專家介紹：

Stephen Bustin是Anglia Ruskin大學分子醫學專業的教授和MIQE（實時熒光定量PCR實驗流程及數據處理的國際化標準及語言規則）的起草人。他的團隊將使用快速而又可靠的ECO48熒光定量PCR儀來處理一系列實驗。

PCRmax:在診斷中，使用qPCR時主要有什么挑戰？

Prof Bustin:在過去的15年里，qPCR已經貫穿了臨床診斷的一個很普通的技術。隨著qPCR儀器的技術進步，使用者已經不需要處理大量的原始數據，但是這會使實驗人員難于驗證實驗的可靠性與真實有效性。因此，在樣品分析之前，全面的質量評價測定成為了關鍵，我們需要保證整個實驗流程是符合MIQE規范的，同時也要快速而有效的。

PCRmax：你能舉一個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以及是怎樣克服它的嗎？

Prof Bustin:我們不斷開展了一些關于潛在患病者細胞的mRNA分子標記物的定量實驗，這有時還包括了一些由傳統的終點檢測PCR轉化而來的熒光定量PCR實驗。而且我認為，在處理大范圍龐大的不同特征的mRNA時，不應該再使用傳統的PCR實驗手段，因其之后的電泳檢測還存在著交叉污染的風險。

 

Eco 48是一個理想的實驗發展和優化的系統，因為它的運行速度相當的快速而且不受數據量的影響。這臺設備有四個熒光檢測通道，可以同時處理四個不同標記的探針，并且它可以在40min內完成一個40個循環的實驗（快速模式下可以達到16min），且實驗符合MIQE規范。此外，對我們來說這個軟件是很容易操作的，且為開展的工作減少了時間和所需的資源。

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:實驗速度的加快使得ECO48成為我們研究的關鍵設備。舉個例子，最近我們正在進行一個在乳腺癌患者的臨床手術中應用qPCR檢測的前沿性研究，主要是對患者淋巴液的檢測。當我們獲得患者的前哨淋巴結后，立即會在實驗室中提取RNA。然后進行RT-qPCR實驗，我們就可以了解上皮細胞mRNA轉移的標記了。

 

這項能力應用相當重要，當它應用在手術中時，我們可以通過快速的實驗得到已經病變的組織細胞的判斷，從而我們可以在一次手術中完成病灶的切除，節省了手術的時間和多次手術的風險，大大的減少了病人的外傷和治療的費用。使用這臺儀器的一個明顯的好處就是Eco 48熒光定量PCR儀完成這個試驗的時間，從抽出物質到分析的時間已經減少到僅僅只有29分鐘了。

 

Eco 48通過快速而又精確的檢測引領了這次業界的技術進步。革命性的熱塊技術改進使得整個實驗可以在40min中完成，同時孔間的溫度均一性還可以達到0.1℃，這使得每一次溫度變化后的實驗誤差不至于變大。同時，高性能的光學系統與易于使用的軟件結合，提高了Eco 48對所有qPCR實驗的分析效率。

PCRmax是比比科技旗下的公司，專門從事為生命科技實驗室提供實時儀器，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.
 

 

 

廣州語特儀器科技有限公司專注于攪拌器/分散乳化機等實驗室樣品制備等通用儀器， 熔點儀/光度計/冰點儀等分析儀器，以及PCR等生命科學儀器。 作為英國比比（Bibby ）在中國南方的首代，廣東，廣西，四川，重慶，云南，海南，貴州和西藏是我司的服務范圍。語特公司也是德國ART， 德國CAT，瑞士Gerber Instruments 在中國的首代。

 

●英國BIBBY 成立于上個世紀50年代，作為英國最大的實驗室科學儀器生產商，  旗下有4個子品牌：Stuart，Techne，Jenway，Electrothermal. 專注于樣品前處理等通用實驗室儀器（如：熔點儀, 攪拌器, 混勻器，搖床, 培養箱，干浴器/氮吹儀，水浴，菌落計數器, 純水蒸餾器），分子生物學研究設備(基因擴增儀PCR，熒光定量，雜交箱)；分光光度計/超微量紫外等分析儀器，及平行反應工作站相關產品。    

   

●德國ART 成立于上個世紀，是德國乃至全球最專業的分散乳化專家。頂級分散乳化產品從實驗室儀器，中試產品到工業設備， 分散頭種類組合高達上百種；應用領域覆蓋了化工，化妝品，制藥，食品，環保等各大領域。

 

●德國CAT 成立于上個世紀50年代，是德國樣品制備儀器方面的專家之一, 以”品質穩定”而聞名。其頂置式攪拌器種類多樣，從手持式，教學用，到科研通用型，高粘度型，是CAT的代表產品線。

 

●瑞士Gerber Instruments 有超過120的歷史，是專注于乳食品行業的典型代表。其產品冰點儀, 乳脂離心機, 食品專用PH計, 流出式粘度計等, 風靡歐洲及其它大陸國家。