Recombinase polymerase amplification

Last updated

Recombinase polymerase amplification (RPA) is a single tube, isothermal alternative to the polymerase chain reaction (PCR). [1] By adding a reverse transcriptase enzyme to an RPA reaction, it can detect RNA as well as DNA, without the need for a separate step to produce cDNA. [2] [3] [4] Because it is isothermal, RPA can use much simpler equipment than PCR, which requires a thermal cycler. Operating best at temperatures of 37–42 °C and still working, albeit more slowly, at room temperature means RPA reactions can in theory be run quickly by simply holding a tube in the hand. This makes RPA an excellent candidate for developing low-cost, rapid, point-of-care molecular tests. An international quality assessment of molecular detection of Rift Valley fever virus performed as well as the best RT-PCR tests, detecting less concentrated samples missed by some PCR tests and an RT-LAMP test. [5] RPA was developed and launched by TwistDx Ltd. (formerly known as ASM Scientific Ltd), a biotechnology company based in Cambridge, UK.

Contents

Technique

The RPA process employs three core enzymes – a recombinase, a single-stranded DNA-binding protein (SSB) and strand-displacing polymerase.

Recombinases are capable of pairing oligonucleotide primers with homologous sequence in duplex DNA. [1]

SSB bind to displaced strands of DNA and prevent the primers from being displaced.

Finally, the strand displacing polymerase begins DNA synthesis where the primer has bound to the target DNA.

By using two opposing primers, much like PCR, if the target sequence is indeed present, an exponential DNA amplification reaction is initiated. No other sample manipulation such as thermal or chemical melting is required to initiate amplification. At optimal temperatures (37–42 °C), the reaction progresses rapidly and results in specific DNA amplification from just a few target copies to detectable levels, typically within 10 minutes, for rapid detection of viral genomic DNA or RNA, [2] [3] [4] [6] [7] [8] pathogenic bacterial genomic DNA, [9] [10] as well as short length aptamer DNA. [11]

The three core RPA enzymes can be supplemented by further enzymes to provide extra functionality. Addition of exonuclease III allows the use of an exo probe for real-time, fluorescence detection akin to real-time PCR. [1] Addition of endonuclease IV means that an nfo probe can be used for lateral flow strip detection of successful amplification. [1] [6] [12] If a reverse transcriptase that works at 37–42 °C is added then RNA can be reverse transcribed and the cDNA produced amplified all in one step. Currently only the TwistAmp exo version of RPA is available with the reverse transcriptase included, although users can simply supplement other TwistAmp reactions with a reverse transcriptase to produce the same effect. As with PCR, all forms of RPA reactions can be multiplexed by the addition of further primer/probe pairs, allowing the detection of multiple analytes or an internal control in the same tube.

Relationship to other amplification techniques

RPA is one of several isothermal nucleic acid amplification techniques to be developed as a molecular diagnostic technique, frequently with the objective of simplifying the laboratory instrumentation required relative to PCR. A partial list of other isothermal amplification techniques include LAMP, NASBA, helicase-dependent amplification (HDA), and nicking enzyme amplification reaction (NEAR). The techniques differ in the specifics of primer design and reaction mechanism, and in some cases (like RPA) make use of cocktails of two or more enzymes. Like RPA, many of these techniques offer rapid amplification times with the potential for simplified instrumentation, and reported resistance to substances in unpurified samples that are known to inhibit PCR. With respect to amplification time, modern thermocyclers with rapid temperature ramps can reduce PCR amplification times to less than 30 minutes, particularly for short amplicons using dual-temperature cycling rather than the conventional three-temperature protocols. [13] In addition, the demands of sample prep (including lysis and extraction of DNA or RNA, if necessary) should be considered as part of the overall time and complexity inherent to the technique. These requirements vary according to the technique as well as to the specific target and sample type.

