Recombinase Polymerase Amplification Reaction Research Articles

A wearable microfluidic device for rapid detection of HIV-1 DNA using recombinase polymerase amplification

Although isothermal nucleic acid amplification is advantageous in pathogen detection in resource-limited settings, an electricity-dependent heating module is often required. Here, we developed a wearable microfluidic device combined with recombinase polymerase amplification (RPA) for simple and rapid amplification of HIV-1 DNA using human body heat. The human body temperature at the human wrist varied from 33 to 34 °C in the ambient environment, which is sufficient to perform RPA reactions. With the aid of a cellphone-based fluorescence detection system, this device detected HIV-1 DNA quantitatively ranging from 102 to 105 copies/mL with a log linearity of 0.98 in 24 min. These results demonstrate that this wearable point-of-care (POC) nucleic acid testing method is advantageous over traditional PCR and other isothermal nucleic acid amplification methods in terms of time, portability and independence on electricity. This wearable microfluidic device in conjunction with a cellphone-based fluorescence detection system can be potentially used for the detection of HIV-1 and adapted for POC detection of a broad range of infectious pathogens in resource-limited settings.

Multiplex Detection of Nucleic Acids Using Recombinase Polymerase Amplification and a Molecular Colorimetric 7-Segment Display.

Nucleic acid analysis has become highly relevant for point-of-care (POC) diagnostics since the advent of isothermal amplification methods that do not require thermal cycling. In particular, recombinase polymerase amplification (RPA) combined with lateral flow detection offers a rapid and simple solution for field-amenable low-resource nucleic acid testing. Expanding POC nucleic acid tests for the detection of multiple analytes is vital to improve diagnostic efficiency because increased multiplexing capacity enables higher information density combined with reduced assay time and costs. Here, we investigate expanding RPA POC detection by identifying a generic multiplex RPA format that can be combined with a generic multiplex lateral flow device (LFD) to enable binary and molecular encoding for the compaction of diagnostic data. This new technology relies on the incorporation of molecular labels to differentiate nucleic acid species spatially on a lateral flow membrane. In particular, we identified additional five molecular labels that can be incorporated during the RPA reaction for subsequent coupling with LFD detection. Combined with two previously demonstrated successful labels, we demonstrate potential to enable hepta-plex detection of RPA reactions coupled to multiplex LFD detection. When this hepta-plex detection is combined with binary and molecular encoding, an intuitive 7-segment output display can be produced. We note that in all experiments, we used an identical DNA template, except for the 5′ label on the forward primer, to eliminate any effects of nucleic acid sequence amplification bias. Our proof-of-concept technology demonstration is highly relevant for developing information-compact POC diagnostics where space and time are premium commodities.

Betaine-assisted recombinase polymerase assay with enhanced specificity

Recombinase polymerase amplification (RPA) is a widespread isothermal amplification method and regarded as an excellent candidate to replace polymerase chain reaction. However, the specificity of RPA is not always satisfactory when the sample contains amounts of background DNA. Herein, we report a novel RPA method named betaine-assisted RPA (B-RPA) that uses inexpensive betaine to avoid nonspecific amplification effectively. Result show that nonspecific amplification is prone to occur in RPA if the primers have not been rigorously refined, especially in detecting samples with large amounts of background DNA. This problem has been addressed by adding betaine to the RPA reactions. Our data show that the addition of 0.8 M betaine can significantly increase specificity and efficiency simultaneously. This B-RPA method is also used to detect hepatitis B virus DNA in clinical plasma samples, thereby demonstrating the clinical practicability of B-RPA.

