Homogeneous DNA Sensor Based On Cascade Amplification For Ultrasensitive Intelligent Detection Of HIV/HCV DNA

As is known to all,human who are infected with the human immunodeficiency virus(HIV)will suffer from acquired immune deficiency syndrome(AIDS).With the successful application of antiretroviral therapy(ART),the incidence and mortality of AIDS have been greatly reduced.However,few people know that the route of transmission of hepatitis C virus(HCV)and HIV are almost consistent.Therefore,patients who are infected with HIV are also more likely to be carriers of HCV.If not discovered and treated in time,patients will suffer from liver fibrosis.The mortality rate of patients who suffer from liver fibrosis are much higher than AIDS.Currently,early screening is still the most effective way to reduce the mortality of HIV/HCV co-infected patients.However,in the early stage of infection,the virus content in patients is very low,so it is urgent to develop a sensitive and rapid method to detect HIV/HCV.In order to solve the above issue,we propose a cascading signal amplification platform combining Exo Ⅲ-assisted target recycle amplification(ERA)and rolling circle amplification(RCA)for ultrasensitive detection of HIV/HCV DNA,so as to achieve early diagnosis of diseases related to HIV/HCV.In the first chapter of this paper,we first introduce AIDS and its pathogen HIV,and then we briefly introduce the principle and application of nucleic acid amplification technologies(for signal amplification),metal nanoclusters synthesis(for signal output)and DNA logic gates(for intelligent detection)related to viral DNA detection.In the second chapter of this paper,a fluorescence system based on ERA and RCA is developed to realize the ultrasensitive detection of HIV/HCV DNA.Firstly,the target DNA is hybridized with the hairpin DNA to form a blunt end.At this time,the hairpin DNA will be gradually digested by Exo Ⅲ to release the target DNA and generate a large number of single-stranded DNA to achieve the first amplification of ERA.Secondly,the generated single-stranded DNA can be used as primers to hybridize with the padlock probes to form circular DNA under the action of ligase.Under suitable conditions,the RCA reaction will be initiated to achieve the second amplification.Because the circular DNA of RCA is designed to be rich in guanine,the amplification products contain a large number of cytosine sequences that is complementary to the circular DNA.Finally,the polycytosine regions of the amplified products are incubated at low temperature with AgNO3 and reduced by NaBH4 to synthesize fluorescent silver nanoclusters in situ,thus realizing the output of detection signal.The detection limits of HIV DNA and HCV DNA are as low as 1.4 fM and 2.1 fM respectively.Therefore,the fluorescence system has great potential in the diagnosis of diseases related to HIV/HCV.In the third chapter of this paper,we propose the combination of DNA logic gates and fluorescence sensor mentioned in the previous chapter to realize the intelligent detection of HIV/HCV DNA.Firstly,we design hairpin DNA with specific hybridization of HIV DNA,HCV DNA and both HIV/HCV DNA respectively.Later,by combining different hairpin DNA in the system,AND logic gates,OR logic gates and combined logic gates are constructed to realize the intelligent detection of HIV and HCV in the samples.