Why is recombinase polymerase amplification becoming so popular?

Although PCR remains the gold-standard technique for molecular diagnostics assays, PCR misses the mark for applications that require fast results or environments where a sophisticated, cumbersome thermal cycler is impractical.¹ As a result, a wide range of isothermal amplification techniques have emerged to bring rapid testing into clinical or field settings. 

LAMP (loop-mediated isothermal amplification) is the most commonly used and widely recognised isothermal technique. However, RPA (recombinase polymerase amplification) has been the fastest-growing isothermal approach in the academic literature over the last five years. 

Figure 1. Number of publications in PubMed published each year that mention the listed techniques. 

A one-pot, isothermal technique 

TwistDx developed RPA in 2006 to advance the development of portable and widely accessible amplification assays.² This technique uses a recombinase, a single-strand DNA binding protein and a strand-displacing polymerase to achieve the same amplification as PCR, but at a constant temperature, typically 37-42 °C:  

1. The recombinase enables the primers to anneal to the complementary sequence in the double-stranded DNA template without the melting stage needed in PCR.  

2. The single-strand DNA binding protein prevents the primer from being replaced by binding to the displaced DNA strands. For this to work well, RPA primers tend to be longer than those used in PCR, typically 32 to 35 nucleotides.³ 

3. The strand-displacing polymerase then extends the primer to produce the amplicons. 
   

Figure 2. Schematic of the RPA reaction. The forward and reverse primers form recombinase nucleoprotein filaments. During strand invasion, the D-loop is stabilised by the single-strand binding protein. The DNA polymerase synthesises amplicons that are used as target DNA for further rounds of amplification. 

Unlike PCR, RPA cannot use Taq polymerase, as the 5′→3′ exonuclease activity digests the displaced strand. Instead, thermostable strand-displacing polymerases like Bsu or Bst are used.^4,5 This means that RPA requires a different structure for probes that do not rely on hydrolysis for real-time fluorescence detection. 

The exo probe contains a tetrohydrofuran (THF) as an abasic nucleotide analogue that is cleaved by exonuclease III when the probe hybridises to its complementary sequence. The fluorophore and black hole quencher groups that flank the THF are then separated, generating a fluorescent signal. Exo probes are often 46-52 nucleotides long.⁴  

The fpg probe (formamidopyrimidine DNA glycosylase) is typically 32-35 bases, i.e. shorter than the exo probe. It has a fluorophore attached to the ribose of an abasic nucleotide via a C-O-C linker, which can be cleaved by the Fpg nuclease. TwistDx has demonstrated that fpg probes, supplied by LGC Biosearch Technologies, can be used in lateral flow strips, further extending the settings and applications for testing.⁶ 

RPA reagents are available in a lyophilised format, making it simple to store and transport the kit wherever it is needed. The reagents can be stored for up to three weeks at 45°C without losing assay sensitivity.⁷ In addition to its versatility, RPA can yield results in less than 10 minutes.⁸ Like PCR, RPA can also be used to amplify RNA targets by adding a reverse transcriptase to the mix.⁴ 

Table 1. Comparison of the properties of RPA versus PCR. 

RPA works across a vast array of samples 

RPA assays using probes from Biosearch Technologies have been used to detect a wide range of pathogens across agriculture and human healthcare.  

• Yeh et al. developed a microfluidic chip that utilises RPA to detect MRSA directly from human blood samples within half an hour.⁹ 

• Larrea-Sarmiento et al. created a multiplex RPA assay for clavibacter – the first for any plant pathogen.¹⁰  

• Salazer et al. developed an RPA technique to detect Anaplasma to help improve detection in regions with limited access to laboratory equipment.¹¹   

• Choi et al. created a multiplex assay for bacteria related to food poisoning. This could allow on-site analysis that is fast and sensitive.¹² 

• Miles et al. developed an RPA assay for the plant pathogen phytophthora that could help make it quicker and easier to deploy testing in the field.¹³  

• Boyle et al. created an RPA assay for tuberculosis that could be developed into a point-of-care test for resource-constrained settings.¹⁴ 

RPA has been used to directly analyse a variety of crude samples, such as plant tissue extract, sap, soil, food and vaginal swab lysate.¹⁵ 

Popularity is driving continuous development 

RPA continues to develop further as researchers find new ways to extend its capabilities. In 2015, Abd El Wahed et al. miniaturised an RPA-based diagnostics kit to fit into a suitcase.¹⁶ The kit, containing a solar panel and power pack, was used to monitor outbreaks of avian influenza A (H7N9), dengue virus, Ebola and Leishmania donovani.¹⁷ 

Developing more complex single-tube multiplexed assays is limited by the difficulties in avoiding non-specific interactions between the various oligos. One way to overcome this was demonstrated by Song et al., who used a combination of RPA and LAMP on a microchip to conduct a 16-plex assay for pathogen detection.¹⁸

Feng Zhang’s research group at the Broad Institute used RPA combined with the CRISPR-Cas13 system to create the SHERLOCK platform to increase the sensitivity and specificity for detecting single base pair differences in RNA.¹⁹ This approach uses RPA to amplify the target input before transcribing it into RNA. Then, when CRISPR-Cas13 binds to the target sequence, it triggers widespread ‘collateral’ RNase activity that cleaves a reporter molecule, releasing the fluorescent signal.  

