Isothermal Nucleic Acid Amplification Method Research Articles

Specific Detection of Influenza A and B Viruses by CRISPR-Cas12a-Based Assay.

A rapid and accurate on-site diagnostic test for pathogens including influenza viruses is critical for preventing the spread of infectious diseases. Two types of influenza virus, A and B cause seasonal flu epidemics, whereas type A can cause influenza pandemics. To specifically detect influenza A (IAV) and B (IBV) viruses, we developed a clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated (Cas) system-based assay. By coupling reverse transcription recombinase polymerase amplification (RT-RPA) and reverse transcription loop-mediated isothermal amplification (RT-LAMP), a CRISPR-Cas12a DNA endonuclease-targeted CRISPR trans-reporter (DETECTR) detected IAV and IBV titers as low as 1 × 100 plaque forming units (PFUs) per reaction without exhibiting cross-reactivity. Only 75 to 85 min were required to detect IAV and IBV, depending on isothermal nucleic acid amplification methods, and results were verified using a lateral flow strip assay that does not require additional analytic equipment. Taken together, our findings establish RT-RPA and RT-LAMP-coupled DETECTR-based diagnostic tests for rapid, specific and high-sensitivity detection of IAV and IBV using fluorescence and lateral flow assays. The diagnostic test developed in this study can be used to distinguish IAV and IBV infections, a capability that is necessary for monitoring and preventing the spread of influenza epidemics and pandemics.

Efficient, Rapid, and Sensitive Detection of Plant RNA Viruses With One-Pot RT-RPA-CRISPR/Cas12a Assay.

Most viruses that infect plants use RNA to carry their genomic information; timely and robust detection methods are crucial for efficient control of these diverse pathogens. The RNA viruses, potexvirus (Potexvirus, family Alphaflexiviridae), potyvirus (Potyvirus, family Potyviridae), and tobamovirus (Tobamovirus, family Virgaviridae) are among the most economically damaging pathogenic plant viruses, as they are highly infectious and distributed worldwide. Their infection of crop plants, alone or together with other viruses, causes severe yield losses. Isothermal nucleic acid amplification methods, such as loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and others have been harnessed for the detection of DNA- and RNA-based viruses. However, they have a high rate of non-specific amplification and other drawbacks. The collateral activities of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated nuclease Cas systems such as Cas12 and Cas14 (which act on ssDNA) and Cas13 (which acts on ssRNA) have recently been exploited to develop highly sensitive, specific, and rapid detection platforms. Here, we report the development of a simple, rapid, and efficient RT- RPA method, coupled with a CRISPR/Cas12a-based one-step detection assay, to detect plant RNA viruses. This diagnostic method can be performed at a single temperature in less than 30 min and integrated with an inexpensive commercially available fluorescence visualizer to facilitate rapid, in-field diagnosis of plant RNA viruses. Our developed assay provides an efficient and robust detection platform to accelerate plant pathogen detection and fast-track containment strategies.

Strategies for Isothermal Amplification of Nucleic Acids: are they Ready to be Applied in Point of Care Diagnosis of Mycosis?

The early detection of invasive fungal infection (IFD) is significant in order to decrease mortality in susceptible patients. There is, therefore, a need for sensitive and specific fungal species detection assays in a clinical laboratory for early targeted therapy. The isothermal amplification method may be useful for the screening of fungal isolates, especially in resource-poor settings. Therefore, our aim was to review the isothermal nucleic acid amplification methods and their applications in fungal pathogen detection. Out of 50 reported studies, 28, 12, 6, 2, and 2 studies used the isothermal-based assays of a loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), rolling circle amplification (RCA), multiple displacement amplification (MDA) and polymerase Spiral Reaction (PSR), respectively. Thirty-two studies used clinical samples, 18 pure culture, and four environmental samples. The diagnostic accuracy of isothermal nucleic acid amplification testing for pathogenic fungal was reported as high (sensitivity 0.89–1.0 and specificity 0.63–1.0) in all studies irrespective of the sample tested. Although the isothermal-based assays showed high sensitivity and specificity in reported studies, it is still poorer than that of PCR assays. However, improving the assay to make it simpler, more effective, and inexpensive compared with newer PCR methods are still needed.

