Isothermal Nucleic Acid Amplification Method Research Articles

Integration of Isothermal Enzyme-Free Nucleic Acid Circuits for High-Performance Biosensing Applications.

The isothermal enzyme-free nucleic acid amplification method plays an indispensable role in biosensing by virtue of its simple, robust, and highly efficient properties without the assistance of temperature cycling or/and enzymatic biocatalysis. Up to now, enzyme-free nucleic acid amplification has been extensively utilized for biological assays and has achieved the highly sensitive detection of various biological targets, including DNAs, RNAs, small molecules, proteins, and even cells. In this Review, the mechanisms of entropy-driven reaction, hybridization chain reaction, catalytic hairpin assembly and DNAzyme are concisely described and their recent application as biosensors is comprehensively summarized. Furthermore, the current problems and the developments of these DNA circuits are also discussed.

Retraction Note: Polymerase Spiral Reaction (PSR): A novel isothermal nucleic acid amplification method

Retraction Note: Polymerase Spiral Reaction (PSR): A novel isothermal nucleic acid amplification method

Ultrasensitive detection of exosomes by microchip electrophoresis combining with triple amplification strategies

The analysis of exosomes is significant as they can be used for various pathophysiological processes, especially cancer related intercellular communication. Therefore, a convenient, reliable, and sensitive detection method is urgently needed. Strand displacement amplification (SDA) and catalytic hairpin assembly (CHA) are two kinds of effective isothermal nucleic acid amplification methods. In this article, an efficient quantitative MCE method for detecting human breast cancer cell (MCF-7) exosomes assisted by triple amplification strategies combining cholesterol probe (Chol-probe) with SDA-CHA was first developed. CD63 aptamer was immobilized on the avidin magnetic beads to specifically capture exosomes and then Chol-probe with high affinity was spontaneously inserted into the exosome membrane, which was the first step of amplification strategy to improve detection sensitivity. After magnetic separation, Chol-probe could complement ssDNA and trigger SDA, producing a large number of DNA sequences (Ta) to trigger CHA, achieving SDA-CHA amplification. Under optimal conditions, the detection limit (LOD) for MCF-7 exosomes was as low as 26 particle/μL (S/N = 3). This method provides an effective approach for sensitive and accurate quantification of tumor exosomes, and can be expected to detect exosomes in clinical samples.

Comparison of ID NOW COVID-19 with RT-PCR for Diagnosis and Management of COVID-19 Cases

ABSTRACT Introduction: Rapid diagnosis of SARS-CoV-2 is a challenging issue for healthcare personnel. Abbott’s ID NOW COVID-19 assay is a rapid instrument based on isothermal nucleic acid amplification method for qualitative estimation of SARS-CoV-2 in nasal, nasopharyngeal, and throat swabs. Aim and objective: The performance of ID NOW was compared with standard reference RT-PCR in 609 patients. Results and discussion: The two methods agreed in 90.6% of the cases and disagreed in 9.4% of the cases, and this agreement was statistically significant (Cohen’s Kappa = 0.727, P < 0.001). The diagnostic performance of ID NOW in predicting RT-PCR positivity was as follows: sensitivity of 78%; specificity of 94%; positive predictive value (PPV) of 80%; negative predictive value (NPV) of 94%; and diagnostic accuracy of 91%. Conclusion: Hence ID NOW can be used as point-of-care testing in the diagnosis of SARS-CoV-2 and thereby immediately isolate positive cases from negative cases.

Isothermal nucleic acid amplification and its uses in modern diagnostic technologies.

