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Results for “Isothermal amplification”

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At least 19 recordsLinked to original sources

PubMed2026

Rapid and reliable diagnosis of mucormycosis using colorimetric loop-mediated isothermal amplification.

Current diagnostic approaches for mucormycosis are often limited by low sensitivity and prolonged turnaround times, which result in delayed treatment and poor clinical outcomes. We developed a novel diagnostic method utilizing a colorimetric loop-mediated isothermal amplification (LAMP) assay for the rapid and sensitive detection of mucormycosis. The assay incorporates specifically designed primers capable of detecting as low as 0.001 picograms (pg) of spiked genomic DNA from Mucorales fungi. This LAMP assay demonstrated a high sensitivity of 98% and a 100% specificity of detecting fungal ribosomal DNA (rDNA) in bronchoalveolar lavage (BAL) samples collected from mice infected with Mucorales fungi (n = 48) or from an uninfected control group (n = 15). To align the assay with clinical antifungal therapy, a subset of infected mice was treated with either liposomal amphotericin B (LAMB) or a combination of LAMB and a humanized monoclonal antibody (VX-01) targeting the Mucorales-specific surface protein CotH3. Consistent with the treatment efficacy, the LAMP assay detected significantly lower fungal burdens in BAL samples from mice receiving the combination therapy compared to those treated with LAMB alone or placebo. Further validation was conducted using BAL samples from patients diagnosed with mucormycosis (n = 24) or aspergillosis (n = 17). The assay demonstrated a sensitivity of 79% and a specificity of 94%. These findings highlight the diagnostic potential of this LAMP-based assay as a point-of-care. Its high sensitivity, specificity, and rapid turnaround time position this assay as a promising tool for early and accurate detection of mucormycosis, with the potential to improve patient management and clinical outcomes.IMPORTANCEMucormycosis is a rapidly progressive and fatal fungal infection. Timely diagnosis is critical for effective treatment, yet current diagnostic tools are slow, insensitive, or require complex laboratory procedures. In this study, we developed and validated a colorimetric loop-mediated isothermal amplification (LAMP) assay that enables rapid and reliable detection of Mucorales DNA directly from bronchoalveolar lavage (BAL) specimens. The assay demonstrated high sensitivity and specificity in both experimental mouse models and clinical samples, producing results within 1 h without the need for sophisticated equipment. This simple, robust, and cost-effective molecular diagnostic tool holds great potential for early detection of mucormycosis, facilitating prompt antifungal therapy and improving patient survival.

Mucormycosis↗

PubMed1992

Strand displacement amplification--an isothermal, in vitro DNA amplification technique.

Strand Displacement Amplification (SDA) is an isothermal, in vitro nucleic acid amplification technique based upon the ability of HincII to nick the unmodified strand of a hemiphosphorothioate form of its recognition site, and the ability of exonuclease deficient klenow (exo- klenow) to extend the 3'-end at the nick and displace the downstream DNA strand. Exponential amplification results from coupling sense and antisense reactions in which strands displaced from a sense reaction serve as target for an antisense reaction and vice versa. In the original design (G. T. Walker, M. C. Little, J. G. Nadeau and D. D. Shank (1992) Proc. Natl. Acad. Sci 89, 392-396), the target DNA sample is first cleaved with a restriction enzyme(s) in order to generate a double-stranded target fragment with defined 5'- and 3'-ends that can then undergo SDA. Although effective, target generation by restriction enzyme cleavage presents a number of practical limitations. We report a new target generation scheme that eliminates the requirement for restriction enzyme cleavage of the target sample prior to amplification. The method exploits the strand displacement activity of exo- klenow to generate target DNA copies with defined 5'- and 3'-ends. The new target generation process occurs at a single temperature (after initial heat denaturation of the double-stranded DNA). The target copies generated by this process are then amplified directly by SDA. The new protocol improves overall amplification efficiency. Amplification efficiency is also enhanced by improved reaction conditions that reduce nonspecific binding of SDA primers. Greater than 10(7)-fold amplification of a genomic sequence from Mycobacterium tuberculosis is achieved in 2 hours at 37 degrees C even in the presence of as much as 10 micrograms of human DNA per 50 microL reaction. The new target generation scheme can also be applied to techniques separate from SDA as a means of conveniently producing double-stranded fragments with 5'- and 3'-sequences modified as desired.

