06/02/2023
Amplification method: PCR vs Isothermal
Amplifying DNA samples prior to sequencing them is an important step that has become routine and sometimes even automated. PCR is the most common and widely known amplification method, but isothermal amplification is another option. Temperature changes are the main difference between isothermal and PCR amplification methods. In PCR, a thermal cycler changes the reaction temperatures repeatedly to affect the actions of the temperature-dependent reagents; in contrast, an isothermal amplification reaction occurs at a single temperature.
Isothermal amplification methods share the ability to amplify DNA at one temperature, as well as the use of strand-displacing DNA polymerases to “unzip” strands as they move along double-stranded DNA (rather than using heat to denature it as in PCR), allowing primers access to templates. Types of isothermal amplification methods include strand-displacement amplification (SDA), rolling-circle amplification (RCA), whole-genome amplification (WGA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), and multiple displacement amplification (MDA), among others. This article looks at the advantages of isothermal and PCR amplification methods, and when one method might be preferable over another.

PCR vs isothermal amplification:

Choosing an amplification method depends on the individual application and available equipment. While PCR is widely used for amplification, it requires access to a thermal cycler. This can be a disadvantage/deal-breaker for point-of-care and diagnostic applications. But if a thermal cycler is available, PCR amplification is a relatively straightforward process, and can be a better choice for rare transcripts. In contrast, isothermal amplification is ideal for situations in which there is no access to a thermal cycler (e.g., clinics, field work, home testing kits), and can sometimes be faster, simpler, and more cost-effective.
Sometimes isothermal amplification methods can have lower sensitivity and/or specificity when compared to PCR, but this can be ameliorated by optimizing reactions. However, isothermal amplification is quickly improving and driving innovation in COVID-19 testing.
Indeed, there are some situations where PCR amplification is still preferable—it can be a simpler means of detecting a target of interest exclusively. This is in contrast to Tecan’s Single Primer Isothermal Amplification (SPIA), a whole-transcriptome approach that allows users to detect the target of interest as well as other relevant sample information, such as co-pathogens or host information. The decision between SPIA and PCR amplification is dependent on the level of information that is required, If only limited information is required, then PCR may be sufficient, but if in-depth information about the sample is required, then SPIA can provide a better solution.
Isothermal amplification applications
Researchers use SPIA for rare transcript detection in microbiome and infectious disease applications to identify early signs of meningitis infection in cerebrospinal fluid. Researchers at Imperial College London and the University of Leicester used an allele-specific LAMP assay to detect a common driver mutation in breast cancer. Identifying this mutation early can aid clinicians and patients in making important treatment decisions.
Researchers are also using SPIA to study mosquito saliva, which is well known as a viral vector for Zika and dengue viruses. Noise suppression can remove high background, such as mosquito rRNA, enhancing the viral information recovered from the mosquito saliva.
Researchers have also been applying isothermal amplification methods to innovations in COVID-19 testing. For example, a researcher from Portugal recently developed a RT-LAMP-based colorimetric COVID-19 test that can be performed in one tube within 30 minutes, using nasopharyngeal swab or saliva samples, and is suitable for point-of-care or low-resource settings as it uses resources unlikely to encounter bottlenecks. While not quite as sensitive as the RT-PCR COVID-19 test, the RT-LAMP test is sensitive enough for surveillance and screening of infectious people who are asymptomatic or presymptomatic. The ability to curb the epidemic while not relying on scarce lab supplies will be advantageous.


References:
1. Ozay, B., et al. A review of reaction enhancement strategies for isothermal nucleic acid amplification reactions. Nov 2021. Sensors and Actuators Rep 3:100033
2. Kalofonou, M., et al. A novel hotspot specific isothermal amplification method for detection of the common PIK3CA p.H1047R breast cancer mutation. March 2020. Sci Rep 10:4553
3. Amaral, C., et al. Amaral, C., Antunes, W., Moe, E. et al. A molecular test based on RT-LAMP for rapid, sensitive and inexpensive colorimetric detection of SARS-CoV-2 in clinical samples. August 2021. Sci Rep 11, 16430.
4. Ludwig, K.U., et al. LAMP-Seq enables sensitive, multiplexed COVID-19 diagnostics using molecular barcoding. June 2021. Nat Biotechnol 39, 1556–1562.

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