The Analysis Of Deoxyribonucleic Acids And The Lipid Profiling Of Cells In Situ With Matrix Assisted Laser Desorption Ionization Mass Spectrometry

In the world,cervical cancer is one of the most common disease,and the incidence and mortality rate of cervical cancer is the second in the world.Human papillomavirus(HPV)persistent infection is the primary cause of cervical cancer.The early detection and prevention is an effective means to prevent cervical cancer.Therefore,it is particularly important to develop a rapid,accurate and highthroughput cervical cytology screening method.In this study,cellular DNA and cell surface lipids of high risk human papillomavirus 16 and 18 are as the study object.The pretreatment and analysis methods of high risk hpv16 type and 18 type deoxyribonucleic acid and lipids on cell were established and optimized.The high throughput HPV genotyping by matrix assisted laser desorption/ionization mass spectrometry is conducted.In situ lipids profiling of high risk types is established to distinguish high-risk hpv16 type and hpv18 type.A based assay for HPV genotyping which is combined matix-assisted laser desorption/ionization-time of flight mass spectrometry(MALDI-TOF MS)and the restriction fragment mass polymorphism(RFMP)assay is established.The DNA is amplified by PCR in two cycles separately with the universal and specific primers.The use of a Type IIS restriction endonuclease cleavage makes the RFMP assay indendent of sequence.After PCR amplification,samples are used to restriction enzyme gigestion with FOKI and BtsCI,and desalting using Millipore.Then the purified samples are analysis by MALDI-TOF MS.The characterictc peaks of restriction enzyme mass polymorphism of HPV16 type were 2177.4 Da,2191.4 Da,3692.4 Da,3796.4 Da,4003.6 Da,4006.6 Da.The characterictc peaks of restriction enzyme mass polymorphism of HPV18 were 2177.4 Da,2222.4 Da,3715.4 Da,3740.4 Da,3991.6 Da,4018.6 Da.The method has high sensitivity,good reproducibility,high sensitivity,strong specificity and rapid detection.It is easier to operate than the gene chip method and can be used in high throughput detection for early screening.In situ lipid analysis is a rapid method to distinguish hpv16 and 18 cell with MALDI-TOF MS.Using ITO glass as sample plate keeps the cells in the original state,and matrix is spraied with matrix sprayer,which can obtain high quality lipid profiles map to distinguish between two different cell types.Then,the principal component analysis is used to reduce the dimension and the random forest is used to classify,the result can distinguish two kinds of cells obviously,the classification accuracy is up to 95%.The two methods of 16 and 18 type are compared.The first method is difficult to operate.It has high sensitivity and The second method is simple and reproducible.The in situ analysis of lipids method is applied to different parts of plant seeds,using peanut and soybean seeds as samples,the significant results were obtained.601.7Da is the four parts of peanut seeds common lipid.The characteristic molecular ion peaks of 759.18 Da,496.37 Da,706.85 Da,496.37 Da were respectively peanut testa,endoleura,germ and cotyledon.The characteristic molecular ion peaks of cotyledons of 617.20 Da,706.54 Da,463.77 Da,650.29 Da,are characteristic molecular ion peaks of soybean testa,endoleura,germ and cotyledon respectively.The random forest classification is carried out separately.The accuracy rate of soybean four classification is 100%,peanut four classification accuracy is 92%.The accuracy is very high.Therefore,the in situ analysis method has universal applicability.