REVIEW

Helicase-dependent isothermal amplification: a novel toolin the development of molecular-based analytical systemsfor rapid pathogen detection

Susana Barreda-García1 & Rebeca Miranda-Castro1 & Noemí de-los-Santos-Álvarez1 &

Arturo J. Miranda-Ordieres1 & María Jesús Lobo-Castañón1

Received: 9 June 2017 /Revised: 18 August 2017 /Accepted: 1 September 2017 /Published online: 20 September 2017# Springer-Verlag GmbH Germany 2017

Abstract Highly sensitive testing of nucleic acids is essentialto improve the detection of pathogens, which pose a majorthreat for public health worldwide. Currently available molec-ular assays, mainly based on PCR, have a limited utility inpoint-of-need control or resource-limited settings.Consequently, there is a strong interest in developing cost-effective, robust, and portable platforms for early detectionof these harmful microorganisms. Since its description in2004, isothermal helicase-dependent amplification (HDA)has been successfully applied in the development of novelmolecular-based technologies for rapid, sensitive, and selec-tive detection of viruses and bacteria. In this review, we high-light relevant analytical systems using this simple nucleic acidamplification methodology that takes place at a constant tem-perature and that is readily compatible with microfluidic tech-nologies. Different strategies for monitoring HDA amplifica-tion products are described. In addition, we present techno-logical advances for integrating sample preparation, HDA am-plification, and detection. Future perspectives and challengestoward point-of-need use not only for clinical diagnosis butalso in food safety testing and environmental monitoring arealso discussed.

Keywords Nucleic acid amplification . Helicase .

Isothermal . Pathogen detection .Molecular test

Introduction

Pathogens are microorganisms that act as invaders, attack ourbodies, and are capable of causing disease. They are respon-sible for the spread of communicable diseases prone to causeepidemics, and according to the World Health Organization(WHO), an important contributor to human morbidity andmortality [1]. Take, for example, the recent cases of Zika out-break in America and Ebola epidemics in West Africa. Theseoverlapping global crises were caused by already known path-ogens that can now spread around the world much faster thanever as a result of the increased population mobility, the in-tensified trade in goods and services, the climate change, andother factors linked to globalization [2]. High death rates frominfectious diseases commonly appear in the context of pover-ty, poor diets, and limited infrastructure found in developingcountries. Water, food, and environmental quality are key el-ements to prevent and control infectious diseases in a global-izing world. In this context, rapid and cost-effective detectionof pathogens at the Bpoint-of-need^ is a critical demand, notonly for clinical diagnosis but also for food safety testing andenvironmental monitoring.

Traditional methods for the detection of pathogens are wellestablished microbiological assays based on culturing andmeasuring the growth of individual microorganisms, whichrequire viable pathogens for replication in different culturemedia such as pre-enrichment and selective plating media[3]. Although these methods are usually inexpensive, theyare time consuming, space occupying, and laborious, involv-ing 2 to 3 days for preliminary identification and even morethan 1 week for confirmation of the specific pathogenic mi-croorganism. As an alternative, there are different molecularmethods that entail near-time or real-time pathogen detection,among them immunoassays and tests for nucleic acids specificof the pathogen.

Published in the topical collection celebrating ABCs 16th Anniversary.

* María Jesús Lobo-Castañónmjlc@uniovi.es

1 Departamento de Química Física y Analítica, Universidad deOviedo, Julián Clavería 8, 33006 Oviedo, Spain

Anal Bioanal Chem (2018) 410:679–693DOI 10.1007/s00216-017-0620-3

The advent of polymerase chain reaction (PCR) as a tool forin vitro nucleic acid amplification has led to a new paradigm forthe direct detection of pathogen-specific nucleic acids, givingrise to assays that are exquisitely sensitive, and relatively rapid.These methods involve multiple thermocycling steps in high-precision instruments difficult to miniaturize, requiring strin-gent conditions of laboratory compartmentalization and thusalmost exclusively performed in centralized laboratories withhigh-end instrumentation [4]. An ideal test for point-of-needpathogen detection should meet the ASSURED—affordable,sensitive, specific, user-friendly, rapid and robust, equipment-free, and delivered to those who need it—criteria outlined bythe WHO [5]. With an incremental knowledge on the complexin vivo nucleic acid amplification processes, which are essen-tial in biological systems [6], novel isothermal nucleic acidamplification methods are gaining their importance for the de-velopment of analytical devices aimed to meet those idealcriteria. Isothermal amplification methods can be easily inte-grated into simple and low-energy consumption microsystems,which may in the future outperform PCR for pathogen detec-tion. These methods include loop-mediated isothermal ampli-fication (LAMP), recombinase polymerase amplification(RPA), nucleic acid sequence-based amplification (NASBA),helicase-dependent amplification (HDA), rolling circle ampli-fication (RCA), signal-mediated amplification of RNA technol-ogy (SMART), and strand displacement amplification (SDA).Previously published reviews [6] gave a broad description ofthese methods and their potential applications. This review fo-cuses in one of these emerging methodologies, HDA, giving alook into the most recent HDA-based analytical methods forthe detection of pathogens. We endeavored to highlight newmethods for the detection of HDA amplification products aswell as relevant systems to integrate sample treatment, ampli-fication, and detection, with the future trends forecasted toconclude.

Principle of helicase-dependent amplification (HDA)

HDA is one of the simplest approaches for isothermal nucleicacid amplification that mimics an in vivo process of DNAreplication, using a helicase to isothermally unwind DNA du-plexes instead of heat, as is the case in PCR. Vincent et al.developed this method in 2004 [7], which was subsequentlypatented in 2007 [8]. The initial studies employed UvrDhelicase from E. coli, a well-studied helicase II that unwindsDNA in a 3’ to 5’ direction, using the energy from the hydro-lysis of adenosine triphosphate to break the hydrogen bondsbetween complementary bases in duplex DNA [9]. In E. colicells, this protein is involved in the repair of mismatches inDNA, working in coordination with methyl-directed mis-match repair (MutL) protein that stimulates the helicase activ-ity. Consequently, the original HDA system requires, in

