Development of a rapid PCR-compatible, instrument-free and point-of-care applicable nucleic acid sample preparation method

Abstract

Molecular diagnostics and particularly polymerase chain reaction (PCR) assays have become a standard method of pathogen detection and disease diagnosis. They have boasted high sensitivity and specificity but been largely confined to laboratory settings due to instrumentation requirements. However, over the past decade several point-of-care (POC) PCR applications have entered the market and the trend of POC nucleic acid amplification testing has been on the rise. Clinical samples contain components known as PCR inhibitors which may inhibit and hinder PCR assays. The nucleic acids are not readily available either. Due to this, clinical samples must be processed by lysing the cells or particles to free the nucleic acids, the nucleic acids must be extracted and purified from the PCR inhibitive compounds and often due to low pathogen amounts they must be concentrated before they can be used as a sample in a PCR assay. The aim of the Master’s thesis was to develop a user-operated, PCR- compatible sample preparation method that in a POC-applicable fashion lyses the sample and purifies and concentrates the target nucleic acids into a ready-to-use PCR sample. The basic principle of the method was developed and optimized and its performance was tested and compared to a commercially available sample preparation method. The developed sample preparation tube method is capable of extracting and concentrating samples in the span of 5 minutes utilizing a tube, reagents, a neodymium magnet and a pipette. The method was demonstrated to be comparable to the reference commercial extraction method in the extraction of MS2 bacteriophage ribonucleic acid from urine sample matrices. With further research, a compact cassette- based POC sample preparation method that can be integrated into POC PCR platforms could be developed.