Base Sequence↗

PubMed2026

SCAN: A sample-to-answer cross-priming isothermal assay for on-site virus detection with RT-qPCR sensitivity and genomically similar virus differentiation specificity.

Genomically similar viruses often differ in pathogenicity and host tropism due to specific mutations, and failure to distinguish them risks misdiagnosis and ineffective control. Molecular methods can differentiate such viruses but require laboratory settings and skilled personnel, while field-deployable immunological methods suffer from cross-reactivity. To address this challenge, we developed SCAN (Sample-to-answer Cross-priming isothermal amplification Assay with Nucleic acid strip), a general framework for on-site detection of genomically similar viruses. Comparative bioinformatics of isolation and sequencing data identifies key conserved differential determinants for primer design, ensuring specificity and reducing non-specific amplification. A one-tube cross-priming isothermal amplification (CPA) enables rapid target amplification without thermal cycling, and the products are visually detected on a nucleic acid strip. All steps are integrated into a handheld, lightweight device (9.9 × 4.4 × 3.3 cm, <200 g) that also prevents aerosol contamination. Using transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV), the latter a natural mutant of TGEV, as a model, SCAN achieves a detection limit of 102 copies/μL with sensitivity comparable to RT-qPCR and supports sample-to-answer testing within 80 min and simple operations. With verified high sensitivity, specificity, and accuracy, as well as field usability, SCAN provides a generalizable route for developing point-of-care tests (PoCT) that require precise field differentiation of closely related pathogens.

Cross-priming isothermal amplification↗

PubMed2026

Molecular diagnostics and integrated management challenges of tobacco streak virus: Current status and future perspectives.

Tobacco streak virus (TSV) is an economically important viral pathogen causing severe yield and quality losses in several agricultural, horticultural and medicinal crops worldwide. Its complex epidemiology involving sap transmission, infected pollen and pollen-feeding thrips, together with symptom similarity to other necrosis-inducing pathogens, frequently results in misdiagnosis and delayed disease management. This review critically evaluates recent advances in TSV diagnostics and integrated disease management strategies. Particular emphasis is placed on the transition from conventional biological and serological assays to advanced molecular diagnostics including reverse transcription polymerase chain reaction (RT-PCR), quantitative real-time PCR, multiplex PCR and emerging isothermal amplification technologies such as recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP). The review also highlights emerging innovations including CRISPR/Cas-based diagnostics in addition, integrated management approaches involving phytosanitation, weed reservoir management, vector ecology-based, host resistance breeding, RNA interference (RNAi) and genome editing technologies are critically analysed. Major challenges including inadequate field validation, limited multiplex capability, poor assay standardization and scarcity of resistant cultivars are discussed. Future objectives to develop quick, field-adaptable and durable TSV detection and management methods are additionally discussed.

CRISPR/Cas diagnostics↗

PubMed1992

Isothermal in vitro amplification of DNA by a restriction enzyme/DNA polymerase system.