addition to the helicase to unwind dsDNA, two accessoryproteins: MutL and a single-stranded DNA-binding protein(SSB) to stabilize the ssDNA preventing the re-associationof the complementary strands. Once DNA is isothermally un-wound and stabilized, two oligonucleotide primers hybridizeto the 5′ and 3′ borders of the target sequence and a DNApolymerase extends the annealed primers by addingdeoxynucleotide-triphosphates (dNTPs) to generate double-stranded amplification products (Fig. 1), in a scheme verysimilar to PCR but without thermal cycling. The process isrepeated, giving rise to an exponential amplification of thetarget dsDNA. The dNTPs mixture must be enriched withdATP, which acts as cofactor of helicase, and Mg2+, cofactorfor both helicase and polymerase. It is very important thathelicase and polymerase work in a coordinated way,preventing polymerase to be displaced by helicase, as thehelicase-catalyzed reaction is the rate-limiting step, and un-wound DNA strands do not re-anneal before they are copiedby polymerase. It is for this reason that a pair helicase/polymerase working together in natural systems must beemployed. In the original work, the E. coli UvrD helicase/DNA polymerase I Klenow fragment pair is used [7] to expo-nentially amplify DNA at 37 °C, mesophilic form of HDA(mHDA). Later, a thermostable helicase Tte-UvrD fromThermoanaerobacter tengcongenesis in combination withBacillus stearothermophilus DNA polymerase (Bst-DNApolymerase) allowed performing the amplification at highertemperatures (60–65 °C), called the thermophilic form ofHDA (tHDA) [10]. Unlike mHDA, the tHDA system doesnot require the MutL protein, thus simplifying the reactioncomponents. At the same time, the efficiency and selectivityof the amplification reaction is improved.

Design of primers

Similar to PCR, HDA amplifies nucleic acids exponentiallyusing two flanking primers. To find the oligonucleotideprimers pair that leads to high yield of a target-specificamplicon, several primer combinations are often tailored byusing online programs originally devised to design PCRprimers (Primer3 [11] and PrimerQuest [12]). For that, specif-ic recommendations for the isothermal technique should beborne in mind, namely: (a) primer size ranging from 24 to33 nucleotides, (b) melting temperature, Tm, from 60 to 74°C, and (c) GC% within the interval 35-60% [13]. However,successful HDA amplification has been achieved whenrelaxing some of these constraints as well. Afterwards, primercandidates are examined for potential dimer and self-dimerformation, first in silico employing DNA folding algorithms[14], and then experimentally screened. Likewise, BLASTanalysis [15] must be carried out to exclude homology tonontarget sequences.

680 Barreda-García S. et al.

Even taking these cautions, the mix of primers along withDNA polymerase and the building blocks (dNTPs) are likelyto do mischief, giving rise to high background signal, and lowsensitivity and selectivity. These problems, mainly attributedto unwanted primer dimers and off-target interactions, aremore prevalent in isothermal amplification techniques due tothe lack of an annealing step from high to low temperatures toform Watson-Crick base pairing. A particular feature of HDAis, however, the recommended long size of HDA primers withrespect to PCR counterparts, which feeds confusion betweenspecific amplicons and spurious products, and hence falsepositive results, as a consequence of their similar size [16].The risk of primer-dimer artifacts would therefore justify thelower primer concentrations used in HDA (75–100 nM) whencomparing with PCR (0.1–1 μM).

To circumvent undesired side products arising fromprimers, a series of actions have been reported; some of themconstitute assay strategies themselves and they will bediscussed below, whereas others are more general, involvingprimer design. Given that DNA polymerase synthesizes newDNA strands in a 5’–3’ direction, interactions between orwithin the primers are due to overlapping 3’-ends, even littleapparent complementarity, and corrective actions have beenfocused in this direction.

Use of chemically inactivated primers containing a 3’blocking group as well as a ribonucleotide moiety near the3’-end has been proposed to prevent elongation by DNA po-lymerase. Unblockage and activation of the primers only takeplace once they hybridize with the target DNA and the ribo-nucleotide linkage is subsequently cleaved by a hot-startRNase H2. At 65 °C, optimal temperature for HDA, RNase

H2 becomes fully active and releases a DNA fragment with a3′-hydroxyl group able to initiate the amplification [17]. Withthis approach, primer-based side reactions are eliminated,while HDA amplification speed and efficiency are not signif-icantly affected; however, it cannot be implemented in RNAamplification.

Inefficient consumption of primers, not leading to the de-sired product, can be also attenuated by self-avoiding molec-ular-recognition system (SAMRS). SAMRS draws on a set ofdNTP analogues (A*, T*, G*, and C*) that have been de-signed to pair with natural nucleobases but not with eachother, based on the number of hydrogen bonds [18].Therefore, primers built from SAMRS hybridize with the tar-get but they do not interact with each other, avoiding primer-dimer artifacts. Thermophilic enzymes (polymerase andhelicase) have demonstrated their capability to work withSAMRS-structurally altered nucleotides by just adjustingMg2+ concentration [19], albeit the best yield has been report-ed when four SAMRS components are placed at the 3′-end ofthe primers, followed by one terminal natural nucleotide.Moreover, the addition of a thermostable reverse transcriptasehas allowed RNA target amplification to benefit from thesechimeric primers.

On the other hand, the influence of the nucleobases placedat the 5′-end of the primers has been also studied, finding animproved efficiency in HDAwhen these termini are enrichedin adenine or cytosine [20].

Many primers designed for PCR amplification do not meetthe recommended Tm needed for HDA. In those cases, it ispossible to modify the existing primer set by adding a custom-ized 22-nt tail at 5′-end [21]. Though this tail is not specific of

Step 3

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Helicase

DNA polymerase Forward primer

Reverse primer

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Fig. 1 Scheme of the helicase-dependent amplification system,adapted from reference [7]. (1)Unwinding dsDNA by helicaseand stabilization of ssDNA bySSB; (2) annealing of primers,and (3) elongation of primers byDNA polymerase

Helicase-dependent isothermal amplification: a novel tool in the development of molecular-based... 681

the target gene sequence, it increases the Tm after the firstamplification and also the length of the amplicon, which iscritical in this isothermal technique. These can be considered ageneral strategy for adapting PCR primers to HDA amplifica-tion, especially for comparison with PCR. Under these condi-tions both sensitivity and selectivity of HDA are improved,and HDA reaches identical limit of quantification (1 f. of plantvirus cDNA) to end-point, SYBR Green, and TaqMan PCR.

