Nucleic Acid Quantification with Amplicon Yield in Recombinase Polymerase Amplification

Abstract

Amplification-based quantitative polymerase chain reaction (qPCR) provides accurate and sensitive nucleic acid quantification. However, the requirement of temperature cycling and real-time monitoring limits its translation to many settings. Quantitative isothermal amplification methods alleviate the need for thermal cyclers; however, they still require continuous monitoring of the nucleic acid amplification on sophisticated readers. Here, we adapted an isothermal recombinase polymerase amplification (RPA) reaction to develop a semiquantitative method that relies on the final amplicon yield to estimate the initial target nucleic acid copy number. To achieve this, we developed a phenomenological model that captures the essential RPA dynamics. We identified reaction conditions that constrained the reaction yield corresponding to the starting DNA template concentration. We validated these predictions experimentally and showed that the amplicon yields at the end of the RPA reaction correlated well with the starting DNA concentration while reducing nonspecific amplification robustly. We demonstrate this approach, termed quantitative endpoint RPA (qeRPA), to detect DNA over five log orders with a detection limit of 100 molecules. Using a linear regression model of the normalized endpoint intensity (NEI) standard curve, we estimate the viral load from the serum of dengue virus-infected patients with comparable performance to qPCR. Unlike the conventional isothermal quantitative methods, qeRPA can be employed for robust and sensitive nucleic acid estimation at close to room temperature without real-time monitoring and can be beneficial for field deployment in resource-limited settings.

Tuning the Dynamic Reaction Balance of CRISPR/Cas12a and RPA in One Pot: A Key to Switch Nucleic Acid Quantification

“…Importantly, the state of dynamic reaction could be identified by calculating the R 2 values from linear fitting curves. When R 2 values were higher than 0.9, ,, the dynamic reaction was thought to be at a near-quantifiable state that might be utilized to quantify DNA targets. Remarkably, other factors such as dNTP mix, reporter, and reaction volume also influence the detection performance .…”

“…The detection of nucleic acids plays a key role in monitoring microbe abundance and identifying pathogen or virus infections. Precise and accurate nucleic acid quantification is crucial for assessing disease risks and formulating correct treatments in medical diagnostics, food processing and safety, and environmental monitoring. − Quantitative real-time polymerase chain reaction (qPCR) has been regarded as a gold standard over the past decade . However, the demand for sophisticated laboratory thermal cycling equipment and trained personnel becomes a roadblock in areas of low resources. , Therefore, it is still necessary to develop nucleic acid detection technologies with absolute quantification, easy operation, low cost, and robust repeatability for further and widespread applications.…”

Glow-in-the-Dark Infectious Disease Diagnostics Using CRISPR-Cas9-Based Split Luciferase Complementation

“…This method provided excellent sensitivity, showing an increase in blue luminescence for inputs down to 2 copies of target DNA, detecting 3.3 aM in a 1 μL sample (Figure B). The assay response reached a plateau at an input ≥200 copies, which suggests that the RPA reaction was exhausted, yielding the same maximal amplicon output for higher input copy numbers …”

“…The assay response reached a plateau at an input ≥200 copies, which suggests that the RPA reaction was exhausted, yielding the same maximal amplicon output for higher input copy numbers. 42 Next we explored whether RPA and LUNAS amplicon detection could be performed simultaneously in a one-pot reaction. For this, the LUNAS reaction mixture including substrate was added to the RPA reaction with RPA components at default concentrations, and luminescence was monitored while incubating the 20 μL mixture at 40 °C (Figure 3C).…”

Rapid Isothermal Detection of Pathogenic Clostridioides difficile Using Recombinase Polymerase Amplification

“…In comparison, in almost exponential PCR, the natural variation of C T values equates to ±1.43 C T , which calculates to a 2–3 fold difference in absolute quantification values . Recently, it was shown that by using Twist RPA pellet chemistry diluted by 1:2 quantitative RPA can be achieved . This essential slowing down of the RPA reaction is also reflected by various mathematical approaches that have been put forward to allow calculating quantitative RPA results from unchanged RPA reactions. − The natural variation of RPA in a slowed-down mode might be very similar to that of PCR, but this remains to be demonstrated.…”