An isothermal in vitro DNA amplification method was developed based upon the following sequence of reaction events. Restriction enzyme cleavage and subsequent heat denaturation of a DNA sample generates two single-stranded target DNA fragments (T1 and T2). Present in excess are two DNA amplification primers (P1 and P2). The 3' end of P1 binds to the 3' end of T1, forming a duplex with 5' overhangs. Likewise, P2 binds to T2. The 5' overhangs of P1 and P2 contain a recognition sequence (5'-GTTGAC-3') for the restriction enzyme HincII. An exonuclease-deficient form of the large fragment of Escherichia coli DNA polymerase I (exo- Klenow polymerase) [Derbyshire, V., Freemont, P. S., Sanderson, M. R., Beese, L., Friedman, J. M., Joyce, C. M. & Steitz, T. A. (1988) Science 240, 199-201] extends the 3' ends of the duplexes using dGTP, dCTP, TTP, and deoxyadenosine 5'-[alpha-thio]triphosphate, which produces hemiphosphorothioate recognition sites on P1.T1 and P2.T2. HincII nicks the unprotected primer strands of the hemiphosphorothioate recognition sites, leaving intact the modified complementary strands. The exo- Klenow polymerase extends the 3' end at the nick on P1.T1 and displaces the downstream strand that is functionally equivalent to T2. Likewise, extension at the nick on P2.T2 results in displacement of a downstream strand functionally equivalent to T1. Nicking and polymerization/displacement steps cycle continuously on P1.T1 and P2.T2 because extension at a nick regenerates a nickable HincII recognition site. Target amplification is exponential because strands displaced from P1.T1 serve as targets for P2 and strands displaced from P2.T2 serve as targets for P1. A 10(6)-fold amplification of a genomic sequence from Mycobacterium tuberculosis or Mycobacterium bovis was achieved in 4 h at 37 degrees C.

Base Sequence↗

PubMed2022

Engineered helicase replaces thermocycler in DNA amplification while retaining desired PCR characteristics.

Polymerase Chain Reaction (PCR) is an essential method in molecular diagnostics and life sciences. PCR requires thermal cycling for heating the DNA for strand separation and cooling it for replication. The process uses a specialized hardware and exposes biomolecules to temperatures above 95 °C. Here, we engineer a PcrA M6 helicase with enhanced speed and processivity to replace the heating step by enzymatic DNA unwinding while retaining desired PCR characteristics. We name this isothermal amplification method SHARP (SSB-Helicase Assisted Rapid PCR) because it uses the engineered helicase and single-stranded DNA binding protein (SSB) in addition to standard PCR reagents. SHARP can generate amplicons with lengths of up to 6000 base pairs. SHARP can produce functional DNA, a plasmid that imparts cells with antibiotic resistance, and can amplify specific fragments from genomic DNA of human cells. We further use SHARP to assess the outcome of CRISPR-Cas9 editing at endogenous genomic sites.

Humans↗

PubMed2026

Gravity-driven millifluidic platform for magnetic solid-phase extraction of Enterocytozoon hepatopenaei DNA from complex shrimp hepatopancreas.

Effective detection of Enterocytozoon hepatopenaei (EHP) in aquaculture is currently hindered by the lack of field-deployable extraction methods capable of processing complex, inhibitor-rich hepatopancreatic tissue. This study presents a gravity-driven millifluidic platform for the rapid extraction of EHP genomic DNA using an optimized, surfactant-compatible magnetic solid-phase extraction (MSPE) chemistry. Utilizing 5% PEG 8000 and 2.0 M NaCl, the platform facilitates the selectively capture of DNA from inhibitor-rich crustacean lysates. The 3D-printed device employs a tilting rocking plate to generate passive, gravity-driven flow, maintaining homogeneous magnetic bead suspension and maximizing solid-phase capture efficiency without external pumps. The integrated platform achieved a DNA yield of 2804.33 ± 15.31 ng/μL, a 5.9-fold increase over manual magnetic bead extraction. TaqMan quantitative PCR (qPCR) validation targeting the EHP SSU rRNA gene was developed. Using a standard curve spanning 101 to 107 plasmid copies (Ct = -3.611 log10 [copy] + 42.309, R2 = 0.998, amplification efficiency 89.2%), the on-chip MSPE achieved a validated analytical limit of detection (LOD) of 1 spore per reaction (100% detection rate, n = 21), whereas a commercial CTAB-based DNA extraction kit failed to achieve a validated LOD even at 10 spores (85.7%, 18/21). Nested PCR targeting the SWP gene was employed for field evaluation. A pilot study across two cohorts (N = 40) demonstrated consistent detection of confirmed EPH-positive cases; however, the small sample size precludes definitive diagnostic accuracy claims. With a total processing time under 30 min, this platform provides a high-efficiency extraction module. Future work will couple the device with isothermal amplification (e.g., LAMP or RPA) to realize a sample-to-answer system for resource-limited aquaculture.