Improvements in HDA amplification

Despite its simplicity, the utility of HDA is often hampered bytemplate-independent primer interactions that give rise to anonspecific amplification phenomenon. This causes false pos-itive results and adversely affects the detectability and robust-ness of the methods based on this amplification reaction.Although a careful primer design can reduce primer-dimerformation, this process and unspecific amplification are morepronounced in HDA than in PCR [16], and fundamentallyoccur because no hot-start polymerase for HDA is currentlyavailable. This is especially a problem for the detection of lowcopy number targets due to the long time required for ampli-fication, and in the design of multiplex amplifications wherevarious pairs of primers must function together. Besides theHDA modification explained before, termed blocked-primerHDA, in which DNA polymerase cannot extend the primersuntil they are cleaved by a thermostable RNase [17] givingrise to a quasi-hot-start amplificationmethod, selectivity of theamplificationmay bemodulated by careful optimization of thereaction conditions. The use of additives frequently employedin PCR, such as DMSO, betaine, and sorbitol, which reduceDNA secondary structures facilitating primer annealing, hasproven to be effective in HDA amplification [22], improvingits yield and specificity. However, these additives can alsogreatly reduce the activity of polymerase. Therefore, it is im-portant to test different concentrations to find the best condi-tions for each amplification system. Similarly, macromolecu-lar crowding agents (polyethylene glycol, dextran, Ficoll) thatmimic the conditions for amplification in natural systems,increase the speed of polymerase and affect the structure ofDNA, improving yield, robustness, and specificity [23]. Theoptimization of reaction enzymes mixture is another importantissue; for example, it has been demonstrated that, surprisingly,lowering the concentration of both ATP and dNTPs with re-spect to that usually employed improves the efficiency andselectivity of HDA, suppressing primer-dimer formation,and thus reduces the background amplification problems[24]. All these measures may be combined, and it is of para-mount importance to experimentally determine the optimalamount of all additives and reagents for each amplificationsystem.

A different strategy that improves the sensitivity and robust-ness of HDA is the digestion with specific restriction

endonucleases during amplification. Mimicking the naturalprocess of mismatch repair in which helicase is involved, re-striction enzymes are selected to cleave a sequence near theplace where primers anneal, generating blunt ends that helprecruiting and loading the helicase [23]. This improves theeffective loading of the helicase and thus accelerates theHDA reaction. However, it renders the design more challeng-ing since it is necessary to find an enzyme capable of cutting thedsDNA close to the target sequence, without fragmenting it.

It is also possible to expand the HDA system to RNAamplification by incorporating into the reactionmixture a ther-mostable reverse transcriptase, RT-HDA [25]. This enzymeconverts the RNA target into DNA, which is subsequentlyexponentially amplified by HDA reaction. Importantly, be-cause the UvrD helicase is able to unwind DNA-RNA hybridsmore efficiently than DNA duplex, the RT-HDA system isable to perform one million-fold amplification of RNA in10 min in a single step at a constant temperature [25].

The efficiency of HDA to amplify long amplicons is lowdue to the limited speed and processivity of the chosenhelicases [7]. This is related to the mechanism of action ofUvrD helicase that must be on high molar excess in relationto the dsDNA substrate to unwind it. To increase the speed ofDNA synthesis, a new bifunctional protein, termedhelimerase, has been engineered by linking the UvrD helicasewith Bst-DNA polymerase [26]. This complex demonstratedincreased apparent processivity, and allowed the amplificationof fragments significantly longer (up to 2.3 kb) than that withthe two individual proteins. However, this new enzyme is notcommercially available, and further studies should be under-taken to simplify and make its production more cost-effective.

Another possibility to improve the efficiency of HDA is toemploy gold nanoparticles (AuNPs) in the reaction media[27]. AuNPs bind to ssDNA with higher affinity than todsDNA [28], and consequently similar to SSB they may im-prove the denaturation efficiency of helicases. In this way notonly sensitivity but also selectivity of HDA has been im-proved [27]. This is a very recent discovery that opens newopportunities for improvingHDA, and undoubtedly it requiresfurther investigations.

Strategies for monitoring HDA

Similar to end-point PCR, the successful performance ofHDA is usually accomplished by agarose gel electrophoresisand staining with fluorescent DNA binders such as ethidiumbromide or safer and more environmentally friendly alterna-tives (SimplySafe, GelRed, or GelGreen). This rapid analysisdoes not allow either quantitation or discrimination of unspe-cific amplicons of similar length, a very common pitfall ofsuboptimal isothermal amplification designs. Nevertheless,HDA is compatible with different detection mechanisms,

682 Barreda-García S. et al.

and significant improvements have been made in monitoringHDA amplicons both at the end-point of the amplificationreaction and in real time.

Real-time HDA

Fluorescence detection

Monitoring the amplification in real time is the most straight-forward solution. Real-time HDAwas first developed using anon-mutagenic non-cytotoxic fluorescent intercalator(EvaGreen) that is only fluorescent when bound to dsDNA,which ensures low background signals [29]. Besides, it showsless amplification inhibition than SYBR Green [30]. Since notemperature cycles are performed, the analytical signal is thetime needed to exceed an established threshold in fluorescenceintensity. This time decreases with target concentration, asexpected, and with the primer concentration used. This meansthat the amplification speed is boosted by the amount ofprimers in solution. However, an enhancement in primer-dimer amplification occurs, limiting the improvement in de-tectability [31]. In fact, detailed comparison of HDA and PCRsuggests that the primer concentration to obtain the lowestlimit of detection (1 f. for oligonucleotide template) is 75nM. For PCR, higher primer concentration, above 200 nM,permitted reaching a smaller limit of detection, 100 aM. Fromreal-time experiments, HDA has been claimed to be fasterthan PCR [16] but no good linearity was obtained. More re-cently, HDAwas proven to be slightly slower than PCR [32]and doubling times (equivalent to one cycle in PCR) are re-ported to consistently range from 50 s [33] to 78 s [17, 32, 34],sometimes longer than usual PCR cycles. Good linearityachieved for real-time HDA assays allowed the detection oftwo human papillomavirus types using plasmids in clinicalsamples [29].

Electrochemical detection

Electrochemical monitoring of the HDA is also possible byreplacing the fluorescent probe with an electroactiveintercalator [35]. The redox activity decreases after intercala-tion because of the inability to reach the electrode surface, sothis detection strategy is more prone to false positive results. Asconventional real-time amplifications, high-throughput mea-surements can be carried out in disposable electrochemical mi-croplates. Similar detection limits and reproducibility to fluo-rescent real-time HDAwere obtained for a specific sequence ofE. coli plasmid but amplification rate was slower, probably dueto stronger inhibition by redox probe than by EvaGreen.

