In Vitro System for the Detection of Prostate Cancer Markers via Loop-Mediated Isothermal Amplification
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In Vitro System for the Detection of Prostate Cancer Markers via Loop-Mediated Isothermal Amplification K. A. Fomichevaa, * and J. A. Makarovaa, b, c, ** a
Hertsen Moscow Oncology Research Institute, National Center of Medical Radiological Research, Moscow, 125284 Russia bEngelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991 Russia c Faculty of Biology and Biotechnologies, Higher School of Economics National Research University, Moscow, 101000 Russia *e-mail: [email protected] **e-mail: [email protected] Received November 15, 2019; revised December 3, 2019; accepted February 5, 2020
Abstract—Loop-mediated isothermal amplification (LAMP) of nucleic acids enables the detection of amplification products 10–20 min after the beginning of the reaction. An intraoperative method for the detection of metastases in lymph nodes based on LAMP, one-step nucleic acid amplification (OSNA), allows the rapid detection of the epithelial-specific marker gene KRT19 in lymph nodes. The standard protocol for OSNA was developed more than 10 years ago to detect breast cancer metastases in lymph nodes. Since then, a new version of the key enzyme involved in the reaction (Bst-polymerase) was obtained, but its use in OSNA has remained unexplored. Moreover, the time has come to apply OSNA to the detection of other cancer types, in particular, prostate cancer. The first step is to create an in vitro system that allows LAMP to be carried out on prostate-cancer cell lines. In this work, the LAMP protocol was developed for the new Bst 3.0 polymerase with optimized dNTP concentrations and without reverse transcriptase, and cell lines were selected (DU-145 for prostate cancer and Molt-4 for negative control). As a result, a new, in vitro system for the LAMP-based detection of prostate cancer with the marker gene KRT19 was developed. Keywords: LAMP, OSNA, prostate cancer DOI: 10.1134/S0003683820090057
INTRODUCTION Prostate cancer (PC) is one of the most widespread malignant neoplasms in men. The presence or absence of metastases in the pelvic lymph nodes (LNs) is an important prognostic factor of the disease and determines the treatment strategy [1–3]. At present, there is an intraoperative method that was developed for the detection of metastases in LNs based on a rapid assay of mRNA for cytokeratin-19, which is encoded by the epithelium-specific KRT19 gene via one-step nucleic acid amplification (OSNA). This approach uses the loop-mediated amplification technique (LAMP) [4]. The OSNA protocol in this case is as follows. The biopsy material taken during surgery is lyzed, and the lysate is subjected to reverse transcription reaction, followed by LAMP (in the same tube). Bst-polymerase from a thermophilic bacteria Geobacillus stearothermophilus that synthesizes DNA via chain displacement is used in LAMP at a constant temperature (65°C), which makes the DNA Abbreviations: dNTP—deoxyribonucleoside triphosphate; FBS—fetal bovine serum; LAMP—loop-mediated isothermal amplification; LN—lymph
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