Aquaculture diagnostics↗

PubMed2026

Saliva-based RT-LAMP assays support heat shock protein 70 as a promising transcript marker for estrus identification in buffaloes.

Buffaloes do not exhibit overt estrus signs particularly during summer, leading to a significant economic loss to farmers. Previous studies have identified several candidate transcripts (HSP70, TIMP1, TLR4 and HSD17B1), abundant in buffalo saliva during estrus stage. However, there is no widely applicable technology for estrus detection targeting these transcripts. Therefore, the present study aimed to develop reverse transcription loop mediated isothermal amplification (RT-LAMP) assays for these candidate transcripts using buffalo saliva. Saliva samples were collected from 10 cyclic buffaloes and RT-LAMP assays were optimized for salivary RNA as well as direct saliva. Among the four candidate transcripts, HSP70 showed a statistically significant colour change (p-value = 0.0191) at the estrus stage compared to the diestrus stage. This abundance of HSP70 was also supported in large simulated population datasets (10,000 animals) generated using R. Further, the RT-LAMP assays were tested using direct saliva without RNA isolation, and the colour change in the samples during estrus suggested the feasibility of estrus identification using direct saliva, overcoming the tedious step of RNA isolation. The detection of HSP70 using either direct saliva or salivary RNA indicated its potential as a marker for estrus identification. Similarly, TLR4 appeared to be another potential biomarker for RT-LAMP reaction using direct saliva, but it needs further validation in both RNA and direct saliva samples. Overall, the proof-of-concept on RT-LAMP assays optimized for salivary transcripts in the present study would be useful for estrus identification in tropical production systems following further validation on a larger sample size.

Animals↗

PubMed2026

CRISPR-Cas-based diagnostics for point-of-care detection of sexually transmitted infections: a laboratory development and evaluation study.

BACKGROUND: Timely, point-of-care diagnosis of sexually transmitted infections (STIs) is crucial for enabling prompt treatment and reducing transmission. We aimed to develop a portable, multiplexed, CRISPR-based assay panel for the detection of Neisseria gonorrhoeae (including the ciprofloxacin resistance marker gyrA S91F), Chlamydia trachomatis, Treponema pallidum, and herpes simplex virus (HSV). METHODS: In this laboratory development and evaluation study, we developed and optimised four multiplexed, CRISPR-based, diagnostic STI assays for point-of-care use. The complete assay panel comprised a CRISPR TP-HSV (cTP-HSV) panel for the detection of T pallidum and pan-HSV, with reflex testing to distinguish HSV-1 from HSV-2, and a CRISPR NG-CT (cNG-CT) panel for the detection of N gonorrhoeae and C trachomatis, with reflex testing to detect N gonorrhoeae using two additional genome regions and to identify the gyrA S91F mutation. Each pathogen was targeted at two independent genomic regions by isothermal amplification and CRISPR-Cas reaction using Cas12a and Cas13a, each with distinct fluorescent reporters. Analytical specificity and limits of detection (LODs) were determined, and a retrospective, masked concordance study was conducted on genomic DNA from 900 clinical samples (400 for cTP-HSV and reflex testing and 500 for cNG-CT and reflex testing), using quantitative PCR as the reference standard. The diagnostic accuracy of the test was assessed by analysis of receiver operating characteristic curves. FINDINGS: The overall sensitivity of the TP-HSV CRISPR assay was 82·5% (95% CI 74·0-88·7) for T pallidum and 94·4% (90·2-97·0) for pan-HSV; LODs were 6·2 copies per μL for T pallidum and 7·8 copies per μL for HSV. Reflex testing gave sensitivities of 97·0% (91·1-99·3) for HSV-1 and 96·0% (89·7-98·7) for HSV-2. The NG-CT CRISPR assay had an overall sensitivity of 80·0% (74·0-84·9) for N gonorrhoeae and 73·0% (65·5-79·3) for C trachomatis, with a LOD of 3·9 copies per μL for both pathogens. Reflex testing for the detection of the gyrA S91F mutation in N gonorrhoeae showed an overall sensitivity of 63·1% (55·1-70·4); however, this was dependent on sample type, with a sensitivity of 85·7% (46·7-99·5) in genital samples and 61·2% (52·8-68·9) in extragenital samples. For all pathogens, assay sensitivity was positively correlated with pathogen load. Area under the curve (AUC) values were 0·90 for T pallidum and 0·99 for pan-HSV in the TP-HSV assay, with values of 0·99 for HSV-1 and 0·97 for HSV-2 obtained in the reflex HSV-1-HSV-2 assay. For the cNG-CT assay, AUC values were 0·90 for N gonorrhoeae and 0·85 for C trachomatis, with a value of 0·72 obtained for gyrA S91F in the reflex cNG-gyrA assay. INTERPRETATION: Our multiplexed, CRISPR-based, point-of-care platform achieved performance consistent with WHO target product profiles for N gonorrhoeae and T pallidum. Proof-of-concept detection of the gyrA S91F resistance marker highlights its potential for resistance-guided therapy. Although optimisation is required before large-scale deployment, this suite offers a promising approach for rapid, decentralised, and resistance-informed STI diagnosis, particularly in resource-limited settings. FUNDING: Victorian Government Department of Health, Australian Government Department of Health, Disability and Ageing and Aged Care, and Australian Research Council.

