Helicase-dependent amplification

Introduction

 Traditional methods for the detection of pathogens are well established microbiological assays and are usually inexpensive. But, they are time consuming, space occupying, and laborious. It takes 2 to 3 days for preliminary identification and even more than 1 week for confirmation. The advent of polymerase chain reaction (PCR) has led to a new paradigm for the detection of pathogens, giving rise to assays that are exquisitely sensitive, and relatively rapid. Polymerase chain reaction is the most widely used method for in vitro DNA amplification and  requires thermocycling to separate two DNA strands.  It involves multiple thermocycling steps in high precision instruments. It is difficult to miniaturize and require stringent conditions of laboratory compartmentalization. Thus it is exclusively performed in centralized laboratories with high-end instrumentation. Hence, the technique suffers from being less affordable, user friendly and robust to be an ideal test for point-of-care pathogen detection.  In vivo, DNA is replicated by DNA polymerases with various accessory proteins, including a DNA helicase that acts to separate duplex DNA. Novel Isothermal amplification methods are gaining importance for the development of analytical devices aimed to meet those ideal criteria. It can be easily integrated into simple and low-energy consumption microsystems. Helicase-dependent amplification (HDA) is one of them and it utilizes a DNA helicase to generate single-stranded templates for primer hybridization and subsequent primer extension by a DNA polymerase. HDA does not require thermocycling.  In addition, it offers several advantages over other isothermal DNA amplification methods by having a simple reaction scheme and being a true isothermal reaction that can be performed at one temperature for the entire process. These properties offer a great potential for the development of simple portable DNA diagnostic devices to be used in the field and at the point-of-care.

 The polymerase chain reaction (PCR) revolutionized capabilities to do biological research, and it has been widely used in biomedical research and disease diagnostics. Hand-held diagnostic devices, which can be used to detect pathogens in the field and at point-of-care, are demanded currently. However, the need for power-hungry thermocycling limits PCR application in such a situation. Several isothermal target amplification methods have been developed. These methods include loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), rolling circle amplification (RCA), strand displacement amplification (SDA) and helicase-dependent amplification (HDA).

Loop mediated isothermal amplification

 An isothermal method which relies on autocycing strand displacement DNA synthesis by Bst DNA Polymerase and a set of 4-6 priers. Two inner and two outer primers are added to increase the sensitivity of the reaction. The final product of the LAMP reaction are DNA molecules with a cauliflower like structure of multiple loops consisting of repeats of the target sequence. The products can be analysed in real time by monitoring of turbidity in the reaction tube resulting from production of magnesium pyrophosphate precipitate during the DNA amplification. Can also be visualized in agarose gel and staining with ETBR or SYBR green. As its an isothermal process, positive reaction can be detected by simple turbidity measurements or visualized directly with naked eye as it requires no expensive instrument.

 recombinase polymerase amplification

 Recombinase polymerase amplification (RPA) is another isothermal technique that, like HDA, does not require an initial heating step to denature the target DNA . It relies on an enzymatic activity to separate the dsDNA in order to assist primer binding to the target sequences. The reaction begins with the integration of a recombinase protein with the primers prior to their annealing to specific sequences in the target. Following primer annealing, the recombinase dissociates from the primers and leaves their 3’ end accessible to the DNA polymerase to initiate the amplification. This creates a d-loop which is stabilized by a single stranded binding protein (SSB) to keep the DNA open as a DNA polymerase with strand displacement activity continues the amplification. Using RPA, billions of DNA copies can be generated efficiently in 60 min with an incubation temperature between 37◦C and 42◦C. The low incubation temperature and short reaction time (15– 30 min) make RPA a suitable assay for use in point-of-care diagnostic applications. Furthermore, primer design is simple without consideration of annealing temperature as they form a complex with the recombinase to target the homologous sequences. RPA is highly sensitive with a detection limit as low as 6.25 fg of genomic DNA input with a specificity >95% (Boyle D.S. et al., 2014). However, RPA has some drawbacks as it can only amplify small DNA fragments (<500 bp). This makes it unsuitable for cases where amplification of full length genes is required. In addition, the longer primers (30–35 nt) required for RPA are prone to produce non-specific amplification at low temperature. Furthermore, the primers used in the RPA reaction frequently generate high background noise on negative and non-template control samples due to the formation of primer dimers thus affecting the sensitivity of the assay.

