Hot Cell-Direct PCR Aimed at Specific Cell Detection

Abstract

Since the polymerase chain reaction (PCR) was proposed, it has become an essential method in the field of biological gene analysis, providing a method to amplify DNA sequences of interest. To detect and/or analyze genes in cells, the gene or expressed gene must first be extracted before PCR. This procedure takes time and may result in the loss of samples. In order to avoid such drawbacks, two methods, hot cell-direct PCR and reverse transcription-PCR (RT-PCR), were invented, to detect genes in cells. Using hot cell-direct PCR, specific genes in microbial cells such as invA in Salmonella enterica have been easily detected and applied to discriminate Archaea from bacteria. As hot celldirect PCR and RT-PCR are fairly simple processes, they can be applied to detect genes in single cells. We developed an original compact disc (CD)-shaped microfluidic device with microchambers for single-cell isolation and a detection system for expressed genes in isolated single cells in a microchamber on the device. We succeeded in the detection of PCR and RT-PCR products in individual cells and successfully detected S. enterica cells by hot cell-direct PCR. Expressed genes in Jurkat cells-human leukemia T cells-were analyzed by this method.

Detection of Salmonella Enterica in Egg Yolk by PCR on a Microfluidic Disc Device Using Immunomagnetic Beads

“…After cells are entrapped in the microchambers, cells are lysed in the chambers by heat treatment at 95 • C. Without taking out cells or genes from the cells after lysis, PCR of a specific gene of the entrapped cells can be performed on the device. This process is referred to as hot cell-direct PCR [17]. Using a fluorescent probe for the PCR product, a PCR amplicon can be detected under an epifluorescent microscope [18].…”