Isothermal Gene Amplification Research Articles

Design and Optimization of Isothermal Gene Amplification for Generation of High-Gain Oligonucleotide Products by MicroRNAs

Design and Optimization of Isothermal Gene Amplification for Generation of High-Gain Oligonucleotide Products by MicroRNAs

Feedback on the detection of SARS-CoV-2 by the point-of-care testing

In order to improve the detection and rapid diagnosis of the SARS-CoV-2 coronavirus, we evaluated the ID NOW™ COVID-19 isothermal gene amplification technique in parallel with the real-time PCR technique (Diasorin) routinely used in the laboratory during a prospective study in the 2020 season. As this technique showed satisfactory sensitivity and specificity of 98% and 97.5% respectively, we then proposed to implement the detection of SARS-CoV-2 coronavirus in the emergency department and maternity as a point-of-care test (POCT) for the 2020-2021 season and to evaluate its clinical and organizational impact. This article summarizes the results obtained and highlights the advantages and limitations of this strategy implemented in the emergency department, particularly in terms of time spent in the department, hospitalization rates, anticoagulant treatment and early isolation of patients, as well as the organizational impact on the maternity unit. Based on this experience, we report on the regulatory constraints that apply when setting up a POCT and the steps required to validate the accreditation in accordance with standard NF EN ISO 22870.

Highly Multiplexed Reverse-Transcription Loop-Mediated Isothermal Amplification and Nanopore Sequencing (LAMPore) for Wastewater-Based Surveillance.

Wastewater-based surveillance (WBS) has gained attention as a strategy to monitor and provide an early warning for disease outbreaks. Here, we applied an isothermal gene amplification technique, reverse-transcription loop-mediated isothermal amplification (RT-LAMP), coupled with nanopore sequencing (LAMPore) as a means to detect SARS-CoV-2. Specifically, we combined barcoding using both an RT-LAMP primer and the nanopore rapid barcoding kit to achieve highly multiplexed detection of SARS-CoV-2 in wastewater. RT-LAMP targeting the SARS-CoV-2 N region was conducted on 96 reactions including wastewater RNA extracts and positive and no-target controls. The resulting amplicons were pooled and subjected to nanopore sequencing, followed by demultiplexing based on barcodes that differentiate the source of each SARS-CoV-2 N amplicon derived from the 96 RT-LAMP products. The criteria developed and applied to establish whether SARS-CoV-2 was detected by the LAMPore assay indicated high consistency with polymerase chain reaction-based detection of the SARS-CoV-2 N gene, with a sensitivity of 89% and a specificity of 83%. We further profiled sequence variations on the SARS-CoV-2 N amplicons, revealing a number of mutations on a sample collected after viral variants had emerged. The results demonstrate the potential of the LAMPore assay to facilitate WBS for SARS-CoV-2 and the emergence of viral variants in wastewater.

Fluorometric Detection of SARS-CoV-2 Single-Nucleotide Variant L452R Using Ligation-Based Isothermal Gene Amplification.

Since the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant was first discovered, several variants showing different infectivity and immune responses have emerged globally. As the conventional method, whole-genome sequencing following polymerase chain reaction (PCR) is currently used for diagnosis of SARS-CoV-2 mutations. However, these conventional PCR-based direct DNA sequencing methods are time-consuming, complicated, and require expensive DNA sequencing modules. Here, we developed a fluorometric method for the accurate detection of a single missense mutation of U to G in the spike (S) gene that changes leucine to arginine (L452R) in SARS-CoV-2 genomic RNA. Our method for the detection of single-nucleotide mutations (SNM) in the viral RNA genome includes RNA sequence-dependent DNA ligation and tandem isothermal gene amplification methods, such as strand displacement amplification (SDA) and rolling circle amplification (RCA) generating G-quadruplex (GQ). In the presence of SNM in the viral RNA, ligation of both ends of the probe DNAs occurs between 5'-phosphorylated hairpin DNA and linear probe DNA that can discriminate a single base mismatch. The ligated DNAs were then extended to generate long-stem hairpin DNAs that are subjected to the first isothermal gene amplification (SDA). SDA produces multitudes of short ssDNA from the long-stem hairpin DNAs, which then serve as primers by annealing to circular padlock DNA for the second isothermal gene amplification (RCA). RCA produces a long stretch of ssDNA containing GQ structures. Thioflavin T (ThT) is then intercalated into GQ and emits green fluorescence, which allows the fluorometric identification of SARS-CoV-2 variants. This fluorometric analysis sensitively distinguished SNM in the L452R variant of SARS-CoV-2 RNA as low as 10 pM within 2 h. Hence, this fluorometric detection method using ligation-assisted tandem isothermal gene amplification can be applied for the diagnosis of SARS-CoV-2 SNM variants with high accuracy and sensitivity, without the need for cumbersome whole-genome DNA sequencing.