  In the rolling circle amplification (RCA), a DNA polymerase extends a primer on a circular template, generating tandemly linked copies of the complementary sequence of the template. However, these isothermal nucleic acid amplification methods also have their limitations. Most of them have complicated reaction schemes. In addition, they are incapable of amplifying DNA targets of sufficient length to be useful for many research and diagnostic applications.

 Strand-displacement amplification (SDA) combines the ability of a restriction endonuclease to nick the unmodified strand of its target DNA and the action of an exonuclease-deficient DNA polymerase to extend the 30 end at the nick and displace the downstream DNA strand. Transcription mediated amplification (TMA) uses a RNA polymerase to make RNA from a promoter engineered in the primer region, a reverse transcriptase to produce complementary DNA from the RNA templates and RNase H to remove the RNA from cDNA.

 HDA is one of the simplest approaches for isothermal nucleic acid amplification that mimics an in vivo process of DNA replication. In living organisms, a DNA helicase is used to separate two complementary DNA strands during DNA replication. The method developed by Vincent et al. in 2004, uses helicase to isothermally unwind DNA duplexes instead of heat, which was subsequently patented in 2007. HDA uses a DNA helicase to separate double-stranded DNA (dsDNA) and generates single-stranded templates for primer hybridization and subsequent extension. As the DNA helicase unwinds dsDNA enzymatically, the initial heat denaturation and subsequent thermocycling steps required by PCR can all be omitted. Thus, HDA provides a simple DNA amplification scheme: one temperature from the beginning to the end of the reaction. The initial studies employed UvrD helicase from E. coli, a well-studied helicase II that unwinds DNA in a 3’ to 5’ direction. In the process it uses energy from the hydrolysis of adenosine triphosphate to break the hydrogen bonds between complementary bases in duplex DNA. In E. coli cells, this protein is involved in the repair of mismatches in DNA, working in coordination with methyl-directed mismatch repair (MutL) protein that stimulates the helicase activity. In the below given methodology use of UvrD-based HDA system on Escherichia coli has been used to demonstrate the working of this system. The process can achieve over a millionfold amplification.

Material

  • T4 gene 32 protein
  • Adenosine 50-triphosphate (ATP)
  • Primer-175-LUX
  • exo-Klenow fragment DNA polymerase
  • pTYB1 vector
  • pTYB3 vector
  • pTXB1 vector
  • dNTPs and oligodeoxynucleotides

  Cloning and purification of UvrD helicase and MutL from E. coli

 uvrD and mutL genes were amplified from E. coli K12 genomic DNA using PCR and cloned into the NcoI and SapI sites of pTYB3 and NdeI and SapI sites of pTYB1, respectively, to construct C-terminal fusions with a self-cleavable affinity tag

 Cloning and purification of gene protein 32 from bacteriophage RB 49

 Gene 32 from Genbank was amplified from RB49 genomic DNA using PCR and cloned into the NdeI and SapI sites of pTXB1 to construct C-terminal fusions with a self-cleavable affinity tag

