Development of an Ultrasensitive HIV-1 DNA Detection Assay Based on an Automated πCode End-Point PCR System

doi:10.33696/aids.1.010

Cited by 4 publications

(5 citation statements)

References 70 publications

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“…16 In our study, we also measured the Suzuki K, et al 17 demonstrated that HIV-1 DNA detection with the novel "πCode end-point PCR assay" is 100% sensitive, while the real-time PCR showed 92.3% in a head-to-head comparison in samples from HIV-1 infected individuals. 17 We have also focused the real time RT PCR assay to detect one copy of proviral HIV-1 DNA with 100% sensitivity.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of a Quantitative Taqman Real-Time PCR Assay to Measure Proviral load from Human Immunodeficiency Virus Type 1 individuals

Suguna¹,

Saikumar²,

Tilton³

et al. 2022

J. Pure Appl. Microbiol.

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Human Immunodeficiency Virus (HIV) is a virus belonging to the family Retroviridae. HIV – 1 is found to be predominant in India and many parts of Africa. The intention of this study was to quantify the HIV Proviral Deoxyribonucleic Acid (DNA) from newly infected HIV-1 individuals. Fifty patients who were tested positive for HIV were included in this study. Proviral Ribo Nucleic Acid (RNA) was extracted by QIAmp® RNA Mini Kit (QIAGEN, Germany) method. Complementary Deoxyribo Nucleic Acid (cDNA) was synthesized by using Invitrogen Superscript III cDNA synthesis Kit (USA). This cDNA was subjected to Polymerase Chain Reaction (PCR) and Gene cloning by transformation method. The quantification of Real time PCR was done by Applied Bio-System (ABI)-Prism 7700. A linear standard curve was obtained 10 copies to 106 copies per reaction. The assay had good analytic sensitivity and linear dynamic range greater than 6 logs. From the results obtained in this study, It was concluded that Taqman Real-Time PCR Assay plays a major role in monitoring the HIV infected patients in routine diagnostics and clinical practice.

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Section: Discussionmentioning

confidence: 99%

Evaluation of a Quantitative Taqman Real-Time PCR Assay to Measure Proviral load from Human Immunodeficiency Virus Type 1 individuals

Suguna¹,

Saikumar²,

Tilton³

et al. 2022

J. Pure Appl. Microbiol.

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“…The corresponding concentration plot reveals that increased variations in the statistical distribution of the samples can lead to fluctuations in the absolute value when the same sample ( Bacillus subtilis 16S rDNA, 619 bp) is repeatedly quantified. The reliability of the automated threshold classification provided by the QuantaSoft companion software from the Bio-Rad QX100 Digital Droplet PCR system is questionable. − As shown in Figure S1, estimating the center of the positive and negative clusters is easy. However, it is difficult to classify the partitions that lie between those centers.…”

Section: Introductionmentioning

confidence: 99%

A Poisson-Independent Approach to Precision Nucleic Acid Quantification in Microdroplets

Ganguly,

Lee

2024

ACS Appl. Bio Mater.

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Digital PCR (dPCR) has become indispensable in nucleic acid (NA) detection across various fields, including viral diagnostics and mutant detection. However, misclassification of partitions in dPCR can significantly impact accuracy. Despite existing methods to minimize misclassification bias, accurate classification remains elusive, especially for nonamplified target partitions. To address these challenges, this study introduces an innovative microdroplet-based competitive PCR platform for nucleic acid quantification in microfluidic devices independent of Poisson statistics. In this approach, the target concentration (T) is determined from the concentration of competitor DNA (C) at the equivalence point (E.P.), where C/T is 1. Competitive PCR ensures that the ratio of target to competitor DNA remains constant during amplification, reflected in the resultant fluorescence intensity, allowing the quantification of target DNA concentration at the equivalence point. The unique amplification technique eliminates Poisson distribution, addressing misclassification challenges. Additionally, our approach reduces the need for post-PCR procedures and shortens analytical time. We envision this platform as versatile, reproducible, and easily adaptable for driving significant progress in molecular biology and diagnostics.

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“…Real-time PCR is providing the same Yes/No result for the presence/absence of a short length nucleotide sequence that endpoint PCR can provide at lower cost and higher throughput. Endpoint PCR has been demonstrated in forensic science 27 and for detection of other infectious diseases such as Hepatitis B, 28 Ebola, 29 and HIV 30 with some authors indicating endpoint methods are more sensitive than real-time. 30 Here, we investigated endpoint PCR as an alternative to real-time PCR for COVID-19 (SARS-CoV-2 RNA) testing.…”

Section: Introductionmentioning

confidence: 99%

“…Endpoint PCR has been demonstrated in forensic science 27 and for detection of other infectious diseases such as Hepatitis B, 28 Ebola, 29 and HIV 30 with some authors indicating endpoint methods are more sensitive than real-time. 30 Here, we investigated endpoint PCR as an alternative to real-time PCR for COVID-19 (SARS-CoV-2 RNA) testing. We compare the two techniques on authentic positive samples collected at Kent hospitals during the pandemic and report detection limits using hospital patient samples and a dilution series using synthetic SARS-COV-2 sequences.…”

Section: Introductionmentioning

confidence: 99%

Endpoint PCR Detection of Sars-CoV-2 RNA

Moses

Warren

Robinson³

et al. 2020

Preprint

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Quantitative real-time PCR methods have been used to perform approximately 278 million tests for COVID-19 up to mid-July 2020. Real-time PCR involves a rate limiting step where the samples are measured in situ during each PCR amplification cycle. This creates a bottleneck limiting scalability and as a consequence reducing access to inexpensive reliable testing at national and international scales. We investigated endpoint PCR for the qualitative detection of SARS-CoV-2 sequences on synthetic RNA standards and hospital patient samples. The endpoint PCR detection limit is constrained only by the stochastics of low copy numbers and reliably detected single copies of synthetic RNA standards. On a set of 30 patient samples, endpoint PCR found one additional positive sample and was able to confirm an indeterminate sample as negative. These results were found using 4 μl reagent and 1 μl of sample representing an 80% reduction relative to the NHS protocol (20 μl reagent and 5 μl sample). These results indicate that endpoint PCR should be the method of choice for large scale testing programmes. Based on the experience from ultra-high throughput genotyping efforts a single workflow using 384-well plates has similar PCR capacity (250 Million) to that required for all testing done worldwide during the first 7 month of the pandemic.

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Development of an Ultrasensitive HIV-1 DNA Detection Assay Based on an Automated πCode End-Point PCR System

Cited by 4 publications

References 70 publications

Evaluation of a Quantitative Taqman Real-Time PCR Assay to Measure Proviral load from Human Immunodeficiency Virus Type 1 individuals

Evaluation of a Quantitative Taqman Real-Time PCR Assay to Measure Proviral load from Human Immunodeficiency Virus Type 1 individuals

A Poisson-Independent Approach to Precision Nucleic Acid Quantification in Microdroplets

Endpoint PCR Detection of Sars-CoV-2 RNA

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