Rapid authentication of mutton products by recombinase polymerase amplification coupled with lateral flow dipsticks

Shish kebabs and mutton slices are two of the most favorite foods in China, which are often adulterated with low-cost animal meat such as chicken or duck meat. Currently, many methods have been applied to mutton adulteration detection. However, existing methods based on protein or polymerase chain reaction (PCR) are time-consuming and tedious, making them unfit for rapid and on-site detection of counterfeit mutton products in restaurants. In this study, a visual lateral flow dipstick (LFD) method in combination with recombinase polymerase amplification (RPA) was developed for authentication of mutton slices or heat-processed shish kebabs. In this method, sheep-specific RPA primer was designed against the cytochrome b (Cyt b) gene. RPA reaction could be performed under 37–42 °C and finished within 15 min. Then, the products were added onto the LFD sample pad until the final results could be observed by naked eyes within 5 min. The total time for authenticating mutton products was only about 30 min, including 5 min for DNA extraction. In addition, only a portable and inexpensive dry bath incubator was required for the authentication. The proposed method was therefore a simple and fast approach, which was also specific and sensitive, and it could be used for on-site mutton product fraud detection by food safety inspection authorities.

Precise definition of starting time by capillary-based chemical initiation of digital isothermal DNA amplification

Digital polymerase chain reaction is a sensitive and reproducible method to assess the presence or absence of the amplification of individual target molecules confined in isolated compartments; this is used for the quantification of nucleic acids. In this paper, the precise definition of the start time of recombinase polymerase amplification (RPA) is proposed to achieve the digital quantification of nucleic acids at the single molecule level. RPA is a sequence-specific isothermal amplification method. Since the reaction will start immediately albeit slowly at room temperature following the addition of the chemical initiator (magnesium acetate), the number of false positives in digital RPA is increased if all reagents are mixed prior to compartmentalization. A capillary-based setup is described here to control the initiation of RPA reactions by encapsulating the chemical initiator to each reaction compartment using shear force when passed through a cross connector. Thousands of independent compartments are generated. The performance of digital droplet RPA (ddRPA) was validated by counting the positive application results of target molecules (Avian virus DNA) confined in the partitions. The ddPRA capillary-based setup provides a simple nucleic acid quantification method without thermal cycling. Potential applications in clinical and academic research under resource-limited settings can be envisaged. The ability to initiate chemical reaction compartments by the encapsulation of a chemical initiator using similar capillaries can be applied to a broader range of applications.

Sensitive and rapid visual detection of Salmonella Typhimurium in milk based on recombinase polymerase amplification with lateral flow dipsticks

Salmonella Typhimurium (S. Typhimurium) can cause serious foodborne diseases. In this study, an assay combining recombinase polymerase amplification (RPA) with lateral flow dipsticks (LFD) was developed to detect S. Typhimurium in milk. The RPA forward primers STF1, STF2, STF3, the reverse primer STR labeled with digoxin, and the probe STProb labeled with FAM were designed and screened to produce RPA products for LFD detection. The RPA reaction volume, temperature, and time were then optimized, and the sensitivity and specificity of the developed method were analyzed. Finally, the RPA–LFD method was evaluated using milk artificially contaminated with S. Typhimurium. Results indicated that the primer pair STF1/STR is the optimal combination for detecting the bacterium. The minimum volume, shortest time, and optimal temperature of the RPA reaction were 10 μL, 10 min, and 40–42 °C, respectively. The limit of detection of RPA-LFD for detecting the genomic DNA of S. Typhimurium was 1 fg, which is 5 and 10 times lower than the corresponding limits of RPA–agarose gel electrophoresis (AGE) and PCR–AGE, respectively. Testing with 29 other foodborne bacteria as controls revealed that RPA–LFD was highly specific for S. Typhimurium. RPA–LFD can detect S. Typhimurium at concentrations as low as 1.95 CFU/mL in artificially inoculated milk samples and is thus 10 times more sensitive than PCR. Hence, the RPA–LFD assay established in this study could be a potential point-of-care/need test for S. Typhimurium, especially in areas with limited resources.

Review: a comprehensive summary of a decade development of the recombinase polymerase amplification.