 HDA reactions for amplifying target sequence

 Two HDA buffers were prepared. The 10 x HDA buffer A contains 350mM Tris-acetate (pH 7.5) and 100mM dithiothreitol and the 10 x HDA buffer B contains 10mM Tris-acetate (pH 7.5), 1 mg/ml bovine serum albumin and 100mM magnesium acetate. HDA reaction component A (30 ml) was prepared by combining 5 ml of 10 x HDA buffer A, template (plasmid DNA, genomic DNA, cells, processed human blood sample), 10–20 pmol of each target-specific primer, 20 nmol dNTPs and dH2O. The reaction component A was heated for 2–10 min at 95oC to denature the template and 1–4 min at 37oC. Reaction component B (20 ml) was freshly prepared by mixing 5 ml of 10x HDA buffer B, 150 nmol ATP, 5U exo-Klenow fragment, 100 ng UvrD helicase, 400–800 ng MutL protein, 4.5 mg T4 gp32 or 5.8 mg RB49 gp32, and dH2O. Component B was then added to component A. The reaction was continued for 1–3 h at 37oC and was then terminated by addition of 12.5 ml of stop buffer (0.1% sodium dodecyl sulphate, 50mM Na2EDTA, 15% Ficoll and 0.2% orange G). Reaction products were analyzed on a 2% GPG LMP agarose gel containing ethidium bromide. The HDA reaction without heat denaturation was set up by combining all the elements mentioned above in the same tube and incubating directly for 2 h at 37oC. To monitor HDA in real time, fluorescent primers were used (primer-175- LUX). The amplification products were detected by measuring fluorescent signals at 490nM at 5 min intervals using a PCR thermal cycler.

 HDA design

 In this system, strands of duplex DNA are separated by a DNA helicase and coated by single-stranded DNA (ssDNA)-binding proteins. Two sequence-specific primers hybridize to each border of the target DNA. DNA polymerases extend the primers annealed to the templates to produce a dsDNA. The two newly synthesized dsDNA products are then used as substrates by DNA helicases, entering the next round of the reaction. Thus, a simultaneous chain reaction proceeds resulting in exponential amplification of the selected target sequence.  E. coli UvrD helicase was chosen as the DNA helicase for our first HDA system because it can unwind blunt-ended DNA fragments. The SSB in the HDA reaction is either bacteriophage T4 gene 32 protein or RB 49 gene 32 protein.

Expected Results

 Amplification of a target sequence from plasmid DNA

 Two M13/pUC19 universal primers (1224 and 1233) were used in an HDA reaction to amplify selectively a 110 base pair (bp) target sequence from a derivative of pUC19 plasmid. In a first step, substrate DNA was mixed with the primers for heat denaturation and subsequent annealing. The component B mixture containing key enzymes, such as E. coli UvrD helicase plus its accessory protein MutL, phage T4 gene 32 protein and the exo-Klenow fragment of DNA polymerase I, was then added into component A. After a 1 hr incubation period at 37oC, a 110-bp amplification product was observed on a 2% agarose gel . Sequencing results confirmed that it matched the target DNA sequence. To determine the essential elements in the HDA reaction, each key component was omitted from the reaction. In the absence of UvrD helicase, no amplification was observed, confirming that helicase is required for the amplification. In the absence of accessory protein MutL, no amplification product was observed, suggesting that UvrD helicase mediated amplification requires MutL. In vivo, MutL, the master coordinator of mismatch repair, recruits UvrD helicase to unwind the DNA strand containing the replication error. MutL stimulates UvrD helicase activity more than tenfold by loading it onto the DNA substrate. In the absence of T4 gene 32 protein, again no amplification product was observed, indicating that SSB is required in this reaction, probably to prevent reassociation of the complementary ssDNA templates at 37oC. In the absence of ATP, no amplification product was detected, indicating that the helicase cofactor is essential for HDA. Target sequences up to 400 bp can be efficiently amplified from plasmid DNA, beyond which the yield drops markedly.

 Amplification of target sequences from genomic DNA

 The E. coli UvrD-based HDA system was used to amplify a 123-bp fragment from an oral pathogen, Treponema denticola. A restriction endonuclease gene encoding a homologue of earIR (GenBank accession number: TDE0228) was chosen as the target gene. The amplification power of the current HDA system was also determined by decreasing the amount of T. denticola genomic DNA. The amount of template was varied from 107 to 103 copies of the T. denticola genome. In general, the intensities of the HDA product decreased as the initial copy number was lowered. With 103 copies of initial target, about 10 ng of products were generated, which corresponds to 1010 molecules of the 123-bp fragment. Thus, the current HDA system described  here is capable of achieving over ten million-fold amplification. The negative control, containing no T. denticola genomic DNA, showed no trace of amplified products, proving the specificity and reliability of HDA.