SHERLOCK can be used as a one-pot method, allowing detection within 15 minutes of femtomolar targets, or as a more sensitive two-step process. The reagents can be lyophilised and the technique can be deployed in the field, detecting Zika and Dengue virus directly from urine and serum samples.²⁰ Similarly, the DETECTR method developed by Jennifer Doudna’s lab uses RPA and Cas12a to identify single nucleotide variants in DNA.²¹ 

The rapid growth in popularity of RPA clearly demonstrates the need for fast, simple and versatile nucleic acid detection. Monitoring outbreaks of disease in particular requires tests that can be used at the point of need without specialist equipment or training. A single tube of readily available enzymes carrying out the succession of steps at body temperature will be an appealing prospect for many circumstances. The ongoing advancements in the field indicate a promising future for strengthening our ability to respond to health crises and improve outcomes. 

References 

1. Soroka M et al. (2021) Loop-Mediated Isothermal Amplification (LAMP): The Better Sibling of PCR? Cells. 10(8): 1931. doi: 10.3390/cells10081931

2. Piepenburg O et al. (2006) DNA Detection Using Recombination Proteins. PLOS Biology. 4(7): e204. doi: 10.1371/journal.pbio.0040204

3. Li J et al. (2019) Review: a comprehensive summary of a decade development of the recombinase polymerase amplification. Analyst. 144, 31-67 doi: 10.1039/C8AN01621F  

4. Daher RK et al. (2016) Recombinase Polymerase Amplification for Diagnostic Applications. Clinical Chemistry. 62(7): 947-958 doi: 10.1373/clinchem.2015.245829  

5. Kojima K et al. (2021) Solvent engineering studies on recombinase polymerase amplification. Journal of Bioscience and Bioengineering. 131(2): 219-224. doi: 10.1016/j.jbiosc.2020.10.001 

6. Powell ML et al. (2018) New Fpg probe chemistry for direct detection of recombinase polymerase amplification on lateral flow strips. Anal Biochem. 543:108-115. doi: 10.1016/j.ab.2017.12.003 

7. Lillis L et al. (2016) Factors influencing Recombinase polymerase amplification (RPA) assay outcomes at point of care. Molecular and Cellular Probes 30(2):74-78. doi: 10.1016/j.mcp.2016.01.009  

8. Aebischer A et al. (2014) Rapid Genome Detection of Schmallenberg Virus and Bovine Viral Diarrhea Virus by Use of Isothermal Amplification Methods and High-Speed Real-Time Reverse Transcriptase PCR. J Clin Microbiol. 52(6): 1883–1892. doi: 10.1128/JCM.00167-14  

9. Yeh EC et al. (2017) Self-powered integrated microfluidic point-of-care low-cost enabling (SIMPLE) chip. Science Advances 3(3) doi: 10.1126/sciadv.1501645 

10. Larrea-Sarmiento A et al. (2021) Multiplex recombinase polymerase amplification assay developed using unique genomic regions for rapid on-site detection of genus Clavibacter and C. nebraskensis. Scientific Reports 11: 12017 doi: 10.1038/s41598-021-91336-7  

11. Salazar A et al. (2021) Recombinase polymerase amplification (RPA) with lateral flow detection for three Anaplasma species of importance to livestock health. Scientific Reports 11:15962. doi: 10.1038/s41598-021-95402-y  

12. Choi G et al. (2016) A centrifugal direct recombinase polymerase amplification (direct-RPA) microdevice for multiplex and real-time identification of food poisoning bacteria. Lab on a Chip 16:2309-2316 doi: 10.1039/C6LC00329J  

13. Miles TD et al. (2015) Development of Rapid Isothermal Amplification Assays for Detection of Phytophthora spp. in Plant Tissue. Phytopathology, 105(2):265-278. doi:10.1094/PHYTO-05-14-0134-R

14. Boyle DS et al. (2014) Rapid Detection of Mycobacterium tuberculosis by Recombinase Polymerase Amplification. PLOS One. 9(8): e103091. doi: 10.1371/journal.pone.0103091  

15. Li J et al. (2019) Review: a comprehensive summary of a decade development of the recombinase polymerase amplification. Analyst, 144:31-67. doi:10.1039/C8AN01621F  

16. Abd El Wahed A et al. (2015) Diagnostics-in-a-Suitcase: Development of a portable and rapid assay for the detection of the emerging avian influenza A (H7N9) virus. Journal of Clinical Virology, 69, 16-21. doi:10.1016/j.jcv.2015.05.004 

17. Abd El Wahed A et al. (2015) Recombinase polymerase amplification assay for rapid diagnostics of Dengue infection. PLOS One 10(6), e0129682 doi:10.1371/journal.pone.0129682 

18. Song J et al. (2017) Two-Stage Isothermal Enzymatic Amplification for Concurrent Multiplex Molecular Detection. Clinical Chemistry, 63(3), 714-722. doi:10.1373/clinchem.2016.263665 

19. Kellner MJ et al. (2019) SHERLOCK: Nucleic acid detection with CRISPR nucleases. Nat Protoc 14(10):2986-3012 doi: 10.1038/s41596-019-0210-2  

20. Gootenberg JS et al. (2017) Nucleic acid detection with CRISPR-Cas13a/C2c2. Science 356(6336):438-442 doi: 10.1126/science.aam9321https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5526198/ 

21. Chen JS et al. (2019) CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science 360(6387):436-439 doi: 10.1126/science.aar6245  

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