New design, development, and optimization of an in-house quantitative TaqMan Real-time PCR assay for HIV-1 viral load measurement

2018 ◽  
Vol 19 (2) ◽  
pp. 61-68 ◽  
Author(s):  
Hassan Noorbazargan ◽  
Seyed Alireza Nadji ◽  
Siamak Mirab Samiee ◽  
Mahdi Paryan ◽  
Samira Mohammadi-Yeganeh
Keyword(s):  
Viral Load ◽  
Real Time ◽  
Real Time Pcr ◽  
Design Development ◽  
Viral Load Measurement ◽  
Pcr Assay ◽  
Load Measurement ◽  
Hiv 1

scholarly journals Performance of a True Point-of-Care Assay for HIV-1/2 Viral Load Measurement at Antenatal and Postpartum Services

2021 ◽  
Vol Publish Ahead of Print ◽  
Author(s):  
B. Meggi ◽  
T. Bollinger ◽  
A. Zitha ◽  
C. Mudenyanga ◽  
A. Vubil ◽  
...  
Keyword(s):  
Viral Load ◽  
Point Of Care ◽  
Viral Load Measurement ◽  
Load Measurement ◽  
Hiv 1 ◽  
True Point ◽  
Point Of Care Assay

Quantitation of HIV-1 RNA levels in plasma and CSF of asymptomatic HIV-1 infected patients from South India using a TaqMan real time PCR assay

2007 ◽  
Vol 39 (1) ◽  
pp. 9-15 ◽  
Author(s):  
Anupa Kamat ◽  
V. Ravi ◽  
Anita Desai ◽  
P. Satishchandra ◽  
K.S. Satish ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
South India ◽  
Pcr Assay ◽  
Hiv 1 ◽  
Asymptomatic Hiv ◽  
Rna Levels

scholarly journals A Simplified Quantitative Real-Time PCR Assay for Monitoring SARS-CoV-2 Growth in Cell Culture

mSphere ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Christian Shema Mugisha ◽  
Hung R. Vuong ◽  
Maritza Puray-Chavez ◽  
Adam L. Bailey ◽  
Julie M. Fox ◽  
...  
Keyword(s):  
Cell Culture ◽  
Real Time ◽  
Real Time Pcr ◽  
Rna Extraction ◽  
Quantitative Real Time Pcr ◽  
Pcr Assay ◽  
Dna Viruses ◽  
Culture Supernatants ◽  
Hiv 1 ◽  
Rna And Dna

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected millions within just a few months, causing severe respiratory disease and mortality. Assays to monitor SARS-CoV-2 growth in vitro depend on time-consuming and costly RNA extraction steps, hampering progress in basic research and drug development efforts. Here, we developed a simplified quantitative real-time PCR assay that bypasses viral RNA extraction steps and can monitor SARS-CoV-2 growth from a small amount of cell culture supernatants. In addition, we show that this approach is easily adaptable to numerous other RNA and DNA viruses. Using this assay, we screened the activities of a number of compounds that were predicted to alter SARS-CoV-2 entry and replication as well as HIV-1-specific drugs in a proof-of-concept study. We found that E64D (inhibitor of endosomal proteases cathepsin B and L) and apilimod (endosomal trafficking inhibitor) potently decreased the amount of SARS-CoV-2 RNA in cell culture supernatants with minimal cytotoxicity. Surprisingly, we found that the macropinocytosis inhibitor ethylisopropylamiloride (EIPA) similarly decreased SARS-CoV-2 RNA levels in supernatants, suggesting that entry may additionally be mediated by an alternative pathway. HIV-1-specific inhibitors nevirapine (a nonnucleoside reverse transcriptase inhibitor [NNRTI]), amprenavir (a protease inhibitor), and allosteric integrase inhibitor 2 (ALLINI-2) modestly inhibited SARS-CoV-2 replication, albeit the 50% inhibitory concentration (IC50) values were much higher than that required for HIV-1. Taking the data together, this simplified assay will expedite basic SARS-CoV-2 research, be amenable to mid-throughput screening assays (i.e., drug, CRISPR, small interfering RNA [siRNA], etc.), and be applicable to a broad number of RNA and DNA viruses. IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of the coronavirus disease 2019 (COVID-19) pandemic, is continuing to cause immense respiratory disease and social and economic disruptions. Conventional assays that monitor SARS-CoV-2 growth in cell culture rely on costly and time-consuming RNA extraction procedures, hampering progress in basic SARS-CoV-2 research and development of effective therapeutics. Here, we developed a simple quantitative real-time PCR assay to monitor SARS-CoV-2 growth in cell culture supernatants that does not necessitate RNA extraction and that is as accurate and sensitive as existing methods. In a proof-of-concept screen, we found that E64D, apilimod, EIPA, and remdesivir can substantially impede SARS-Cov-2 replication, providing novel insight into viral entry and replication mechanisms. In addition, we show that this approach is easily adaptable to numerous other RNA and DNA viruses. This simplified assay will undoubtedly expedite basic SARS-CoV-2 and virology research and be amenable to use in drug screening platforms to identify therapeutics against SARS-CoV-2.