Nucleic acids are prominent biomarkers for diagnosing infectious pathogens using nucleic acid amplification techniques (NAATs). PCR, a gold standard technique for amplifying nucleic acids, is widely used in scientific research and diagnosis. Efficient pathogen detection is a key to adequate food safety and hygiene. However, using bulky thermal cyclers and costly laboratory setup limits its uses in developing countries, including India. The isothermal amplification methods are exploited to develop miniaturized sensors against viruses, bacteria, fungi and other pathogenic organisms and have been applied for in situ diagnosis. Isothermal amplification techniques have been found suitable for POC techniques and follow WHO's ASSURED criteria. LAMP, NASBA, SDA, RCA and RPA are some of the isothermal amplification techniques which are preferable for POC diagnostics. Furthermore, methods such as WGA, CPA, HDA, EXPAR, SMART, SPIA and DAMP were introduced for even more accuracy and robustness. Using recombinant polymerases and other nucleic acid-modifying enzymes has dramatically broadened the detection range of target pathogens under the scanner. The coupling of isothermal amplification methods with advanced technologies such as CRISPR/Cas systems, fluorescence-based chemistries, microfluidics and paper-based sensors has significantly influenced the biosensing and diagnosis field. This review comprehensively analyzed isothermal nucleic acid amplification methods, emphasizing their advantages, disadvantages and limitations.

Expanded targeted screening for congenital cytomegalovirus infection.

An early diagnosis and intervention for congenital cytomegalovirus infection can reduce long-term disability; however, the introduction of universal neonatal screening has been controversial worldwide. The present study clarified the outcome of a targeted screening protocol for detecting congenital cytomegalovirus infection based on suggestive perinatal conditions. In addition, the positive rate was compared to those from the reported studies and the validity of the targeted screening criteria was discussed. A total of 2121 newborn infants were admitted to our hospital between October 2018 and October 2021. Cytomegalovirus DNA was examined by the isothermal nucleic acid amplification method for urine samples from newborns with any of the following: microcephaly, abnormal ultrasound findings in the brain and visceral organs, repeated failure in neonatal hearing screening, suspicious maternal cytomegalovirus infection during pregnancy, and other abnormal findings suggestive of congenital cytomegalovirus infection. Among 2121 newborns, 102 (4.8%) were subject to the urine cytomegalovirus DNA test based on the abovementioned criteria. Of them, three were cytomegalovirus DNA-positive. According to the protocol, the cytomegalovirus DNA-positive rates were 0.14% among the total enrollment of 2121 newborns and 2.9% (3/102) among the targeted newborns. This protocol may overlook congenital cytomegalovirus infection that is asymptomatic or exhibits inapparent clinical manifestations only at birth; however, it is feasible and helps lead to the diagnosis of congenital cytomegalovirus infection that may otherwise be overlooked.

Nested Phosphorothioated Hybrid Primer-Mediated Isothermal Amplification for Specific and Dye-Based Subattomolar Nucleic Acid Detection at Low Temperatures.

Developing dye-based isothermal nucleic acid amplification (INAA) at low temperatures such as 37 °C remains a technical challenge. Here, we describe a nested phosphorothioated (PS) hybrid primer-mediated isothermal amplification (NPSA) assay which only utilizes EvaGreen (a DNA-binding dye) to achieve specific and dye-based subattomolar nucleic acid detection at 37 °C. The success of low-temperature NPSA essentially depends on employing Bacillus smithii DNA polymerase, a strand-displacing DNA polymerase with wide range of activation temperature. However, the NPSA's high efficiency entails nested PS-modified hybrid primers and the additives of urea and T4 Gene 32 Protein. To address the inhibition of urea on reverse transcription (RT), one-tube two-stage recombinase-aided RT-NPSA (rRT-NPSA) is established. By targeting human Kirsten rat sarcoma viral (KRAS) oncogene, NPSA (rRT-NPSA) stably detects 0.2 aM of KRAS gene (mRNA) within 90 (60) min. In addition, rRT-NPSA possesses subattomolar sensitivity to detect human ribosomal protein L13 mRNA. The NPSA/rRT-NPSA assays are also validated to obtain consistent results with PCR/RT-PCR methods on qualitatively detecting DNA/mRNA targets extracted from cultured cells and clinical samples. As a dye-based, low-temperature INAA method, NPSA inherently facilitates the development of miniaturized diagnostic biosensors.

Elucidating the Role of CRISPR/Cas in Single-Step Isothermal Nucleic Acid Amplification Testing Assays.

