Loop-Mediated Isothermal Amplification (LAMP) For Detection Of Three Enteric Viruses And Its Application In Water Samples

As the water environment deteriorating, water pollution problem has become the focus of attention and research. Now there are many records about large scope of disease outbreak because of drinking water polluted by pathogenic microorganism, waterborne pathogenic microorganisms include bacteria, viruses and protozoans primarily, conventional chlorine disinfection can kill bacteria effectively, but the virus in water are hardy and can't effectively removed by conventional water treatment technology. Therefore, the detection of virus in water is a valuable work for preventing waterborne disease,evaluating water quality and environmental sanitation. This paper has developed loop-mediated isothermal amplification(LAMP) technology for rapidly detecting three enteric viruses, designed private primers for every virus, optimized amplification conditions (magnesium ions concentration, betaine concentration, reaction time, reaction temperature) and testing its specificity and sensitivity. Then, optimized LAMP were used for detection of three enteric viruses in 12 dringking water and 12 reclaimed water samples in beijing,respectively. The main conclusions are as follows:1.The optimal amplification condition for astrovirus was at 65℃for 90 min with 4mmol/L magnesium ions and 1 mol/L Betaine. The specificity of LAMP assay was further validated by cross-reaction with different virus (rotavirus and norovirus) and restriction analysis of the amplified products. The sensitivity of the LAMP and PCR were 5.04 copies/μLand 5.04×10 copies/μL, respectively, The sensitivity of LAMP are 10 times higher than PCR2. The optimal amplification condition for rotavirus was at 65℃for 90 min with 3mmol/L magnesium ions and 1 mol/L Betaine. The specificity of LAMP assay was further validated by cross-reaction with different virus (astrovirus and norovirus) and restriction analysis of the amplified products. The sensitivity of the LAMP and PCR were 5.08 copies/μLand 5.08×102 copies/μL, respectively, The sensitivity of LAMP are 100 times higher than PCR. 3. The optimal amplification condition for norovirus was at 63℃for 90 min with 4mmol/L magnesium ions and 10mmol/L Betaine. The specificity of LAMP assay was further validated by cross-reaction with different virus (astrovirus and rotavirus) and restriction analysis of the amplified products. The sensitivity of the LAMP and PCR were 2.168 copies/μL and 2.168×102 copies/μL, respectively, The sensitivity of LAMP are 100 times higher than PCR4. Improved RT-LAMP was used for detection of three enteric viruses in 12 dringking water and 12 reclaimed water samples of beijing. The detection rate of three enteric viruses were:drinking water (astrovirus was not detected, rotavirus 16.7%, norovirus 16.7%); reclaimed water(astrovirus 41.7%, rotavirus 25%, norovirus 16.7%)All these results suggested that LAMP was a simple, high sensitivity and specificity method which was potential for rapid detection of enteric viruses.