In both fluorescent and electrochemical approaches, a meltcurve after amplification is compulsory to check the presenceof unspecific amplicons. To improve selectivity and allowmultiplex assays, specific probes such as molecular beacons,

FRET, TaqMan, or Scorpions probes are specifically designedto hybridize with a short fragment of the target strand (Fig. 2a,right panel). TaqMan MGB (minor groove binder) probeswere first used in combination with two DNA polymeraseswith 5′ to 3′ exonuclease activity [36]. These probes areshorter than conventional TaqMan probes because of the spe-cial quencher that binds to the minor groove increasing theoverall binding energy expected for a probe of the same size.The probes were designed to have a melting temperature closeto the HDA optimized one (63–65 °C). The Gst DNA poly-merase was very efficient in cleaving the probe, which in-creased dramatically the signal-to-noise ratio and, as a conse-quence, reduced the threshold time (transformed into thresh-old cycle, Ct, by analogy with PCR). Taq polymerase was alsoefficient under isothermal conditions, even though it isintended to work after each denaturation step at 95 °C.Asymmetric amplification is not needed but it can speed upthe reaction when using one of the primers at concentrationabove the recommended 75 nM provided no unspecific am-plification is promoted. Multiplex detection is possible usinghydrolysis probes but optimization is critical for target se-quences with very different copy number and requires a dena-turing step, so it is not entirely isothermal. In any case, theamplification is slower than for individual amplifications.

More recently, hairpin probes with a fluorophore and aquencher at each end where also successfully used in a 4-plex real- t ime HDA assay for two sequences ofC. trachomatis and one of N. gonorrhoeae in addition to theinternal control [22]. However, end-point detection was final-ly selected after careful optimization of components in themultiplex assay. The hairpin probe is not fluorescent in solu-tion but it fluoresces after hybridization with the amplifiedtarget. Unlike TaqMan probes, polymerases with no exonu-clease activity are required in order to maintain the probehybridized to the amplified strand. Asymmetry ratio of 1:3,with concentration above 25 nM for the limiting one, ampli-fied preferentially the strand complementary to the probe, im-proving the end-point signal. Initial alkaline denaturation at 65°C was more effective than the thermal shock at 95 °C, mak-ing this method completely isothermal. HDA was inhibitedwith 50–100 ng of human genomic DNA but this limitationwas overcome with a sequence-specific isolation and purifi-cation of the bacterial DNA. This method is based on hybrid-ization with RNA oligonucleotides complementary to the an-alyte far from the target sequence and entrapment andpreconcentration on magnetic beads covered with antibodiesspecific for RNA-DNA hybrids. The purification thus pro-vides ssDNA as an input sequence for HDA using probes witha fluorophore and a quencher for detection (Fig. 2b and leftpanel). In such a way, as low as 25 cells/mL of two differentbacteria were detected simultaneously, 2.5–5 times higherthan for individual amplifications, in about 3 h including sam-ple preparation [37].

Helicase-dependent isothermal amplification: a novel tool in the development of molecular-based... 683

Dedicated real-time instrumentation for HDA (Solana fromQuidel [38]) and other isothermal amplifications are nowcommercially available although hand-held devices are stillunder development [39].

Amplicon capturing approaches

Lateral flow devices

In order to achieve low-cost DNA detection assays, real-timemonitoring is not adequate. Since the beginning, lateral flowdevices (LFD) were developed to reveal the isothermal ampli-fication. In symmetric amplifications a dual-labeled ampliconis obtained by incorporating a biotinylated primer and theother primer bound to a fluorescein tag. The LFD containsstreptavidin-coated gold nanoparticles that bind the biotinyl-ated amplicon, and the complex flows until the detection line,where immobilized antibodies capture the fluorescein-labeledamplicons forming a visible red-colored line (Fig. 3).

Asymmetric amplification is more widely used and relieson a preferential amplification of the strand arisen from abiotinylated primer. The limiting primer is not labeled. A

specific complementary short probe labeled with a hapten(fluorescein, digoxigenin or 2,4-dinitrophenyl) at the 3’-endto avoid extension is also added to the amplification solution.This probe hybridizes with the biotinylated strand, forming apartial duplex. This particular hybridization decreases thechance of detecting unspecific products. Then, the partial du-plex enters the LFD where it binds to streptavidin-conjugatedcolor particles and is finally captured by the appropriate anti-body on the detection line (Fig. 3). This way, duplex amplifi-cations (two targets in a single experiment) can be carried out,one of them being the internal control of amplification [40,41]. Commercial kits for some pathogen are currentlymarketed [42]. The assay for Herpes simplex virus (HSV)-1and -2 in genital and oral lesions was cleared by FDA in 2011and it does not require nucleic acid extraction [43]. A simpledilution allows detection of as low as 5.5 and 34.1 copies ofHSV-1 and HSV-2, respectively, per reaction within 1.5 h[44]. Manual extraction amenable to resource-limited placesreached 470 copies/reaction of HIV-1 DNA from dried bloodspots with LFD [45]. The identification at species level ofPlasmodium malaria parasite is possible by careful selectionof the hybridization probes and primers [46]. Three probes

Entrapment&

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protein

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exonuclease activity TaqManprobeprobe

Fig. 2 Right panel: schematic representation of HDA assays withhomogeneous fluorescent monitoring, by using dual-labeled probes (a),TaqMan (b) hairpin ones. Left panel: a previous sequence-specific sample

preparation employing RNA probes along with magnetic particlesmodified with an antibody specific for RNA-DNA hybrids boosts assayperformance in clinical samples

684 Barreda-García S. et al.

with different labels were designed for a single HDA assay;one for all Plasmodium species, a second one specific forP. falciparum, and the third as an internal control. A secondHDA amplification with a specific primer for P. vivax and thefirst and third probes allowed the detection of this species inLFD cartridges. No extraction of DNA from the protozoon isneeded. The sensitivity is 5 to 10 times lower when applied topatient samples (200 parasites/μL) instead of spiked humanblood. Microtiter plate version of these LFD with colorimetricenzymatic detection was also demonstrated for detecting 10copies of the ureC gen of Helicobacter pylori [47]. Sincesymmetric amplification was carried out, a step of thermaldenaturation was needed before hybridization in a separatetube, which lengthens the assay.

Hybridization-based capture of amplicons

Undoubtedly, an extra level of selectivity is needed when re-vealing HDA amplicons. Hybridization-based target captureon surfaces is a simpler and cost-effective alternative toimmunocapturing. A specific short capture probe partiallycomplementary to asymmetrically amplified biotinylated-strand is usually anchored on the surface. Further enzymaticlabeling allows optical measurement (Fig. 4). A variety ofsurface chemistries have been applied to construct the DNAchip from the simplicity of polypropylene sheets that onlyrequire aminated probes and UV-cross-linking [48] to siliconnitride wafers that are not biologically compatible and needtwo additional layers of polydimethylsiloxane andpoly(phenylalanine-lysine), respectively [17, 33]. Those layersintroduce a large number of amine groups for further DNAbinding, achieving an optimal DNA coverage of 1.5×1012

molecules/cm2 and 100% hybridization efficiency [49].Hydrazine-terminated DNA capture probes were cross-linked using N-succinimidyl 4-formylbenzoate and thesemodified surfaces are stable up to 9 months at 37 °C [34]. Inboth assays, asymmetric HDA is performed (1:4 and 1:2, re-spectively) and the biotinylated strand is captured on the chipsurface. It is worth noting the high concentration of primerused in the second approach (above 100 nM).