Humans↗

PubMed2026

Multiplexed RT-LAMP Assays in Handheld Devices for In-Situ Detection of Chikungunya, Dengue, Mayaro, and Zika Viruses.

Mosquito-borne viruses pose a significant global health challenge, particularly in resource-limited settings where multiple viruses often cause illnesses with similar symptoms that require different treatment. We introduce the first 7-plex reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay in a hand-held device capable of detecting the presence of Chikungunya virus (CHIKV), dengue virus serotypes (DENV 1-4), Mayaro virus (MAYV), and Zika virus (ZIKV) in a single test. If the result is positive from the single-plex device for the 7-plex assay, 3-plex and 4-plex devices are then used to identify the exact virus within a specimen. In-situ detection is achieved by integrating valve-enabled, paper-based sample preparation with fluorescence detection using a blue LED flashlight as a light source and a yellow plastic film as a filter, allowing visual discrimination between positive and negative samples by the naked eye or by recording images using a smart phone. The detection limits ranged from 2 genome equivalents (GE)/reaction (for ZIKV) to 92 GE/reaction (for DENV-3) across 7 types of viruses when 1 μL of viral RNA was used. We observed 90% overall agreement between the point-of-care (POC) device and lab-based reverse transcription polymerase chain reactions (RT-PCR) when blinded clinical specimens were tested. This assay and device have a potential to address critical surveillance gaps in endemic regions, enabling timely detection of multiple mosquito-borne viruses to guide appropriate clinical management and public health countermeasures in settings where laboratory resources are scarce.

Nucleic Acid Amplification Techniques↗

PubMed2026

Artificial intelligence in molecular diagnostics for pandemic preparedness.

INTRODUCTION: Molecular diagnostics focusing on the detection and analysis of nucleic acids are indispensable tools for early pathogen identification, transmission monitoring, and genomic surveillance during pandemics. Recent technological advances have broadened the diagnostic landscape, incorporating PCR-based methods, isothermal amplification, high-CRISPR-based amplification detection, and sequencing. Despite their diagnostic potential, widespread implementation remains limited by high validation costs, time and logistical constraints, the need for specialized professional knowledge, and a lack of adaptability in resource-limited settings. Artificial intelligence (AI) is increasingly recognized as a promising but challenging approach, offering tools that streamline assay development, automate data interpretation, and optimize real-time diagnostic performance. AREAS COVERED: This review introduces recently published AI tools with potential to enhance the in-silico design validation process of oligonucleotides for molecular assays. These cover tools for initial assay design and optimization to validation and continuous assay updates. The limitations, including concerns regarding data accuracy, the lack of transparency in data processing ('black box' models), and unresolved licensing and regulatory issues, are highlighted for each tool and as expert opinion. EXPERT OPINION: Collectively, these challenges currently confine most AI-based approaches to research settings and prevent their routine implementation in clinical molecular diagnostics. Their widespread adoption depends on addressing remaining technical, regulatory, and practical challenges.