Organizational Impact of an ID NOW™ COVID-19 Point-Of-Care Testing for SARS-Cov2 Detection in A Maternity Ward

Background: SARS-CoV-2 has been responsible for more than 676 million cases of COVID-19 worldwide. RT-PCR is considered the “gold standard” for the diagnosis of patients suspected of having COVID-19. During the heightened waves of the pandemic, more rapid tests have been required. Point-of-care tests (POCT) for COVID-19 include antigen tests, serological tests, and other molecular-based platforms. The ID NOW™ COVID-19 assay (Abbott) performs an isothermal gene amplification of a target encoding the RNA-dependent RNA polymerase of SARSCoV-2. The main objective of this study was to evaluate the organizational impact following the implementation of a POC testing platform ID NOW™ in a maternity ward.

Point-of-care COVID-19 testing: colorimetric diagnosis using rapid and ultra-sensitive ramified rolling circle amplification.

In this paper, we report a molecular diagnostic system—combining a colorimetric probe (RHthio-CuSO4) for pyrophosphate sensing and isothermal gene amplification (ramified rolling circle amplification)—that operates with high selectivity and sensitivity for clinical point-of-care diagnosis of SARS-CoV-2. During the polymerase phase of the DNA amplification process, pyrophosphate was released from the nucleotide triphosphate as a side product, which was then sensed by our RHthio-CuSO4 probe with a visible color change. This simple colorimetric diagnostic system allowed highly sensitive (1.13 copies/reaction) detection of clinical SARS-CoV-2 within 1 h, while also displaying high selectivity, as evidenced by its discrimination of two respiratory viral genomes (human rhino virus and respiratory syncytial virus) from that of SARS-CoV-2. All of the reactions in this system were performed at a single temperature, with positive identification being made by the naked eye, without requiring any instrumentation. The high sensitivity and selectivity, short detection time (1 h), simple treatment (one-pot reaction), isothermal amplification, and colorimetric detection together satisfy the requirements for clinical point-of-care detection of SARS-CoV-2. Therefore, we believe that this combination of a colorimetric probe and isothermal amplification will be useful for point-of-care testing to prevent the propagation of COVID-19.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00216-022-04156-7.

Detection of SARS-CoV-2 RNA through tandem isothermal gene amplification without reverse transcription

Diagnosis of SARS-CoV-2 infection through rapid, accurate, and sensitive testing is the most important and fundamental step in coping with the COVID-19 epidemic. We have developed a sensitive fluorometric assay to detect SARS-CoV-2 viral RNA without thermal cycling. This assay system, based on tandem isothermal gene amplification (TIGA), is composed of ternary rolling circle amplification (t-RCA) and subsequent strand displacement amplification (SDA) coupled with G-quadruplex-generating RCA (SDA/GQ-RCA). Without the need to convert viral RNA into cDNA, viral RNA forms a ternary complex composed of hairpin primer (HP) and dumbbell padlock DNA during the t-RCA process. t-RCA generates a long chain of single-stranded DNA (ssDNA) with tandemly repeated hairpin structures that are subjected to SDA. SDA produces multiple short ssDNAs from t-RCA products, which then serve as primers for the second RCA reaction. A long ssDNA harboring repeated copies of the G-quadruplex is produced in the second round of RCA. Emission of enhanced fluorescence by thioflavin T, which intercalates into the G-quadruplex, allows fluorometric detection of amplified viral genes. This fluorometric analysis sensitively detected SARS-CoV-2 RNA as low as 5.9 aM, with a linear range between 0.2 fM and 200 fM within 1 h. Hence, this isothermal gene amplification method without reverse transcription of viral RNA can be applied to diagnose COVID-19 with high sensitivity and accuracy as an alternative to current PCR-based diagnosis.