Rapid detection of peach latent mosaic viroid by reverse transcription recombinase polymerase amplification

Reverse transcription recombinase polymerase amplification (RT-RPA), an isothermal nucleic acid amplification and detection method, was developed to detect peach latent mosaic viroid (PLMVd) in pollen and peach leaves. Results showed that this RT-RPA detection method can be performed at 42 °C and completed in approximately 5 min, and there was no cross-reactivity with other common peach viruses. A sensitivity assay showed that the RT-RPA assay was 1000-fold more sensitive than a regular RT-PCR. Moreover, the method was successfully applied to test field-collected samples. The newly developed RT-RPA assay is rapid, sensitive, and reliable method for detection of PLMVd in peach pollen and leaves and can be utilized as an effective technique in quarantine and viroid-free certification processes.

Real‐time analysis of switchable nanocomposites of magnesium pyrophosphates and rolling circle amplification products

AbstractRolling circle amplification (RCA) is a robust isothermal nucleic acid amplification method producing nanocomposites of DNA and inorganic magnesium pyrophosphate precipitates. Although the conformation and structure of such nanocomposites impact applications, most studies of them have been performed at the end‐point after exposure to a treatment. Here, we use real‐time optomagnetic measurements of the hydrodynamic size of magnetic nanoparticles (MNPs) to study the growth of RCA products grafted onto MNPs as well as the effect of post‐RCA temperature annealing and chemical treatment. As a key result, we show that secondary structures in the RCA products trap and partially protect magnesium pyrophosphate precipitates and that these are reversibly released upon heating above a characteristic temperature defined by the sequence of the RCA product. These findings provide a deeper mechanistic understanding of the synthesis and structure of DNA nanocomposites, which impacts applications of DNA nanocomposites in sensing and drug delivery.

A wearable microfluidic device for rapid detection of HIV-1 DNA using recombinase polymerase amplification

Although isothermal nucleic acid amplification is advantageous in pathogen detection in resource-limited settings, an electricity-dependent heating module is often required. Here, we developed a wearable microfluidic device combined with recombinase polymerase amplification (RPA) for simple and rapid amplification of HIV-1 DNA using human body heat. The human body temperature at the human wrist varied from 33 to 34 °C in the ambient environment, which is sufficient to perform RPA reactions. With the aid of a cellphone-based fluorescence detection system, this device detected HIV-1 DNA quantitatively ranging from 102 to 105 copies/mL with a log linearity of 0.98 in 24 min. These results demonstrate that this wearable point-of-care (POC) nucleic acid testing method is advantageous over traditional PCR and other isothermal nucleic acid amplification methods in terms of time, portability and independence on electricity. This wearable microfluidic device in conjunction with a cellphone-based fluorescence detection system can be potentially used for the detection of HIV-1 and adapted for POC detection of a broad range of infectious pathogens in resource-limited settings.

Evaluation of a novel micro/nanofluidic chip platform for the detection of influenza A and B virus in patients with influenza-like illness

We introduced a novel micro/nanofluidic chip platform (MNCP), which is based on an isothermal nucleic acid amplification method. This study aimed to evaluate the MNCP method for influenza A and B viruses detecting and subtyping using throat swab samples from patients with influenza-like illness (ILI). A total of 266 throat swab samples from 266 non-repeated patients with ILI were tested for influenza A and B viruses using three methods, MNCP, a rapid influenza diagnostic test (RIDT), and real-time reverse transcription polymerase chain reaction (rRT-PCR). The results of MNCP were compared to those obtained by rRT-PCR and RIDT and the performance of MNCP was further evaluated. Compared with rRT-PCR results, the rates of sensitivity, specificity, overall concordance, and the kappa value of MNCP were 98.89%, 96.97%, 97.65%, and 0.95 for influenza A virus; 94.95%, 99.38%, 97.68%, and 0.95 for influenza B virus, respectively. Subtypes of influenza A viruses, e.g., A(H1N1)pdm09, A(H3N2), and A(not subtyped), and influenza B viruses could be distinguished in one MNCP assay within 1 h. Compared with rRT-PCR and MNCP, RIDT showed poor clinical sensitivity for influenza virus detection. This study showed MNCP is rapid, sensitive and versatile detecting system with potential for clinical application in pathogen diagnosis for patients with ILI.