Polypropylene chips use streptavidin-HRP conjugates forlabeling and detect silver enzymatically deposited. In this way,the plant pathogen Phytophthora was detected at the specieslevel using three different sets of primers in individual HDAassays on chips containing probes for each species. All fivespecies were detected but two of them could not be distin-guished because they differ in a single nucleotide [50].Combining hybridization and labeling in a single step, timeis reduced to 60 min without loss of detectability [48].

The silicon wafer approach uses anti-biotin antibody-perox-idase conjugate and interference-based detection. This unusualstrategy relies on the apparent color change caused by the de-position of the enzymatic product on the thin film. The viabilityof multiplex HDA was demonstrated for Staphylococcus spe-cies [33]. Two genes were amplified in the tube and then de-tected on a chip containing 13 different probes (1 for genusidentification, 11 for each species, and an additional for highlyhomolog bacteria) after a quick hybridization and labeling steps(5 min each). The limit of detection for pure species is 1 cfu/reaction (about 3000 cfu/mL) but increases 10 to 30 times inthe presence of several species. The time turnaround is short-ened to 30 min when performing a symmetric HDA in thehybridization chip [34]. Interestingly, unlike HDA in a tube,no artifacts were observed in the chip, suggesting beneficial

DNA sample

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Fig. 3 Schematic of lateral flowHDA assay (left), symmetric(right), asymmetric DNAamplification

Helicase-dependent isothermal amplification: a novel tool in the development of molecular-based... 685

surface/volume conditions. Blocked primer HDA combinedwith these chips permitted the detection of one copy of geno-micM. tuberculosis in 40 min with single nucleotide polymor-phism selectivity [17].

Magnetic particles are very easy to handle and facilitate thewashing steps, so they are more and more incorporated intocommercial assays. This platform was first used to develop asandwich assay to capture the denatured and purifiedamplicons obtained in a symmetric HDA assay [51].Aminated magnetic beads were modified with thiolated par-tially complementary probes through a heterobifunctionalcrosslinker. Then the DNA sandwich assay was completedby hybridization with AuNPs covered by another partiallycomplementary probe. The duplexes on the beads were placedonto disposable carbon electrodes where Au was dissolvedand oxidized at 1.25 V. Finally the cathodic current of Aureduction was measured by differential pulse voltammetry(DPV). However, the assay is long (6 h excluded DNA ex-traction) and semi-quantitative for M. tuberculosis.

More recently this pathogen was detected at attomolar levelalso using magnetic beads for capturing the unpurified bio-tinylated strands amplified by asymmetric HDA [31].Fluorescein-labeled probes hybridize with the anchored am-plified strand at the center region and are detected after enzy-matic labeling on the disposable carbon electrode bychronoamperometry. Minimization of unspecific productsusing as low as 5 nM of the limiting primer was essential.Reduction of the salt concentration during the hybridizationstep from 1 to 0.1 M NaCl was also needed to achieve a limitof detection of 0.5 aM of a synthetic specific sequence (15copies). This value rivals the detectability of an analogousassay using PCR (11 copies) [32]. Symmetric HDAwas faster(30 min versus 90 min) than the asymmetric one but 10 timesless sensitive when combined with the hybridization assayeven after a denaturation step to separate the duplex. Theamplification factor of asymmetric HDA was calculated tobe 106 respective to the genomagnetic assay, which explainsthe ultrasensitivity. Genomic DNA from a variety of clinical

samples was also tested with concordant results in comparisonwith real-time PCR and cultures.

Labels other than enzymes can also be exploited in hybrid-ization assays but until now only fluorophores were applied toHDA assays. Quantum dots covered by DNA short specificcapture probes were immobilized on paper after oxidation thecellulosic fibers with periodate to generate aldehyde groups onsurface that can react with aminopropylimidazol forming aSchiff base subsequently reduced to yield stable covalent C–N bonds [52]. Imidazol moieties bind to CdSeS/ZnS-probeoligonucleotides to complete the sensing surface.Optimization of quantum dot concentration improved the limitof detection up to 7.5 times. Paper modification is stable for atleast 2 wk and reusable up to five times [53]. This platformwas combined with a sandwich hybridization assay, whichwas performed with hybridization probes with an acceptorfluorophore in the nearest end to the quantum dot platform.FRET-based transduction allows the detection of the decreas-ing emission of the donor QD, the increasing emission of theacceptor fluorophore, or, as in this work, the ratio of bothsignals, which is more powerful to discriminate low concen-trations. SNP selectivity was reached by adjusting the concen-tration of formamide and the ionic strength in the hybridiza-tion step. An amplification factor of 107 was estimated per-mitting the detection of 37 zmol of a synthetic sequence cor-responding to a diagnostic marker of E. coli in 3 h [52].However, this time could be shortened if asymmetric HDAwas performed avoiding the time- and reagent-consumingmagnetic separation step employed for generating singlestranded sequences from HDA amplicons.

Integration of HDA and other steps in the analyticalprocess

Nucleic acid-based pathogen analysis involves three mainsteps: sample preparation (cell lysis, nucleic acid extractionand purification), amplification, and detection, all of which

N×1×

(1)

(2) (3)

S P

E E E

colored

polymerase

helicase SSB protein

primer Biotinylated primer

Enzyme conjugate

Captureprobe

Fig. 4 Hybridization-based on-surface capture of HDAamplicons: (1) homogeneousasymmetric HDA, (2) specificcapture of biotinylated ss-amplicon, and (3) enzymelabeling and optical detection ofthe enzymatic product

686 Barreda-García S. et al.

are of paramount importance for obtaining reliable results.There are different attempts in the literature toward the designof platforms that integrate HDA amplification and at least oneof these steps in order to obtain cost-effective, robust, auto-mated, and user-friendly systems useful for point-of-needpathogen control. The development of such fully integrated,low cost devices to detect DNA from pathogens fuels thedevelopment of isothermal amplification schemes as HDA.Beyond the format scheme, determined steps are being takento make this technology amenable to decentralize analysis inremote locations without electricity supply.

