Mass Spectrometry Genotyping And Detection Of Human Papillomavirus(HPV) Based On Nanoparticles

Cervical cancer is the fourth most common cancer among women worldwide.Studies have shown that persistent infection with high-risk HPV is a necessary condition for cervical lesions.Therefore,HPV typing plays an important role in the screening of early cervical cancer.However,many high-risk HPV infections are automatically eliminated by the immune system.Without cervical-related lesions,HPV L1 protein detection can understand whether HPV-DNA is integrated into host cells.In the chromosome,to judge benign or malignant lesions caused by HPV infection.Different levels of cervical cancer are different from HPV infection types.The determination of viral load alone often misleads people to predict the development of cervical cancer into cervical cancer.Therefore,in the assessment of persistent infection of HPV,it is not only necessary to detect the viral load of HPV.It is also necessary to determine which type of HPV it belongs to.Detection of HPV L1 egg white has a certain guiding value for the prognosis of cervical human papillomavirus infection,and it is more meaningful to combine HPV typing.The combination of HPV typing and HPV L1 protein detection will maximize the shortcomings of each other,improve the accuracy of cervical lesion diagnosis,and reduce the incidence of missed diagnosis.Based on the characteristics of nanomaterials,MALDI TOF MS was used to establish high-risk HPV 16,18-type detection method and HPV 16 L1 detection method.The method has the advantages of high sensitivity,high throughput and rapidity,and has been successfully applied to the detection of clinical samples.1.Spermine was successfully modified onto nanodiamond by EDC coupling reaction.By comparing the modified nano-diamonds of 100 nm and 5 nm,it is found that the 5 nm nanodiamond has a larger specific surface area and the modification effect is better.SP-NDs are coated with a large amount of spermine.The characteristics of polyamines in acidic conditions can be positively charged.It can effectively attract negatively charged DNA phosphate backbone by static electricity.It is found that SP-NDs are oligonucleotides.With excellent adsorption(184 mg/g),the study also found that SP-NDs can not only extract and enrich oligonucleotides in dilute solutions,but also from complex matrices such as sodium dodecylbenzene sulfonate and The extraction of oligonucleotides in a urea solution is an excellent solid adsorbent for oligonucleotides.2.The PCR product directly detected by MALDI MS is limited by two aspects.One is that the molecular weight of the oligonucleotide is too large to be detected by MALDI,and the other is that the PCR buffer solution contains enzymes and salts to inhibit the ionization of the oligonucleotide.Therefore,a complicated desalination purification process is required.For one problem,we used nested PCR amplification,embedded in the endonuclease site,and then digested into short oligonucleotide fragments by restriction endonucleases,and each HPV type can be cleaved into six different molecular weights.The DNA fragment,which analyzes the results of the test,increases reliability.In response to the second problem,we found that SP-NDs can not only extract oligonucleotides from complex matrices,but also extract oligonucleotides directly from PCR products.More importantly,SP-NDs can be used.HPV-specific DNA fragments were selectively extracted from the PCR-RFMP enzymatic digestion products for MS analysis,and good results were obtained in clinical samples.SP-NDs This method avoids the traditional purification scheme,simplifies the analysis process and improves sensitivity.As a solid phase adsorbent,SP-NDs can be used to extract and purify oligonucleotides before MALDI analysis,replacing traditional methods of purification by electrophoresis or liquid chromatography.Based on the PCR-RFMP method,SP-NDs were enriched,extracted and isolated and analyzed by MALDI MS for DNA methylation,single nucleotide polymorphism and other virus typing.3.Laser desorption ionization mass spectrometry(LDI MS)was established for HPV 16 L1 protein based on non-covalent competitive adsorption between HPV16 L1 nucleic acid aptamer(APTHPV16L1)and mass tag on gold nanoparticles(AuNP).A new method of sensitive detection.In our design,APTHPV16 L1 competes with the mass label(melamine)for occupying the non-valence interaction sites on the surface of AuNPs.When APTHPV16L1 is present,it preferentially occupies the non-valence interaction sites on the surface of AuNPs.Quality labels were not detected on the surface of AuNPs.With the addition of HPV16 L1 protein,there is a more specific interaction between HPV16 L1 protein and APTHPV16L1,which causes APTHPV16L1 to detach from the surface of AuNPs.At this time,the exposed reaction sites are mass-labeled.occupy.The signal of the mass label was directly detected by LDI-TOF MS after centrifugation.Thus,the mass spectrometry signal intensity of the AuNPs surface quality label can reflect the content of HPV 16 L1 protein.Quantitatively determined by internal standard method,HPV 16 L1 protein has a linear relationship in the concentration range of 2?80 ng/mL,the correlation coefficient can reach 0.998,the detection limit(LOD)is 58.8 pg/mL,and the method is successfully used in clinical and The detection of HPV 16 L1 protein in vaccine samples has the advantages of high sensitivity,low detection limit and high flux.It is expected to be applied to the early clinical diagnosis and prognosis of cervical cancer.4.According to the nucleic acid aptamer,the stability of the graphene oxide colloid solution can be enhanced under high salt conditions,and can also be detached from the surface of the graphene oxide by specifically binding to the target,and the exposed graphene oxide is easily aggregated under high salt.Change the absorbance at 230 nm.We found a linear relationship between the absorbance and the logarithm of the HPV16 L1 protein concentration to quantify the HPV16 L1 protein.The method is short in time,high in sensitivity,and low in reagent consumption.It can be detected only by a simple ultraviolet spectrophotometer.More importantly,the test results for clinical samples are consistent with the ELISA and the method described in Chapter 4,and have good specificity.The recovery rate of the method was 87%?102%,and the detection limit was 2 pg/mL.The innovation of this research is that fluorescently labeled aptamers are not used,thus avoiding fluorescent bleaching.Based on the optical properties of graphene oxide itself,we have established a highly sensitive,simple and rapid method for detecting HPV 16 L1 protein,which reduces the cost of the experiment and may become an alternative method for the detection of conventional HPV16 L1 protein.