Corrigendum: Akkermansia muciniphila as a Model Case for the Development of an Improved Quantitative RPA Microbiome Assay.

[This corrects the article DOI: 10.3389/fcimb.2018.00237.].

Detection of Chlamydia trachomatis and Neisseria gonorrhoeae Using Multiplex Strand Invasion Based Amplification (mSIBA).

Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) are among the most prevalent causes of sexually transmitted infections (STIs) worldwide. Timely and accurate diagnosis plays an important role in deciding appropriate treatment and preventing the spread of the infection. Strand invasion based amplification (SIBA), is an established isothermal nucleic acid amplification method for the rapid and accurate detection of infectious diseases. SIBA was applied for the simultaneous detection of CT and NG in less than 1h. The multiplex SIBA (mSIBA) method displayed high analytical sensitivity and specificity for the detection of CT and NG. Since the method is performed at low and constant temperature, it can therefore be run on portable instruments. SIBA enables rapid screening for CT and NG within point-of-care or central laboratory settings.

Flap endonuclease-initiated enzymatic repairing amplification for ultrasensitive detection of target nucleic acids.

A new isothermal nucleic acid amplification method termed FERA (Flap endonuclease-initiated Enzymatic Repairing Amplification) is developed for the ultrasensitive detection of target nucleic acids. In the FERA method, flap endonuclease (FEN) catalyzes the hydrolytic cleavage at the junction of single- and double-stranded DNAs which is formed only in the presence of target nucleic acids, and releases short oligonucleotides to promote the cyclic enzymatic repairing amplification (ERA) combined with FEN-based amplification. As a result, a large amount of single- and double-stranded DNAs are generated under the isothermal conditions, leading to the high fluorescence intensity from the SYBR I green dye. Relying on the powerful amplification method, we successfully determined the target nucleic acids with a limit of detection as low as 15.16 aM, which corresponds to approximately 180 molecules in 20 μL reaction volume, and verified the practical applicability by detecting long target nucleic acids derived from Chlamydia trachomatis.

A simple isothermal nucleic acid amplification method for the effective on-site identification for adulteration of pork source in mutton

As meat adulteration issue is becoming increasingly prominent worldwide, it is crucial to realize on-site detection with simple equipment. Conventional nucleic acid amplification methods are reliable but the requirement of complex equipment, skilled technicians and long operation time limit their on-site use. Here, a simple denaturation bubble-mediated strand exchange amplification method (SEA) requiring only a pair of primers and one polymerase was first reported for identifying adulteration of pork source by targeting the specie-specific mitochondrial DNA sequence. The SEA method displayed good specificity for pork and could detect as low as 30 pg/μL pork DNA. In binary mixtures, the SEA method could detect 1% pork meat total DNA by both colorimetric and fluorescence determination, fulfilling the requirement of artificial meat adulteration. Excitedly, the whole detection process could be finished within 1 h by coupling with fast tissue DNA extraction method, only requiring a simple heating block. Therefore, with simplicity and rapidity, SEA method will be suitable for on-site meat species identification.

