Application Of Three PCR Techniques In HIV Diagnosis And Efficacy Monitoring

Part OneApplication of Quantitative Detection of HIV RNA in the HIV-1 DiagnosisBackgroundThe pathogen of AIDS is human immunodeficiency virus.In February 2017,the nationwide survey(Hong Kong,Macao and Taiwan excluded)reported a total of 485649 cases of legal infectious diseases,1409 cases of death,in which deaths of AIDS were 1097(77.9%).Early detection and early diagnosis are particularly important for the treatment of HIV infection,but also can reduce mortality and improve the quality of life of infected people.Commonly,AIDS detection methods are mainly antibody detection and virus detection.Virus detection includes p24 antigen detection,cell culture(virus isolation)and nucleic acid detection.Antibody detection is usually carried out 3 to 8 weeks after infection,and nucleic acid detection can be used within 2 weeks after infection shortening the window period.Moreover,nucleic acid detection is considered to be a meaningful method for early diagnosis of HIV due to its high sensitivity and specificity.According to National AIDS Testing Technical Specifications revised in 2015,antibody confirmation test and HIV-1 nucleic acid test are used to be supplementary test of strategy,showing that nucleic acid detection plays an important role in the diagnosis of AIDS.Quantitative detection of nucleic acids can determine the viral load of peripheral blood.Compared with the qualitative detection,quantitative detection is more sensitive and convenient.In this study,we used the In-house nested PCR method which built by Chinese Center for Disease Control and Prevention STD and AIDS Center reference laboratory for the nucleic acid qualitative detection.All human cells contain ?-actin gene which could be the internal reference for the PCR experiments.Only when the ?-actin gene successfully amplified,can we confirm that the nucleic acid extraction is valid.In the meanwhile,the HIV-specific gene amplified with 6 pairs of primers(pol region,gag region,env region)by nested PCR,and the amplification results determined that the HIV qualitative detection was positive or negative.Using NucliSENS EasyQ HIV-1 v2.0 directly detected HIV RNA in the plasma for the quantitative detection of nucleic acids.To evaluate the availability of HIV-1 nucleic acid quantification for the diagnosis of HIV-1 infection,we will compare the feasibility of qualitative test of HIV-1 nucleic acid with which of quantitative test of HIV-1 nucleic acid.ObjectiveTo investigate availability of the quantitative detection of HIV nucleic acids for HIV-1 diagnosis.MethodsSelecting 216 samples,including 136 babies born to HIV-1 positive mothers and 80 HIV-1 positive adults from Dehong,Yunnan province,all of them were both carried out qualitative and quantitative assays of HIV-1 nucleic acid,the results of two assays were analyzed by synthesis.ResultsAfter analyzing the results,216 samples showed an excellent consistency between qualitative and quantitative assays.1.53 samples(50 adults and 3 babies)were nucleic acid positive,whose plasma viral load>5000CPs/ml.In which 50 adults' follow-up results of WB confirmatory test were positive for HIV-1 antibody,showing that the qualitative and quantitative assays of HIV-1 nucleic acid were both meaningful;2.163 samples(30 adults and 133 babies)were nucleic acid negative,Plasma viral load was lower than the detection limit.ConclusionIn this study,we could conclude that when the plasma viral load>5000CPs/ml,quantitative detection of nucleic acids is an efficient method to diagnose the HIV-1 infection.Part TwoApplication of PCR-Fluorescent Probing for Detecting HIV-1 Provirus in Adults Received Long-term HAARTBackgroundAfter invading the human body,the virus can integrate with the host genes and hide in the tissues or organs,such as lymphoid tissues,the brain cells and so on.This is where the virus reservoir of HIV-1 coming from.HAART can't clear the virus away from the virus reservoir.Provirus mean the virus don't spread in the infected cells and hide from the immune system elimination.But the virus will replicate again on some conditons.The extensive application of HAART has significantly reduced the mortality of AIDS caused by the Human Immunodeficiency Virus.HAART can suppress the virus relication efficiently.After the treatment,plasma viral loads of the AIDS patients or HIV infected persons were lower than the detection limit and the number of CD4+ T lymphocytes rised up,rebuilding the human immunologic function.But because of the persistent exist of virus reservoir,we can't cure the disease once and for all.In this study,we use PCR-Fluorescent Probing to detect the level of HIV-1 provirus in PBMCs of patients who received the long-term HAART treatment.We were trying to explore the availability of HIV-1 virus reservoir as an indicator of the effectiveness of HAART and the progression of disease,laying the foundation of exploration of new treatments targeting the virus complete elimination in the future.ObjectiveThe aim of this study was to investigate changes of the virus reservoirs of HIV/AIDS patients who received highly active antiretroviral therapy(HAART)using HIV-1 DNA Detection Kit,and