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Chapter 3.4. Nucleic acid amplification technology: advantages and disadvantages of PCR and isothermal amplification

Authors: 

Faizul Rahman Sjafri, Nor Amalina Zulkiply and Aziah Ismail

Art work
Title: 
On the outskirts
Artist: 
Uli Reinhardt
In poor resource settings the availability of efficient diagnostic methods at the point of care could avoid 
delays in the implementation of treatment without the need of the existence of laboratories and highly skilled staff.
The essence of global health equity is the idea 
that something so precious as health might 
be viewed as a right.
Paul Farmer

Introduction

Infectious diseases are responsible for the greatest health burden in developing countries. Each year, approximately 1 million people die from malaria, 4.3 million from acute respiratory infections, 2.9 million from enteric infections, and 5 million from AIDS and tuberculosis [1]. These diseases affect the health and quality of life of the world’s population. The disability-adjusted life year (DALY) is used by the World Health Organization (WHO) to measure the overall disease burden as the number of years lost owing to ill-health, disability, or early death, and it also includes a factor to allow for a reduction in quality of life due to disability [1]. One of the factors that affects people’s health is how quickly and reliably diagnostic tests are used in their location.

It can be time-consuming to culture organisms for the identification of pathogens, and the process can have low sensitivity. Furthermore, culturing pathogens depends on the skills of the technician for certain steps, and in some cases, the cells appear to be transiently in a non-culturable state [2].

Serological methods, in contrast, are used to screen for antibodies. Serological testing depends on the quality of the antigen used in the assay, and quality control of the antigen preparation is required to ensure consistent performance from one batch to another. In most cases, when the test is not reproducible, the quality of the prepared antigen has not been properly controlled. Thus, the quality of the antigen affects the interpretation of the results [3]. Reported sensitivity to IgM is low, and paired serum samples are needed to detect an increase in the IgG antibody titer for diagnosing Mycoplasma pneumoniae infection [4]. If the antigen is not specific or is poorly standardized, the results are difficult to interpret. For example, in the Widal test, the difficulty is in establishing a steady-state baseline titer for a given population because it may differ from non-endemic areas to endemic areas as a result of repeated exposures to Salmonella enterica Typhi in endemic areas. Furthermore, some antigens are not specific to S. enterica Typhi or S. enterica Paratyphi; previous studies have demonstrated cross-reactivity with other non-Salmonella organisms, and found that the test results were not reproducible. Nucleic acid testing is one of the most reliable tests for detecting infectious diseases because it is rapid, simple, specific, and sensitive compared with the culture method and serological tests.

Nucleic acid amplification technology (NAAT)

Nucleic acid amplification technology (NAAT) is a common platform for amplifying and detecting low levels of nucleic acids. This technology can be used to detect the presence of microorganisms in the human body by amplifying specific target regions. NAAT involves three main steps: i) sample preparation; ii) DNA amplification; and iii) detection of the amplified products. Sample preparation aims to simultaneously isolate nucleic acids, reduce the inhibitory substances present in samples, and increase the number of target organisms by culturing in suitable media [5]. The enrichment step promotes the growth of bacteria in the medium to a detectable level [6]. After enrichment, the extraction of genomic DNA from bacterial cells can be performed using the boiling method, a commercial kit, or automated DNA preparation prior to amplification.

Polymerase chain reaction (PCR)

The polymerase chain reaction (PCR) is commonly used to amplify DNA. PCR is an in vitro process for generating a large number of copies of a specific DNA fragment [7]. DNA amplification via PCR is based on three steps: denaturation, annealing, and extension, including enzymatic amplification [8]. This amplification occurs in the presence of a pair of primers targeting a specific region of the genome and a DNA polymerase enzyme to synthesize a new DNA strand. Enzymatic amplification involves the synthesis by DNA polymerase of deoxyribonucleotide triphosphate (dNTP) molecules into new DNA strands based on the target DNA sequences, with the aid of a pair of primers, through repeated cycles of denaturation, annealing, and extension. In each cycle, the double-stranded target DNA is separated to allow for binding of the primers to the target DNA and attachment of DNA polymerase to synthesize new strands that are complementary to the targeted DNA sequence. The cycles are then continuously repeated from denaturation until extension to produce billions of copies of DNA. The total number of DNA fragments produced is measured as 2n, where n is the number of cycles [7].

