Amplification Based Methods For Detection Of Infectious Dieases Biology

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Methods for the diagnosis of infectious diseases have crawled in the last 2-3 decades. Many tests that form the backbone of the "modern" microbiology laboratory are based on very old and labour-intensive technologies such as microscopy. Innovation and implementation of PCR had lead to major advances in area of research and diagnostics. However, it requires a thermocycler and longer time to separate two DNA strands and amplify the required fragment. Burning need of the hour include more rapid tests without sacrificing sensitivity, value-added tests, and point-of-care tests for both high- and low-resource settings. Over the period of last few years, research has been focused on alternative methods to improve the diagnosis of infectious diseases. These include various isothermal amplification-based molecular approaches.

In this article, we review these isothermal nucleic acid amplification technologies and their applications along with some of the merits and demerits of these tests.



In vitro Nucleic Acid amplification was for the first time described in 1971 (Kleppe). Followed by synthesis of tRNA gene by primer-directed DNA repair and this was not exponential amplification. In 1983, Kary Mullis postulated the concept of the polymerase chain reaction (PCR) but remained theoretical until 1985 when Saiki published the first application of PCR on beta-Globin. Thereafter, polymerase chain reaction became backbone of existing research and diagnostic world. Innovations in biotechnology that combine molecular biology, microfabrication and bioinformatics are moving nucleic acid technologies from futuristic possibilities to common laboratory techniques and modes for disease diagnoses. In this way, amplification of nucleic acids is widely used in research, forensics, medicine, and agriculture [1].

One of the most widely used amplification methods is the polymerase chain reaction (PCR), which is a target amplification method [2]. A PCR reaction typically utilizes two oligonucleotide primers, which are hybridized to the 5'and 3'ends of the target sequence, and a DNA polymerase, which can extend the annealed primers by adding on deoxyribonucleoside-triphosphates (dNTPs) to generate double-stranded products .By raising and lowering the temperature of the reaction mixture, the two strands of the DNA product are separated and can serve as templates for the next round of annealing and extension, and the process is repeated[3]. Although PCR has been widely used by researchers, but it is labour intensive, requires expensive thermocycling machine and expertise.

Several isothermal amplification techniques have been developed in the last two decades without using thermocycler machine. These non-PCR based methods have exploited the high fidelity polymerase of phages and some accessory proteins for in vitro nucleic acid amplification.

All these methods do not require temperature cycling, operate at a constant temperature, and offer potential advantages including cost, speed, portability and reduced sensitivity to inhibitors over PCR.

We here describe the best known isothermal amplification methods (such as transcription mediated amplification (TMA) or self-sustained sequence replication (3SR), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), loop-mediated isothermal amplification of DNA (LAMP), helicase-dependent amplification (HDA), circular helicasedependent amplification (cHDA)), rolling circle amplification (RCA), single primer isothermal amplification (SPIA), signal mediated amplification of RNA technology (SMART) and isothermal multiple displacement amplification (IMDA) and their applications in molecular diagnosis.


Transcription mediated amplification (TMA) is RNA transcription amplification system which uses RNA polymerase (T7 RNA polymerase) to make RNA from a RNA promoter sequences engineered in the primer region, a reverse transcriptase (M-MuLV) to produce complementary DNA from the RNA templates (Guatelli et al, 1990). This RNA amplification technology has been further improved by introducing a third enzymatic activity, Rnase H, to remove the RNA from cDNA without the heat-denatured step (Fig No.2). Thus, the thermocycling step has been eliminated, generating an isothermal amplification method named self-sustained sequence replication (3SR)[5]. It is single-tube reaction, amplifies either DNA or RNA, and produces RNA amplicons, in contrast to most other nucleic acid amplification methods that only produce DNA. It has very rapid kinetics resulting in a billion fold amplification within 15-30 minutes.

The end products of TMA can be detected using gel electrophoresis, fluorescence probes and colorimetric assay. TMA has been used for detection of N.gonorrhoeae and C. trachomatis (Hobbs ) HCV Ferraro et al. 2008, Gelderblom et al.2007, West Neil fever (Ziermann 2008).