Developing assays that combine CRISPR/Cas and isothermal nucleic acid amplification has become a burgeoning research area due to the novelty and simplicity of CRISPR/Cas and the potential for point-of-care uses. Most current research explores various two-step assays by appending different CRISPR/Cas effectors to the end of different isothermal nucleic acid amplification methods. However, efforts in integrating both components into more ideal single-step assays are scarce, and poor-performing single-step assays have been reported. Moreover, lack of investigations into CRISPR/Cas in single-step assays results in incomplete understanding. To fill this knowledge gap, we conducted a systematic investigation by developing and comparing assays that share the identical recombinase polymerase amplification (RPA) but differ in CRISPR/Cas12a. We found that the addition of CRISPR/Cas12a indeed unlocks signal amplification but, at the same time, impedes RPA and that CRISPR/Cas12a concentration is a key parameter for attenuating RPA impediment and ensuring assay performance. Accordingly, we found that our protospacer adjacent motif (PAM)-free CRISPR/Cas12a-assisted RPA assay, which only moderately impeded RPA at its optimal CRISPR/Cas12a concentration, outperformed its counterparts in assay design, signal, sensitivity, and speed. We also discovered that a new commercial Cas12a effector could also drive our PAM-free CRISPR/Cas12a-assisted RPA assay and reduce its cost, though simultaneously lowering its signal. Our study and the new insights can be broadly applied to steer and facilitate further advances in CRISPR/Cas-based assays.

Advances in isothermal nucleic acid amplification methods for hepatitis B virus detection.

Hepatitis B virus (HBV) infection is a major global health problem of widespread concern. Clinically, serological assays are the most widely used diagnostic tests for HBV infection, with the presence of HBsAg in the serum being indicative of acute and chronic hepatitis B infection. However, increased identification of HBV DNA positive but HBsAg negative cases has greatly promoted the use of molecular assays for more accurate HBV diagnosis. Over the past few decades, especially since the outbreak of COVID-19, significant advancements have been made in the techniques and devices for nucleic acid testing (NAT). Nowadays, the mainstream NAT techniques can broadly be split into two categories: PCR-based methods and non-PCR-based isothermal amplification methods. As achieving point-of-care testing (POCT) or on-site testing is an important development tendency for the next-generation NAT, non-PCR-based isothermal amplification methods like nucleic acid sequence-based amplification (NASBA), rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), and recombinase polymerase amplification (RPA) have garnered significant attention in recent years. In this review, we provide a comprehensive overview of the nucleic acid isothermal amplification technologies currently used for HBV detection. The analytical performances of different methods are compared and their integration with microfluidics, lateral flow assays, and CRISPR/Cas systems is also discussed.

Development of a multi-recombinase polymerase amplification assay for rapid identification of COVID-19, influenza A and B.

The coronavirus disease 2019 (COVID‐19) pandemic caused extensive loss of life worldwide. Further, the COVID‐19 and influenza mix‐infection had caused great distress to the diagnosis of the disease. To control illness progression and limit viral spread within the population, a real‐time reverse‐transcription PCR (RT‐PCR) assay for early diagnosis of COVID‐19 was developed, but detection was time‐consuming (4–6 h). To improve the diagnosis of COVID‐19 and influenza, we herein developed a recombinase polymerase amplification (RPA) method for simple and rapid amplification of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS‐CoV‐2), the causative agent of COVID‐19 and Influenza A (H1N1, H3N2) and B (influenza B). Genes encoding the matrix protein (M) for H1N1, and the hemagglutinin (HA) for H3N2, and the polymerase A (PA) for Influenza B, and the nucleocapsid protein (N), the RNA‐dependent‐RNA polymerase (RdRP) in the open reading frame 1ab (ORF1ab) region, and the envelope protein (E) for SARS‐CoV‐2 were selected, and specific primers were designed. We validated our method using SARS‐CoV‐2, H1N1, H3N2 and influenza B plasmid standards and RNA samples extracted from COVID‐19 and Influenza A/B (RT‐PCR‐verified) positive patients. The method could detect SARS‐CoV‐2 plasmid standard DNA quantitatively between 102 and 105 copies/ml with a log linearity of 0.99 in 22 min. And this method also be very effective in simultaneous detection of H1N1, H3N2 and influenza B. Clinical validation of 100 cases revealed a sensitivity of 100% for differentiating COVID‐19 patients from healthy controls when the specificity was set at 90%. These results demonstrate that this nucleic acid testing method is advantageous compared with traditional PCR and other isothermal nucleic acid amplification methods in terms of time and portability. This method could potentially be used for detection of SARS‐CoV‐2, H1N1, H3N2 and influenza B, and adapted for point‐of‐care (POC) detection of a broad range of infectious pathogens in resource‐limited settings.

