Development of Enzyme-Linked Oligonucleotide Assay (ELONA) for Pathogenic Microorganisms Detection in Aquatic samples

摘要

Norovirus (NoV) is a non-enveloped single-stranded RNA virus, which is known to be the main cause of viral gastroenteritis in the world. It can affect people of all ages and small children and senior citizens are more vulnerable. NoV outburst could lead an entire society into sudden sabotage. Therefore, it is vital to prevent NoV infection in the first hand. The objective of this study is to develop an Enzyme-Linked Oligonucleotide Assay (ELONA) to detect NoV RNA in environmental samples. In this study, the artificial DNA including a section of NoV sequence (a-NoV, 89 bp), was prepared. The a-NoV is captured by an amine modified COG2R primer, which is immobilized on to 96 micro-well plate. The a-NoV hybridizes with a biotin-modified second DNA probe (b-QNIF2d) which bind to streptavidin-conjugated horseradish peroxide (SA-HRP). SA-HRP react with the substrate to produce a colour change so that the absorbance of the substrate is changed. Using this method, a calibration curve with a 150 pM of limit of detection was generated for the a-NoV spiked in a DNA pool extracted from a wastewater sample. Optimizing the ELONA method and increasing the sensitivity of this system is currently under consideration.