A rapid real-time PCR assay for the detection of HIV-1 proviral DNA using double-stranded primer

2010 ◽  
Vol 164 (1-2) ◽  
pp. 55-62 ◽  
Author(s):  
Chou-Pong Pau ◽  
Susan K. Wells ◽  
Donna L. Rudolph ◽  
S. Michele Owen ◽  
Timothy C. Granade
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pcr Assay ◽  
Proviral Dna ◽  
Hiv 1

scholarly journals Development of a fluorescent quantitative real-time polymerase chain reaction assay for the detection of novel-goose parvovirus in vivo

2021 ◽  
Author(s):  
Haibin Ma ◽  
Yahui Li ◽  
Junzheng Yang
Keyword(s):  
Viral Load ◽  
Real Time ◽  
Real Time Pcr ◽  
Bursa Of Fabricius ◽  
Pcr Assay ◽  
Standard Curve ◽  
Goose Parvovirus ◽  
Detection And Quantification

Objectives: To develop a sensitive, highly specific fluorescent quantitative real-time PCR assay for accurate detection and quantification of novel-goose parvovirus (N-GPV) in vitro and in vivo. Methods: Specific primers was designed based on N-GPV inverted terminal repeats region; virus RNA (DFV, NDV, AIV, DHV-1, DHV-3) and virus DNA (MDPV, GPV, N-GPV) were extracted, cDNA (DFV, NDV, AIV, DHV-1, DHV-3) were prepared from viral RNAs using M-MLV Reverse Transcriptase, and prepared cDNA (DFV, NDV, AIV, DHV-1, DHV-3) and DNA (MDPV, GPV, N-GPV) amplified by real-time PCR; the sensitivity, specificity and reproducibility of established real-time PCR methods were evaluated, and finally we validated the reliability of real-time PCR methods in ducklings models in vivo. Results: The standard curve of established real-time PCR had a good linearity (slope was -0.3098, Y-intercept was 37.865, efficiency of standard curve was 0.995); the detection limit of established real-time PCR for N-GPV was 10 copies/reaction. The sensitivity of real-time PCR was 10 copies/uL, which was 1000 times higher than conventional gel-based PCR assay. The results of intra-assay CVs (0.04-0.74%) and inter-assay CVs (0.16-0.53%) showed that the real-time PCR assay had an excellent repeatability. This method also could efficiently detect viral load in heart, liver, spleen, lung, kidney, pancreas, bursa of Fabricius, brain, blood and excrement from ducklings models after N-GPV infection from 6h to 28 days, which could provided us a dynamic distribution observation of N-GPV viral load using this real-time PCR assay in vivo. Conclusion: In the study, we developed a high sensitive, specific and reproducible real-time PCR assay for N-GPV detection. The established real-time PCR assay was suitable for parvovirus detection and quantification simultaneously, no matter sample obtained from blood, internal organs or ileac contents; the present work may provide insight into the pathogenesis of N-GPV and will contributes to better understanding of this newly emerged novel GPV related virus in cherry valley ducks.

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