Recent Advances in Molecular Point of Care Tests

Molecular point of care tests has become promising methods for the global control of infectious diseases in recent years. Different kinds of point of care testing devices have been introduced into the market in the last decade. They are mainly based on miniaturization and integration of sample processing, nucleic acid amplification, and detection systems. These devices must offer a low-cost, sensitive, specific, and practical analysis to be used especially in low-resource settings. Microfluidics has high potential for handling very small volumes of fluids on a single platform. The key design features for molecular point of care tests in resource-limited settings include isothermal nucleic acid amplification methods to eliminate the need for a thermocycler, lyophilized reagents for long-term stability at high temperature and relatively simple test procedures. CRISPR-Cas-based new generation molecular point of care tests have been developed in recent years. In these tests Cas enzymes are used as highly specific target sequence recognition elements. Collateral cleavage activity of these enzymes cleaves both target sequence and labeled RNA in the mixture and a signal is generated. The ongoing Coronavirus 2019 pandemic has shown the importance of rapid diagnostic tests for the prevention of further spread. Although real-time polymerase chain reaction method was used successfully for the rapid diagnosis during the pandemic, additional rapid and practical tests that could be performed outside the laboratories would provide even faster diagnosis and lighten the burden of test load in large central laboratories. Molecular point of care tests are considered to be one of the best candidates to fill the this gap in the near future. The future technology challenges will be the standardization of the methods and further miniaturization of the system components. In this review article, point-of-care tests adapted from nucleic acid amplification methods with a large number of studies in the literature and tests based on the CRISPR-Cas system which constitutes the newest group and which is among the point-of-care molecular tests based on new nextgeneration sequencing have been discussed.

Dual Rolling Circle Amplification-Assisted Single-Particle Fluorescence Profiling of Exosome Heterogeneity for Discriminating Lung Adenocarcinoma from Pulmonary Nodules

Dual Rolling Circle Amplification-Assisted Single-Particle Fluorescence Profiling of Exosome Heterogeneity for Discriminating Lung Adenocarcinoma from Pulmonary Nodules

Advances in Nucleic Acid Amplification-Based Microfluidic Devices for Clinical Microbial Detection

Accurate and timely detection of infectious pathogens is urgently needed for disease treatment and control of possible outbreaks worldwide. Conventional methods for pathogen detection are usually time-consuming and labor-intensive. Novel strategies for the identification of pathogenic nucleic acids are necessary for practical application. The advent of microfluidic technology and microfluidic devices has offered advanced and miniaturized tools to rapidly screen microorganisms, improving many drawbacks of conventional nucleic acid amplification-based methods. In this review, we summarize advances in the microfluidic approach to detect pathogens based on nucleic acid amplification. We survey microfluidic platforms performing two major types of nucleic acid amplification strategies, namely, polymerase chain reaction (PCR) and isothermal nucleic acid amplification. We also provide an overview of nucleic acid amplification-based platforms including studies and commercialized products for SARS-CoV-2 detection. Technologically, we focus on the design of the microfluidic devices, the selected methods for sample preparation, nucleic acid amplification techniques, and endpoint analysis. We also compare features such as analysis time, sensitivity, and specificity of different platforms. The first section of the review discusses methods used in microfluidic devices for upstream clinical sample preparation. The second section covers the design, operation, and applications of PCR-based microfluidic devices. The third section reviews two common types of isothermal nucleic acid amplification methods (loop-mediated isothermal amplification and recombinase polymerase amplification) performed in microfluidic systems. The fourth section introduces microfluidic applications for nucleic acid amplification-based detection of SARS-CoV-2. Finally, the review concludes with the importance of full integration and quantitative analysis for clinical microbial identification.