Compared to PCR, the guidelines for primer and probe design for RPA are less established, and may take a certain degree of trial and error, although recent results indicate that standard PCR primers can work as well. [14] The general principle of a discrete amplicon bounded by a forward and reverse primer with an (optional) internal fluorogenic probe is similar to PCR. PCR primers may be used directly in RPA, but their short length means that recombination rates are low and RPA will not be especially sensitive or fast. Typically 30–38 base primers are needed for efficient recombinase filament formation and RPA performance. This is in contrast to some other techniques such as LAMP which use a larger number of primers subject to additional design constraints. Although the original 2006 report of RPA describes a functional set of reaction components, the current (proprietary) formulation of the TwistAmp kit is "substantially different" [15] and is available only from the TwistDx supplier. This is in comparison to reaction mixtures for PCR which are available from many suppliers, or LAMP or NASBA for which the composition of the reaction mixture is freely published, allowing researchers to create their own customized "kits" from inexpensive ingredients.

Published scientific literature generally lacks detailed comparison of the performance of isothermal amplification techniques such as RPA, HDA, and LAMP relative to each other, often rather comparing a single isothermal technique to a "gold standard" PCR assay. This makes it difficult to judge the merits of these techniques independently from the claims of the manufacturers, inventors, or proponents. Furthermore, performance characteristics of any amplification technique are difficult to decouple from primer design: a "good" primer set for one target for RPA may give faster amplification or more sensitive detection than a "poor" LAMP primer set for the same target, but the converse may be true for different primer sets for a different target. An exception is a recent study comparing RT-qPCR, RT-LAMP, and RPA for detection of Schmallenberg virus and bovine viral diarrhea virus, [16] which effectively makes the point that each amplification technique has strengths and weaknesses, which may vary by the target, and that the properties of the available amplification techniques need to be evaluated in combination with the requirements for each application. As with PCR and any other amplification technique, there is obviously a publication bias, with poorly performing primer sets rarely deemed worthy of reporting.

Related Research Articles

<span class="mw-page-title-main">Complementary DNA</span> DNA reverse transcribed from RNA

In genetics, complementary DNA (cDNA) is DNA that was reverse transcribed from an RNA. cDNA exists in both single-stranded and double-stranded forms and in both natural and engineered forms.

<span class="mw-page-title-main">Polymerase chain reaction</span> Laboratory technique to multiply a DNA sample for study

The polymerase chain reaction (PCR) is a method widely used to make millions to billions of copies of a specific DNA sample rapidly, allowing scientists to amplify a very small sample of DNA sufficiently to enable detailed study. PCR was invented in 1983 by American biochemist Kary Mullis at Cetus Corporation. Mullis and biochemist Michael Smith, who had developed other essential ways of manipulating DNA, were jointly awarded the Nobel Prize in Chemistry in 1993.

<span class="mw-page-title-main">Primer (molecular biology)</span> Short strand of RNA or DNA that serves as a starting point for DNA synthesis

A primer is a short, single-stranded nucleic acid used by all living organisms in the initiation of DNA synthesis. A synthetic primer may also be referred to as an oligo, short for oligonucleotide. DNA polymerase enzymes are only capable of adding nucleotides to the 3’-end of an existing nucleic acid, requiring a primer be bound to the template before DNA polymerase can begin a complementary strand. DNA polymerase adds nucleotides after binding to the RNA primer and synthesizes the whole strand. Later, the RNA strands must be removed accurately and replace them with DNA nucleotides forming a gap region known as a nick that is filled in using an enzyme called ligase. The removal process of the RNA primer requires several enzymes, such as Fen1, Lig1, and others that work in coordination with DNA polymerase, to ensure the removal of the RNA nucleotides and the addition of DNA nucleotides. Living organisms use solely RNA primers, while laboratory techniques in biochemistry and molecular biology that require in vitro DNA synthesis usually use DNA primers, since they are more temperature stable. Primers can be designed in laboratory for specific reactions such as polymerase chain reaction (PCR). When designing PCR primers, there are specific measures that must be taken into consideration, like the melting temperature of the primers and the annealing temperature of the reaction itself. Moreover, the DNA binding sequence of the primer in vitro has to be specifically chosen, which is done using a method called basic local alignment search tool (BLAST) that scans the DNA and finds specific and unique regions for the primer to bind.