Nucleic acid amplification has permeated every field in the life sciences since the introduction of the classic polymerase chain reaction (PCR) method in 1983. Yet, despite its fundamental reach, PCR has been constrained within the walls of a laboratory, due to its requirement for a sophisticated thermocycling machine, limiting external application in low-resource settings. New isothermal amplification strategies are seeking to break through traditional laboratory boundaries by providing nucleic acid replication at constant temperatures. Of these methods, recombinase polymerase amplification (RPA) is one of the fastest developing, experiencing rapid uptake and market, even though it was introduced comparatively late. Critically, RPA's technology potentiates highly accessible and sensitive nucleic acid amplification outside of laboratory, and even self-testing. Here we provide a comprehensive review of the equipment-free simplicity of RPA over its first decade of development. Our review includes key knowledge of RPA technology, such as its reaction components, mechanism, sensitivities and specificities, and distinctive detection methods. The review also provides know-how for developing RPA assays, and information about commercially available RPA reaction kits and accessories. We summarise critical RPA experimental tips and issues available through data mining the published literature, to assist researchers in mastering the RPA reaction. We also outline influential hotspots of RPA development, and conclude with outlooks for future development and implications for eclipsing PCR and further revolutionising the life sciences.

Recombinase polymerase amplification assay for rapid detection of the root-knot nematode Meloidogyne enterolobii

Summary Rapid diagnosis tools for detection of root-knot nematodes play an important role in the disease control and eradication programme. Recombinase polymerase amplification (RPA) assays were developed targeting the IGS rRNA gene of the pacara earpod tree root-knot nematode, Meloidogyne enterolobii. The RPA assays using TwistAmp® Basic and TwistAmp® exo kits allowed detection of M. enterolobii from gall tissues and crude nematode extracts of all stages of target species without a DNA extraction step. The results of real-time RPA assays using a real-time fluorescent detection of a series of crude nematode extracts showed reliable detection with sensitivity of 1/10 of a second-stage juvenile in a RPA reaction tube after 15-20 min. The RPA assay provides affordable, simple, fast and sensitive detection of M. enterolobii.

Adenoviral detection by recombinase polymerase amplification and vertical flow paper microarray

Respiratory viral infections often mimic the symptoms of infections caused by bacteria; however, restricted and targeted administration of antibiotics is needed to combat growing antimicrobial resistance. This is particularly relevant in low-income settings. In this work, we describe the use of isothermal amplification of viral DNA at 37 °C coupled to a paper-based vertical flow microarray (VFM) setup that utilizes a colorimetric detection of amplicons using functionalized gold nanoparticles. Two oligonucleotide probes, one in-house designed and one known adenoviral probe were tested and validated for microarray detection down to 50 nM using a synthetic target template. Furthermore, primers were shown to function in a recombinase polymerase amplification reaction using both synthetic template and viral DNA. As a proof-of-concept, we demonstrate adenoviral detection with four different adenoviral species associated with respiratory infections using the paper-based VFM format. The presented assay was validated with selected adenoviral species using the in-house probe, enabling detection at 1 ng of starting material with intra- and inter-assay %CV of ≤ 9% and ≤ 13%. Finally, we validate our overall method using clinical samples. Based on the results, the combination of recombinase polymerase amplification, paper microarray analysis, and nanoparticle-based colorimetric detection could thus be a useful strategy towards rapid and affordable multiplexed viral diagnostics.

Single-Step Recombinase Polymerase Amplification Assay Based on a Paper Chip for Simultaneous Detection of Multiple Foodborne Pathogens.