CASCADE: Naked eye-detection of SARS-CoV-2 using Cas13a and gold nanoparticles

The COVID-19 pandemic has brought to light the need for fast and sensitive detection methods to prevent the spread of pathogens. The scientific community is making a great effort to design new molecular detection methods suitable for fast point-of-care applications. In this regard, a variety of approaches have been developed or optimized, including isothermal amplification of viral nucleic acids, CRISPR-mediated target recognition, and read-out systems based on nanomaterials.Herein, we present CASCADE (CRISPR/CAS-based Colorimetric nucleic Acid DEtection), a sensing system for fast and specific naked-eye detection of SARS-CoV-2 RNA. In this approach, viral RNA is recognized by the LwaCas13a CRISPR protein, which activates its collateral RNase activity. Upon target recognition, Cas13a cleaves ssRNA oligonucleotides conjugated to gold nanoparticles (AuNPs), thus inducing their colloidal aggregation, which can be easily visualized. After an exhaustive optimization of functionalized AuNPs, CASCADE can detect picomolar concentrations of SARS-CoV-2 RNA. This sensitivity is further increased to low femtomolar (3 fM) and even attomolar (40 aM) ranges when CASCADE is coupled to RPA or NASBA isothermal nucleic acid amplification, respectively. We finally demonstrate that CASCADE succeeds in detecting SARS-CoV-2 in clinical samples from nasopharyngeal swabs.In conclusion, CASCADE is a fast and versatile RNA biosensor that can be coupled to different isothermal nucleic acid amplification methods for naked-eye diagnosis of infectious diseases.

A novel isothermal method for amplification of long specific amplicon from linear template

Isothermal nucleic acid amplification methods have been successfully developed and applied for diagnostic purpose, especially for detection of pathogens. However, amplicon size of such methods is relatively short (< 500 bp) to limit their application for long amplicon production that can be used for various downstream applications including genomic surveillance of pathogens. To fill the gap, we developed a method for specific amplification of kilobases-long target sequence from RNA templates. This method, named CREA, utilizes sequence specific recombination of Cre recombinase to generate circular intermediate template for subsequent RCA reaction. CREA with SARS-CoV-2 spike template could amplify ~ 2.9 kb target and up to ~ 1.9 kb amplicon was able to produce in sufficient amount for general cloning. Each step of CREA procedure was thoroughly analyzed to provide directions for further optimizations. Furthermore, we evaluated a variation of CREA which utilized DNA ligase.

Rapid Molecular Diagnostics in the Field and Laboratory to Detect Plant Pathogen DNA in Potential Insect Vectors.

A variety of sensitive and specific molecular diagnostic assays has been described for detecting nucleic acids in biological samples that may harbor pathogens of interest. These methods include very rapid, isothermal nucleic acid amplification methods that can be deployed outside of the laboratory environment, such as loop-mediated isothermal DNA amplification (LAMP) and recombinase-polymerase amplification (RPA). However, all molecular diagnostic assays must be preceded by nucleic acid extraction from the biological samples of interest, which provides suitable template molecules for the assays. To exploit the features of the amplification assays and be utilized outside of the lab, these methods must be rapid and avoid the need for typical laboratory chemicals and equipment. We describe a protocol for the extraction of DNA from field-collected insects that can be implemented at the point of collection and used to detect the presence of DNA sequences from potential plant pathogens that may be vectored by the insects. This protocol provides template DNA that is suitable for PCR, LAMP, and RPA. The FTA PlantSaver card-based DNA extraction product was also confirmed to amplify the mitochondrial cytochrome oxidase 1 (CO1) universal barcode that could later be sequenced to identify any insect. Lastly, we provide an example using field-collected insects, Neokolla (Graphocephala) heiroglyphica, and demonstrate the detection of the plant pathogen Xylella fastidiosa in carrier insects using PCR, RPA, and LAMP.

Clinical evaluation of an innovative isothermal amplification detection system for COVID-19 diagnosis.

