随着技术的进步，PCR技术因其易用性已经在诊断上大量应用了，但是要想完成一个传递可靠、重现性好以及结果有意义的实验还是一个具挑战的。这里，我们很荣幸的邀请了英国著名的PCR专家Stephen Bustin教授，他试用了来自PCRmax公司的ECO48荧光定量PCR仪去检测快速繁殖的高致病性的细菌与真菌。以下为Stephen Bustin教授使用ECO48后与PCRmax公司之间的访谈记录

专家介绍：

Stephen Bustin是Anglia Ruskin大学分子医学专业的教授和MIQE（实时荧光定量PCR实验流程及数据处理的国际化标准及语言规则）的起草人。他的团队将使用快速而又可靠的ECO48荧光定量PCR仪来处理一系列实验。

PCRmax:在诊断中，使用qPCR时主要有什么挑战？

Prof Bustin:：在过去的15年里，qPCR已经贯穿了临床诊断的一个很普通的技术。随着qPCR仪器的技术进步，使用者已经不需要处理大量的原始数据，但是这会使实验人员难于验证实验的可靠性与真实有效性。因此，在样品分析之前，全面的质量评价测定成为了关键，我们需要保证整个实验流程是符合MIQE规范的，同时也要快速而有效的。

PCRmax：你能举一个你最新研究的例子并讲一下你在研究的过程中所遇到的难点以及是怎样克服它的吗？

Prof Bustin:我们不断开展了一些关于潜在患病者细胞的mRNA分子标记物的定量实验，这有时还包括了一些由传统的终点检测PCR转化而来的荧光定量PCR实验。而且我认为，在处理大范围庞大的不同特征的mRNA时，不应该再使用传统的PCR实验手段，因其之后的电泳检测还存在着交叉污染的风险

Eco 48是一个理想的实验发展和优化的系统，因为它的运行速度相当的快速而且不受数据量的影响。这台设备有四个荧光检测通道，可以同时处理四个不同标记的探针，并且它可以在40min内完成一个40个循环的实验（快速模式下可以达到16min），且实验符合MIQE规范。此外，对我们来说这个软件是很容易操作的，且为开展的工作减少了时间和所需的资源。

PCRmax:对于你的研究来说，荧光定量PCR仪有多重要，特别是Eco 48？

Prof Bustin:实验速度的加快使得ECO48成为我们研究的关键设备。举个例子，最近我们正在进行一个在乳腺癌患者的临床手术中应用qPCR检测的前沿性研究，主要是对患者淋巴液的检测。当我们获得患者的前哨淋巴结后，立即会在实验室中提取RNA。然后进行RT-qPCR实验，我们就可以了解上皮细胞mRNA转移的标记了。

这项能力应用相当重要，当它应用在手术中时，我们可以通过快速的实验得到已经病变的组织细胞的判断，从而我们可以在一次手术中完成病灶的切除，节省了手术的时间和多次手术的风险，大大的减少了病人的外伤和治疗的费用。使用这台仪器的一个明显的好处就是Eco 48荧光定量PCR仪完成这个试验的时间，从抽出物质到分析的时间已经减少到 仅仅只有29分钟了。

研究后的创新

Eco 48通过快速而又精确的检测引领了这次业界的技术进步。革命性的热块技术改进使得整个实验可以在40min中完成，同时孔间的温度均一性还可以达到0.1℃，这使得每一次温度变化后的实验误差不至于变大。同时，高性能的光学系统与易于使用的软件结合，提高了Eco 48对所有qPCR实验的分析效率。

PCRmax是比比科技旗下的公司，专门从事为生命科技实验室提供实时仪器，耗材和试剂。广州语特仪器科技有限公司是其中国大南方区的总代。

40 cycles in 40 minutes
Rapid Eco 48 Thermal Cycler critical to qPCR expert Professor Stephen Bustin's assay development

Despite substantial advances in the accessibility and ease-of-use of PCR for diagnostics, generating an assay that delivers reliable, reproducible and meaningful results is still a challenging task. 
Here,eminent PCR expert Professor Stephen Bustin examines how the Eco 48 real-time quantitative PCR (qPCR) Thermal Cycler from PCRmax allows the rapid development of robust bacterial and fungal assays.

Introducing the Assay Expert.

Stephen Bustin is Professor of Molecular Medicine, Anglia  Ruskin  University and lead-author of the "Minimum Information for Publication of Quantitative real-time PCR Experiments" (MIQE) guidelines. His team relies on the speed and precision of the Eco 48 for a range of assay applications, from quality assurance through to intra-operative diagnostics.

PCRmax: What are the major challenges for users of qPCR in diagnostics?

Prof Bustin: Over the past 15 years qPCR has become a commonplace technique throughout clinical diagnostics. As qPCR instrumentation has advanced, however, users have become more distanced from their raw data, making it harder to assess the true efficiency and reliability of the technique. Robust quality assessment (QA) of an assay is therefore critical before sample analysis. Since assessment and validation are intensive processes, the practical challenge of qPCR is to ensure the procedure is fast and cost effective while complying with the MIQE framework.

PCRmax: Can you give us an example of your current research and how you have overcome some of the challenges you just highlighted?

Prof Bustin: We are constantly developing new assays for the quantification of potential cellular mRNA biomarkers as well as the diagnosis of fungal and bacterial pathogens; this sometimes involves the conversion of less reliable conventional endpoint PCR assays to the qPCR format. I believe that, where possible, such legacy assays should no longer be used as they are poor at detecting the large expression range differences characteristic of cellular mRNA, while the need to run a gel means there is always the potential for contamination

The Eco 48 package is an ideal system for assay development and optimisation because it performs runs so quickly without any impact on data quality. The machine has four channels for multiplex development which enables us to run process 40 cycles in around 16 minutes, with performance aligned to the MIQE guidelines. Moreover the operating software is easy to use, reducing the time and resources required for developmental work.

PCRmax: How important has qPCR instrumentation, and particularly the Eco 48, been to your research?

Prof Bustin: The importance of speed in assay development has made the Eco 48 a key tool throughout our research. For example our team has recently worked on a project aimed at developing a qPCR-based test for the intra-operative assessment of lymph nodes in patients with breast cancer. Here, the sentinel lymph node is removed from the patient whilst in theatre and rushed to the lab for RNA extraction. A reverse transcription (RT)-qPCR reaction is then performed to screen for epithelial cell mRNA markers indicative of metastasis.

The ability to perform inter-operative diagnosis is extremely valuable because it cuts time in the operating theatre and can avoid the need for an additional operation, greatly reducing patient trauma as well as the cost of treatment. With the Eco 48 Thermal Cycler the time taken to perform the entire assay from extraction to analysis has been reduced to just 29 minutes, a clear benefit of employing this instrument over alternative techniques.

The innovations behind the research

The Eco 48 delivers this industry leading speed and precision through a variety of advanced features. Innovative block technology achieves complete heating uniformity and rapid cycles in under 40 minutes, with the sensitivity to deliver ±0.1oC uniformity across the whole block instantly after every temperature change for precise amplification. Combined with a high-performance optical system and easy-to-use software, the Eco 48 improves analytical productivity for practically all qPCR applications. 

PCRmax is a Bibby Scientific company specialising in providing real time instrumentation, consumables and reagents for the life science laboratory.