Highly Sensitive Detection For HBV DNA And YMDD Drug Resistance Mutation Based On CRISPR

Chronic hepatitis B(CHB)is one of the most prominent global public health issue currently,seriously threatens human health.Although hepatitis B vaccination efficiently reduced the morbidity,10-30 million people get infected with hepatitis B virus(HBV)each year,and estimated 1 million people die annually due to HBV related complications.In order to control HBV transmission effectively and ensure patients get treatment timely,efficient detection of HBV is required.Nucleic acid amplification testing(NAT)has been widely used in HBV diagnosis,monitoring,prognosis and blood screening in transfusion due to its high sensitivity and specificity.However,low-level HBV DNA that hardly detected by current methods was still existed in part of infected patients in clinical practice.These situations not only increased a potential risk for chronic hepatitis,liver cirrhosis and primary liver cancer,but may also lead to HBV infection in transfusion and liver transplantation.In addition,detection and monitoring of HBV drug resistance mutations can prevent related antiviral therapy failure.Owing to the existence of drug resistant quasi-species,partial patients have extremely low level of HBV drug resistant variants,which is difficult to detect.Under certain circumstance as liver transplant and HCV coinfection,those undetected variants may lead to significant clinical deterioration like HBV infection and liver failure.Therefore,developing a highly sensitive technology for HBV DNA and drug resistance mutation detection has great significance for prevention and treatment of HBV.Recently,a new RNA-targeting effector CRISPR-Cas13a was developed to a highly sensitive and specific detection technology even for trace nucleic acid.By combining with recombinase polymerase amplification(RPA),this technology can accurately detect single-copy nucleic acid and distinguish single base mutation.Different with the high-cost and low-stability isothermal amplification technology,polymerase chain reaction(PCR)based nucleic acid method is more practical in clinical detection.In this study,we established a highly sensitive and specific detection platform(PCR-CRISPR)for HBV DNA and drug resistance mutation based on PCR amplification and CRISPR-Cas13a system for the first time,which can detect as low as one copy HBV DNA.For serum sample detection,this method showed higher sensitivity than qPCR and even achieved the sensitivity that over droplet digital PCR(ddPCR).Moreover,we established highly sensitive detedtion method for HBV YMDD drug resistance mutation based on technologies that mentioned before,which can specifically detect single copy variant.For those serum samples with extremely low-level viral load,PCR-CRISPR identified YMDD drug resistance mutation that not detected by qPCR.1.Establishment a highly sensitive detection method for HBV DNA based on PCR-CRISPRAfter the process of induction,expression,purification and activity identification,LwCas13a protein with satisfactory RNase activity and 96%purity was obtained.By comparing the gene sequences of different HBV genotypes in polymerase region(P region),we designed and screened an efficient crRNA targeting conserved region for HBV DNA detection.Based on the steps of PCR amplification,transcription,crRNA recognition and LwCas13a reactivation,report RNA cleavage and fluorescence signal measurement,we established a new nucleic acid detection method:PCR-CRISPR.Using this method,we can detect as low as single-copy HBV DNA with high sensitivity.2.Serum HBV DNA detection with PCR-CRISPR technologyUsing PCR-CRISPR method,we got the accordant results(16 HBV positive samples and 16 HBV negative samples)with qPCR for HBV DNA detection in 32 alknown serum samples.We further explored the sensitivity and specificity for serum HBV DNA detection in 280 blinded clinical samples.Based on the Real Time HBV DNA quantification results,90 HBV-positive and 180 HBV-negative samples were successfully detected by PCR-CRISPR.For the rest 10 samples HBV-negative by qPCR,both PCR-CRISPR and ddPCR detected trace HBV DNA(concentration range from 1.2to 5.4 copies per test of HBV DNA).Next,10 technical replicates were tested for each samples by the two methods.On average,it could be detected 7 times out of 10replicates by PCR-CRISPR but only 3 times by ddPCR.By referring to the clinical information of those 10 patients,we found 6 patients with CHB history and 2 patients infected with HBV.The results showed that the new method exhibited higher sensitivity than qPCR and even achieved the sensitivity that over ddPCR.3.Establish a detection method for HBV YMDD mutation based on PCR-CRISPRAfter sequence conservation analysis of the YMDD region,we designed and screened two crRNAs for YVDD and YIDD mutation detection respectively,and established PCR-CRISPR method for those two kinds of mutations.This assay can detect as low as1 copy YVDD mutant and 100 copies YIDD mutant in 10 minutes.Through the difference of fluorescence signal,PCR-CRISPR can effectively distinguish 1 copy YVDD variant with 10~6 copies wild strain.Research in this part demonstrated that,the newly-established method can detect YMDD drug resistance variants with high sensitivity and short time.4.Serum HBV YMDD mutation detection with PCR-CRISPR technologyDirect sequencing,qPCR and PCR-CRISPR were applied to 424 serum samples for YMDD mutation detection.As the results showed,27 serum samples with HBV DNA levels of>100 IU/mL were detected harboring YMDD drug resistance mutations(5samples with YVDD and 22 samples with YIDD)by all three methods.In addition,18samples with YMDD drug resistance mutation(5 samples with YVDD and 13 samples with YIDD)had been detected both by qPCR and PCR-CRISPR,but not detected by direct sequencing.Moreover,PCR-CRISPR detected 12 additional drug resistance samples with HBV DNA levels of<100 IU/mL(4 samples with YVDD and 8 samples with YIDD)that not detected by neither qPCR nor direct sequencing.The above results proved that,PCR-CRISPR can be applied for highly sensitive detection of HBV YMDD drug resistance variant in serum samples,particularly suitable in low level HBV DNA samples.Overview,this study combined PCR amplification and CRISPR-Cas13a system for the first time,and established a highly sensitive and specific detection platform for HBV DNA and YMDD drug resistance mutation based on PCR-CRISPR.This method can detect the low-level HBV DNA in serum samples,which is of great clinical significance in reducing the risk of HBV infection in blood transfusion and liver transplant,decreasing the recurrence rate after antiviral drugs withdrawal and making suitable treatment program.In addition,this research provided theoretical and basis and technology foundation for highly sensitive and specific nucleic acid detection of other pathogen.