A pre-integrated system design intended for a point-of-care (POC) and sample-to-result diagnostic platform with nucleic acid amplification has been developed, which is equipment/electricity-free without any permanent instruments or manual sample processing. This semi-integrated system focuses on pandemic situations that are suitable for the Affordable, Sensitive, Specific, User-friendly, Robust and rapid, Equipment-free, and Deliverable to the end-user "ASSURED" concept recommended by the World Health Organization (WHO). Nucleic acid amplification is an essential rate-limiting factor in the performance of integrated systems that involve sample preparation and detection. The ORF1ab (RdRp) gene of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been targeted by RT-LAMP optimization and evaluation using a commercial hot-pack as a heat source that successfully achieves a femto-scale (<6.8 × 102 copies per rxn) limit of detection (LOD) within 40 min (except for the RNA extraction step). Therefore, the prototype system was assessed using COVID-19-suspected clinical samples (eighty eight) and compared with the results of a commercial real-time reverse transcription polymerase chain reaction (RT-qPCR) assay (Allplex SARS-CoV-2 Assay kit (Seegene, Seoul, Republic of Korea)). These innovative approaches achieved over 95% sensitivity and specificity. In conclusion, the developed system using a hot-pack as a heat source is a promising tool that enables the rapid identification of infectious diseases in the real world.

A high-specificity flap probe-based isothermal nucleic acid amplification method based on recombinant FEN1-Bst DNA polymerase

The COVID-19 pandemic has unfortunately demonstrated how easily infectious diseases can spread and harm human life and society. As of writing, pandemic has now been on-going for more than one year. There is an urgent need for new nucleic acid-based methods that can be used to diagnose pathogens early, quickly, and accurately to effectively impede the spread of infections and gain control of epidemics. We developed a flap probe-based isothermal nucleic acid amplification method that is triggered by recombinant FEN1-Bst DNA polymerase, which—through enzymatic engineering—has both DNA synthesis, strand displacement and cleavage functions. This novel method offers a simpler and more specific probe–primer pair than those of other isothermal amplifications. We tested the method's ability to detect SARS-CoV-2 (both ORF1ab and N genes), rotavirus, and Chlamydia trachomatis. The limits of detection were 10 copies/μL for rotavirus, C. trachomatis, and SARS-CoV-2 N gene, and 100 copies/μL for SARS-CoV-2 ORF1ab gene. There were no cross-reactions among 11 other common pathogens with characteristics similar to those of the test target, and the method showed 100% sensitivity and 100% specificity in clinical comparisons with RT-PCR testing. In addition to real-time detection, the endpoint could be displayed under a transilluminator, which is a convenient reporting method for point-of-care test settings. Therefore, this novel nucleic acid senor has great potential for use in clinical diagnostics, epidemic prevention, and epidemic control.

Establishment of a visualized isothermal nucleic acid amplification method for on-site diagnosis of acute hepatopancreatic necrosis disease in shrimp farm.

Acute hepatopancreatic necrosis disease (AHPND) is a significant deadly infectious disease in the shrimp farming industry, causing serious economic losses globally every year. Because of the rapid progress speed, lack of effective treatment and high mortality rate of AHPND, monitoring with frequent diagnostic tests is vital for a successful prevention. The conventional histopathological diagnosis fell far short of the requirement for efficient monitoring, and the polymerase chain reaction (PCR)-based molecular diagnostic methods that rely on sophisticated thermocycler and trained personnel are hardly applicable in the field. Combining the recombinase polymerase amplification (RPA) and the lateral flow strips (LFSs), a diagnostic method suitable for on-site everyday monitoring of AHPND has been established in this study. This RPA-LFS method targeted the binary toxic photorhabdus insect-related genes PirA and PirB on a virulence plasmid of the AHPND-causative Vibrio parahaemolyticus strains. The diagnostic test was completed within 30min at 37°C and showed good specificity and good sensitivity of 20fg DNA of the AHPND shrimp or one colony-forming unit of the causative bacterium per reaction, which was better than the administration-approved standard AP4 assay. Crude templates from sample boiling could be directly used. Tests of clinical samples showed 100% consistency of this method with the standard AP4 assay. This RPA-LFS method can be a good choice for on-site diagnosis of AHPND with quick response time, easy procedure and low demand for resources, and should have significant value for the control of spreading of this dangerous disease in farmed shrimp.

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.