NASBA was developed by J. Compton in 1991who defined it as "a primer-dependent technology that can be used for the continuous amplification of nucleic acids in a single mixture at one temperature."

NASBA is particularly suited to detection of genomic, ribosomal or messenger RNA. The product of NASBA is ss RNA of the original target. 108 - fold amplification of the target may be obtained in 30 min. It utilises activity of 3 enzymes RNA polymerase (T7 RNA polymerase)(RNA dependent RNA Polymerase activity) to make RNA from a RNA promoter sequences engineered in the primer region, a reverse transcriptase (AMV) to produce complementary DNA from the RNA templates and RNAse H to degrade RNA from DNA-RNA hybrid followed by formation of cDNA by reverse transcriptase enzyme. Again RNA polymerase (DNA dependent RNA polymerase activity) to make RNA copies from double stranded hence functional T7 RNA promoter sequences (Fig No. 3).

The end products of NASBA can be detected using gel electrophoresis, fluorescence probes (NASBA real time) and colorimetric assay (NASBAELISA) [6-8]. Food and Drug Administration office of United States of America (FDA) has approved the technique in NucliSence formulation (NASBAECL) for molecular detection of some microorganisms such as HCV and HIV-1[9, 10]. Around 500 articles for identification and detection of microorganism employing NASBA have been reported.


Strand-displacement amplification (SDA) is an isothermal technique first introduced by Walker et al. 1992. It 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 3' end at the nick and displace the downstream DNA strand. The displaced strand serves as a template for an antisense reaction and vice versa, resulting in exponential amplification of the target DNA (Figure 5). In the originally designed SDA, a target DNA sample is heat denatured. Four primers (B1, B2, S1, and S2), present in excess, and bind the target strands at positions flanking the sequence to be amplified. Primers S1 and S2 have HincII recognition sequences (5' GTTGAC 3') located 5' to the target complementary sequences. The four primers are simultaneously extended by exo- klenow using dGTP, dCTP, TTP, and dATP(αS). Extension of B1 displaces the S1 primer extension product, S1-ext. Likewise, extension of B2 displaces S2-ext. B2 and S2 bind to displaced S1-ext. B, and S1 bind to displaced S2-ext. Extension and displacement reactions on templates S1-ext and S2-ext produce two fragments with a hemiphosphorothioate HincII at each end and two longer fragments with a hemiphosphorothioate HincII site at just one end. HincII nicking and exo-kle now extension/displacement reactions initiate at these four fragments, automatically entering the SDA reaction cycle. These reaction steps continuously cycle during the course of amplification. Present in excess are two SDA primers (S1 and S2).

The 3'end of S1 binds to the 3'end of the displaced target strand T1, forming a duplex with 5'overhangs. Likewise, S2 binds T2. The 5'overhangs of S1 and S2 contain the HincII recognition sequence (5'- GTTGAC -3'. Exo- klenow extends the 3'ends of the duplexes using dGTP, dCTP, TTP, and dATP(αS), which produces hemiphosphorothioate recognition sites on S1:T1 and S2:T2. HincII nicks the unmodified primer strands of the hemiphosphorothioate recognition sites, leaving intact the modified complementary strands. Exo- klenow extends the 3'end at the nick on S1:T1 and displaces the downstream strand that is equivalent to T2. Likewise, extension at the nick on S2:T2 results in displacement of T1. Nicking and polymerization/displacement steps cycle continuously on S1:T1 and S2:T2 because extension at a nick regenerates a nickable HincII recognition site. Target amplification is exponential because strands displaced from S1:T1 serve as target for S2 while strands displaced from S2:T2 serve as target for S1 [16]. SDA technology has been used mainly for clinical diagnosis of infectious diseases such as chlamydia and gonorrhoea [17-20]. This technique can be used for isothermal amplification of RNA templates in RT-SDA format by adding reverse transcriptase to the original process [21, 22]. SDA has been performed on C. trachomatis Verteramo et. al. 2009, N. gonorrhoeae and C.trachomatis Van et al. 2001 Cosentino, et al. 1999, M. tuberculosis Hellyer, et al. 1999.


LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LAMP) is a novel method that amplify DNA with high specificity, efficiency, rapidity at isothermal conditions and relies on auto cycling strand displacement DNA synthesis by Bst DNA polymerase first develop by, Notomi, T. et al. in 2000. It can directly be performed on clinical samples (DNA Isolation is optional). Bst polymerase is thermostable DNA polymerase from Bacillus stearothermophilus (N3468) and contains the 5´ → 3´ polymerase activity, strand displacement activity but lacks 5´ →3´ exonuclease activity, 3' →5'proof reading activity. It uses 4 primers.Two inner primers (FIP and BIP) and two outer primers (F3 and B3) [29]. The amplification products are stem-loop DNA structures with several inverted repeats of the target and cauliflower-like structures with multiple loops (Figure 7).

The LAMP method is also a highly efficient amplification method that allows the synthesis of large amounts of DNA in a short time. As a result, pyrophosphate ions are produced in large amounts and form white precipitates of magnesium pyrophosphate. Judging the presence or absence of this white precipitate allows easy distinction of whether nucleic acid was amplified by the LAMP method [30]. However the other formats such as gel electrophoresis, real-time turbidimetry, and fluorescence probes have been used for detection of LAMP products [31, 32]. This technology has widely been used for molecular detection of several microorganisms by researchers and it can be a suitable choice for design and development of rapid molecular tests in the field [33, 34]. There has been successful report of 10493 articles for detection and identification of bacteria, parasites, viruses and fungi by LAMP.


Rolling circle amplification (RCA) generates multiple copies of a sequence for the use in vitro DNA amplification adapted from in vivo rolling circle DNA replication [23, 24]. In its original formulation, the RCA reaction involves numerous rounds of isothermal enzymatic synthesis in which phi 29 DNA polymerase extends a circle-hybridized primer by continuously progressing around the circular DNA probe of several dozen nucleotides to replicate its sequence over and over again (Figure 6) [25, 26]. The single stranded nature of amplicons in case of linear RCA may be beneficial for subsequent manipulations with these DNAs towards their detection [24].

This reaction is widely used for diagnostic purposes in direct or indirect detection of different DNA/RNA, protein, and other biomarkers via a set of various bimolecular recognition events. A similar reaction was described for RNA polymerases as well, but the RNA-generated process does not require any hybridization-dependent priming [27]. Therefore, the latter is only used to produce functional RNA sequences, such as RNA ladders and self-processing ribozymes.

Recently, RCA has been further developed in a technique, named multiply-primed rolling circle amplification (multiply-primed RCA) that uses the unique properties of phi29 DNA polymerase and random primers to achieve a 10,000-fold amplification (Figure 6). The process allows amplification of circular DNA directly from cells or plaques, generating, or cloning [28]. RCA-based approaches have recently been attracting attention of diagnostics-oriented biotech companies and research centers for gene tests and immunoassays, SNP scoring and sequencing template preparation, single-cell analysis systems, and gene expression studies[26]. Johne et al. 2009 and Rector et al.2004 optimized RCA for Begomovirus and type 16 Human papilloma virus respectively.


Helicase-dependent amplification (HDA) is based on the unwinding activity of a DNA helicase [41]. This process uses a helicase, rather than heat, to separate the two strands of a DNA duplex generating single-stranded templates for the purpose of in vitro amplification of a target nucleic acid [42]. Sequence-specific primers hybridize to the templates and are then extended by DNA polymerases to amplify the target sequence. This process repeats itself so that exponential amplification can be achieved at a single temperature (Figure 9). This process allows multiple cycles of replication to be performed at a single incubation temperature, completely eliminating the need for thermo cycling equipment [3]. The HDA amplicons can be detected using gel electrophoresis, real-time format, and enzyme-linked immunosorbent assay (ELISA).[41-45]HDA has been used for identification of S. aureus, MRSA, Goldmever et al 2008, M. tuberculosis Gill, et al. 2007, H. pylori Gill, et al. 2007.