<span class="mw-page-title-main">Reverse transcriptase</span> Enzyme which generates DNA

A reverse transcriptase (RT) is an enzyme used to convert RNA genome to DNA, a process termed reverse transcription. Reverse transcriptases are used by viruses such as HIV and hepatitis B to replicate their genomes, by retrotransposon mobile genetic elements to proliferate within the host genome, and by eukaryotic cells to extend the telomeres at the ends of their linear chromosomes. Contrary to a widely held belief, the process does not violate the flows of genetic information as described by the classical central dogma, as transfers of information from RNA to DNA are explicitly held possible.

Viral load, also known as viral burden, is a numerical expression of the quantity of virus in a given volume of fluid, including biological and environmental specimens. It is not to be confused with viral titre or viral titer, which depends on the assay. When an assay for measuring the infective virus particle is done, viral titre often refers to the concentration of infectious viral particles, which is different from the total viral particles. Viral load is measured using body fluids sputum and blood plasma. As an example of environmental specimens, the viral load of norovirus can be determined from run-off water on garden produce. Norovirus has not only prolonged viral shedding and has the ability to survive in the environment but a minuscule infectious dose is required to produce infection in humans: less than 100 viral particles.

<span class="mw-page-title-main">Reverse transcription polymerase chain reaction</span> Laboratory technique to multiply an RNA sample for study

Reverse transcription polymerase chain reaction (RT-PCR) is a laboratory technique combining reverse transcription of RNA into DNA and amplification of specific DNA targets using polymerase chain reaction (PCR). It is primarily used to measure the amount of a specific RNA. This is achieved by monitoring the amplification reaction using fluorescence, a technique called real-time PCR or quantitative PCR (qPCR). Confusion can arise because some authors use the acronym RT-PCR to denote real-time PCR. In this article, RT-PCR will denote Reverse Transcription PCR. Combined RT-PCR and qPCR are routinely used for analysis of gene expression and quantification of viral RNA in research and clinical settings.

Helicase-dependent amplification (HDA) is a method for in vitro DNA amplification that takes place at a constant temperature.

Gene amplification refers to a number of natural and artificial processes by which the number of copies of a gene is increased "without a proportional increase in other genes".

<span class="mw-page-title-main">Real-time polymerase chain reaction</span> Laboratory technique of molecular biology

A real-time polymerase chain reaction is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR, not at its end, as in conventional PCR. Real-time PCR can be used quantitatively and semi-quantitatively.

<span class="mw-page-title-main">Rolling circle replication</span> DNA synthesis technique

Rolling circle replication (RCR) is a process of unidirectional nucleic acid replication that can rapidly synthesize multiple copies of circular molecules of DNA or RNA, such as plasmids, the genomes of bacteriophages, and the circular RNA genome of viroids. Some eukaryotic viruses also replicate their DNA or RNA via the rolling circle mechanism.

Nucleic acid sequence-based amplification, commonly referred to as NASBA, is a method in molecular biology which is used to produce multiple copies of single stranded RNA. NASBA is a two-step process that takes RNA and anneals specially designed primers, then utilizes an enzyme cocktail to amplify it.

Potato virus Y (PVY) is a plant pathogenic virus of the family Potyviridae, and one of the most important plant viruses affecting potato production.

Loop-mediated isothermal amplification (LAMP) is a single-tube technique for the amplification of DNA for diagnostic purposes and a low-cost alternative to detect certain diseases. LAMP is an isothermal nucleic acid amplification technique. In contrast to the polymerase chain reaction (PCR) technology, in which the reaction is carried out with a series of alternating temperature steps or cycles, isothermal amplification is carried out at a constant temperature, and does not require a thermal cycler. LAMP was invented in 1998 by Eiken Chemical Company in Tokyo. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) combines LAMP with a reverse transcription step to allow the detection of RNA.

Webtag is an on-line bioinformatics tool providing oligonucleotide sequences that are absent from a specified genome. These tags can be appended to gene specific primers for reverse transcriptase polymerase chain reaction (RT-PCR) experiments, circumventing genomic DNA contamination.

