Advances in Quantification for Isothermal Diagnostic Systems

Qualitative “yes-or-no” assays can provide simple yet powerful diagnostic detection solutions, with their utility exemplified during the COVID-19 pandemic. However, there are cases where quantification is required, including applications in personalised medicine. For example, quantifying hepatitis B viral load allows clinicians to tailor treatment dosages, resulting in improved clinical outcomes with reduced side-effects. Ideally, these tests should have the capacity to operate in Point-of-Care (PoC) settings and deliver results within a short amount of time (>30 mins) to facilitate real-time treatment decisions. Quantitative assays also have applications outside the clinical diagnostics arena, assisting in environmental testing such as water quality screening, food safety, and agriculture.

Polymeric chain reaction (PCR), and its variants qualitative PCR (qPCR) and real time PCR (RT-PCR), are considered the gold-standard for DNA amplification, particularly for quantification.

Advancements in rapid PCR techniques have greatly reduced overall assay run-time, however PCR still faces limitations including reliance on complex and expensive thermocyclers often requiring off-site sample processing.[1]

While PCR remains the gold-standard for qualitative amplification isothermal techniques may offer advantages including:

• High sensitivity

• Simplified workflows, reducing contamination risk

• Reduced run time

• Decreased processing time

• Decreased susceptibility to inhibitors [2,3]

In addition, isothermal amplification techniques, including LAMP and RPA, run at a constant temperature eliminating the need for complex thermocycling equipment. A major drawback of isothermal assays, however, is the lack of reliable quantification methods.

Download the poster below to see how Axxin instruments demonstrate the use of temperature ramping over a narrow range to facilitate the quantification of isothermal assays.

D014268v1 Axxin Poster, New Advances in Quantification for Isothermal Diagnostic Systems.

Download • 3.09MB

References:

[1] Zhu, H., Zhang, H., Xu, Y., Laššáková, S., Korabečná, M., & Neužil, P. (2020). PCR Past, Present and Future, BioTechniques, 69(4), 317–325. doi:10.2144/btn-2020-0057

[2] Soroka, M., Wasowicz  B., & Rymaszewska, A. (2021). Loop-Mediated Isothermal Amplification (LAMP): The Better Sibling of PCR?, Cells, 10, 1931. doi:10.3390/cells10081931

[3] Bodulev, O. L., & Sakharov, I. Yu. (2020). Isothermal Nucleic Acid Amplification Techniques and Their Use in Bioanalysis, Biochemistry, 85(2), 147-166. doi:10.1134/S0006297920020030