circular Helicase-dependent amplification (cHDA) is used for amplifying nucleic acids from a circular DNA template. This system combines a DNA polymerase and a helicase preparation to amplify a target sequence as well as the entire circular DNA template containing the target sequence [50]. The technique is based on the T7 replication machinery, which includes the processive T7 helicase, an exonuclease-deficient T7 DNA polymerase (T7 sequenase) and the T7 Gp2.5 single-stranded DNA binding (SSB) protein. After the duplex DNA template is unwound by T7 helicase, specific primers anneal to the separated DNA strands and T7 sequenase extends the 3'end of each primer by a rolling circle mechanism to amplify not only a region defined by the primers but also continuous concatemers of the template (Figure 11). The process can be carried out at one temperature (25â-¦C) for the entire process. Amplification can be performed using purified plasmid DNA or crude cell lysate can amplify inserts as large as 10 kilo base pairs [50]. Xu, et al. 2006 performed cHDA on E. coli .


SMART is based on the formation of a three-way junction (3WJ) structure. The method relies on signal amplification and does not require thermal cycling or involve the copying of target sequences. The assay generates a signal that is highly target dependent and is appropriate for the detection of DNA or RNA targets [11].

It consists of two single-stranded oligonucleotide probes extension probe and template probe, each probe includes one region that can hybridize to the target at adjacent positions and another, much shorter, region that hybridizes to the other probe. The two probes are annealed to each other in the presence of the specific target, so forming a 3WJ (Figure 4A).

After 3WJ formation, Bst DNA polymerase extends the short (extension) probe by copying the opposing template probe to produce a double stranded T7 RNA polymerase promoter sequence (Figure 4B). This double stranded hence functional promoter sequences allows T7 RNA polymerase to bind and generate multiple copies of an RNA amplicons and therefore being produced only when a specific target is present to allow 3WJ formation. Each RNA amplicons may itself be amplified by binding to a second template oligonucleotide (probe for amplification) and is extended by DNA polymerase to generate a double-stranded promoter, leading to transcription which increases the RNA amplicons can be detected by an enzyme linked oligosorbent assay (ELOSA) or in real time format[12,13]. This process is in fact a signal amplification method that the target sequence is not itself amplified [14]. SMART has been used for detection of cyanophages (Hall, et al. 2002) and E. coli (Wharam, et al. 2001).


Isothermal Multiple Displacement Amplification (IMDA) is based on strand displacement replication of the nucleic acid sequences by multiple primers [35]. Two sets of primers are used, a right set and a left set (Figure 8). The primers in the right set are complementary to one strand of the nucleic acid molecule to be amplified and the primers in the left set are complementary to the opposite strand. The 5'ends of primers in both sets are distal to the nucleic acid sequence of interest where the primers have hybridized to the nucleic acid sequence molecule to be amplified. Amplification is done with the help of phi29 DNA polymerase is a highly processive enzyme that incorporates at least 70,000 nucleotides in one binding event alone with 3'-5'proof reading and strand displacement activity proceeds by replication initiated at each primer and continuing through the nucleic acid sequence of interest. A key feature of this method is the displacement of intervening primers during replication by the polymerase.

In another preferred form of the method, referred to as whole genome strand displacement amplification, a random set of primers is used to randomly prime a sample of genomic nucleic acid [36, 37]. Amplification proceeds by replication with a highly processive polymerase initiated at each primer and continuing until spontaneous termination. In this way, multiple overlapping copies of the entire genome to be synthesized in a short time [38-40]. IMDA has been successfully used for whole genome amplification from Plasma Lu, et al. 2005, Blood, bone marrow aspirates, tissue biopsy Luthra, et al. 2004, Blood Hosono, et al. 2003.