<span class="mw-page-title-main">Nucleic acid test</span> Group of techniques to detect a particular nucleic acid sequence

A nucleic acid test (NAT) is a technique used to detect a particular nucleic acid sequence and thus usually to detect and identify a particular species or subspecies of organism, often a virus or bacterium that acts as a pathogen in blood, tissue, urine, etc. NATs differ from other tests in that they detect genetic materials rather than antigens or antibodies. Detection of genetic materials allows an early diagnosis of a disease because the detection of antigens and/or antibodies requires time for them to start appearing in the bloodstream. Since the amount of a certain genetic material is usually very small, many NATs include a step that amplifies the genetic material—that is, makes many copies of it. Such NATs are called nucleic acid amplification tests (NAATs). There are several ways of amplification, including polymerase chain reaction (PCR), strand displacement assay (SDA), transcription mediated assay (TMA), and loop-mediated isothermal amplification (LAMP).

The versatility of polymerase chain reaction (PCR) has led to modifications of the basic protocol being used in a large number of variant techniques designed for various purposes. This article summarizes many of the most common variations currently or formerly used in molecular biology laboratories; familiarity with the fundamental premise by which PCR works and corresponding terms and concepts is necessary for understanding these variant techniques.

Multiplex polymerase chain reaction refers to the use of polymerase chain reaction to amplify several different DNA sequences simultaneously. This process amplifies DNA in samples using multiple primers and a temperature-mediated DNA polymerase in a thermal cycler. The primer design for all primers pairs has to be optimized so that all primer pairs can work at the same annealing temperature during PCR.

<span class="mw-page-title-main">Reverse Transcription Loop-mediated Isothermal Amplification</span>

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a one step nucleic acid amplification method to multiply specific sequences of RNA. It is used to diagnose infectious disease caused by RNA viruses.

<span class="mw-page-title-main">Transcription-mediated amplification</span>

Transcription-mediated amplification (TMA) is an isothermal, single-tube nucleic acid amplification system utilizing two enzymes, RNA polymerase and reverse transcriptase.

<span class="mw-page-title-main">RNase H-dependent PCR</span> Laboratory technique

RNase H-dependent PCR (rhPCR) is a modification of the standard PCR technique. In rhPCR, the primers are designed with a removable amplification block on the 3’ end. Amplification of the blocked primer is dependent on the cleavage activity of a hyperthermophilic archaeal Type II RNase H enzyme during hybridization to the complementary target sequence. This RNase H enzyme possesses several useful characteristics that enhance the PCR. First, it has very little enzymatic activity at low temperature, enabling a “hot start PCR” without modifications to the DNA polymerase. Second, the cleavage efficiency of the enzyme is reduced in the presence of mismatches near the RNA residue. This allows for reduced primer dimer formation, detection of alternative splicing variants, ability to perform multiplex PCR with higher numbers of PCR primers, and the ability to detect single-nucleotide polymorphisms.

