A system for rapid point-of-use nucleic acid (NA) analysis based on PCR techniques is described. The extraction and concentration of DNA from test samples has been accomplished utilizing silicon fluidic microchips with high surface-area-to-volume ratios. Short (500 bp) and medium size (48,000 bp) DNA have been captured, washed, and eluted using the silicon dioxide surfaces of these chips. Chaotropic (GuHCl) salt solutions were used as binding agents. Wash and elution agents consisted of ethanol-based solutions and water, respectively. DNA quantities approaching 40 ng/cm2 of binding area were captured from input solutions in the 100-1000 ng/mL concentration range. For dilute samples of interest for pathogen detection, PCR and gel electrophoresis were used to demonstrate extraction efficiencies of about 50 percent, and concentration factors of about 10x using bacteriophage lambda DNA as the target. Rapid, multichannel PCR thermal cycling modules with integrated solid-state detection components have also been demonstrated. These results confirm the viability of utilizing these components as elements of a compact, disposable cartridge system for the detection of NA in applications such as clinical diagnostics, biowarfare agent detection, food quality control, and environmental monitoring.
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.
A rapid, fully automated QRT-PCR assay definitively characterizes breast cancer SLN with accuracy equal to conventional pathology. This approach is superior to intraoperative SLN analysis and can provide standardized, objective results to assist in pathologic diagnosis.
Thorough groin RLNDs have a predictable LN yield. LN ratio is the strongest predictor of outcome. Because pelvic LNs are frequently positive ilioinguinal dissection should be considered for all patients, especially those with macroscopic metastases to groin LNs.
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