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数字PCR技术先驱选择Bio-Rad公司的QX100微滴式数字PCR系统开发慢性粒细胞白血病的诊断方法

  1. Bio-Rad公司的QX100
  2. 数字PCR技术

来源:BIO-RAD 2013-05-31 16:57

近日,数字PCR技术的先驱Alec Morley教授选择了Bio-Rad公司的QX100微滴式数字PCR系统,用以开发慢性粒细胞白血病(CML)的诊断方法。 早在1992年,澳大利亚弗林德斯大学的Morley教授及其团队首次提出了一种称为"有限稀释PCR"法的核酸靶分子定量检测手段。为验证这一技术理念,他们运用该方法成功地对急性白血病中的标记基因突变进行了定量检测。

 

近日,数字PCR技术的先驱Alec Morley教授选择了Bio-Rad公司的QX100微滴式数字PCR系统,用以开发慢性粒细胞白血病(CML)的诊断方法。

早在1992年,澳大利亚弗林德斯大学的Morley教授及其团队首次提出了一种称为"有限稀释PCR"法的核酸靶分子定量检测手段。为验证这一技术理念,他们运用该方法成功地对急性白血病中的标记基因突变进行了定量检测。其基本思路是通过对DNA样品进行适度稀释,再等量分散到若干个独立的反应孔,使得每个反应孔中只存在一个或两个拷贝靶标分子,随后对这些分子进行PCR扩增。以这样的方式,Morley的研究小组能从160,000个正常基因组背景中检测出2个拷贝的白血病DNA。

随后他们在《柳叶刀》杂志上发表文章,阐述了利用有限稀释PCR可高灵敏度地定量分析白血病细胞,从而评估疗效,进而预测白血病的预后。在之后的工作中,Morley的实验室尝试利用实时定量PCR来开发一种高灵敏的方法,可分离并定量检测典型的与CML相关的染色体易位。

因为每位患者的易位点不同,可能会使用患者各异的引物,所以实时定量PCR条件也可能有差异,从而产生不同的检查结果。如今,Morley教授的实验室又回到了数字PCR。Morley教授认为:"数字PCR的进步让我们能够克服定量PCR扩增效率的差异,也让我们摒弃了标准曲线。"

2002年,弗林德斯大学和Morley团队的研究人员共同组建了Monoquant公司,以推进科研成果的商业化进程。Monoquant公司最近使用Bio-Rad的QX100系统来建立和完善CML新的临床诊断技术。QX100微滴式数字PCR平台不仅实现极高的检测灵敏度,还避免了使用患者特异的PCR引物所带来的扩增效率差异,而这正是向FDA提交申请时的障碍之一。Monoquant希望QX100微滴式数字PCR仪获得的结果能有助于FDA的快速审批。

"得知我们曾参与创建的技术如今正在推动我们目前的工作,这感觉真是太棒了!" Morley教授表示。

 

 

Monoquant researchers are developing a more sensitive, reliable test for chronic myeloid leukemia using the QX100? Droplet Digital? PCR System.

Hercules, CA - January 4, 2013 - Professor Alec Morley, a pioneer in digital PCR, has chosen Bio-Rad Laboratories' QX100 Droplet Digital PCR system to develop a diagnostic test for chronic myeloid leukemia (CML).

In 1992, Prof. Morley and his lab at Flinders University and Medical Center in Adelaide, South Australia, published a general method called "limiting dilution PCR" for quantifying PCR targets. As a proof of concept, they used this method for the quantification of marker mutations in acute leukemia. By diluting DNA samples so that only one or two copies per well were present and then amplifying those copies with PCR, Morley's team was able to detect two copies of leukemic DNA against a background of 160,000 normal genomes.

They subsequently reported in The Lancet that the outcome of acute leukemia can be predicted by measuring the response to treatment using limiting dilution PCR to quantify the leukemic cells at high sensitivity. In later work, the Morley Lab used real-time quantitative PCR (qPCR) to develop a highly sensitive method for isolating and quantifying the chromosomal translocation that is typically associated with CML.

Using droplet digital PCR to diagnose leukemia

Because the translocation point for each patient is different in CML, real-time PCR conditions may vary from patient to patient and may therefore produce different results. The lab has now returned to digital PCR.

"Advancements in digital PCR have given us the ability to overcome variations in real-time PCR amplification efficiency and have also enabled us to do away with using a standard curve," Prof. Morley

said.

Monoquant, a company associated with Flinders University, recently used Bio-Rad's QX100 system to refine the new clinical test for CML. Not only does the instrument offer high sensitivity, it also removes variability in amplificationefficiency that results from using patient-specific PCR primers, a traditional sticking point for the FDA. Monoquant hopes the results from the QX100 system will fast-track the FDA approval process for its test.

"It's a great feeling knowing that something we helped create is propelling our work today," Prof. Morley said. "We are hoping that this new test we're developing will offer a better degree of monitoring and better disease management for patients by tracking the progression or remission of CML."

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