Technology for automated, rapid, and quantitative PCR or reverse transcription-PCR clinical testing
Technology for automated, rapid, and quantitative PCR or reverse transcription-PCR clinical testing
复制标题
DOI：
10.1373/clinchem.2004.046474
复制
发表时间：
2005-05-01
影响因子：
9.3
通讯作者：
Godfrey, TE
中科院分区：
文献类型：
--
作者：
Raja, S;Ching, J;Godfrey, TE
背景：基于PCR的检测可以改善临床护理，但它们仍然是技术要求高和劳动密集型的。我们描述了一种新的仪器，GeneXpert（R），进行自动化核酸分离，逆转录和荧光定量PCR在类似的35分钟。方法：产量和完整性的RNA分离的GeneXpert进行了比较与Qiagen为基础的提取平行样品（5 μ m冷冻组织切片）。通过对10种组织进行重复（n = 10）分析，使用两种基因组合的双链（靶和内源性对照）逆转录-PCR反应，确定自动化RNA分离、逆转录和定量PCR的重现性。然后使用GeneXpert对黑色素瘤、乳腺癌和肺癌患者的淋巴结进行快速分析，并对转移到肺的黑色素瘤、原发性肺腺癌和健康肺组织进行分析。通过凝胶电泳测定，RNA完整性与Qiagen分离的RNA相当。对于黑色素瘤样本，新测量的Δ Ct的95%预测区间为+/- 1.54个周期，对于乳腺癌样本，新观察到的Δ Ct的区间为+/- 1.40个周期。GeneXpert成功检测了淋巴结中转移性黑色素瘤、乳腺癌和肺癌的存在，并区分了转移性黑色素瘤、肺腺癌和健康肺。结论：GeneXpert的RNA产量和完整性与台式方法相当。GeneXpert数据的重现性与我们手中的手动方法相似，但在某些应用中可能需要改进。GeneXpert可以在大约35分钟内进行RNA分离、逆转录和定量PCR，因此可以在适用时用于术中检测。(c)2005年美国临床化学协会。
Background: PCR-based assays can improve clinical care, but they remain technically demanding and labor-intensive. We describe a new instrument, the GeneXpert (R), that performs automated nucleic acid isolation, reverse transcription, and fluorescence-based quantitative PCR in similar to 35 min.Methods: Yield and integrity of RNA isolated on the GeneXpert were compared with Qiagen-based extraction for parallel samples (5-mu m frozen tissue sections). The reproducibility of automated RNA isolation, reverse transcription, and quantitative PCR was determined by replicate (n = 10) analysis of 10 tissues, using duplex (target and endogenous control) reverse transcription-PCR reactions for two gene combinations. The GeneXpert was then used to perform rapid analysis of lymph nodes from melanoma, breast cancer, and lung cancer patients and analysis of melanoma metastatic to the lung, primary lung adenocarcinoma, and healthy lung tissue.Results: On the GeneXpert, RNA was recovered in slightly over 6 min, and the yield was similar to 70% of that from parallel Qiagen reactions. The RNA integrity was comparable to that of Qiagen-isolated RNA as determined by gel electrophoresis. For the melanoma samples, the 95% prediction interval for the Delta Ct for a new measurement was +/- 1.54 cycles, and for breast cancer samples, the interval for a newly observed Delta Ct was +/- 1.40 cycles. GeneXpert assays successfully detected the presence of metastatic melanoma, breast cancer, and lung cancer in lymph nodes and also differentiated among metastatic melanoma, lung adenocarcinoma, and healthy lung.Conclusions: RNA yield and integrity on the GeneXpert are comparable to benchtop methods. Reproducibility of the GeneXpert data is similar to that seen with manual methods in our hands but may need improvement for some applications. The GeneXpert can perform RNA isolation, reverse transcription, and quantitative PCR in similar to 35 min and could therefore be used for intraoperative testing when applicable. (c) 2005 American Association for Clinical Chemistry.