scholarly journals Quadruplex qPCR for qualitative and quantitative analysis of the HIV-1 latent reservoir

2019 ◽  
Author(s):  
Christian Gaebler ◽  
Julio C. C. Lorenzi ◽  
Thiago Y. Oliveira ◽  
Lilian Nogueira ◽  
Victor Ramos ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Quantitative Polymerase Chain Reaction ◽  
Specific Antigen ◽  
Latent Reservoir ◽  
Next Generation ◽  
Sequencing Method ◽  
Generation Sequencing ◽  
Hiv 1 ◽  
Next Generation Sequencing Method

AbstractHIV-1 infection requires life-long therapy with anti-retroviral drugs due to the existence of a latent reservoir of transcriptionally inactive integrated proviruses. The goal of HIV-1 cure research is to eliminate or functionally silence this reservoir. To this end there are numerous ongoing studies to evaluate immunologic approaches including monoclonal antibody therapies. Evaluating the results of these studies requires sensitive and specific measures of the reservoir. Here we describe a relatively high throughput combined quantitative polymerase chain reaction (qPCR) and next generation sequencing method. Four different qPCR probes covering the packaging signal (PS), group-specific antigen (gag), polymerase (pol), and envelope (env) are combined in a single multiplex reaction to detect the HIV-1 genome in limiting dilution samples followed by sequence verification of individual reactions that are positive for combinations of any 2 of the 4 probes (Q4PCR). This sensitive and specific approach allows for an unbiased characterization of the HIV-1 latent reservoir.SummaryHIV-1 cure research seeks to decrease or eliminate the latent reservoir. The evaluation of such curative strategies requires accurate measures of the reservoir. Gaebler et al. describe a combined multicolor qPCR and next generation sequencing method that enables the sensitive and specific characterization of the HIV-1 latent reservoir.

scholarly journals Combination of quadruplex qPCR and next-generation sequencing for qualitative and quantitative analysis of the HIV-1 latent reservoir

2019 ◽  
Vol 216 (10) ◽  
pp. 2253-2264 ◽  
Author(s):  
Christian Gaebler ◽  
Julio C.C. Lorenzi ◽  
Thiago Y. Oliveira ◽  
Lilian Nogueira ◽  
Victor Ramos ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Specific Antigen ◽  
Antiretroviral Drugs ◽  
Latent Reservoir ◽  
Next Generation ◽  
Packaging Signal ◽  
Sequencing Method ◽  
Multiplex Reaction ◽  
Generation Sequencing ◽  
Hiv 1

HIV-1 infection requires lifelong therapy with antiretroviral drugs due to the existence of a latent reservoir of transcriptionally inactive integrated proviruses. The goal of HIV-1 cure research is to eliminate or functionally silence this reservoir. To this end, there are numerous ongoing studies to evaluate immunological approaches, including monoclonal antibody therapies. Evaluating the results of these studies requires sensitive and specific measures of the reservoir. Here, we describe a relatively high-throughput combined quantitative PCR (qPCR) and next-generation sequencing method. Four different qPCR probes covering the packaging signal (PS), group-specific antigen (gag), polymerase (pol), and envelope (env) are combined in a single multiplex reaction to detect the HIV-1 genome in limiting dilution samples followed by sequence verification of individual reactions that are positive for combinations of any two of the four probes (Q4PCR). This sensitive and specific approach allows for an unbiased characterization of the HIV-1 latent reservoir.

scholarly journals Development of a Next-Generation Sequencing Method for BRCA Mutation Screening

2012 ◽  
Vol 14 (6) ◽  
pp. 602-612 ◽  
Author(s):  
Maurice Chan ◽  
Shen Mo Ji ◽  
Zhen Xuan Yeo ◽  
Linda Gan ◽  
Eric Yap ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Brca Mutation ◽  
Mutation Screening ◽  
Next Generation ◽  
Sequencing Method ◽  
Generation Sequencing ◽  
Next Generation Sequencing Method

scholarly journals Subacute Thyroiditis is Associated with HLA-B*18:01, -DRB1*01 and -C*04:01—The Significance of the New Molecular Background

2020 ◽  
Vol 9 (2) ◽  
pp. 534
Author(s):  
Magdalena Stasiak ◽  
Bogusław Tymoniuk ◽  
Renata Michalak ◽  
Bartłomiej Stasiak ◽  
Marek L. Kowalski ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Human Leukocyte ◽  
Subacute Thyroiditis ◽  
Control Group ◽  
Next Generation ◽  
Sequencing Method ◽  
Triggering Factor ◽  
Almost All ◽  
Generation Sequencing ◽  
Next Generation Sequencing Method

Subacute thyroiditis (SAT) is a thyroid inflammatory disease whose pathogenesis is still not completely defined. Previous viral infection is considered to be a triggering factor in genetically predisposed individuals. In about 70% of patients, susceptibility to SAT is associated with the HLA-B*35 allele. The correlation between SAT and other human leukocyte antigens (HLA) has not yet been unequivocally demonstrated and the genetic background is still unknown in about 30% of patients. The purpose of our study was to perform HLA genotyping using a next-generation sequencing method, to find out whether alleles other than HLA-B*35 are correlated with SAT morbidity. HLA-A, -B, -C, -DQB1, -DRB1 were genotyped using a next-generation sequencing method in 1083 subjects, including 60 SAT patients and 1023 healthy controls. Among 60 patients diagnosed with SAT, 81.7% of subjects were identified as having allele HLA-B*35, 23.3% had HLA-B*18:01, 28.3% had HLA-DRB1*01 and 75.5% had HLA-C*04:01. These alleles occurred in the control group at frequencies of 10.2%, 7.2%, 12.9% and 12.5%, respectively. The differences were statistically significant, with p < 0.05. In addition to its previously described relationship with HLA-B*35, genetic susceptibility to SAT was associated with the presence of HLA-B*18:01, DRB1*01 and C*04:01. The alleles HLA-B*18:01 and DRB1*01 were independent SAT risk factors. The assessment of these four alleles allows the confirmation of genetic predisposition in almost all patients with SAT.

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