Technology for Automated, Rapid, and Quantitative PCR or Reverse Transcription-PCR Clinical Testing

Abstract

Background: PCR-based assays can improve clinical care, but they remain technically demanding and labor-intensive. We describe a new instrument, the GeneXpert ® , that performs automated nucleic acid isolation, reverse transcription, and fluorescence-based quantitative PCR in ϳ35 min. Methods: Yield and integrity of RNA isolated on the GeneXpert were compared with Qiagen-based extraction for parallel samples (5-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 ϳ70% 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 ⌬Ct for a new measurement was ؎1.54 cycles, and for breast cancer samples, the interval for a newly observed ⌬Ct was ؎1.40 cycles.

Evaluation of the Cepheid GeneXpert BCR-ABL Assay

“…This selfcontained automated instrument integrates microfluidic sample preparation with RT-PCR-based, real-time fluorescent signal detection. 13 Virtually all of the reagents used for both the RNA preparation and the subsequent RT-PCR and PCR steps are lyophilized into a disposable multichambered cartridge, potentially decreasing both reagent-and pipette-related bias. The sample is transferred through the chambers during the extraction process by the pumping action of a central piston; subsequently, the purified RNA reaches the detection chamber where the reverse transcription, the multiplexed amplification steps, and the fluorescent signal detection take place.…”

Biomarker selection for detection of occult tumour cells in lymph nodes of colorectal cancer patients using real-time quantitative RT–PCR

“…It can now be concluded that real-time qRT -PCR assay for biomarker mRNA, particularly when used with a specific probe and RNA copy standard, is a superior method for micrometastases detection because it is objective, highly sensitive and quantitative, and has a very wide measuring range (Godfrey et al, 2001;Ho et al, 2004;Ö berg et al, 2004). Moreover, this technology lends itself to automation and rapid test performance suitable for a clinical setting (Raja et al, 2005).…”

Evaluation of rapid GeneXpert MTB/RIF method using DNA tissue specimens of vertebral bones in patients with suspected spondylitis TB

“…The system will be running on the GeneXpert platform (Cepheid, Sunnyvale, CA) by using disposable cartridge that contains a previously-determined reagent. 13 GeneXpert provided as a simple method that works automatically, take two hours, and is not require an expert to perform this examination. Several reports described the success of GeneXpert on detecting every pulmonary TB cases among all suspects, including more than 90% of suspects with negative results in acid fast staining examination.…”