 In addition to T. denticola, the E. coli UvrD-based HDA system can amplify target sequences from various genomic DNAs isolated from Helicobacter pylori, E. coli, Neisseria gonorrhoeae, Brugia malayi and human cells. As helicases are able to unwind duplex DNA enzymatically, we tested whether the entire HDA reaction could be carried out at one temperature without prior heat denaturation. Another region (102 bp) of the earIR homologue gene was chosen as target. Component B was added to A either immediately or after a denaturation step. The yield of the one-step HDA amplification was about 40–60% of the two-step HDA reaction. Nevertheless, enough product is generated to be detected. This demonstrates that HDA is able to amplify a target sequence from bacterial genomic DNA at one temperature for the entire process.

 Amplification of a target sequence from T. denticola cells

 To test whether HDA can be used on crude samples, the reaction was carried out directly on bacterial cells. A 111-bp sequence within T. denticola glycogen phosphorylase gene was chosen as target. A specific product was obtained when using 107 to 104 cells as template. As the initial cell number was lowered, the intensity of the HDA-specific product decreased and other products of lower molecular weight were observed. These products are non-target specific as they could also be detected for the negative control. They result from a nonspecific amplification and are most probably derivates of primer-dimers. Primer-dimers can be generated by the HDA reaction when the template amount is very low; they also occur in the PCR reaction. Nevertheless, the negative control allows us to distinguish the target-specific from the non-target-specific products. The current HDA system can work on crude samples, such as whole bacterial cells with only a tenfold loss of sensitivity compared with the purified genomic DNA.

 

Detection of B. malayi DNA in blood

To test the possibility of using HDA on real samples, a pathogen’s DNA sequence was amplified in the presence of human blood. A 99-bp fragment of the HhaI repeat of the filarial parasite B. malayi was chosen as target. First reported to comprise 10–12%, and then 1% of the Brugia genome, this highly repeated sequence became a target of choice for the detection of B. malayi. Decreasing amounts of B. malayi genomic DNA were added to human blood samples. After extraction and dialysis, the samples were used as templates for HDA reactions. A specific product was detected for samples containing as low as 5 pg of B. malayi DNA, which corresponds to 500 copies of the genome. These results demonstrate the feasibility of using HDA to detect a pathogen in a real sample.

 Real-time HDA

A real-time detection system using a LUXTM primer specific to the earIR homologue gene in T. denticola. Two identical HDA reactions along with a negative control were performed. After 35 min, product accumulation generated a typical sigmoid curve. A semi-logarithmic plot of the increase in fluorescence in the early phase of the reaction revealed an initial first-order reaction with a rate of amplification (V) of 0.23 RFU/min, which corresponds to a doubling time of 3 min. Following the log-linear phase, the reaction slowed, entering a transition phase (between 45 and 80 min), eventually reaching the plateau phase.

 Advantages

HDA has a significant advantage over PCR in that it eliminates the need for an expensive and power-hungry thermocycler. HDA also offers several advantages over existing isothermal DNA amplification methods. First, it has a simple reaction scheme, in which a target sequence can be amplified by two flanking primers, similar to PCR. In contrast, other isothermal DNA amplification techniques have complicated reaction mechanisms and experimental designs. For example, SDA uses four primers to generate initial amplicons and modified deoxynucleotides to provide strand-specific nicking. TMA needs three different enzymatic steps (transcription/cDNA synthesis/RNA degradation) to accomplish an isothermal RNA amplification. This complexity and the inefficiency in amplifying long targets limit their use in biomedical research. These isothermal amplification techniques are primarily used in specifically designed diagnostic assays, and PCR remains the only protocol used by researchers to amplify specific targets of DNA. Unlike other isothermal methods, HDA uses only two oligonucleotide primers to initiate the exponential DNA synthesis needed to reach high analytical sensitivity. In addition, HDA can yield a surplus of one of the two strands of DNA by using a different ratio of the two amplification primers. In this manner it allows for the use of hybridization probes to confirm the legitimacy of the nucleic acid amplification products without a denaturation heating step. By enabling the use of probes under isothermal conditions (i.e., 65◦C), HDA allows for the inclusion of competitive internal controls (CIC) in its assays. The CIC is a spike in template DNA that shares the same primer binding sequence as the target sequence, but has a different internal sequence such that it can be detected with a separate probe. The use of a CIC allows users to distinguish between true negative assay results from invalids due to the presence of amplification inhibitors found in some. Second, HDA is a true isothermal DNA amplification method. As DNA helicase can melt double-stranded target DNA at the beginning of the reaction, the entire HDA reaction can be performed at one temperature. In contrast, other isothermal methods, such as SDA, still need an initial heat denaturation step at a high temperature followed by amplification at a lower temperature. Third, HDA is at its early development stage. The current UvrD system can achieve over a million-fold amplification. A pathogen genomic DNA can even be detected in a human blood sample. This demonstrates that HDA can be performed on crude samples and has the potential to be used as a diagnostic tool. Methods for the early and sensitive detection of pathogenic bacteria suited to low-resource settings could impact diagnosis and management of diseases.  Helicase-dependent isothermal amplification (HDA) is an ideal tool for this purpose, especially when combined with a sequence-specific detection method able to improve the selectivity of the assay.  The implementation of this approach requires that its analytical performance is shown to be comparable with the gold standard method, polymerase chain reaction (PCR).