Detection of human rhinoviruses by reverse transcription strand invasion based amplification method (RT-SIBA)

Background: Rhinovirus (RV), a major cause of respiratory infection in humans, imposes an enormous economic burden due to the direct and indirect costs associated with the illness. Accurate and timely diagnosis is crucial for deciding the appropriate clinical approach and minimizing unnecessary prescription of antibiotics. Diagnosis of RV is extremely challenging due to genetic and serological variability among its numerous types and their similarity to enteroviruses.Objective: We sought to develop a rapid nucleic acid test that can be used for the detection of Rhinovirus within both laboratory and near patient settings.Study design: We developed and evaluated a novel isothermal nucleic acid amplification method called Reverse Transcription Strand Invasion-Based Amplification (RT-SIBA) to rapidly detect Rhinovirus from clinical specimens.Result: The method, RT-SIBA, detected RV in clinical specimens with high analytical sensitivity (96%) and specificity (100%). The time to positive result was significantly shorter for the RV RT-SIBA assay than for a reference RV nucleic acid amplification method (RT-qPCR).Conclusion: The rapid detection time of the RV SIBA assay, as well as its compatibility with portable instruments, will facilitate prompt diagnosis of infection and thereby improve patient care.

Nucleic Acid Detection by a Target-Assisted Proximity Proteolysis Reaction.

Nucleic acid analysis plays an important role in diagnosing diseases as well as understanding biology. Despite advances in technology, there is still a need to develop a rapid and simple method to detect specific nucleic acids, especially in remote locations and low-resource cases. Here, we proposed a proximity proteolysis reaction in which the reaction between protease and zymogen is enhanced in the presence of a target molecule. The pair of proteins was site-specifically modified with oligonucleotides, and the conjugates were used to develop a method of detecting nucleic acids. Target DNA and RNA could be detected in less than 1 h at sub-nanomolar concentrations based on an absorbance signal. The assay method was resistant to interference by biological matrixes, and its sensitivity could be improved when combined with an isothermal nucleic acid amplification method. The results demonstrated the feasibility of this proximity proteolysis reaction as a new platform technology for detecting specific nucleic acid sequences.

PrimedRPA: primer design for recombinase polymerase amplification assays.

SummaryRecombinase polymerase amplification (RPA), an isothermal nucleic acid amplification method, is enhancing our ability to detect a diverse array of pathogens, thereby assisting the diagnosis of infectious diseases and the detection of microorganisms in food and water. However, new bioinformatics tools are needed to automate and improve the design of the primers and probes sets to be used in RPA, particularly to account for the high genetic diversity of circulating pathogens and cross detection of genetically similar organisms. PrimedRPA is a python-based package that automates the creation and filtering of RPA primers and probe sets. It aligns several sequences to identify conserved targets, and filters regions that cross react with possible background organisms.Availability and implementationPrimedRPA was implemented in Python 3 and supported on Linux and MacOS and is freely available from http://pathogenseq.lshtm.ac.uk/PrimedRPA.html.Supplementary information Supplementary data are available at Bioinformatics online.

Akkermansia muciniphila as a Model Case for the Development of an Improved Quantitative RPA Microbiome Assay.

Changes in the population levels of specific bacterial species within the gut microbiome have been linked to a variety of illnesses. Most assays that determine the relative abundance of specific taxa are based on amplification and sequencing of stable phylogenetic gene regions. Such lab-based analysis requires pre-analytical sample preservation and storage that have been shown to introduce biases in the characterization of microbial profiles. Recombinase polymerase amplification (RPA) is an isothermal nucleic acid amplification method that employs commercially available, easy-to-use freeze-dried enzyme pellets that can be used to analyze specimens rapidly in the field or clinic, using a portable fluorometer. Immediate analysis of diverse bacterial communities can lead to a more accurate quantification of relative bacterial abundance. In this study, we discovered that universal bacterial 16S ribosomal DNA primers give false-positive signals in RPA analysis because manufacturing host Escherichia coli DNA is present in the RPA reagents. The manufacturer of RPA reagents advises against developing an RPA assay that detects the presence of E. coli due to the presence of contaminating E. coli DNA in the reaction buffer (www.twistdx.co.uk/). We, therefore, explored four strategies to deplete or fragment extraneous DNA in RPA reagents while preserving enzyme activity: metal-chelate affinity chromatography, sonication, DNA cleavage using methylation-dependent restriction endonucleases, and DNA depletion using anti-DNA antibodies. Removing DNA with anti-DNA antibodies enabled the development of a quantitative RPA microbiome assay capable of determining the relative abundance of the physiologically-important bacterium Akkermansia muciniphila in human feces.