Ligation-free isothermal nucleic acid amplification

In this study, we uncover a ligation-free DNA extension method in two adjacent fragmented probes, which are hybridized to target RNA, for developing a ligation-free nucleic acid amplification reaction. In this reaction, DNA elongation occurs from a forward probe to a phosphorothioated-hairpin probe in the presence of target RNA regardless of ligation. The second DNA elongation then occurs simultaneously at the nick site of the phosphorothioated probe and the self-priming region. Therefore, the binding site of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) 12a is repeatedly amplified, inducing a fluorescence signal in the presence of CRISPR-Cas12a. This ligation-free isothermal gene amplification method enables the detection of target RNA with 49.2 fM sensitivity. Moreover, two types of mRNA detection are feasible, thus, demonstrating the potential of this method for cancer companion diagnostics. Notably, the proposed method also demonstrates efficacy when applied for the detection of mRNA extracted from human cells and tumor-bearing mouse tissue and urine samples. Hence, this newly developed ligation-free isothermal nucleic acid amplification system is expected to be widely used in a variety of gene detection platforms.

Retroreflection-based sandwich type affinity sensing of isothermal gene amplification products for foodborne pathogen detection.

Loop-mediated isothermal amplification (LAMP) is an outstanding method for molecular diagnostics, as the rapid, specific, and sensitive amplification of target genes is possible. However, it is necessary to measure fluorescence in the quantitative analysis of LAMP products, so a sophisticated optical setup is required. This study tried to develop a novel sensing method that can quantify target analytes with simple equipment, such as nonspectroscopic white light and a CMOS camera. To achieve this, a retroreflective Janus particle (RJP) as a probe and specially designed loop primers, fluorescein isothiocyanate (FITC)- and biotin-modified loop primers, were introduced into the LAMP system. By performing LAMP in the presence of designed primers, double-stranded amplicons possessing FITC and biotin labels at each end are generated in proportion to the quantity of the target pathogen. Using the anti-FITC antibody-modified sensing surface and streptavidin-conjugated RJP probes, the amplicons can be captured in sandwich-configuration and detected under nonspectroscopic conditions composed of white light and a camera. To confirm the feasibility of the sensing system, the invA gene of Salmonella was selected as the target. It was possible to quantitatively analyze the Salmonella concentration from 0 to 106 colony-forming units, sufficiently covering the required detection range. In addition, quantitative analyses of pathogens in contaminated food sources, including milk and chicken meat, were successfully conducted with a limit of detection of 10 CFU.

A Real-Life Approach for Evaluation of Rapid Ag Testing in Sars-Cov2 Infection

Keywords: Respiratory infections;SARS-CoV2;Ag rapid test Received: March 19, 2021, Accepted: April 03, 2021, Published: April 03, 2021 Introduction Biological diagnosis of respiratory infections by SARS-CoV2 uses molecular tools targeting viral genes by RT-PCR analysis or isothermal gene amplification. PCR-positive samples RT-PCR Thermo gave positive results on 86 specimens. * Ct ranged from 10.74 to 40.92 for S target (with 10 lacking of S response due to variations). * Ct ranged from 12.59 to 32.97 for N target. * Ct ranged from 10.41 to 33.68 for Orf1ab target. * For 70 of 86 specimens, all the 3 Ct were <26.00. [...]unexpected negative results by Ag testing (although Ct<26.00 by RT-PCR) were 5 with RAPID TEST 1, 7 with RAPID TEST 2, 2 with AllTest and 2 with Acro Biotech. Variants of SARS-CoV2 Among the 86 RT-PCR positive specimens, there was 10 variants of SARS-COV2: * Two of them (UK and del 69-70 variants) gave a strong and quick positive result by each of the Ag testing device. * Three variant samples gave a negative result by each of the Ag testing device (low burden for two of them: UK and del 69-70 variants;high burden for the other: del 69-70 variant). * Three variant samples gave positive results (even slight or late) for some of the device: RAPID TEST 1 with one ZA variant, RAPID TEST 2 with one UK variant, both RAPID TEST 1 and RAPID TEST 2 with another UK variant. * Two variant samples gave a negative result by one of the 4 devices (AllTest with a del 69-70 variant, low burden) or by two (RAPID TEST 1 and RAPID TEST 2 with a UK variant, high burden).