 Critical Parameters

Primer design is the most critical difference between HDA and PCR. Primers intended for HDA have less tolerance to secondary structures that could lead to potential primer-dimer amplicon formation. This is due to the fact that in HDA, amplicons are continuously formed, whereas in PCR, periods of synthesis are segregated to incubations following the annealing temperature. As such, amplification products that are more efficiently replicated during HDA can outrun slower amplification products more easily than in PCR. HDA reaction assembly, amplification, and gel electrophoresis–based detection steps should be carried out in physically separated locations. Test operators should always wear gloves, open reaction tubes only when adding reagents into them during reaction setup, and keep tubes closed at all other times. The HDA and qHDA assays are extremely sensitive to changes in magnesium and salt concentrations.

Time Considerations

HAD amplification is highly primer dependent. Some reactions are completed with extremely short incubation durations such as <20 min. A number of additives, such as water-exclusion agents like Ficoll, can also enhance the speed of the HDA reaction. In addition, the purity of the input sample containing the target nucleic acids can influence the duration of the incubation. Unprocessed clinical samples, such as whole blood, may require longer incubation times. For e.g. 5 μl of unprocessed whole blood takes 120 min. Once optimal amplification duration is identified, the reproducibility with other similar samples is relatively good.

A particular feature of HDA is the recommended long size of HDA primers with respect to PCR counterparts. This leads to confusion between specific amplicons and spurious products, and hence false positive results, as a consequence of their similar size. HAD uses lower primer concentrations (75–100 nM) compared to PCR (0.1–1 μM) to avoid the risk of primer-dimer artifacts. Different research group utilized different assay strategies to avoid undesired side products arising from primers. Given that DNA polymerase synthesizes new DNA strands in a 5’–3’ direction, interactions between or within the primers are due to overlapping 3’-ends, even little apparent complementarity, and corrective actions have been focused in this direction. Chemically inactivated primers containing a 3’ blocking group as well as a ribonucleotide moiety near the 3’-end has been proposed to prevent elongation by DNA polymerase. Unblockage and activation of the primers only take place once they hybridize with the target DNA and the ribonucleotide linkage is subsequently cleaved by a hot-start RNase H2. At 65 °C, optimal temperature for HDA, RNaseH2 becomes fully active and releases a DNA fragment with a 3′-hydroxyl group able to initiate the amplification. With this approach, primer-based side reactions are eliminated, not affecting HDA amplification speed and efficiency. However, it cannot be implemented in RNA amplification. Inefficient consumption of primers, not leading to the desired product, can be also attenuated by self-avoiding molecular-recognition system (SAMRS). SAMRS draws on a set of dNTP analogues (A*, T*, G*, and C*) that have been designed to pair with natural nucleobases but not with each other, based on the number of hydrogen bonds. Therefore, primers built from SAMRS hybridize with the target but they do not interact with each other, avoiding primer dimer artifacts. Research groups have demonstrated thermophilic enzymes (polymerase and helicase) capability to work with SAMRS-structurally altered nucleotides by just adjusting Mg2+ concentration. Moreover, the addition of a thermostable reverse transcriptase has allowed RNA target amplification to benefit from these chimeric primers. On the other hand, the influence of the nucleobases placed at the 5′-end of the primers has been also studied, finding an improved efficiency in HDA when these termini are enriched in adenine or cytosine.