Humans↗

PubMed2026

Potato Black Scurf and Stem Canker: Pathogen Biology, Global Distribution, and Traditional and Modern Diagnostics.

Rhizoctonia solani is a soil- and seed-borne fungal pathogen of potatoes. It is a persistent threat to potato production worldwide. The symptoms appear as black scurf on tubers and stem canker, causing severe yield and quality losses of potatoes. The pathogen reproduces asexually via hyphae and sclerotia. Its genetic diversity is organized into anastomosis groups (AGs), with AG3-PT being the predominant group on potato. The global trade of seed potatoes is very important for agricultural development; however, it has facilitated the dissemination of the pathogen across regions. Moreover, disease development is affected by environmental and agronomic factors, causing variable symptom severity and differential economic impacts. Given the pathogen's genetic complexity, accurate diagnosis is very important, necessitating a transition from traditional culture-based and biochemical methods toward molecular, genomic, and emerging digital technologies. Methods such as PCR, isothermal amplification, sequencing, sensor-based biosensing, and artificial intelligence-driven imaging have improved the detection, quantification, and noninvasive monitoring of the pathogen. Combining these diagnostic methods into a tiered framework will be helpful for precision disease surveillance, informed disease management decision-making, and the development of sustainable potato production systems.

black scurf↗

PubMed2025

The impact of Schistosoma haematobium hybridization on molecular diagnosis of schistosomiasis: A review with emphasis on female genital schistosomiasis.

Female genital schistosomiasis (FGS) is a gynecological manifestation of urinary schistosomiasis in female genitals. FGS is a neglected tropical disease; not only are most patients unaware of the condition, but healthcare workers and policymakers have inadequate knowledge about it. The treatment and control of FGS relies on current guidelines for controlling and eliminating schistosomiasis without rigorous focus on clinical evidence of the presence of FGS. Neglect of FGS has led to the misconception that the disease is sexually transmitted. Diagnosing FGS remains challenging as there is no widely accepted reference assay. Urine examination, which is the gold standard in urogenital schistosomiasis has some limitations in diagnosing FGS as the demonstration of Schistosoma haematobium and/or eggs alone does not necessarily indicate FGS. In order to overcome challenges with the biopsy and colposcopy approach, some studies have evaluated the potential of PCR-based assays and isothermal amplification of Schistosoma DNA. Recent studies have reported hybridization between S. haematobium and other livestock schistosomes, but little is known about the impact of hybridization on schistosomiasis diagnosis. These hybrids not only affect livestock and humans but also have their genomes modified, and in some cases, abnormal egg morphology due to Schistosoma hybridization might affect the actual prevalence estimation. Herein, we highlight the potential impacts of S. haematobium hybridization on molecular diagnosis of schistosomiasis, with an emphasis on FGS.

Humans↗

PubMed1990

Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication.

A target nucleic acid sequence can be replicated (amplified) exponentially in vitro under isothermal conditions by using three enzymatic activities essential to retroviral replication: reverse transcriptase, RNase H, and a DNA-dependent RNA polymerase. By mimicking the retroviral strategy of RNA replication by means of cDNA intermediates, this reaction accumulates cDNA and RNA copies of the original target. Product accumulation is exponential with respect to time, indicating that newly synthesized cDNAs and RNAs function as templates for a continuous series of transcription and reverse transcription reactions. Ten million-fold amplification occurs after a 1- to 2-hr incubation, with an initial rate of amplification of 10-fold every 2.5 min. This self-sustained sequence replication system is useful for the detection and nucleotide sequence analysis of rare RNAs and DNAs. The analogy to aspects of retroviral replication is discussed.

Base Sequence↗

PubMed1990

Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication.