One-step single-tube accelerated quantitative nucleoprotein gene-specific reverse transcription loop-mediated isothermal gene amplification (RT-LAMP) assay for rapid, real-time & reliable clinical detection of Ebola virus.

Background & objectives:Due to the absence of specific drugs or vaccines for Ebola virus disease, rapid, sensitive and reliable diagnostic methods are required to control the transmission chain of the disease and for better patient management. Isothermal amplification of nucleic acids has emerged as a promising alternative in which rapid and efficient amplification is achieved at a constant temperature without the thermal cycling required in PCR.Methods:A one-step single-tube accelerated quantitative reverse trascription loop-mediated isothermal amplification (RT-LAMP) assay was developed by targeting the NP gene of 2014 Zaire Ebola virus (ZEBOV). The RT-LAMP assay was found to be specific for ZEBOV, without having any cross-reactivity with related haemorrhagic fever viral agents.Results:The comparative evaluation of Ebola virus NP gene-specific RT-LAMP assay with reverse transcription (RT) - PCR and TaqMan real-time RT-PCR demonstrated that RT-LAMP was 10-1000 folds more sensitive than TaqMan real-time RT-PCR and conventional RT-PCR, respectively, with a detection limit of 1 copy number. In the absence of real-world clinical samples, the feasibility of Ebola virus RT-LAMP assay for clinical diagnosis was evaluated with different body fluids including serum, urine, saliva, semen and stool samples from healthy human volunteers spiked with gamma-irradiated ZEBOV 2014 obtained from Robert Koch Institute, Berlin, Germany, through the European Network for Diagnostics of Imported Viral Diseases. The Ebola virus RT-LAMP assay could correctly be picked up the spiked samples up to 1 copy of viral RNA without having any matrix interference. The monitoring of gene amplification can also be visualized with the naked eye by using SYBR Green I fluorescent dye.Interpretation & conclusions:Thus, due to easy operation without a requirement of sophisticated equipment and skilled personnel, the RT-LAMP assay reported here is a valuable tool as a point-of-care diagnosis for the rapid and real-time detection of Ebola virus in resource-limited healthcare settings of developing countries.

Urinary exosomal mRNA detection using novel isothermal gene amplification method based on three-way junction

Exosomal messenger RNA (mRNA) has emerged as a valuable biomarker for liquid biopsy-based disease diagnosis and prognosis due to its stability in body fluids and its biological regulatory function. Here, we report a rapid one-step isothermal gene amplification reaction based on three-way junction (3WJ) formation and the successful detection of urinary exosomal mRNA from tumor-bearing mice. The 3WJ structure can be formed by the association of 3WJ probes (3WJ-template and 3WJ-primer) in the presence of target RNA. After 3WJ structure formation, the 3WJ primer is repeatedly extended and cleaved by a combination of DNA polymerase and nicking endonuclease, producing multiple signal primers. Subsequently, the signal primers promote a specially designed network reaction pathway to produce G-quadruplex probes under isothermal conditions. Finally, G-quadruplex structure produces highly enhanced fluorescence signal upon binding to thioflavin T. This method provides a detection limit of 1.23 pM (24.6 amol) with high selectivity for the target RNA. More importantly, this method can be useful for the sensing of various kinds of mRNA, including breast cancer cellular mRNA, breast cancer exosomal mRNA, and even urinary exosomal mRNA from breast cancer mice. We anticipate that the developed RNA detection assay can be used for various biomedical applications, such as disease diagnosis, prognosis, and treatment monitoring.

Utilizing multiplex fluor LAMPs to illuminate multiple gene expressions in situ.

