Development And Application Evaluation Of Magnetic Capture Hybridization And Quantatitative PCR For HBV CccDNA

PART1Preparation and identification for specific functionalizednanoparticles of HBV CccDNAObjective: To prepare sepcific nanoparticles for riching andseparating HBV CccDNA and lay the foundation for establishing a novelmethod of HBV CccDNA assay with magnetic capture hybridization in thenext part.Methods: Initially, Fe3O4nanoparticles were synthesized by chemicalco-precipitation. Then silica nanoparticles were synthesized by sol-geltechnique and silica-coated nanoparticles were modified by streptavidin.Moreover, oligonucleotides for specific HBV CccDNA capture, locating onboth sides of RcDNA gap, which could selectively capture CccDNA, weredesigned and labelled by biotin. And oligonucleotides labeled withfluorescein isothiocyanate (FITC), which were completely complementaryto capture probe, were synthesized and captured by functionalizationalnanoparticles for validating the characteristic of functionalizationalnanoparticles. Results:(1) Photograph of the single-phase nanopowders revealed that therewas a uniform distribution of particles with particle sizes between8and25nm (average size13.2nm) which was in agreement with results thatpreviously mentioned. Silica-coated nanoparticles were cubic and highlyuniform in size and the particle sizes. In addition, the silica-coated particleshad significantly better dispersion than that of Fe3O4nanoparticles.(2) The figure of infrared spectrum2,471cm-1showed that theFe-O-Fe stretching vibration,1104cm-1was the stretching vibration of Si-O.Therefore, main peaks showed that synthetic particles contained Fe3O4andsilica. Furthermore, hysteresis loop of the Fe3O4magnetite nanoparticles atroom temperature indicated that the saturation magnetization of thenanoparticles is about55emu/g, which was less than the saturationmagnetization of bulk magnetite (92emu/g). Moreover, as could be seenapparently from the figure that the magnetic nuclear showed non-hysteresisand remanence phenomena, displaying obvious superparamagneticphenomenon that existed magnetism only when there was the externalmagnetic field, after the removal of the external magnetic field, themagnetic completely disappear.(3) The characteristic absorptions at260nm changed significantlybefore and after the bioconjugation reaction. After coupling the nucleic acid,the nucleic concentrations of supernatant were significantly decreased. Inaddition, streptavidin-modified nanoparticles had a high oligonucleotide binding capacity; its average amount of nucleic acid fixed was about3200pmol/mg.(4) After FITC-labeled sequences adding to buffer and hybridizingwith functionalized nanoparticles, the fluorescence intensity of supernatantwas decreased obviously compared with that of pre-hybridization.Moreover,per millgram functionalized nanoparticles could capture about2000pmolFITC-labelled sequences.Conclusion: Specific functionalized nanoparticles of CccDNA caneffectively capture the complementary sequences labelled by FITC, and thecapture amount enough to capture CccDNA in routine sample. PART2Preparation and quantity of HBV CccDNA standardObjective: To prepare the CccDNA standard by constructingrecombinant plasmid of HBV complete genome and provide the standardand control material to establish a quantitative method and validate theperformance for CccDNA.Methods: HBV DNA was extracted from sera of chronic hepatitis Bpatients and HBV complete genome was amplified with high fidelity PCRmethod. Then recombinant plasmids of HBV complete genome wereconsrtucted by T-A cloning. And CccDNA standards were prepared bymeans of enzyme digestion, connections, enrichment and recycling on the basis of recombinant plasmids of HBV complete genome.Results:(1) About3.2kb DNA fragments were sucessfully amplified byhigh fidelity PCR method from the serum of CHB patients.The3.2kb DNAwas inserted into pMD18-T and the recombinant plasmids of HBVcomplete genome were sucessfully constructed. Furthermore, therecombinant plasmids were digested by Hind III and two target fragmentswere identified, which lengths were the same as expected.(2) It was comfirmed that the two recombinant plasmids werebelonging to HBV genetype B and C respectively via sequencing. And4probes and the forward primer designed in the DR1and DR2could matchwith the sequences of recombinant plasmids.