PCR has potential from a clinical diagnostic perspective because it facilitates the detection of multiple pathogens from a clinical specimen [9]. For example, previous studies have shown the success of multiplex PCR in detecting S. enterica Typhi, S. enterica Paratyphi A, S. enterica Paratyphi B, and Salmonella serovars using various target genes [10-12].

PCR is a common molecular method that requires a thermal cycler. The thermal cycler can be programmed to generate the temperature changes required for denaturing the double-stranded DNA template into single-stranded DNA, annealing the primers to the single-stranded DNA template, and synthesizing the new strands of DNA in the presence of dNTPs and Taq DNA polymerase. The automated heating and cooling processes in the three steps are necessary to ensure the complete cycling of the amplification process and the successful generation of many copies of DNA. This replicated DNA is detected at the end point of the amplification process, i.e., the plateau phase, when the DNA amplification graph levels off and production is no longer exponential. This method requires both a heating and cooling system in a single unit, increasing the cost of the thermal cycler.

PCR is divided into two types: i) conventional symmetric PCR, and ii) linear after the exponential (LATE) PCR. LATE-PCR is a novel form of asymmetric PCR licensed by Smith Detection. This method provides considerable technical advantages over symmetric real-time PCR, and facilitates high-quality endpoint analysis. LATE-PCR also introduces new probe design criteria that uncouple hybridization probe detection from primer annealing and extension, increasing probe reliability, improving allele discrimination, and increasing the signal strength by 80–250% relative to symmetric PCR [13].

PCR is a powerful method for detecting pathogens in clinical specimens by amplifying the specific fragments of DNA a billion-fold in a 25- to 35-cycle reaction that lasts < 2 hours. Another advantage of PCR is that the method detects the antigen itself rather than the antibody; therefore, the results provide information on the current infection status and are not dependent on the host's immune response to the infection [14]. The common detection method for PCR amplicons is agarose gel electrophoresis by intercalating double-stranded DNA with either ethidium bromide or SYBR green dye [15, 16]. To avoid laborious work with agarose gel electrophoresis, some researchers have used another approach, the lateral flow assay, which allows interpretation of the results in 15–20 minutes. This assay is a powerful method with the advantages of stability, sensitivity, specificity, and simplicity of use [17].

Real-time PCR

Real-time PCR is another method for detecting the DNA of organisms based on amplicons. There are two types of chemistry involved: TaqMan® and SYBR®-Green. TaqMan®-based detection uses a fluorogenic probe to detect the specific PCR product as it accumulates during the amplification cycles. The fluorescent signal is generated during specific hybridization between the probe and target sequence, which significantly increases the specificity of the amplicons [18]. In contrast, SYBR®-Green-based detection uses a SYBR Green I dye that binds to any double-stranded amplicons. This technique enables monitoring of the amplification steps of any double-stranded DNA sequence. However, SYBR Green I dye also binds to any double-stranded DNA, including non-specific double-stranded DNA (e.g., primer dimers), and may generate false-positive results [19]. Although the detection of PCR amplicons via real-time PCR is more sensitive and specific than agarose gel electrophoresis, this method requires expensive equipment and reagents as well as highly skilled and qualified personnel to operate the machine and perform the test.

Isothermal amplification

PCR requires special equipment called a thermal cycler for amplification. In PCR, a specific temperature is required for every thermal cycling step. Isothermal amplification offers an alternative approach with a single temperature for the entire amplification reaction process, eliminating the need for thermocycling in the PCR assay [20-23]. Therefore, most isothermal amplification methods require only a simple heating device (e.g., a heating block or a water bath) for the amplification process.

The common isothermal amplification techniques used for the detection of microorganisms that cause infectious diseases are nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), and helicase-dependent amplification (HDA) assays [22-26].