 Optimization

Optimization of current HDA systems involves identifying rate limiting steps. In the HDA reaction, the unwinding, primer annealing and extension steps must be coordinated. One of the rate-limiting steps could be the coordination between the helicase and the DNA polymerase. The exo-Klenow fragment can be substituted by other polymerases such as T7 sequenase (USB) or Klenow fragment, but none of these polymerases improved the reaction. A DNA polymerase, which can move with the DNA helicase in a coordinated way, would be an ideal combination. This kind of coordination can be found at the in vivo replication fork where DNA polymerase III interacts with the DnaB helicase. One way to achieve this kind of coordination is to use a helicase/polymerase pair that works together naturally. Another rate-limiting step could be the interaction between SSB and DNA. The essential role of SSB in the HDA reaction is probably to prevent the reassociation of the separated DNA strands. Indeed, no DNA amplification was observed in the absence of SSB. Both T4 gene 32 protein and RB49 gene 32 protein can efficiently support the HDA reaction. They can be substituted by E. coli SSB or T7 gene 2.5 SSB, but the yield of amplification is lower. HDA is often hampered by template-independent primer interactions giving rise to a nonspecific amplification phenomenon. This causes false positive results and adversely affects the detectability and robustness of the method. Primer-dimer and unspecific amplification are more pronounced in HDA than in PCR. A careful primer design can reduce primer-dimer formation, and fundamentally occur because no hot-start polymerase for HDA is currently available. This is especially a problem for the detection of low copy number targets due to the long time required for amplification. Also in case of multiplex amplifications where various pairs of primers must function together. The frequently used additives such as DMSO, betaine, and sorbitol  in PCR are used to reduce DNA secondary structures facilitating primer annealing. They have also proved to be effective in HDA amplification improving its yield and specificity. However, these additives can also greatly reduce the activity of polymerase. The optimization of reaction enzymes mixture is another important issue. Lowering the concentration of both ATP and dNTPs with respect to that usually employed improves the efficiency and selectivity of HAD. It suppresses primer-dimer formation, and thus reduces the background amplification problems. All these measures may be combined, and it is of paramount importance to experimentally determine the optimal amount of all additives and reagents for each amplification system.

The sensitivity and robustness of HDA has been seen to improve with a different strategy that is the digestion with specific restriction endonucleases during amplification. This strategy mimics the natural process of mismatch repair in which helicase is involved. The restriction enzymes are selected to cleave a sequence near the place where primers anneal, generating blunt ends that help recruiting and loading the helicase. This improves the effective loading of the helicase and thus accelerates the HDA reaction. However, it renders the design more challenging since it is necessary to find an enzyme capable of cutting the dsDNA close to the target sequence, without fragmenting it. The efficiency of HDA to amplify long amplicons is low due to the limited speed and processivity of UvrD helicase, that must be on high molar excess in relation to the dsDNA substrate to unwind it.

Helimerase, a new bifunctional protein, has been engineered by linking the UvrD helicase with Bst-DNA polymerase to increase the speed of DNA synthesis. This complex demonstrated increased apparent processivity, and allowed the amplification of fragments significantly longer (up to 2.3 kb) than that with the two individual proteins. However, this new enzyme is not commercially available, and further studies should be undertaken to simplify and make its production more cost-effective. Gold nanoparticles (AuNPs) in the reaction media provide a solution to improve the efficiency of HDA. AuNPs bind to ssDNA with higher affinity than to dsDNA, similar to SSB and they may improve the denaturation efficiency of helicases. In this way not only sensitivity but also selectivity of HDA has been improved. This is a very recent discovery that opens new opportunities for improving HDA, and undoubtedly it requires further investigations.