Amplification and detection are easily integrated byperforming the amplification on suitable materials.Membrane-based devices are well suited for low cost analysis,and several materials were tested [54]. While glass-fiber andnitrocellulose inhibit both PCR and isothermal amplificationsdue to strong enzyme adsorption, polycarbonate impededHDA when carried out within its pores but not when someliquid remains. Polyethersulfone showed the best compatibil-ity with both PCR and isothermal amplification. Cellulose canalso be used after BSA treatment to avoid enzyme adsorption[55]. The mixture of enzymes for HDA amplification is stable,after drying on paper, at room temperature for about 1 month,probably due to the presence of high molecular weight carbo-hydrate in the formulation. A rapid HDA detection ofM. tuberculosis (10 min) was performed in this substrate usingthree times higher concentration of enzymes to compensateadsorption and PicoGreen as fluorescent dye. Amplificationoccurred even at 50 °C achieved with a hand warmer. Thisqualitative assay offers a limit of detection of 100 copies. Easyand efficient extraction of DNA from cell lysate on paperinserted on a pipette was also shown [56]. The isothermalamplification of Chlamydia trachomatis was carried out inthe sealed pipette and detected in a LFD. In this way, an easilyoperated and rapid diagnostic platform useful for point-of-caredetection of sexually transmitted infectious is presented, withimproved limit of detection compared with the immunoassaystypically employed for this purpose. Although not truly inte-grated, sample preparation and amplification are combined byusing a syringe or bicycle pump to remove the cellular lysissolution and transfer the HDA solution after amplification.These pumps combined with toe warmers make this approachelectricity-free. A pump-based extraction with a hand-warmerheated amplification was also employed to detect Clostridiumdifficile in stool samples [57]. Positive amplifications weredetected by PAGE after on-chip HDA for 45 min, includingwarming up. Channels were coated with a costly polymer tolimit enzyme adsorption. Formation of primer-dimers was notnegligible and attributed to the high surface to volume ratio,contrary to the observation of Jenison et al. [34], but might bereduced by adding DMSO. Similarly, a modular device usingchip-based DNA hybridization with optical detection was re-ported without the need of power supply [48].

Only two batteries are the power need of a fully disposableintegrated paper-based device [58]. It consists of threemodules:the extraction module constructed on paper, the more complexamplificationmodule with very thin ceramic heating plates, anda lateral flow strip based onDNA hybridization for detection. Allreagents are dried and stored on the device but long storagerequires 4 °C (2 months) or –20 °C (3 months). The extractionis performed in 2 min and the overall turnaround is 77 min.

The simplicity of these extraction methods contrasts withmicro solid phase extraction on polymer microchannels.Integration of extraction and HDA on a disposablemicrofluidic disposable device was first achieved in 2010[16]. In 2012 a meso-scale fluidic system integrated the entireprocess: from extraction to optical detection (a digital camera)in an analyzer/cartridge device [59]. It was applied to stoolsamples for the detection of Clostridium difficile with a limitof detection of 10 cfu using blocked-primer HDA and siliconwafer chips. However, the manual assay is faster because ofthe motor movements and mechanical calibrations.

Passive mixing is an alternative to pumps for fluid move-ment. When combined with DNA electrostatic entrapmentfrom cell lysate on chitosan membranes, an 80% extractionefficiency compared with a commercial kit was achieved.After DNA extraction, HDAwas performed in a low cost de-vice incorporating simple optical detection [60]. Reducing thecost of optics is also behind the development of the ratiometricFRETassay described above. A UV lamp for excitation and ani-Pad camera permitted obtaining limits of detection in the fMrange comparable to the less cost-efficient epifluorescence mi-croscope [52]. Based on the same electrostatic principle, DNApurification was achieved on polycarbonate modified withpolyethyleneimine in a valve-free fashion in 30 min [61].Device rotation sequentially propels the solutions from eachof three reservoirs (for extraction, washing, and HDA reagents)to the chamber where DNA is electrostatically entrapped toperform the symmetric HDA amplification of E. coli DNA.However, detection was not integrated.

The volume of sample used is decreasing to the nL range.HDAwas performed in 192 nL in a microfluidic device withreal-time detection [30]. However, low fluorescence increaseand variability in threshold time was higher than when using 5μL volume. This technology was applied to the amplificationof a plasmid containing a fragment of SARS virus.

Finally, surface HDA has also been demonstrated as a stepforward to full assay integration on modified glass slides. Theefficiency of on-surface amplification has been reported to bepoorer than in solution phase because of the steric hindranceof the solid surface that reduces the polymerase extensionefficiency, electrostatic repulsion of high-density primers,and attachment of long DNA sequences that prevent accessto the anchored primer [62]. Slightly lower detectability forS. aureus, 5×104 versus 2×104 cells, was achieved in the firston-chip HDA automated assay [63]. The aminated primer was

Helicase-dependent isothermal amplification: a novel tool in the development of molecular-based... 687

linked to an epoxysilanized glass slide. Only one primer wasadded to a minimum of 150 μL of solution, which consumed3-fold amount of reagents. Programmed pump mixing duringthe 120 min of HDA with automatic washing steps at 30 °Cleaves the surface ready for laser scanning of the fluorophore-labeled duplex anchored on the surface. The single assay forN. gonorrhoeae was much less sensitive. Multiplexamplification/detection was feasible using the samefluorophore because of the spatial separation of capture probesspecific of each bacterium.

In order to promote the on-surface amplification, a limitedamount of a shortened version of the anchored primer wasadded to the solution (1:15 ratio) [64]. Under those conditions,it is believed that amplification initiates in solution until al-most complete depletion of the limiting primer. Then the am-plification shifts to the surface promoted by the longer an-chored primer. This pathway is supported by a delay reportedon ITO-modified surfaces, which implied a minimum of90 min for amplicon detection. No differences with blankexperiments were obtained at 75 min. The fluorescein-labeled duplex synthesized on the surface was electrochemi-cally detected by DPV after enzymatic labeling with alkalinephosphatase (ALP). Peroxidase could not be used because theenzymatic product, the oxidized TMB, presents a largeoverpotential that precludes its detection at usual potentials.Transparent ITO surfaces are also compatible with opticaldetection but UV-vis detection offered one order of magnitudehigher limit of detection even though peroxidase, which has ahigher turnover than ALP, was used. Only 10 genomic copiesof Salmonella were detected electrochemically, whichmatches real-time PCR approaches. Interestingly, the ITOmodified surface was stable under dry conditions for 9 monthsprovided that a mixture of BSA and glucose, both at 2.5%,was previously added. A rehydration step is also needed be-fore use. This extended storage stability is highly desirable forelectrochemical DNA-based sensors mostly based on fairlystable self-assembled monolayers on Au.