analyze its relationship with the therapeutic effects.MethodsA group of 91 chronic HIV-1 infected adults who received 5?10 years-HAART treatment,participated in cross-sectional study.After the ethical approval was passed,a volume(40 ml)of peripheral whole blood was obtained from each patient.The HIV-1 RNA viral load was detected by the bDNA method.DNA was extracted using Human DNA Kit.PCR-Fluorescent Probing was used to detect the level of HIV-1 DNA.And the T cells were counted by Flow Cytometry.SPSS software was used to analyze the collected data.Results1.Among 91 patients received 5?10 years HAART treatment,the level of plasma HIV-1 RNA of all the patients were lower than the detection limit;2.HIV-1 DNA was detected in 91 patients' peripheral blood mononuclear cells(PBMCs)[mean(1.77±0.76)logl0copies/106cells]using HIV-1 DNA Detection Kit,3.The ratio of HIV-1 DNA negative patients was 39/91,and the proportion of uncertain patients was 52/91 using In-house nested PCR;4.The average number of CD4+ T lymphocytes was 479.56±188.11/?lConclusionHIV-1 patients showed significant antiviral and immune reconstitution after long-term(5-10 years)HAART treatment.The HIV-1 DNA Detection kit can detect the virus reservoir sensitively,and HIV-1 proviral DNA can play an important indicator of the efficacy of treatment and monitoring the disease progression.PART THREEThe Study of Application of ddPCR and qPCR in HIV Early Infants DiagnosisBackgroundHuman Immunodeficiency Virus is a kind of provirus that can integrate into the DNA of a host cell.The amount of HIV DNA in the host cells is an indicator measuring the size of virus reservoir.HIV DNA is very important for HIV diagnosis and therapeutic monitoring.Currently,the main method for quantitation detection of HIV DNA is real-time quantitation PCR(qPCR).The qPCR can't absolutely quantify the HIV DNA,because it relies on cycle threshold which is prone to be influenced by the amplification efficiency.The droplet digital polymerase chain reaction(ddPCR)is a brand new technology for nucleic acid detection and absolute quantitation.In droplet digital PCR,PCR solution is divided into smaller reactions through a water oil emulsion technique,which are then made to run PCR individually.Every water-in-oil emulsion contains one DNA molecule hybridizing with a fluorescent labeled probe.Then we obtain the absolute quantitation of single molecule DNA detecting every reaction by the droplet detector.The ddPCR has relative high sensitivity and with no need to establish a standard curve.But the application of ddPCR in quantitation detection of HIV nucleic acid isn't wide in China,because it is very expensive.In this study,we both use the ddPCR and qPCR to detect the samples from newborn at different months comparing the sensitivities and specificities of two methods.And evaluate the consistency of two detection methods,laying the foundation of developing the diagnostic testing strategy of HIV-exposed infants in China.ObjectiveTo compare the sensitivities and specificities of ddPCR and qPCR.And evaluate the consistency of two detection methods,MethodsSelecting dried blood spots samples from 116 infants at different months who born to HIV-positive mothers from Yunnan,Sichuan,Xinjiang and Chongqing provinces in the year of 2014-2016.According to the National AIDS Testing Technical Standards(2015),when a infant's two consecutive nucleic acid tests were positive,we diagnosed the infant HIV positive.Otherwise we diagnosed HIV negative.There were 54 HIV positive samples and 62 HIV negative samples in all 116 drid blood samples.This study has been approved by the medical ethics committee and all the subjects have signed the informed consent form.Results1.For 116 dried blood spots samples from infants at different months,the sensitivity of qPCR was 63.0%,specificity was91.9%;the sensitivity of ddPCR was59.2%,specificity was75.8%.And the coincidence rate of results for qPCR and ddPCR is 73.3%,The kappa value is 0.391.2.For those samples collected within the postpartum 24h,the sensitivity of qPCR was 36.3%,specificity was 80.0%;the sensitivity of ddPCR was27.2%,specificity wa73.3%.And the coincidence rate of results for qPCR and ddPCR is 53.8%.The kappa value is-0.173.3.For those samples collected at the age of 4 weeks,the sensitivity of qPCR was 50.0%,specificity was100.0%;the sensitivity of ddPCR was57.1%,specificity wa92.9%.And the coincidence rate of results for qPCR and ddPCR is 78.6%.The kappa value is 0.478.4.For those samples collected at the age of 6 weeks,the sensitivity of qPCR was 78.6%specificity wasl00.0%;the sensitivity of ddPCR was78.6%,specificity wa77.8%.And the coincidence rate of results for qPCR and ddPCR is 87.5%.The kappa value is 0.745.5.For those samples collected at the age of 3 months,the sensitivity of qPCR was 80.0%,specificity was86.7%;the sensitivity of ddPCR was66.7%,specificity was 60.0%.And the coincidence rate of results for qPCR and ddPCR is 66.7%.The kappa value is 0.336.ConclusionThe sensitivities and specificities of ddPCR and qPCR are not high.And the consistency of two detection methods is low.So the ddPCR and qPCR can't provide adequate references for the early diagnostic testing of HIV-exposed infants.