Nucleic acid sequence-based amplification

NASBA was first reviewed by Compton (1991), who described the technology and its application, especially for identifying bacterial and viral RNA in clinical samples. NASBA is a transcription-based isothermal amplification system that specifically amplifies the RNA of the organism. The reaction is performed in a two-stage protocol: the initial phase for denaturation and primer annealing at 65°C and the cycle phase for target amplification at a constant temperature of 41°C. During the initial phase, the reverse primers (P1) that contain a promoter region are elongated via the activity of reverse transcriptase. The RNA-cDNA hybrid is then degraded by RNase H. During the cycle phase, the forward primer (P2) binds with the cDNA sequence to form new templates, which are extended by reverse transcriptase. The double-stranded promoter region is created, and newly synthesized RNA fragments are produced by RNA polymerase, starting the next cycle of amplification [24, 27]. 

The amplification of 106 to 109-fold RNA is achieved within 90 minutes of the NASBA reaction [24]. The single-stranded RNA amplicons generated by NASBA can be detected using agarose gel electrophoresis [28], the molecular beacon-based real-time amplification technique [29], or ELISA [30].

An RNA target is converted to double-stranded cDNA with a promoter region through reverse transcription, followed by RNase H degradation of the original strand and DNA polymerization initiated by a second primer (P2). RNA polymerase amplification generates RNA products that feed back into the cycle reaction [27].

Strand displacement amplification

Strand displacement amplification (SDA) was first described by Walker et al. (1992). Four primers are used in this technique. The first primer set (S1 and S2) is constructed to generate an overhanging restriction enzyme recognition site, and the second primer set (B1 and B2) represents the bumper primers. The DNA target is first denatured by heating at 95°C, and each strand is allowed to anneal with two primers (S1 and B1). The B1 extended product displaces the extension from the S1 primer, which can bind to the opposite strand primers (B2 and S2). Eventually, the newly synthesized DNA from the extended primers is cleaved by a restriction endonuclease, and the polymerase amplifies the fragments and generates new strands [26, 27]. 

Thermophilic SDA (tSDA) is an improvement on conventional SDA in which Bst polymerase (having an optimal temperature ranging from 60°C to 65°C, 5ʹ to 3ʹ polymerase activity, and the capability for strand displacement) and the BsoB1 restriction enzyme are used. Both enzymes were isolated from Bacillus stearothermophilus, allowing for the use of higher temperatures (50°C to 60°C) for amplification. Therefore, this technique increases the stringency of primer hybridization, improves the reaction kinetics, and reduces the amplification time, resulting in 109 to 1010-fold amplification of DNA target sequences in 15 minutes [31]. The amplicons of SDA can be detected by real-time fluorescence and fluorescence resonance energy transfer (FRET) probe assays [32, 33].

Rolling circle amplification

Rolling circle amplification (RCA) was first described by Fire and Xu (1995). The technique involves a DNA primer and padlock probes. The probes consist of two target-complementary portions linked with a connecting sequence that are designed to circularize via ligation with the DNA target. After ligation, circular padlock probes are used as a template and extended by phage phi 29 polymerase, resulting in multiple copies of circular DNA sequences. This polymerase, with high strand displacement activity, can continuously extend around the circle probe and displace the amplified fragment, generating a long, single-stranded DNA product [25, 27]. RCA reaction takes place at 65°C for one hour, and the amplified product can be detected by fluorescence [34], chemiluminescence [35], or electrochemistry [36].

Loop-mediated isothermal amplification

Notomi et al. (2000) first described loop-mediated isothermal amplification (LAMP). This technique involves four to six specific primers targeted at six or eight target regions and extended by the Bst polymerase to synthesize new DNA strands. At the initial stage, the dumbbell-like DNA strands are generated by the extension of outer and inner primer sets from the DNA template. Then, these dumbbell-like DNA strands become the new template DNA for the next step of amplification. The amplification of the dumbbell-like DNA strands produces long-chain, high-molecular-weight DNA products that resemble the laddering pattern on the agarose gel profile. Furthermore, the amplification process can be accelerated by the addition of loop primers [22, 37]. 

