Study On The Detection Of Norovirus By Loop-mediated Isothermal Amplification

The reaction of isothermal nucleic acid amplification is always maintained at a constant temperature,which does not need special instruments with precise temperature controller,and has unique advantages in rapid detection of nucleic acid in vitro.The reaction of loop-mediated isothermal amplification,LAMP,is under the condition of 60-65 ?,and the amplification time is usually within an hour.LAMP is a powerful amplification method and produces a large amount of white pyrophosphate magnesium precipitation,that can be directly determined with naked eyes.But the disadvantage of LAMP is that unexpected non-specific amplifications often occurs and false positive signals generates.In order to improve the accuracy,LAMP amplification was combined with one-step strand displacement(OSD)or hybridization chain reaction(HCR)in my work,and sensitive detection of norovirus was realized using Microplate Reader Flow Cytometer,or Glucometer,respectively.After separation and purification assisted by magnetic beads,30 copies of norovirus DNA can be detected even in additional fecal solutions.The detailed content is shown as follows:LAMP combined with OSD for the detection of norovirus using quantitive real-time PCR: common DNA dyes,such as Evagreen,have no ability of recognizing DNA sequences,while OSD probes can specifically identify the sequences of LAMP products.Therefore,the problem of false positive can be avoided through the coupling of LAMP with OSD under optimized reaction conditions.However,when detection of norovirus with additional added fecal solution or other LAMP products,such as rotavirus,the fluorescence of LAMP-OSD was susceptible.LAMP combined with HCR for the detection of norovirus using Flow Cytometer: we proposed a new technology for LAMP combined with HCR for norovirus detection,and the feasibility,necessity and technical advantages of this method were verified.DNA with fluorescent groups was amplified by LAMP and then HCR reactions directly on magnetic beads.The magnetic beads containing lAMP-HCR products were used to be detected using Flow Cytometer with the detection limit of 30 copies of norovirus genes.Due to the separation and purification of magnetic spheres,even if different percentages of fecal samples(0.1% ~ 2%)had been added,the detection results did not be affected.LAMP combined with HCR for the detection of norovirus using glucometer: DNA conjugated with sucrase was amplified by LAMP-HCR on magnetic beads mentioned above,and then the magnetic beads with amplification products were mixed with sucrose,which would be hydrolyzed by sucrose into glucose that could be measured by a Glucometer.Thirty copies of the norovirus genes could also be detected in 2 % of fecal solution,enabling rapid field diagnosis of norovirus.