New Methods For Detecting And Genotyping Hepatitis C Virus

Hepatitis C virus(HCV) has infected a population of over170million,while the vaccineof HCV is difficult to manufacture.What’s more,there is no specific medicine forit.Therefore,to diagnose HCV at an early time is definitly meaningful.At the presentstage,diagnostic methods for HCV are polymerase chain reaction(PCR) and enzyme-linkedimmunosorbent assay(ELISA).However,both of them are not convenient or time-savingenough.New convenient and sensitive methods are quite needful.Here we report some experiments based on molecular beacon(MB) and goldnanoparticles(AuNP) for directly detecting HCV RNA and genotyping of the virus.First partof this paper is using Duplex-specific nuclease(DSN) as a signal amplifying tool for MB todetect HCV RNA directly.In addition,we used MB to genotype HCV by detecting its PCRproduction.Moreover,AuNP are used to detect HCV RNA sensitively and easily.(1)While the hybridization of MB and RNA could be digested by DSN,the RNA wouldrecyle,which caused the signal being amplified.DSN could make MB2000times sensitivethan before.For JFH1RNA,standard of HCV RNA,we could detect it in a low concentration.And then,we detected the RNA of positive huh7.5cellline.We could distinguish positive andnegative serum RNA samples,the signals of which were basically corresponding to ELISA.(2) If we could use MB to detect the PCR production of RNA,gene orders of the emplatebeside the primer parts would be distinct.In this experiment,we fould the proper method andcondition for MB to detect double-stranded DNA(dsDNA),the PCR production.We designeda MB which targeted the different part of JFH1and H77S,subtypes of HCV,to distinguishthem.Hybridization of MB and the target part of dsDNA could easily distinguish differentgenotypes of virus RNA after their PCR.(3)In a saline solution,gold nanoparticles(AuNP) should adsorb lots of single-strandedDNA(ssDNA) to maintain the colloid state.If enough RNA could hybridize the ssDNA,AuNPwould just coagulate.Using primer,AuNP,saline solution,we could observe the color changingof AuNP(coagulation) or no changing while there was or not HCV RNA.What’s more,detecting the absorption on the UV-VIS spectrophotometer could make it more sensitive.Although not consummate yet,these methods are very simple,time-saving andspecific,which could be new mentalities for detecting HCV.