Applications

Most applications of HDA are devoted to the clinical fieldwhere rapid, cost-efficient devices are more and more in de-mand to cut the increasing cost of diagnosis, especially in thecase of infectious disease. Another market niche is the use atthe point of need with special interest in remote locations or indeveloping countries where electricity is not always taken forgranted. However, some applications for animal and plantviruses as well as for food contamination surveillance arestarting to gain recognition, including amplification of RNAvirus [65]. In Table 1, a summary of the main features of thereported methods is shown.

Clinical samples are contaminated more than desired withlow levels of ubiquitous bacteria such as Streptoccocus orStaphyloccocus. Elimination of unintended contaminationduring sample processing is of utmost importance in clinicaldiagnosis. Addition of a non-target specific sequence at a largecopy number or contaminating the sample with a definedamount of bacteria, the amplification of the contaminant spe-cies can be completely suppressed because of the limitedamount of HDA primers [71]. It is important that the non-target sequences/organisms have some level of homologywith the target to consume the primers. The approach is com-bined with on-chip hybridization. Only the competitive se-quence hybridization is visible under conditions whereprimers are spent in the competitive amplification before de-tectable levels of the contaminants are reached. A mathemat-ical expression to calculate the appropriate amount of suppres-sor is derived and tested against the limit of detection of thetarget bacteria. As expected, it decreases when increasing thesuppressor concentration but it remains below the threshold totrigger clinically relevant blood infection. Although this strat-egy has some limitations, it can be applied to other DNAamplification schemes.

Pros and cons of HDA for pathogen detection

There is no doubt about the urgent need of effective systemsfor rapid, at the point of need detection of pathogens for a widevariety of applications, including clinical diagnostics, foodsafety, and environmental monitoring. Nucleic acid amplifica-tion by PCR revolutionized the field of microbiology, offeringmajor advantages of speed and sensitivity for pathogen detec-tion. Although it is now possible to use PCRwhen there are noclassic lab benches [72], new isothermal amplificationmethods have been designed, which, compared with PCR,retain their advantages while reducing the complexity of anal-ysis, thus resulting in a high potential for a simple integrationinto point-of-care testing and resource-limited places.

Themain pros of HDA applied to the detection of pathogensare common to other molecular techniques, both PCR and iso-thermal amplification methods, i.e., high sensitivity in such away that organisms that are difficult or impossible to grow inthe laboratory can be detected at very low levels, and rapidturnaround time (typically between 30 and 90min). In addition,HDA is easy to use, especially when lateral flow devices areused for detection. AmpliVue assays, commercialized byQuidel (proprietary for HDA technology), are small hand-held cartridge based on HDA-lateral flow devices that are con-sidered CLIA moderately complex and are available for therapid detection of pathogens such as Bordella pertusis,Clostridium difficile, Herpes simples virus, and Trichomonasvaginalis [73] without any other equipment than a heated lid.One of the requirements for a test system to be considered a

688 Barreda-García S. et al.

Table 1 Summary of published helicase-dependent amplification methods for pathogen detection

Pathogen Detection LD (copies/reaction) HDAtime (min)

HDAT (°C) Sample turnaroundtime (min)

Ref

E. coli Gel electrophoresis – 60 65 cultures 90a [61]

Tomato virus Gel electrophoresis 4 pg RNA 60 65 Leaf tissue – [65]

L. reticulatis Gel electrophoresis 1 ng 90 65 Insect infected mixedcereals

– [66]

C. difficile PAGE 0.0125 pg 45 65 Stool – [57]

HPV-16 Real-time 1 90 65 Cervical swabs 90 [29]

HPV-18 Real-time 10 90 65 Cervical swabs 90 [29]

SARS Real-time – 30 62 Plasmid DNA 30 [30]

M.T. Real-time 300 90 65 Plasmid DNA 90 [18]

E. coli Real-time 100 cfu/mL 60 65 Cultures 60 [16]

T. vaginalis Real-time (comercial) – 45 – Vaginal swab/ urine 45 [38]

E. coli Echem real-time 105 80 65 Plasmid DNA 80 [35]

V. cholerae TaqMan real-time 50 60 65 Genomic DNA 60 [36]

B. antracis TaqMan real-time 50 (pXO1 gene) 90 63 Genomic DNA 90 [36]

B. antracis TaqMan real-time 50 (pXO2 gene) 90 63 Genomic DNA 90 [36]

B. antracis TaqMan real-time 50 (multiplex) 90 63 Genomic DNA 90 [36]

M.T. Fluorescence 100 10 65 Artificial sputum – [55]

S. aureus Fluorescence 5 cfu/mL 90 65 Spiked milk powder 120 [67]

50 cfu/mL 90 65 Spiked pork meat 120 [67]

N.G. Fluorescence 5–10 cells 60-120 65 Spiked collection media – [22]

25 cells (multiplex) 90-120 65 Spiked collection media – [22]

C.T. Fluorescence 5–10 cells 60-120 65 Spiked collection media – [22]

25 cells (multiplex) 90-120 65 Spiked collection media – [22]

N.G. LFD 50 20 65 Urine 35 [23]

C. difficile LFD 20 60 65 Stool 75 [40]

S. aureus LFD 50 cfu/mL 60 65 Bacteria blood culture 80 [41]

Strep Group A LFD – 35 64 pharyngeal swab 45 [42]

HSV-1 LFD 5.5 60 64 Genital lesion swab 75 [44]

HSV-2 LFD 34.1 60 64 Genital lesion swab 75 [44]

HIV-1 LFD 470 copies/mL 75 64 Dried blood spots 95 [45]

Plasmodium LFD 50 copies 90 64 genomic DNA 105 [46]

Plasmodium LFD 200 parasites/μL 120 64 Blood samples 135 [46]

C.T. LFD 1000 cells 30 65 Synthetic urine 50 [56]

S. equi LFD 50 90 65 Horse nasal swabs 105 [68]

M. T LFD 2 30 65 Cultures 50 [69]

Salmonella LFD 35–40 cfu 60 64 Bacterial culture 90 [70]

1.3–1.9 cfu/mL 60 64 spiked chicken, infantcereal, milk

90b [70]

S. typhymurium LFD 100 cfu/mL 60 65 waswater / eggs 77a [58]

1000 cfu/mL 60 65 milk /juice

H. pylori Hyb Enzym optic 10 60 60 Gastric biopsies 240 [47]

Staphylococcus Hyb Enzym optic 1 (mecA gene) 45 65 Blood samples 75 [33]

Staphyloccocus Hyb Enzym optic 1–250 cfu (tuf gene) 45 65 Blood samples 75 [33]

M. T. Hyb Enzym optic 1 40 65 Clinical samples – [17]

S. aureus Hyb Enzym optic 10 30 65 genomic DNA 35 [34]

S. aureus Hyb Enzym optic – 40 65 Blood cultures 60a [34]

Phytophthora Hyb Enzym optic – 90 65 --- [50]

P. kernoviae Hyb Enzym optic 10 pg/μL 75 65 Artificially infectedleaf tissue

135 [48]

Helicase-dependent isothermal amplification: a novel tool in the development of molecular-based... 689

CLIA-waiver is that it must be run using a specimen that doesnot require processing [74]. This can be considered anotheradvantage of HDA, which is used for a variety of specimencollections such as vaginal, rectal, or nasopharyngeal swabswith a simple heat treatment for lysing. However, the sampletreatment depends on the pathogen/sample analyzed. In foodanalysis, for example, a culture enrichment step is usually re-quired to reach the legal limits, in many cases absence of thepathogen.