Development and evaluation of a rapid nucleic acid amplification method to detect influenza A and B viruses in human respiratory specimens

Isothermal nucleic acid amplification methods can potentially shorten the amount of time required to diagnose influenza. We developed and evaluated a novel isothermal nucleic acid amplification method, RT-SIBA to rapidly detect and differentiate between influenza A and B viruses in a single reaction tube. The performance of the RT-SIBA Influenza assay was compared with two established RT-PCR methods. The sensitivities of the RT-SIBA, RealStar RT-PCR, and CDC RT-PCR assays for the detection of influenza A and B viruses in the clinical specimens were 98.8%, 100%, and 89.3%, respectively. All three assays demonstrated a specificity of 100%. The average time to positive result was significantly shorter with the RT-SIBA Influenza assay (<20 min) than with the two RT-PCR assays (>90 min). The method can be run using battery-operated, portable devices with a small footprint and therefore has potential applications in both laboratory and near-patient settings.

Rolling circle transcription-based polymeric siRNA nanoparticles for tumor-targeted delivery

RNA, one of the major biological macromolecules, has been considered as an attractive building material for bottom-up fabrication of nanostructures in the past few decades due to advancements in RNA biology, RNA chemistry and RNA nanotechnology. Most recently, an isothermal enzymatic nucleic acid amplification method termed rolling circle transcription (RCT), which achieves a large-scale synthesis of RNA nanostructures, has emerged as one of fascinating techniques for RNAi-based therapies. Herein, we proposed a newly designed RCT method for synthesis of polymeric siRNA nanoflower, referred to ‘RCT and annealing-generated polymeric siRNA (RAPSI)’: (1) Amplification of the antisense strand of siRNA via RCT process and (2) annealing of chimeric sense strand containing 3′-terminal DNA nucleotides that provide enzyme cleavage sites. To verify its potentials in RNAi-based cancer therapy, the newly designed RAPSI nanoflower was further complexed with glycol chitosan (GC) derivatives, and systemically delivered to PC-3 xenograft tumors. The resultant RAPSI nanoparticles exhibited the improved particle stability against polyanion competition or nuclease attack. When the RAPSI nanoparticles reached to the cytoplasmic region, active mono siRNA was liberated and significantly down-regulated the expression of target VEGF gene in PC-3 cells. Excellent tumor-homing efficacy and anti-tumor effects of the RAPSI nanoparticles were further demonstrated. Overall, the proposed RCT-based polymeric siRNA nanoflower formulation can provide a new platform technology that allows further functional modifications via an advanced annealing method for systemic cancer RNAi therapy.

Rapid and sensitive real-time assay for the detection of respiratory syncytial virus using RT-SIBA\xae

BackgroundRespiratory syncytial virus (RSV) is one of the most common causes of respiratory tract infections among young children and the elderly. Timely and accurate diagnosis of respiratory tract infections improves patient care and minimizes unnecessary prescriptions of antibiotics. We sought to develop a rapid nucleic acid tests for the detection of RSV within minutes, while retaining the high sensitivity achieved with RT-PCR.MethodsWe developed and evaluated a reverse transcription isothermal nucleic acid amplification method, reverse transcription strand invasion based amplification (RT-SIBA), for the rapid detection of RSV.ResultsThe developed RT-SIBA assay showed good sensitivity by detecting as few as 10 copies of RSV RNA within 20 min compared with reverse transcription polymerase chain reaction, which took approximately 2 h. The performance of the RT-SIBA RSV assay was further investigated using nasopharyngeal swab specimens. The RT-SIBA assay had a sensitivity of 100% (25/25) and a specificity of 100% (15/15).ConclusionRT-SIBA did not require highly purified RNA for the rapid detection of RSV and was therefore compatible with rapid specimen processing methods. This reduces the complexity of specimen preparation and further shortens the total amount of time needed to detect RSV in clinical specimens. The developed RT-SIBA assay for RSV could be a useful tool for prompt management of this infection