(3) CccDNA standards were sucessfully constructed by means ofmoleculer methods and their concentrations were1.05×1010IU/mL and6.19×109IU/mL separately, they were named CccDNA-B and CccDNA-Crespectively.Conclusion: The recombinant plasmid of HBV complete genome andCccDNA standard was smoothly prepared and they can be used as standardand control materials for validating performace and quality control. PART3Development and methodology evaluation of MagneticCapture Hybridization and quantitative PCR for HBV CccDNAObjective: To develop a magnetic capture hybridization andquantitative assay method for HBV CccDNA and verify the methodperformance (sensetivity, specificity, linear, interference, etc).Methods: Primers, locating on both sides of RcDNA gap, whichselectively amplify CccDNA, were designed. Then the magnetic capturehybridization and quantitative PCR method for CccDNA was developed.Moreover,10-fold serial dilutions of HBV CccDNA standard (106,105,104,103,102and101IU/mL) were hybridized with the optimal functionalizednanoparticle capture probe that mentioned in the previous experiment for1,2and3h. To test whether the MCH-qPCR method for CccDNA crosswisereacts with HBV relaxed circular DNA (RcDNA), the specificityexperiments of MCH-qPCR were performed by mixing different contentsof CccDNA standard with total HBV DNA (CccDNA negative). Finally, theculture supernatants of HepG2.2.15cells and10serum samples of CHBpatients were tested by MCH-qPCR.Results:(1) Use of21-mer and35-mer capture probes allowed detection theexpected concentration（105IU/mL）of purified CccDNA in3h. However,the expected concentration was not available by use of25-mer capture eventhe hybridization time was improved from1to3h. Furthermore, reducinghybridization time from3to1did not affect the desired concentration by use of49-mer probes. Thus1h hybridization with the49mer capture probewas selected for all further experiment.(2) Linear and regression between expected values of CccDNA anddetected values by MCH-qPCR could be described by a linear model andthe R2=0.994was consistent with the strong linear relationship betweenadded concentration (Log10) and detected concentration (Log10)(F=507.5,p<0.01). So, the sensetivity and sepecficity of MCH-qPCR meet therequirments.(3) After continuous4d, the concetration of CccDNA in culturesupernatants were4.02×103IU/mL,1.04×102IU/mL,4.38×102IU/mL and5.01×103IU/mL respectively and the concentrations of the controlmaterials, which were tested in the same way as culture supernatants, meetthe expected concentration.(4) The concentrations of HBV DNA were over107IU/mL in10serum samples of CHB patients, and CccDNA were detected byMCH-qPCR in8samples. Furthermore, total DNA was extracted fromserum by Hirt method, then total DNA were directly detected byquantitative PCR for CccDNA, the concentrations were higher than that ofMCH-qPCR, and their difference was significant (p <0.01).Conclusion: The novel MCH-qPCR method is sucessfully developedand it has low limit of detection (102IU/mL), good sepecificity and truness.It could meet the requirment of clinical routine detection. PART4Primary clinical application of MCH-qPCRObjective: To evaluate the clinical application of MCH-qPCR methodfor CccDNA quantitative detection and analyze the correlation between theHBV DNA and CccDNA to understand the changes of CccDNA indifferent samples.Methods: Liver tissues, peripheral blood mononuclear cells (PBMCs)and serum samples of CHB were collected and their total DNA wereextracted by Hirt, then CccDNA was detected by established MCH-qPCR.Results:(1) Twenty eight cases of liver tissues were analyzed for thepresence of CccDNA and HBV DNA. In15/28(53.5%) samples CccDNAand21/28(75%) samples HBV DNA were detectable, it was positivecorrelation between them（p <0.01，R2=0.824）.(2) Twenty one cases of PBMCs of CHB patients were analyzed forthe presence of CccDNA and HBV DNA. In4/21(19.0%) samplesCccDNA were detectable, but their concentration is low (102~103IU/mL).The correlation between PBMCs CccDNA and HBV DNA was notsignificant (p>0.05).(3) Eithty two cases of CHB patient serum were analyzed for thepresence of CccDNA and HBV DNA. In48/81(59.3%) samples CccDNA were detectable,82serum samples show that HBV cccDNA levels weresignifcantly higher in serum samples of HBeAg positive than HBeAgnegative chronic hepatitis B patients, and there was no difference betweensamples of chronic hepatitis B patients with different ALT levels.Conclusion: MCH-qPCR method could detect CccDNA from livertissue, peripheral blood mononuclear cells and serum samples, detectionresults have showen that the detection rate of liver tissue is high and that ofperipheral mononuclear cells is low, HBeAg status is positively correlatedwith the CccDNA.