Stephen Bustin是Anglia Ruskin大學(xué)分子醫(yī)學(xué)專業(yè)的教授和MIQE（實(shí)時(shí)熒光定量PCR實(shí)驗(yàn)流程及數(shù)據(jù)處理的國際化標(biāo)準(zhǔn)及語言規(guī)則）的起草人。他的團(tuán)隊(duì)將使用快速而又可靠的ECO48熒光定量PCR儀來處理一系列實(shí)驗(yàn)。
PCRmax:在診斷中，使用qPCR時(shí)主要有什么挑戰(zhàn)？
Prof Bustin:在過去的15年里，qPCR已經(jīng)貫穿了臨床診斷的一個(gè)很普通的技術(shù)。隨著qPCR儀器的技術(shù)進(jìn)步，使用者已經(jīng)不需要處理大量的原始數(shù)據(jù)，但是這會使實(shí)驗(yàn)人員難于驗(yàn)證實(shí)驗(yàn)的可靠性與真實(shí)有效性。因此，在樣品分析之前，全面的質(zhì)量評價(jià)測定成為了關(guān)鍵，我們需要保證整個(gè)實(shí)驗(yàn)流程是符合MIQE規(guī)范的，同時(shí)也要快速而有效的。
PCRmax：你能舉一個(gè)你最新研究的例子并講一下你在研究的過程中所遇到的難點(diǎn)以及是怎樣克服它的嗎？
Prof Bustin:我們不斷開展了一些關(guān)于潛在患病者細(xì)胞的mRNA分子標(biāo)記物的定量實(shí)驗(yàn)，這有時(shí)還包括了一些由傳統(tǒng)的終點(diǎn)檢測PCR轉(zhuǎn)化而來的熒光定量PCR實(shí)驗(yàn)。而且我認(rèn)為，在處理大范圍龐大的不同特征的mRNA時(shí)，不應(yīng)該再使用傳統(tǒng)的PCR實(shí)驗(yàn)手段，因其之后的電泳檢測還存在著交叉污染的風(fēng)險(xiǎn)。
PCRmax:對于你的研究來說，熒光定量PCR儀有多重要，特別是Eco 48？
Prof Bustin:實(shí)驗(yàn)速度的加快使得ECO48成為我們研究的關(guān)鍵設(shè)備。舉個(gè)例子，最近我們正在進(jìn)行一個(gè)在乳腺癌患者的臨床手術(shù)中應(yīng)用qPCR檢測的前沿性研究，主要是對患者淋巴液的檢測。當(dāng)我們獲得患者的前哨淋巴結(jié)后，立即會在實(shí)驗(yàn)室中提取RNA。然后進(jìn)行RT-qPCR實(shí)驗(yàn)，我們就可以了解上皮細(xì)胞mRNA轉(zhuǎn)移的標(biāo)記了。
Eco 48通過快速而又精確的檢測引領(lǐng)了這次業(yè)界的技術(shù)進(jìn)步。革命性的熱塊技術(shù)改進(jìn)使得整個(gè)實(shí)驗(yàn)可以在40min中完成，同時(shí)孔間的溫度均一性還可以達(dá)到0.1℃，這使得每一次溫度變化后的實(shí)驗(yàn)誤差不至于變大。同時(shí)，高性能的光學(xué)系統(tǒng)與易于使用的軟件結(jié)合，提高了Eco 48對所有qPCR實(shí)驗(yàn)的分析效率。
PCRmax是比比科技旗下的公司，專門從事為生命科技實(shí)驗(yàn)室提供實(shí)時(shí)儀器，耗材和試劑。廣州語特儀器科技有限公司是其中國大南方區(qū)的總代。
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.