In situ gene expression detection is the best way to determine temporal and spatial differences in gene expression. However, in situ hybridization procedures are inherently difficult to execute and typically suffer from degradation of sample tissues, limited sensitivity to genes with low expression, high background, and limitation to single gene detections. We propose to utilize an isothermal gene amplification technique, LAMP (Loop-Mediated Isothermal Amplification), to solve these problems in a novel way. LAMP greatly amplifies the signal of expressed genes and can use multiple sets of primers and different fluorescent-labeled probes to produce multiplex gene detection. LAMP is a rapid, isothermal reaction that reduces the handling and degradation of tissue by cutting down on the washing steps required by other methods. Using this technique, we have successfully amplified 3 target genes, have produced positive fluorescent in situ results simultaneously for two genes. We have also demonstrated that LAMP can be used to exploit standard NBT/BCIP (nitro-blue tetrazolium chloride/5-bromo-4-chloro-3'-indolyphosphate p-toluidine salt) detection of single expression. In situ LAMP is a robust and applicable method that can be exploited for detection of gene expression in plant species, as well as in animals and bacteria.

Point-of-care genetic analysis for multiplex pathogenic bacteria on a fully integrated centrifugal microdevice with a large-volume sample

We present a fully integrated portable centrifugal microsystem for multiplex detection of food poisoning bacteria with a large volume of sample up to 1 mL. The microsystem consists of a portable genetic analyzer and a fully integrated centrifugal microdevice. The centrifugal microdevice is designed with two units: a 3D printed solution-loading cartridge and a centrifugal microfluidic disc. All the essential solutions for loop-mediated isothermal amplification (LAMP) reaction are stored inside the cartridge, and orderly released into centrifugal microdevice by a rotation program. Each unit of the device is designed with 20 reaction chambers for simultaneous detection of food-borne bacteria in one test. To increase the amount of a sample to 1 mL, we incorporated the super absorbent polymer (SAP) in the waste chamber to absorb the sample and the washing solution during the device operation. The whole process was automatically conducted including designated solution release, bead-based DNA extraction, isothermal gene amplification by Eriochrome Black (EBT)-mediated LAMP reaction, and colorimetric and UV–visible detection of amplicons. The ratio between Abs640nm and Abs570nm was used as a criterion to confirm the positive result, and the result was positive upon the condition of Abs640/Abs570 ≥ 1.0. To demonstrate the pathogenic bacteria detection on our proposed microsystem, we targeted three kinds of bacteria (Escherichia coli O157:H7, Salmonella typhimurium, and Vibrio parahaemolyticus) for monoplex and multiplex detection. The whole process from sample to result was completed within 1 h with a low limit of detection (LOD) of 102 cells/mL.

Real-time recombinase polymerase amplification assay for the rapid and sensitive detection of Campylobacter jejuni in food samples

Campylobacter jejuni (C. jejuni), a foodborne pathogen, is a major contributor to human bacterial gastroenteritis worldwide and detrimental to public health. It is crucial for initiating appropriate outbreak control strategies to rapidly detect C. jejuni. As a novel isothermal gene amplification technique, recombinase polymerase amplification (RPA) has been developed for the molecular detection of diverse pathogens. In this study, we developed a real-time RPA assay so as to achieve the rapid and efficient detection of C. jejuni by targeting the hipO gene. The specificity and senstivity of real-time RPA was validated and the practical applicability of the method for the detection of C. jejuni in artificially contaminated milk and chicken breast samples was proved by comparing their reaction time, sensitivity, and efficacy with those of real-time PCR and culture-based methods. Based on the real-time RPA assay, analysis time was reduced to approximately 13 mins from 60 mins and the results were as reliable as those of the real-time PCR assay. Taken together, in terms of the detection of C. jejuni, the real-time RPA method was simple, rapid, sensitive, and reliable.

Development of real-time recombinase polymerase amplification assay for rapid and sensitive detection of canine parvovirus 2

BackgroundCanine parvovirus 2, a linear single-stranded DNA virus belonging to the genus Parvovirus within the family Parvoviridae, is a highly contagious pathogen of domestic dogs and several wild canidae species. Early detection of canine parvovirus (CPV-2) is crucial to initiating appropriate outbreak control strategies. Recombinase polymerase amplification (RPA), a novel isothermal gene amplification technique, has been developed for the molecular detection of diverse pathogens. In this study, a real-time RPA assay was developed for the detection of CPV-2 using primers and an exo probe targeting the CPV-2 nucleocapsid protein gene.ResultsThe real-time RPA assay was performed successfully at 38 °C, and the results were obtained within 4–12 min for 105–101 molecules of template DNA. The assay only detected CPV-2, and did not show cross-detection of other viral pathogens, demonstrating a high level of specificity. The analytical sensitivity of the real-time RPA was 101 copies/reaction of a standard DNA template, which was 10 times more sensitive than the common RPA method. The clinical sensitivity of the real-time RPA assay matched 100% (n = 91) to the real-time PCR results.ConclusionThe real-time RPA assay is a simple, rapid, reliable and affordable method that can potentially be applied for the detection of CPV-2 in the research laboratory and point-of-care diagnosis.