A paper chip device-based recombinase polymerase amplification (RPA) method was developed for highly sensitive and selective single-step detection of foodborne pathogens. A paper chip was manufactured by simply stacking functional papers. RPA reagents and fluorescent probe were dried on the reaction zone of a patterned poly(ether sulfone) membrane. The RPA reaction was initiated by adding pathogen DNAs into an injection hole. Paper chip-based analysis of pathogens showed optimal performance at 37 °C for 20 min and the results were comparable to those obtained with solution-based RPA reactions. Based on the paper chip-based fluorescence signal, Escherichia coli, Staphylococcus aureus, and Salmonella typhimurium were simultaneously detected with detection limits of 102 cfu/mL. The diagnostic utility of the device was demonstrated by the reliable detection of E. coli and S. aureus present in spiked milk. This ready-to-use device could be integrated with simple nucleic acid extraction for food pathogen detection in resource-limited settings.

Isothermal amplification using modified primers for rapid electrochemical analysis of coeliac disease associated DQB1*02 HLA allele

DNA biosensors are attractive tools for genetic analysis as there is an increasing need for rapid and low-cost DNA analysis, primarily driven by applications in personalized pharmacogenomics, clinical diagnostics, rapid pathogen detection, food traceability and forensics. A rapid electrochemical genosensor detection methodology exploiting a combination of modified primers for solution-phase isothermal amplification, followed by rapid detection via hybridization on gold electrodes is reported. Modified reverse primers, exploiting a C18 spacer between the primer-binding site and an engineered single stranded tail, are used in a recombinase polymerase amplification reaction to produce an amplicon with a central duplex flanked by two single stranded tails. These tails are designed to be complementary to a gold electrode tethered capture oligo probe as well as a horseradish peroxidase labelled reporter oligo probe. The time required for hybridization of the isothermally generated amplicons with each of the immobilized and reporter probes was optimised to be 2 min, in both cases. The effect of amplification time and the limit of detection were evaluated using these hybridization times for both single stranded and double stranded DNA templates. The best detection limit of 70 fM for a ssDNA template was achieved using 45 min amplification, whilst for a dsDNA template, just 30 min amplification resulted in a slightly lower detection limit of 14 fM, whilst both 20 and 45 min amplification times were observed to provide detection limits of 71 and 72 fM, respectively, but 30 and 45 min amplification resulted in a much higher signal and sensitivity. The genosensor was applied to genomic DNA and real patient and control blood samples for detection of the coeliac disease associated DQB1*02 HLA allele, as a model system, demonstrating the possibility to carry out molecular diagnostics, combining amplification and detection in a rapid and facile manner.

Washing-Free Electrochemical Detection of Amplified Double-Stranded DNAs Using a Zinc Finger Protein.

Recombinase polymerase amplification (RPA) has been combined with electrochemical detection for simple and rapid point-of-care testing. However, there are two major hindrances to this simple and rapid testing: (i) washing or purification steps are required to remove unbound labeled probes and interfering species in the sample; (ii) it is difficult to quantify double-stranded DNA (dsDNA) electrochemically by using biospecific affinity binding without dsDNA denaturation. In the present study, we describe a wash-free and rapid electrochemical method to detect RPA-amplified dsDNAs using a zinc finger protein, Zif268. Electrochemical detection is achieved using proximity-dependent electron mediation of ferrocenemethanol between a glucose-oxidase (GOx) label and an electrode, which differentiates the specifically electrode-bound and -unbound labels without a washing or purification step. RPA-amplified dsDNA containing a biotin-terminated forward primer is specifically bound to a neutravidin-modified electrode, and GOx-conjugated Zif268 is specifically bound to the dsDNA. The whole detection is performed within 17 min (15 min for the RPA reaction and <2 min for the electrochemical measurement). Electrochemical detection is carried out without an additional incubation period, because the specific binding between Zif268 and the dsDNA occurs during the RPA reaction. The detection method could discriminate between target template DNA of Piscirickettsia salmonis and nontarget DNAs (random sequence and calf thymus DNA). The detection limit for the target DNA is approximately 300 copies in 13.2 μL, indicating that the detection method is ultrasensitive. We believe that the method could offer a promising solution for simple and rapid point-of-care testing.