Despite the above advantages, HDA still need to overcomesome important limitations, again in many cases common toother nucleic acid amplification techniques. Sample contami-nation is one of the typical concerns when using highly sen-sitive methods. HDA can be very useful for pathogen detec-tion, but it can also be easily misinterpreted. Closed and dis-posable devices may contribute to alleviate this problem. Theuse of a constant temperature of amplification is the origin ofanother problem, i.e., the non-specific amplification productsattributable to mispriming that may lead to false positive.Among isothermal methods, low-temperature amplificationssuch as RPA (operating at 37 °C) or LAMP that uses sixdifferent primers are more prone to this problem.Conversely, false negatives may arise because test inhibition,especially in complex biological matrices. The nature andamount of potential inhibitors is different in different samplesand the concentration at which these inhibitors affect HDAmust be empirically obtained. The use of an internal control,as is the case in Solana commercially available assays [75], isa way of minimizing and controlling this problem. Finally,lack of validated assays is another important drawback.

Considering the common advantages and disadvantagesthat isothermal nucleic acid amplification methods possess,it is difficult to recommend one method that may be appliedas universal pathogen test. However, the LAMP technique iscurrently more popular for point of need applications. This isprobably related to a better marketing by developers, withdifferent companies commercializing LAMP products.Conversely, a single company is still proprietary of HDAtechnology, limiting its widespread use. In the future, with ahigh promotion by developers and advancements in the inte-gration of the entire analytical process in lab-on-a-chip sys-tems and wireless communication, HDAwill most likely showa significant increase for point of need pathogen detection. Aneconomic study is also needed to determine whether this ap-proach is going to be truly cost-effective.

Conclusions

Helicase-dependent amplification, a novel nucleic acid ampli-fication strategy inspired by biological processes, is a promis-ing alternative to PCR for pathogen detection in differenttypes of samples. It has been mainly applied in the field ofinfectious disease diagnosis and treatment monitoring, al-though it is also earning popularity for foodborne pathogendetection. Important progress has been made in the past fewyears for developing devices based on HDA amplification andcompatible with point-of-need testing useful for resource-limited settings.

Table 1 (continued)

Pathogen Detection LD (copies/reaction) HDAtime (min)

HDAT (°C) Sample turnaroundtime (min)

Ref

C. difficile Hyb Enzym optic 1 cfu (manual) 45 65 Fecal samples 60a [59]

10 cfu (automated) 45 65 Fecal samples 90a [59]

M. T. Hyb Enzym chronoA 15 90 65 Clinical samples – [31, 32]

M.T. Hibridization DPV 0.1 ng/μL 90 65 Synthetic DNA 360 [51]

E. coli Hibridization FRET 37 zmol 60 65 Synthetic DNA – [52]

S. aureus On-surface fluorescence 50,000 cells 120 65 Genomic DNA – [63]

S. aureus On-surface fluorescence 5×105 cells (multiplex) 120 65 Genomic DNA – [63]

N.G. On-surface fluorescence 1.32×105 cells 120 65 Genomic DNA – [63]

N.G. On-surface fluorescence 1.32×106 cells(multiplex)

120 65 Genomic DNA – [63]

Salmonella On-surface enzymchronoA

10 90 65 Genomic DNA – [64]

Salmonella On-surface enzym optic 100 90 65 Genomic DNA – [64]

C.T.:Clamidia trachomatis, N.G.:Neisseria gonorrhoeae, M.T.:Mycobacterium tuberculosis, HSV: herpes simplex virus, HPV: human papiloma virus,HIV: human immunodeficiency virus, Echem: electrochemical, Hyb: hybridization, Enzym: enzymatic, ChronoA: chronoamperometry, DPV: differen-tial pulse voltammetrya Including extraction.b +2–4 h of enrichment.

690 Barreda-García S. et al.

One of the major advantages of HDA over other isothermalamplification techniques such as loop mediated amplification(LAMP) is that the reaction configuration is very simple, and itsPCR-like reaction scheme allows for adaptation of the currentPCR assays to the HDA system. However, it would be neces-sary to have a systematic study of the modifications in theprimers needed to alleviate the non-specific amplification prob-lems, more pronounced in HDA. The availability of specificsoftware to model the primer modification would be particular-ly useful, accelerating the adoption of the HDA systems at fieldconditions. Another critical challenge is the improvement inmultiplexing capability of paramount importance in infectiousdisease diagnosis and treatment monitoring, especially to copewith the rapid emergence of antibiotic-resistant bacteria.

Early detection of pathogens is an important issue for con-trolling the spread of infectious disease and to rapidly respondto outbreaks of contagious and serious diseases. On-surfaceHDA holds promise in the development of integrated deviceswhere amplification and detection are performed in an automat-ed, miniaturized closed system. However, these platforms haveto be integrated with sample preparation systems and furtherevaluated in both clinical laboratories and different settings atthe point of need before they are effectively marketed and de-ployed in real applications. Different applications have alreadybeen described for the detection of common diseases associatedwith developing countries, such as malaria and AIDS.Nevertheless, the demonstration of cost-effectiveness will be-come a critical factor for their ultimate success. In this sense,the fact that the methodology is under the protection of a patentand that the necessary enzymes are produced, supplied, andlicensed by a single company may be a limiting factor forwidespread use of HDA. Once these rights have expired,HDA-based devices have the potential to become a referencefor nucleic acid amplification at point of need applications, witha profitable commercialization worldwide.

Acknowledgements This work was financially supported by theSpanish Ministerio de Economía y Competitividad (Project no.CTQ2015-63567-R), the Principado de Asturias government (ProjectFC15-GRUPIN14-025), and cofinanced by FEDER funds.

Compliance with ethical standards

Conflict of interest The authors declare that they have no conflict ofinterest

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