Rapid and sensitive detection of canine distemper virus by real-time reverse transcription recombinase polymerase amplification

BackgroundCanine distemper, caused by Canine distemper virus (CDV), is a highly contagious and fatal systemic disease in free-living and captive carnivores worldwide. Recombinase polymerase amplification (RPA), as an isothermal gene amplification technique, has been explored for the molecular detection of diverse pathogens.MethodsA real-time reverse transcription RPA (RT-RPA) assay for the detection of canine distemper virus (CDV) using primers and exo probe targeting the CDV nucleocapsid protein gene was developed. A series of other viruses were tested by the RT-RPA.Thirty-two field samples were further tested by RT-RPA, and the resuts were compared with those obtained by the real-time RT-PCR.ResultsThe RT-RPA assay was performed successfully at 40 °C, and the results were obtained within 3 min–12 min. The assay could detect CDV, but did not show cross-detection of canine parvovirus-2 (CPV-2), canine coronavirus (CCoV), canine parainfluenza virus (CPIV), pseudorabies virus (PRV) or Newcastle disease virus (NDV), demonstrating high specificity. The analytical sensitivity of RT-RPA was 31.8 copies in vitro transcribed CDV RNA, which is 10 times lower than the real-time RT-PCR. The assay performance was validated by testing 32 field samples and compared to real-time RT-PCR. The results indicated an excellent correlation between RT-RPA and a reference real-time RT-PCR method. Both assays provided the same results, and R2 value of the positive results was 0.947.ConclusionsThe results demonstrated that the RT-RPA assay offers an alternative tool for simple, rapid, and reliable detection of CDV both in the laboratory and point-of-care facility, especially in the resource-limited settings.

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

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

Rolling circle amplification as isothermal gene amplification in molecular diagnostics.

Rolling circle amplification (RCA) developed in the mid-1990s has been widely used as an efficient isothermal DNA amplification process for molecular diagnosis. This enzymatic process amplifies target DNA sequences with high fidelity and specificity by using the strand displacing DNA polymerases. The product of RCA is long single-stranded DNA that contains tandem repeat of target sequence. Isothermal reaction amplification condition of RCA has an advantage over conventional polymerase chain reaction, because no temperature cycling devices are needed for RCA. Thus, RCA is suitable tool for point-of-care detection of target nucleic acids as well as facile detection of target genes. Combined with various detection methods, RCA could amplify and detect femtomolar scale of target nucleic acids with a specificity of one or two base discrimination. Herein, RCA technology is reviewed with an emphasis on molecular diagnosis of microRNAs, infectious pathogens, and point mutations.

Fluorometric Detection of MicroRNA Using Isothermal Gene Amplification and Graphene Oxide

We have developed a facile fluorometric system for the detection of microRNA (miRNA), using rolling circle amplification (RCA), graphene oxide (GO), and fluorescently labeled peptide nucleic acid (F-PNA). The padlock probe DNA complementary to a target miRNA was selectively ligated to form circular DNA that was then used as the template for RCA. F-PNAs complementary to the target miRNA were annealed to multiple sites of the isothermally amplified single-stranded RCA product (RCAP) containing multiple target miRNA sequences. This F-PNA/RCAP duplex is less adsorbed onto the GO monolayer, thus attenuating the quenching of F-PNA fluorescence by GO. In the absence of target miRNA (and hence the absence of RCA and duplex formation), the free F-PNA is completely adsorbed onto the GO monolayer and fluorescence quenching ensues. Thus, GO-based fluorescence detection coupled with isothermal gene amplification would be a simple and convenient method for the quantitative detection of miRNA.