The sensitivity and specificity of the LAMP assay depend on good primer design, which can be complicated, especially for new users. The entire LAMP reaction can be performed with a simple heating device (e.g., a heating block or a water bath) at 60–65°C for one hour, and the amplified products can be detected by visual fluorescence, visual assessment of turbidity, or lateral nucleic acid flow [37, 39, 40].

The principle of LAMP amplification is based on the autocyclic strand displacement reaction, which is carried out using Bst polymerase at a constant temperature. The dumbbell-shaped structure of the outer and inner primer sets becomes the new template that enters into the cyclic amplification step [38].

Helicase-dependent amplification

Helicase-dependent amplification (HDA) is based on in vivo DNA replication mechanisms. The technique involves two enzymes—helicase and Bst polymerase—as well as single-stranded binding protein (SSB protein) and primers. The reaction is initiated by unwinding the double-stranded DNA (dsDNA) into single-stranded DNA (ssDNA) using the helicase enzyme. HDA is the only isothermal amplification technique that is similar to PCR. Unlike PCR, HDA uses helicase, instead of heat, to separate the dsDNA, eliminating the need for a denaturation step. The re-annealing of ssDNA is prevented by the presence of SSB protein. The primers are then hybridized to the ssDNA template and subsequently elongated with the Bst polymerase, resulting in DNA amplification [23]. 

Thermophilic HDA (tHDA) is an improvement over mesophilic HDA (37°C) in which a thermostable helicase is derived from Thermoanaerobacter tengcongensis. The optimal temperature for this enzyme ranges from 45°C to 65°C; therefore, the HDA reaction can be performed at the higher temperature of 65°C to reduce the usage of the SSB protein [42]. This higher temperature increases the specificity of the primers, improves the reaction kinetics, and simultaneously increases the yield of the HDA products [41]. The simplicity of the protocol makes HDA very attractive for the development and establishment of portable DNA diagnostic devices and point-of-care testing [27].

Helicases unwind dsDNA, and SSB protein prevents ssDNA from self-annealing. DNA polymerase extends the complementary strand from hybridized primers, and the cycles are repeated 

Advantages and limitations of the isothermal amplification methods

Similar to the PCR assay, isothermal amplification is a rapid, sensitive, specific, and user-friendly assay. However, in isothermal amplification, the reaction can be performed at a single temperature using simple heating devices, such as a heating block or water bath, whereas PCR requires high-precision equipment, such as a thermal cycler [23]. Most isothermal methods (SDA, RCA, LAMP, and HDA) exploit the strand displacement activity of a Bst polymerase to amplify the target in less than one hour. The target template can vary from RNA molecules to double-stranded DNA. DNA is often preferred because it is more stable than RNA and is associated with better sensitivity in the samples that are stored and transported under suboptimal conditions [43].

NASBA is the only isothermal amplification method that uses RNA as a starting material. The major drawback of the assay is that it is prone to ribonuclease (RNase) contamination, which might degrade the target RNA [43].

The RCA method employs the widest range of target molecules from small and circular single-stranded DNA to double-stranded DNA as well as RNA. However, the assay requires an additional heating step (95°C) to allow the dsDNA to separate into ssDNA before the padlock probe can circularize to the target sequence [43].

SDA and LAMP are isothermal amplification methods that require more than one set of primers to amplify a single target gene. The obvious limitation of both assays is their complicated reaction schemes and primer design, especially for new users [23]. In SDA, four primers are used to generate the initial target amplicons, and they require four dNTPs, one of which contains an α-phosphorothioate modification (dCTPα5) to provide strand-specific nicking. In the LAMP reaction, four to six primers are used, which target six or eight different regions within a target DNA for amplification [32, 37]. SDA also requires an initial heat denaturation step (95°C) to denature the DNA template before the isothermal reaction occurs during the amplification process.

HDA has the simplest reaction scheme compared with the other isothermal amplification methods, but the assay can only amplify short DNA sequences (from 70 to 120 bp). However, the assay has been improved to amplify longer target sequences (up to 400 bp) using a thermostable UvrD helicase (Tte-UvrD) enzyme [42].

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