Strategies for monitoring HDA

HDA is compatible with different detection mechanisms, and significant improvements have been made in monitoring HDA amplicons both at the end-point of the amplification reaction and in real time. Similar to end-point PCR, the successful performance of HDA is usually accomplished by agarose gel electrophoresis and staining with fluorescent DNA binders such as ethidium bromide or safer and more environmentally friendly alternatives. A very common pitfall of suboptimal isothermal amplification designs is it does not allow either quantitation or discrimination of unspecific amplicons of similar length.

Real-time HDA

Fluorescence detection

Monitoring of the amplification process in real time is the most straightforward solution. Real-time HDA was first developed using a non-mutagenic, non-cytotoxic fluorescent intercalator (EvaGreen) . It is only fluorescent when bound to dsDNA, which ensures low background signals. Besides, it shows less amplification inhibition than SYBR Green. Since no temperature cycles are performed, the analytical signal is the time needed to exceed an established threshold in fluorescence intensity. This time decreases with target concentration, as expected, and with the primer concentration used. This means that the amplification speed is boosted by the amount of primers in solution. However, an enhancement in primer dimer amplification occurs, limiting the improvement in detectability

Electrochemical detection

Electrochemical monitoring of the HDA is also possible by replacing the fluorescent probe with an electroactive intercalator. Its principle is that the redox activity decreases after intercalation because of the inability to reach the electrode surface, so this detection strategy is more prone to false positive results. As conventional real-time amplifications, high-throughput measurements can be carried out in disposable electrochemical microplates. Similar detection limits and reproducibility to fluorescent real-time HAD were obtained for a specific sequence of E. coli plasmid but amplification rate was slower, probably due to stronger inhibition by redox probe than by EvaGreen.

In both fluorescent and electrochemical approaches, a melt curve after amplification is compulsory to check the presence of unspecific amplicons. To improve selectivity and allow multiplex assays, specific probes such as molecular beacons, FRET, TaqMan, or Scorpions probes are specifically designed to hybridize with a short fragment of the target strand . More recently, hairpin probes with a fluorophore and a quencher at each end where also successfully used in a 4- plex real-time HDA assay for two sequences of C. trachomatis and one of N. gonorrhoeae in addition to the internal control.

Amplicon capturing approaches

Lateral flow devices

In order to achieve low-cost DNA detection assays, real-time monitoring is not adequate. In symmetric amplifications a dual-labeled amplicon is obtained by incorporating a biotinylated primer and the other primer bound to a fluorescein tag. The LFD contains streptavidin-coated gold nanoparticles that bind the biotinylated amplicon. The complex flows until the detection line, where immobilized antibodies capture the fluorescein-labeled amplicons forming a visible red-colored line.

Hybridization-based capture of amplicons

Hybridization-based target capture on surfaces is a simpler and cost-effective alternative to immunocapturing. A specific short capture probe partially complementary to asymmetrically amplified biotinylated strand is usually anchored on the surface. Further enzymatic labeling allows optical measurement. A variety of surface chemistries have been applied to construct the DNA chip from the simplicity of polypropylene sheets that only require aminated probes and UV-cross-linking to silicon nitride wafers that are not biologically compatible and need two additional layers of polydimethylsiloxane and poly(phenylalanine-lysine), respectively. Undoubtedly, it provides an extra level of selectivity to reveal HDA amplicons.

