to dire consequences, especially for therapy with antimicrobial agents. The increase in the frequency of Methicillin-Resistant Staphylococcus aureus (MRSA) as the causal agent of nosocomial infection and the possibility of emergence of resistance to vancomycin demands a quick and trustworthy characterization of isolates and identification of clonal spread within hospitals. Enough information must be generated to permit the implementation of appropriate measures for control of infection, so that outbreaks can be contained. Molecular typing techniques reviewed in this manuscript include: plasmid profile analysis, analysis of chromosomal DNA after enzymatic restriction, Southern blotting, pulsed field gel electrophoresis (PFGE), techniques involving polymerase chain reaction and multilocus sequence typing (MLST). Repetitive DNA Sequence PCR (rep-PCR) may be used for screening due to its practicality, low cost and reproducibility. Because of its high discriminatory power Pulsed-Field Gel Electrophoresis (PFGE) still remains the gold standard for MRSA typing. New techniques with higher reproducibility and discriminatory power, such as Multi-Locus Sequence Typing (MLST), are appearing. These are mostly useful for global epidemiology studies. Molecular typing techniques are invaluable tools for the assessment of putative MRSA outbreaks and so should be extensively used for this purpose.

细菌耐药

Antimicrob Agents Chemother. 2011 Apr;55(4):1701-5. Epub 2011 Jan 24.
Antimicrobial susceptibilities and molecular epidemiology of clinical isolates of Clostridium difficile in taiwan.
Lin YC, Huang YT, Tsai PJ, Lee TF, Lee NY, Liao CH, Lin SY, Ko WC, Hsueh PR.
SourceDepartment of Internal Medicine, Taipei Medical University Hospital, Taipei, Taiwan.

Abstract
The antimicrobial susceptibility and virulence factors of Clostridium difficile clinical isolates in Taiwan have not previously been reported. One hundred and thirteen isolates were collected from two major teaching hospitals in Taiwan from 2001 to 2009. Molecular typing was performed by an automated repetitive extragenic palindromic sequence-based PCR (rep-PCR) method (DiversiLab; Bacterial Barcodes, Inc., Athens, GA) and PCR ribotyping. Detection of tcdA, tcdB, cdtA, and cdtB genes was performed using a multiplex PCR assay, and gyrA and gyrB genes of moxifloxacin-nonsusceptible isolates were sequenced. All isolates were susceptible to vancomycin and metronidazole. Ninety-five (84%) isolates were susceptible to moxifloxacin, and the MIC(90) for nemonoxacin was 4 μg/ml. Tigecycline showed favorable antibacterial activity (MIC(90) of 0.06 μg/ml). Thirteen rep-PCR types were identified as a predominant rep-PCR type (type A; non-North American pulsed-field gel electrophoresis type 1 [NAP1], -NAP7, or -NAP8) accounting for 52.2% (59 isolates). Nine of 18 moxifloxacin-nonsusceptible isolates belonged to the rep-PCR type A. The rep-PCR type A and C isolates were distinct from NAP1 (ribotype 027) and NAP8 (ribotype 078) as determined by PCR ribotyping. Seventy-four (65%) isolates harbored tcdA and tcdB, and 15 (13%) harbored cdtAB encoding binary toxin. Eleven isolates had a gene deletion in tcdC, including a 39-bp deletion (9 isolates) and an 18-bp deletion (2). In conclusion, dissemination of a predominant C. difficile clone in southern and northern Taiwan was noted. However, no NAP1 (ribotype 027) isolate could be discovered in this study.