This amplification technology uses a single chimeric primer for amplification of DNA (SPIA) and RNA (Ribo-SPIA)[46]. SPIA employs a single, target-specific chimeric primer composed of deoxyribonucleotides at the 3- end and ribonucleotides at its 5- end, RNase H, and a DNA polymerase with a strong strand displacement activity. Amplification is initiated by hybridizing the chimeric primer to a complementary sequence in the target DNA molecule. DNA polymerase initiates primer extension of the hybridized primer and extends along the target DNA strand. Following initiation of the primer extension step, the 5' RNA portion of the extended primer (RNA-DNA hybrid) is cleaved by RNase H, thus freeing part of the primer-binding site on the target DNA strand form binding of a new chimeric primer. The newly bound primer competes with the previous primer extension product for binding to the complementary DNA target sequence and is stabilized by binding of DNA polymerase and displaces the 5' end of the previous extension product. As replication is again initiated by primer extension, RNase H cleavage of the 5' RNA portion of the newly extended primer again frees part of the primer binding site for subsequent primer binding and replication cycle is repeated. SPIA amplification can be used for global genomic DNA amplification and for the amplification of specific genomic sequences and synthetic oligonucleotide DNA targets. Ribo-SPIA is similarly suitable for global and target-specific RNA amplification (Figure 10) [47-49]. Ribo-SPIA technology provides an elegant method for linear, isothermal amplification of the mRNA species in a total RNA population. Replication is initiated and repeated up to 10,000 times off of each original transcript. Therefore, this process can be used for amplification of large populations of nucleic acid species, which are limited in biological samples, as are commonly encountered in clinical researches [46].


In this study, we described the well studied isothermal technologies for nucleic acid amplification that offer several advantages over PCR in that they eliminate the need for an expensive and cost-intensive thermocycler.

However, these isothermal amplification technologies have some restrictions that confine their employment in some aspects of molecular biology.

For example, TMAs prerequisite is three different enzymatic steps (transcription/cDNA synthesis/RNA degradation) to accomplish an isothermal RNA amplification,[4] and its starting material is limited to single stranded nucleic acid/RNA.

SDA needs four primers to generate initial amplicons and modified deoxynucleotides to provide strand-specific nicking [15, 16] and it is inefficient at amplifying long target sequences.

LAMP insists upon four to six specific primers that their designs are complicated for new user [51]. Also, its final product is a complex mixture of stem-loop cauliflower-like DNA structures of various sizes. Nagamine et al. have devised extra steps to obtain uniform single-stranded DNA from LAMP products. This is preferable for various hybridization techniques. The advanced method uses the thermo stable TspRI restriction enzyme to digest amplification product,[52] and an additional primer hybridized to the 9-nt 3'overhang at the TspRI cleavage site to displace single-stranded DNA by primer extension [53] . Recently, Kaneko et al. evaluated the tolerance of LAMP to a culture medium and some biological substances [55]. According to their study, the sensitivity of LAMP was less affected by the various components of the clinical samples than was PCR; therefore, DNA purification can be omitted.

On the other hand, some of these methods such as HDA have a simple reaction scheme, in which a target sequence can be amplified by two flanking primers, similar to PCR [41, 42]. One of the most important advantages of the isothermal amplification techniques is related to their tolerances to some inhibitory materials that affect the PCR efficiency.

Another example is about HDA; a pathogen genomic DNA can even be detected in a human blood sample [41]. This demonstrates that HDA can be performed on crude samples and has the potential to be used as a diagnostic tool.

RCA holds a explicit position in DNA diagnostics among other isothermal amplification techniques due to its robustness and simplicity. As compared with RCA, all other isothermal methods of signal, probe, or target DNA amplification, such as transcription-based system, strand displacement approach or loop-mediated techniques are rather complicated and in most cases they require prior assay optimization [26, 54]. Although, SDA and RCA are described as isothermal amplification systems, both methods require an initial heat denaturation step.

Another important advantage for the isothermal amplification techniques is no need to initial heat denaturation at a high temperature followed by amplification at a lower temperature. This property has been reported about some isothermal amplification methods. For example, because there is no necessity for heat denaturation of the template DNAs, LAMP can be used more easily and rapidly in molecular medicine [56]. As DNA helicase can unwind double-stranded target DNA at the beginning of the reaction, the entire HDA reaction can be performed at one temperature [41].

Beyond the shadow of doubt, these isothermal amplification based technologies be at variance with their nature and volume of sample required, processing of specimen, and methods of amplification and detection. In spite, of these many limitations isothermal amplifications based techniques would successfully compete with its widely employed thermal cycler based predecessor (PCR) for the number of diagnostic applications. On the whole, simplicity and isothermal nature of these methods offer great potentials for the development of hand-held DNA diagnostic devices that could be used to detect pathogens at point-of-care or in the field.

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