 Integration of HDA and other steps in the analytical process

 Nucleic acid-based pathogen analysis involves three main steps: sample preparation (cell lysis, nucleic acid extraction and purification), amplification, and detection. Each step is of paramount importance for obtaining reliable results. To obtain cost-effective, robust, automated, and user-friendly systems which can be useful for point-of-need pathogen control different attempts have been made in past toward the design of platforms to integrate HDA amplification. The development of such fully integrated, low cost devices to detect DNA from pathogens fuels the development of isothermal amplification schemes as HDA. In order to make this technology to be used in remote locations without electricity supply different strategies are being developed. Amplification and detection are easily integrated by performing the amplification on suitable materials. While glass-fiber and nitrocellulose inhibit both PCR and isothermal amplifications due to strong enzyme adsorption, polycarbonate impeded HDA when carried out within its pores but not when some liquid remains. Polyethersulfone showed the best compatibility with both PCR and isothermal amplification. Cellulose can also be used after BSA treatment to avoid enzyme adsorption. 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 carbohydrate in the formulation. A rapid HDA detection of M. tuberculosis(10 min) was performed in this substrate using three times higher concentration of enzymes to compensate adsorption and PicoGreen as fluorescent dye. Amplification occurred even at 50 °C achieved with a hand warmer. This qualitative assay offers a limit of detection of 100 copies.

 Troubleshooting

 The HDA reaction is subject to the same limitations as PCR. However, unlike PCR it utilizes Bst DNA polymerase, an enzyme more tolerant to amplification inhibitors than Taq polymerase used in PCR-based platforms. Also much simpler sample processing workflows can be used in IsoAmp assays than in PCR. In addition, HDA may be more vulnerable to amplicon contamination by primer-dimer products that can amplify more efficiently than the legitimate amplicon. Therefore, care must be taken to avoid such contaminations by using the means of detection (real-time or lateral-flow end-point detection) in the alternate protocols preferentially over the Basic Protocol, even if the latter is more easily implemented in a minimally equipped laboratory.

 The analytical sensitivity and specificity of HDA is comparable to that of PCR. A significant number of publications reporting the performance of HDA assays have appeared recently.            Point-of-care (POC) diagnostic assays which do not require sophisticated equipment and can be rapidly and cheaply performed in the plant fields are in high demand. An important amount of agricultural product is lost every year due to multiple diseases. Crop disease management is a priority in agriculturally based economies. The best option is to detect the presence of pathogens in the field as early as possible and thus avoid the onset of the disease. Hence, the effectiveness of many integrated pest management strategies are highly dependent on the availability of fast, sensitive and specific diagnostic methods. PCR based methods have multiple advantages over other technologies. But requirement of an electricity supply to perform a PCR limits its adequacy for POC applications. As an alternative, isothermal DNA amplification methods are ideally suited to overcome this limitation. Isothermal amplification combined with lateral flow strips and portable fluorometers has been successfully used for POC detection of pathogen DNA. Nevertheless, portable fluorometers are expensive and not necessarily suited for use in the field exposed to adverse weather conditions, thus limiting their widespread adoption. A POC diagnostic assay technology integrating the entire process from sample preparation to visualization of results is still elusive. Diagnostic technologies for the detection of plant pathogens with point-of-care capability and high multiplexing ability are an essential tool in the fight to reduce the large agricultural production losses caused by plant diseases. The main desirable characteristics for such diagnostic assays are high specificity, sensitivity, reproducibility, quickness, cost efficiency and high-throughput multiplex detection capability.

 Infections caused by the bacillus Mycobacterium tuberculosis (MTB) are still one of the world’s deadliest communicable diseases. One study compared PCR and HDA for detection of Mycobacterium tuberculosis, by an electrochemical genomagnetic assay. A limitation of the simplest isothermal amplification schemes is their susceptibility to false positives arising from primer-dependent artifacts and non-specific amplification. They performed asymmetric HDA amplification for 84-base-long DNA sequence specific for Mycobacterium tuberculosis HDA, magnetic entrapment of the amplified single-stranded DNA sequences, and postamplification hybridization to develop an electrochemical system able to generate precise detection. To demonstrate the potential of this scheme for improved genetic detection of MTB, they compared its ability to detect both HDA and PCR amplicons. Detailed comparison of the kinetics and efficiency of HDA amplification, both symmetric and asymmetric, with those of similar PCR amplifications supports the suitability of this method as a viable alternative to PCR. Their results indicated the generalizability of the magnetic platform with electrochemical detection for quantifying amplification products without previous purification. Both assays PCR and HAD were applied to the detection of M. tuberculosis in sputum, urine, and pleural fluid samples with comparable results. Simplicity and isothermal nature of HDA offered great potential for the development of point-of-care devices.