References

  1. 1 2 3 4 Piepenburg, Olaf; Williams, Colin H.; Stemple, Derek L.; Armes, Niall A. (2006). "DNA Detection Using Recombination Proteins". PLOS Biology. 4 (7): e204. doi: 10.1371/journal.pbio.0040204 . PMC   1475771 . PMID   16756388.
  2. 1 2 Euler, Milena; Wang, Yongjie; Nentwich, Oliver; Piepenburg, Olaf; Hufert, Frank T.; Weidmann, Manfred (2012). "Recombinase polymerase amplification assay for rapid detection of Rift Valley fever virus". Journal of Clinical Virology. 54 (4): 308–12. doi:10.1016/j.jcv.2012.05.006. PMID   22683006.
  3. 1 2 Amer, H.M.; Abd El Wahed, A.; Shalaby, M.A.; Almajhdi, F.N.; Hufert, F.T.; Weidmann, M. (2013). "A new approach for diagnosis of bovine coronavirus using a reverse transcription recombinase polymerase amplification assay". Journal of Virological Methods. 193 (2): 337–40. doi:10.1016/j.jviromet.2013.06.027. PMC   7113639 . PMID   23811231.
  4. 1 2 Abd El Wahed, Ahmed; El-Deeb, Ayman; El-Tholoth, Mohamed; Abd El Kader, Hanaa; Ahmed, Abeer; Hassan, Sayed; Hoffmann, Bernd; Haas, Bernd; Shalaby, Mohamed A.; Hufert, Frank T.; Weidmann, Manfred (2013). Meng, Xiang-Jin (ed.). "A Portable Reverse Transcription Recombinase Polymerase Amplification Assay for Rapid Detection of Foot-and-Mouth Disease Virus". PLOS ONE. 8 (8): e71642. Bibcode:2013PLoSO...871642A. doi: 10.1371/journal.pone.0071642 . PMC   3748043 . PMID   23977101.
  5. ↑ Escadafal, Camille; Paweska, Janusz T.; Grobbelaar, Antoinette; Le Roux, Chantel; Bouloy, Michèle; Patel, Pranav; Teichmann, Anette; Donoso-Mantke, Oliver; Niedrig, Matthias (2013). De Silva, Aravinda M (ed.). "International External Quality Assessment of Molecular Detection of Rift Valley Fever Virus". PLOS Neglected Tropical Diseases. 7 (5): e2244. doi: 10.1371/journal.pntd.0002244 . PMC   3662703 . PMID   23717706.
  6. 1 2 Boyle, D. S.; Lehman, D. A.; Lillis, L.; Peterson, D.; Singhal, M.; Armes, N.; Parker, M.; Piepenburg, O.; Overbaugh, J. (2013). "Rapid Detection of HIV-1 Proviral DNA for Early Infant Diagnosis Using Recombinase Polymerase Amplification". mBio. 4 (2): e00135–13. doi:10.1128/mBio.00135-13. PMC   3622927 . PMID   23549916.
  7. ↑ Euler, M.; Wang, Y.; Otto, P.; Tomaso, H.; Escudero, R.; Anda, P.; Hufert, F. T.; Weidmann, M. (2012). "Recombinase Polymerase Amplification Assay for Rapid Detection of Francisella tularensis". Journal of Clinical Microbiology. 50 (7): 2234–8. doi:10.1128/JCM.06504-11. PMC   3405570 . PMID   22518861.
  8. ↑ Euler, M.; Wang, Y.; Heidenreich, D.; Patel, P.; Strohmeier, O.; Hakenberg, S.; Niedrig, M.; Hufert, F. T.; Weidmann, M. (2013). "Development of a Panel of Recombinase Polymerase Amplification Assays for Detection of Biothreat Agents". Journal of Clinical Microbiology. 51 (4): 1110–7. doi:10.1128/JCM.02704-12. PMC   3666764 . PMID   23345286.
  9. ↑ Sebastian K, Valentina R, Frank FB, Markus von NR (2014). "Multiplex isothermal solid-phase recombinase polymerase amplification for the specific and fast DNA-based detection of three bacterial pathogens". Microchimica Acta. 181 (13–14): 1715–1723. doi:10.1007/s00604-014-1198-5. PMC   4167443 . PMID   25253912.
  10. ↑ Santiago-Felipe S, Tortajada-Genaro LA, Morais S, Puchades R, Maquieira Á (2015). "Isothermal DNA amplification strategies for duplex microorganism detection". Food Chem. 174: 509–515. doi:10.1016/j.foodchem.2014.11.080. hdl: 10251/66087 . PMID   25529713.
  11. ↑ Loo JF, Lau PM, Ho HP, Kong SK (2013). "An aptamer-based bio-barcode assay with isothermal recombinase polymerase amplification for cytochrome-c detection and anti-cancer drug screening". Talanta. 115: 159–165. doi:10.1016/j.talanta.2013.04.051. PMID   24054573.
  12. ↑ Rohrman, Brittany A.; Richards-Kortum, Rebecca R. (2012). "A paper and plastic device for performing recombinase polymerase amplification of HIV DNA". Lab on a Chip. 12 (17): 3082–8. doi:10.1039/c2lc40423k. PMC   3569001 . PMID   22733333.
  13. ↑ "Rapid PCR". Dna.utah.edu. Retrieved 2014-06-21.
  14. ↑ "PCR primers work using standard RPA reagents". TwistDx. Retrieved 2015-10-19.[ permanent dead link ]
  15. ↑ "Publications". TwistDx. Retrieved 2014-06-21.
  16. ↑ Aebischer, Andrea (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". Journal of Clinical Microbiology. 52 (6): 1883–92. doi:10.1128/JCM.00167-14. PMC   4042763 . PMID   24648561.
This page is based on this Wikipedia article
Text is available under the CC BY-SA 4.0 license; additional terms may apply.
Images, videos and audio are available under their respective licenses.