Rapid Molecular Diagnosis of Bacterial Infection Using Integrated Lab-on-a-disc

Bacterial infection is a disaster leading to high fatal rate in intensive caring unit. Rapid profiling of infectious bacteria is necessary for applying the correct medication. Current gold standard using plate inoculation is inaccurate, time-consuming and labour-intensive. Therefore we have developed a molecular diagnostic approach to target marker DNA of the infectious bacteria for rapid profiling. A micro-fluidic platform lab-on-a-disc (LOAD) has been adopted because using one simple spinning action can provide highly controllable centrifugation drive force for the actuation of samples and reagents anywhere within the boundary of the disc. When centrifugal force-triggered valve is applied, complex sequential flow of liquid can be controlled with various centrifugal force. This will enable parallel execution of many reactions simultaneously with minimal complexity in the design of fluidic pumping and flow control.We report an integrated LOAD for direct sample-to-answer applications - fully automated assay from patient's sample input to detection of signal output. The integrated LOAD with PDMS-made microfludic disc performs three major functions, namely DNA extraction, LAMP reaction and detection. Using microfluidics technology, target bacteria can be detected using as little as 10 μL blood sample loaded into sample loading site. The DNA release after cell lysis in heating site is bound on the silica membrane. After washing, the purified DNA elution is subjected to LAMP reaction to amplify the target genetic sequence. Loop-mediated isothermal amplification (LAMP) is an isothermal nucleic acid amplification method where reaction occurs under 65 oC. The amplified signal is reported by DNA binding fluorescent dye. Our prototype shows high yield and purity of bacterial DNA from clinical samples such as blood. We demonstrated the detection of Acinetobacter baumanii, which is one of the key pathogens resulting in hospital-acquired infections, in clinical blood sample using the LOAD platform. Fast signal detection and active temperature control within the LOAD platform has also enabled real-time LAMP targeting of specific DNA sequences as barcodes to identify infected bacterial species. We found the detection sensitivity of LAMP using DNA is 10-15 g, while that of bacteria concentration is 102 cfu/ml. The system is capable of providing bacterial DNA profiling within 2 hours.In conclusion, our integrated LOAD is a simple (sample-to-answer), specific (specific genetic sequences recognition), robust (automated assay on microfluidic disc) method for rapid molecular diagnosis of bacterial infection. The short turnaround time and the technical advancement of sample-to-answer in one LOAD platform approach for rapid bacterial detection should have much potential in addressing the needs of point-of-care medical diagnosis applications. The simplicity allows the clinical healthcare workers to utilize without needing technical training.

Comprehensive evaluation of molecular enhancers of the isothermal exponential amplification reaction.

The exponential amplification reaction (EXPAR) is an emerging isothermal nucleic acid amplification method with high potential for molecular diagnostics due to its isothermal nature and high amplification efficiency. However, the use of EXPAR is limited by the high levels of non-specific amplification. Hence, methods that can improve the specificity of EXPAR are desired to facilitate its widespread adoption in practice. Herein, we proposed a strategy to improve EXPAR performance by using molecular enhancers. Eight small molecules were investigated, including ethylene glycol, propylene glycol, betaine, dimethyl sulfoxide (DMSO), trehalose, tetramethylammonium chloride (TMAC), bovine serum albumin (BSA) and single-stranded binding (SSB) proteins. A combination of kinetic and end-point analysis was adopted to investigate how these molecules affected EXPAR performance. Trehalose, TMAC, BSA and SSB proteins were found to have positive effects on EXPAR with trehalose being able to increase the efficiency of EXPAR. In contrast, TMAC, BSA and SSB proteins were shown to increase the specificity of EXPAR. We applied our findings to demonstrate the combination of trehalose and TMAC could simultaneously improve both the efficiency and specificity of an EXPAR-based miRNA detection method. The information provided in this study may serve as a reference to benefit the wider isothermal amplification community.