Recombinase polymerase amplification (RPA) combined with lateral flow (LF) strip for detection of Toxoplasma gondii in the environment

Toxoplasma gondii infects all warm-blooded vertebrates, resulting in a great threat to human health and significant economic loss to the livestock industry. Ingestion of infectious oocysts of T. gondii from the environment is the major source of transmission. Detection of T. gondii oocysts by existing methods is laborious, time-consuming and expensive. The objective of the present study was to develop a recombinase polymerase amplification (RPA) method combined with a lateral flow (LF) strip for detection of T. gondii oocysts in the soil and water. The DNA of T. gondii oocysts was amplified by a pair of specific primers based on the T. gondii B1 gene over 15min at a constant temperature ranging from 30°C to 45°C using RPA. The amplification product was visualized by the lateral flow (LF) strip within 5min using the specific probe added to the RPA reaction system. The sensitivity of the established assay was 10 times higher than that of nested PCR with a lower detection limit of 0.1 oocyst per reaction, and there was no cross-reactivity with other closely related protozoan species. Fifty environmental samples were further assessed for the detection validity of the LF-RPA assay (B1-LF-RPA) and compared with nested PCR based on the B1 gene sequence. The B1-LF-RPA and nested PCR both showed that 5 out of the 50 environmental samples were positive. The B1-LF-RPA method was also proven to be sufficiently tolerant of existing inhibitors in the environment. In addition, the advantages of simple operation, speediness and cost-effectiveness make B1-LF-RPA a promising molecular detection tool for T. gondii.

Recombinase Polymerase Amplification Assay—A Simple, Fast and Cost-Effective Alternative to Real Time PCR for Specific Detection of Feline Herpesvirus-1

Feline herpesvirus 1 (FHV-1), an enveloped dsDNA virus, is one of the major pathogens of feline upper respiratory tract disease (URTD) and ocular disease. Currently, polymerase chain reaction (PCR) remains the gold standard diagnostic tool for FHV-1 infection but is relatively expensive, requires well-equipped laboratories and is not suitable for field tests. Recombinase polymerase amplification (RPA), an isothermal gene amplification technology, has been explored for the molecular diagnosis of infectious diseases. In this study, an exo-RPA assay for FHV-1 detection was developed and validated. Primers targeting specifically the thymidine kinase (TK) gene of FHV-1 were designed. The RPA reaction was performed successfully at 39°C and the results were obtained within 20 min. Using different copy numbers of recombinant plasmid DNA that contains the TK gene as template, we showed the detection limit of exo-RPA was 102 copies DNA/reaction, the same as that of real time PCR. The exo-RPA assay did not cross-detect feline panleukopenia virus, feline calicivirus, bovine herpesvirus-1, pseudorabies virus or chlamydia psittaci, a panel of pathogens important in feline URTD or other viruses in Alphaherpesvirinae, demonstrating high specificity. The assay was validated by testing 120 nasal and ocular conjunctival swabs of cats, and the results were compared with those obtained with real-time PCR. Both assays provided the same testing results in the clinical samples. Compared with real time PCR, the exo-RPA assay uses less-complex equipment that is portable and the reaction is completed much faster. Additionally, commercial RPA reagents in vacuum-sealed pouches can tolerate temperatures up to room temperature for days without loss of activity, suitable for shipment and storage for field tests. Taken together, the exo-RPA assay is a simple, fast and cost-effective alternative to real time PCR, suitable for use in less advanced laboratories and for field detection of FHV-1 infection.

Rapid, Affordable and Portable Medium-Throughput Molecular Device for Zika Virus.

