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Updated: Jun 22, 2026
Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays
Published on: September 26, 2011
Low-cost HIV-1 diagnosis and quantification in dried blood spots by real time PCR
Nishaki Mehta1, Sonia Trzmielina, Bareng A S Nonyane
1Department of Pediatrics and Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.
Insights
A new real-time PCR assay using dried blood spots (DBS) offers rapid and affordable HIV-1 diagnosis and viral load monitoring. This method is crucial for early infant diagnosis and treatment in resource-limited settings.
Area of Science:
- Molecular Biology
- Virology
- Public Health
Background:
- Effective management of HIV-1 infection in adults and children, especially in resource-limited settings, requires rapid and cost-effective diagnostic and monitoring tools.
- Early diagnosis and treatment initiation, particularly for perinatally infected infants, are critical for improving outcomes.
- Dried blood spots (DBS) offer a convenient method for sample collection and transport, necessitating a reliable assay for HIV-1 analysis from DBS.
Purpose of the Study:
- To develop and validate a real-time LightCycler (rtLC) PCR assay for the detection and quantification of HIV-1 RNA from DBS.
- To assess the assay's performance characteristics, including speed, cost-effectiveness, accuracy, and reliability across different HIV-1 clades.
Main Methods:
- Development of a one-step, single-tube rtLC PCR assay utilizing primers specific for conserved long-terminal repeat sequences of HIV-1.
- Quantification of HIV-1 RNA using SYBR Green dye and a standard curve generated from known HIV-1 RNA concentrations.
- Validation against established commercial assays (Roche Ultrasensitive and branched DNA) and assessment of performance across various HIV-1 genotypes.
Main Results:
- The developed rtLC DBS assay demonstrated high accuracy and reliability, with a dynamic range of 5 log(10) and low coefficient of variation (<8% up to 4 log(10) dilution).
- The assay showed good correlation with commercial assays (r = 0.91 and r = 0.89) and a lower limit of detection of 136 copies.
- The rtLC DBS assay was found to be 2.5 times faster and 40 times cheaper than existing commercial methods, with similar performance across different real-time PCR systems.
Conclusions:
- The rtLC DBS assay is accurate, reliable, and inclusive of various HIV-1 genotypes.
- Its affordability and requirement for small blood volumes make it highly suitable for early diagnosis and monitoring of pediatric HIV-1 infection in resource-limited settings.
- The assay's rapid turnaround time and cost-effectiveness significantly enhance the potential for timely clinical management of HIV-1.
Background:
Rapid and cost-effective methods for HIV-1 diagnosis and viral load monitoring would greatly enhance the clinical management of HIV-1 infected adults and children in limited-resource settings. Recent recommendations to treat perinatally infected infants within the first year of life are feasible only if early diagnosis is routinely available. Dried blood spots (DBS) on filter paper are an easy and convenient way to collect and transport blood samples. A rapid and cost effective method to diagnose and quantify HIV-1 from DBS is urgently needed to facilitate early diagnosis of HIV-1 infection and monitoring of antiretroviral therapy.
Methods And Findings:
We have developed a real-time LightCycler (rtLC) PCR assay to detect and quantify HIV-1 from DBS. HIV-1 RNA extracted from DBS was amplified in a one-step, single-tube system using primers specific for long-terminal repeat sequences that are conserved across all HIV-1 clades. SYBR Green dye was used to quantify PCR amplicons and HIV-1 RNA copy numbers were determined from a standard curve generated using serially diluted known copies of HIV-1 RNA. This assay detected samples across clades, has a dynamic range of 5 log(10), and %CV <8% up to 4 log(10) dilution. Plasma HIV-1 RNA copy numbers obtained using this method correlated well with the Roche Ultrasensitive (r = 0.91) and branched DNA (r = 0.89) assays. The lower limit of detection (95%) was estimated to be 136 copies. The rtLC DBS assay was 2.5 fold rapid as well as 40-fold cheaper when compared to commercial assays. Adaptation of the assay into other real-time systems demonstrated similar performance.
Conclusions:
The accuracy, reliability, genotype inclusivity and affordability, along with the small volumes of blood required for the assay suggest that the rtLC DBS assay will be useful for early diagnosis and monitoring of pediatric HIV-1 infection in resource-limited settings.
