ABSTRACT
<p><b>OBJECTIVE</b>An approach for analysis of HIV quasispecies using Miseq high-throughput sequencing platform (hereinafter referred to as Miseq platform) was established and applied to contact tracing for a possible case of HIV sexual transmission.</p><p><b>METHODS</b>Four plasma specimens were collected from 2 HIV infections (P1 and P2) suspected to be involved in the sexual transmission and 2 local HIV infections as controls (P3 and P4). The RNAs were extracted from the specimens and then reverse-transcribed into cDNA. After HIV subtyping, Miseq platform was performed to detect and sequence the HIV quasispecies (352 bp) in each specimen. The frequency of quasispecies was counted and ranked. Intrapersonal and interpersonal genetic distance and phylogenetic tree were calculated by using the top 5, 20, 100, 500, and all quasispecies, respectively.</p><p><b>RESULTS</b>The subtypes of HIV from all 4 specimens were CRF01_AE. 23 788 to 37 397 cleaned sequences representing 1 229 to 1 412 unique HIV quasispecies were obtained from these specimens by using Miseq platform. The average genetic distance (3.5%-4.5%) between quasispecies from specimens P2 and P1 was significantly lower than that (10.3%-19.6%) between quasispecies from P2 and the controls (P3 or P4). Phylogenetic tree analysis indicated that sequences from specimens P1 and P2 clustered together while sequences from P3 and P4 exhibited completely independent clusters. When the top 20 or more quasispecies from each specimen were analyzed, sequences from P1 showed a paraphyletic relationship with those from P2, which may indicated that the direction of HIV transmission was from P1 to P2.</p><p><b>CONCLUSION</b>With the feature of convenient and economic operation, Miseq platform has high practical value in contact tracing of possible HIV transmission.</p>
Subject(s)
Humans , Contact Tracing , HIV Infections , HIV Seropositivity , HIV-1 , PhylogenyABSTRACT
ObjectiveTo establish a novel assay for HIV-1 p24 ultrasensitive detection based on Gold Nanoparticle Probe (GNP) and PCR.MethodsSandwich ELISA method was established by a pair of anti-p24 monoclonal antibodies (mAbs),1G12 and 1D4,and was used to detect recombinant HIV-1 p24 antigen.The bio-barcode DNA was 47 bp,selected from genome of Arabidopsis,and formed double-stranded DNA by hybridization with the capture DNA (complementary with bio-barcode DNA) modified with sulfhydryl.Then double-stranded DNA were conjugated on the surface of 1D4-modified gold nanoparticles by sulfhydryl,and the Gold Nanoparticle Probe was produced.1G12 was precoated in the micropaltes,and in the presence of target recombinant HIV-1 p24 protein,a sandwich immuno-complex would form by adding GNP.Then the bio-barcode DNA in the immuno-complex were released by heating as detection signal,and consequently characterized by the polymerase chain reaction (PCR) with synthesized special primers and analyzed by 4％ agar gel electrophoresis,so HIV-1 p24 antigen could be evaluated.The sensitivity comparison between the new assay and ELISA can be done.ResultsSandwich ELISA was used to quantify HIV-1 p24 antigen by monoclonal antibodies 1G12 and 1D4,and the limit of detection (LOD) was 1000 pg/ml.The new GNP assay was established by the same pair of antibodies,combined with PCR and agar gel electrophoresis,and was used to indirectly detect HIV-1 p24 antigen.The band intensity of PCR products paralleled with the quantity of HIV-1 p24 antigen,and the limit of detection (LOD) could reach down to 1 pg/ml.ConclusionThe new assay based on GNP and PCR was efficient in the detection of HIV-1 p24,which is at least 3 orders of magnitude more sensitive than traditional ELISA.
ABSTRACT
Objective To evaluate NucliSens HIV-1 QT(bioMerieux,Netherlands) in quantitating human immunodeficiency virus type 1(HIV-1) RNA in whole semen or seminal plasma from HIV-1-infected people.Methods Five levels of HIV-1 RNA were spiked to whole semen,seminal plasma and blood plasma samples from healthy people,and then measured by NucliSens HIV-1 QT.The same method was used to measure HIV-1 RNA in seminal plasma and blood plasma samples from 15 HIV-1 infected people.Results Nucleic acid amplification inhibitors were found in whole semen but not in seminal plasma when using NucliSens HIV-1 QT.No significant difference was found between normal seminal plasma and blood plasma samples spiked with HIV-1 RNA,and no false positive result was found in 10 normal seminal plasma samples.For 15 cases of HIV-1 infection,HIV-1 was detected in 80%(12/15) of the plasma samples and 40%(6/15) of the seminal plasma samples,with viral loads of