Explore the source record for details and available documents.

Base Sequence↗

PubMed1991

NASBA isothermal enzymatic in vitro nucleic acid amplification optimized for the diagnosis of HIV-1 infection.

Isothermal nucleic acid amplification of target RNA or DNA sequences is accomplished by the simultaneous enzymatic activity of AMV reverse transcriptase, T7 RNA polymerase and RNase H. Amplification factors of the nucleic acid sequence based amplification (NASBA) method range from 2 x 10(6) to 5 x 10(7) after 2.5 h incubation at 41 degrees C. During NASBA there is a major accumulation of specific single stranded RNA. RNA:DNA hybrid and double stranded DNA are also synthesized, although to a minor extent. The system is optimized for the detection of HIV-1 sequences in in vitro infected cells, blood and plasma. Detection levels are 10 molecules of HIV-1 in a model system with in vitro generated HIV-1 RNA as input and 5 infected cells on a background of 5 x 10(4) non-infected cells. Blood and plasma can also be used as the source of nucleic acid for detection of HIV-1 sequences using a specifically developed sample preparation method. Using NASBA it is possible to amplify specifically RNA or DNA from a pool of total nucleic acid, which permits the investigation of the expression of specific genes involved in pathogenesis of infectious agents. The combination of NASBA with a rapid and user-friendly nucleic acid extraction method makes the whole procedure suitable for large scale diagnosis of infectious agents (e.g. HIV-1).

Base Sequence↗

PubMed1991

Self-sustained sequence replication (3SR): an isothermal transcription-based amplification system alternative to PCR.

Explore the source record for details and available documents.

Base Sequence↗

PubMed2025

RT-RPA Assisted CRISPR/Cas12a Based One-Pot Rapid and Visual Detection of the Pan-Dengue Virus.

Globally ≤ 4 billion of the population are at potential risk of contracting dengue virus (DENV) infection. Seasonal outbreaks of dengue are frequently reported causing a high healthcare burden. Undiagnosed DENV can lead to severe morbidity and mortality. Early diagnosis of DENV relies on molecular methods, which are impractical in resource-constrained settings (RCSs). Dengue can be caused by any of the four distinct DENV serotypes. Therefore, a simple method for rapid diagnosis of Pan-DENV serotypes is of utmost importance at RCSs. A fluorescence detection platform for Pan-DENV using RT-RPA and CRISPR/Cas12a was developed targeting nonstructural 1 (NS1) gene for DENV-1, 2, and 3, and envelope (E) gene for DENV-2. Further, crRNA specific to DENV serotypes were designed to facilitate CRISPR/Cas12a detection. Analytical sensitivity was determined using synthetic RNA and DENV serotypes genome. Clinical validation of the assay was performed using RNA extracted from AES/AFI clinical samples. The developed CRISPR/Cas12a-based detection platform can detect all four serotypes of DENV viz 1-4 in a single pot using fluorescence detection. This assay showed the limit of detection ≥ 781 zg reaction- 1, ≥ 1.81 ag reaction-1, ≥ 62.5 fg reaction-1, and ≥ 2.5 pg reaction-1 for synthetic DENV-1, DENV-2, DENV-3, and DENV-4 template, respectively. Our assay demonstrated the analytic sensitivity of ≥ 10 ng reaction-1 for DENV-1 and DENV-4, and ≥ 0.5 ng reaction-1 for DENV-3 and DENV-4 genomes. This assay showed no cross-reactivity with other related etiologies tested causing AFI/AES. With 76 clinical samples (DENV PCR positive = 16, DENV PCR negative = 60), the assay demonstrated 93.7% sensitivity and 100% specificity with an overall accuracy of 98.7% for detection of the Pan-DENV serotypes. Our assay displayed comparable results to that of RT-PCR. The ease of interpretation and rapid detection of the Pan-DENV, represents the potential of the developed assay as an ideal point-of-care test. This assay upon field-deployment could help in reducing healthcare burden, provide differential diagnosis and support initiating early and prompt treatment to patients at RCS.

Dengue Virus↗