Zika virus (ZIKV) has gained global attention as an etiologic agent of fetal microcephaly and Guillain-Barré syndrome. Existing immuno-based rapid tests often fail to distinguish between Zika and related flaviviruses that are common in affected regions of Central and South Americas and the Caribbean. The US CDC and qualified state health department laboratories can perform the reverse transcription polymerase chain reaction (RT-PCR) ZIKV test using highly sophisticated instruments with long turnaround times. The preliminary results of a portable and low-cost molecular diagnostics system for ZIKV infection are reported here. In less than 15 minutes, this low-cost platform can automatically perform high quality RNA extraction from up to 12 ZIKV-spiked urine samples simultaneously. It can also perform reverse transcription recombinase polymerase amplification reaction (RT-RPA) in ≤15 minutes. The fluorescent signal produced from probe-based RT-RPA or RT-PCR assays can be monitored using LEDs and a smartphone camera. In addition, the RT-RPA and RT-PCR assays do not cross-react with dengue and chikungunya viral RNA. This low-cost system lacks complicated, sensitive and high cost components, making it suitable for resource-limited settings. It has the potential to offer simple sample-to-answer molecular diagnostics and can inform healthcare workers of patients’ diagnosis promptly.

Rapid Detection of Black Queen Cell Virus from Honeybee using Reverse Transcription Real-Time Recombinase Polymerase Amplification (RT/RT RPA)

Black Queen Cell Virus (BQCV) is one of pathogenic virus in honeybee which could be detected using reverse transcription real-time PCR (RT/RT-PCR). In this study, for rapid detection of BQCV, Recombinase Polymerase Amplification (RPA) was applied, and BQCV-specific reverse transcription real-time RPA (RT/RT-RPA) method was newly developed based on BQCV-specific RT/RT PCR. The Real-time RPA (RT-RPA) was performed at 37°C isothermal condition for 40 minutes. During the experiments, specific DNA amplifications were real-timely monitored using fluorescent detector. BQCV-specific DNA amplification could be detected from 3 min 26 sec after RPA reaction with specific DNA templates by RT-RPA, while 41 min 42 sec was required by qRTPCR with same quantities of initial templates. With generated cDNA from BQCV-infected honeybee, specific DNA amplification was recognized at 4 min 18 sec using RT-RPA, however, 66 min 5 sec was needed using Real-time PCR. Moreover, with reverse transcriptase and RPA solution, BQCVspecific DNA amplification could be detected at 8 min 36 sec from total RNA of BQCV-infected honeybee using one-step Reverse Transcription/Real-Time RPA (RT/RT-RPA).

P07.06 A low cost, hand-held point of care molecular diagnostic device for sexually transmitted infections

Introduction Rapid and accurate field diagnostics have potential to impact on the burden of STIs in resource poor settings. Microfluidic and nano technologies offer opportunities to create molecular detection platforms but costs may be prohibitive. We present a low cost isothermal amplification, point of care test for rapid identification of sexually transmitted infections. Sample collection integrates directly with a microfluidic device for automated sample preparation, isothermal amplification and optical detection. Methods Cell lysis, within the microfluidic cartridge, is conducted using a chemical method and nucleic acid purification is achieved on activated cellulose membrane. The microfluidic device incorporates passive mixing of lysis-binding buffers and sample using a serpentine channel. Isothermal amplification is conducted using thermophillic helicase dependent amplification (tHDA) and recombinase polymerase amplification (RPA). A low cost real-time isothermal amplification platform has been developed capable of running six amplifications simultaneously. Results Results have shown extraction efficiencies for the new membrane of 69% and 57% compared to commercial Qiagen extraction of 85% and 59.4% for 0.1 ng/µL and 100 ng/µL salmon sperm DNA respectively spiked in phosphate buffered solution. Extraction experiments using the serpentine passive mixer cartridges incorporating lysis and nucleic acid purification showed extraction efficiency around 80% of the commercial Qiagen kit. The platform is capable of detecting 1.32 × 10 6 copies of target DNA through thermophillic helicase dependent amplification and 1 × 10 5 copies of Chlamydia trachomatis genomic DNA within 10 min through RPA. Conclusion We have produced a low cost, rapid nucleic acid extraction, isothermal amplification and detection platform consistent with use remote resource poor settings. The simple optics setup demonstrated high sensitivity and rapid detection of the tHDA and RPA reactions removing the requirement for expensive dichroic filters and lenses. Diagnostic performance of the device is currently being undertaken. Disclosure of interest statement No Disclosure of interest.

Development of a quantitative recombinase polymerase amplification assay with an internal positive control.

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to quantify DNA sample concentration using a standard curve. In this manuscript, a detailed protocol for developing and implementing a real-time quantitative recombinase polymerase amplification assay (qRPA assay) is provided. Using HIV-1 DNA quantification as an example, the assembly of real-time RPA reactions, the design of an internal positive control (IPC) sequence, and co-amplification of the IPC and target of interest are all described. Instructions and data processing scripts for the construction of a standard curve using data from multiple experiments are provided, which may be used to predict the concentration of unknown samples or assess the performance of the assay. Finally, an alternative method for collecting real-time fluorescence data with a microscope and a stage heater as a step towards developing a point-of-care qRPA assay is described. The protocol and scripts provided may be used for the development of a qRPA assay for any DNA target of interest.

Influence of sequence mismatches on the specificity of recombinase polymerase amplification technology

Recombinase polymerase amplification (RPA) technology relies on three major proteins, recombinase proteins, single-strand binding proteins, and polymerases, to specifically amplify nucleic acid sequences in an isothermal format. The performance of RPA with respect to sequence mismatches of closely-related non-target molecules is not well documented and the influence of the number and distribution of mismatches in DNA sequences on RPA amplification reaction is not well understood. We investigated the specificity of RPA by testing closely-related species bearing naturally occurring mismatches for the tuf gene sequence of Pseudomonas aeruginosa and/or Mycobacterium tuberculosis and for the cfb gene sequence of Streptococcus agalactiae. In addition, the impact of the number and distribution of mismatches on RPA efficiency was assessed by synthetically generating 14 types of mismatched forward primers for detecting five bacterial species of high diagnostic relevance such as Clostridium difficile, Staphylococcus aureus, S. agalactiae, P. aeruginosa, and M. tuberculosis as well as Bacillus atropheus subsp. globigii for which we use the spores as internal control in diagnostic assays. A total of 87 mismatched primers were tested in this study. We observed that target specific RPA primers with mismatches (n > 1) at their 3'extrimity hampered RPA reaction. In addition, 3 mismatches covering both extremities and the center of the primer sequence negatively affected RPA yield. We demonstrated that the specificity of RPA was multifactorial. Therefore its application in clinical settings must be selected and validated a priori. We recommend that the selection of a target gene must consider the presence of closely-related non-target genes. It is advisable to choose target regions with a high number of mismatches (≥36%, relative to the size of amplicon) with respect to closely-related species and the best case scenario would be by choosing a unique target gene.

Equipment-free incubation of recombinase polymerase amplification reactions using body heat.

The development of isothermal amplification platforms for nucleic acid detection has the potential to increase access to molecular diagnostics in low resource settings; however, simple, low-cost methods for heating samples are required to perform reactions. In this study, we demonstrated that human body heat may be harnessed to incubate recombinase polymerase amplification (RPA) reactions for isothermal amplification of HIV-1 DNA. After measuring the temperature of mock reactions at 4 body locations, the axilla was chosen as the ideal site for comfortable, convenient incubation. Using commonly available materials, 3 methods for securing RPA reactions to the body were characterized. Finally, RPA reactions were incubated using body heat while control RPA reactions were incubated in a heat block. At room temperature, all reactions with 10 copies of HIV-1 DNA and 90% of reactions with 100 copies of HIV-1 DNA tested positive when incubated with body heat. In a cold room with an ambient temperature of 10 degrees Celsius, all reactions containing 10 copies or 100 copies of HIV-1 DNA tested positive when incubated with body heat. These results suggest that human body heat may provide an extremely low-cost solution for incubating RPA reactions in low resource settings.