Journal of Virological Methods, Journal Year: 2024, Volume and Issue: unknown, P. 115035 - 115035
Published: Sept. 1, 2024
Language: Английский
ACS Omega, Journal Year: 2025, Volume and Issue: unknown
Published: March 26, 2025
Background: Enterovirus (EV) infections encompass a spectrum of diseases, with severe cases often linked to enterovirus A71 (EV-A71), highlighting its significance in EV screening. Although the CRISPR/Cas12a diagnostic system shows promise nucleic acid detection, enhancing specificity and sensitivity when combined reverse transcription-loop-mediated isothermal amplification (RT-LAMP), limitation lies targeting single sequence, thus preventing simultaneous detection both EVs EV-A71. Results: This study presents novel one-tube strategy by integrating HNB (hydroxynaphthol blue)-RT-LAMP for EV-A71 tube. The assay initially detects using an colorimetric approach under natural light, followed specific through blue light. limit was 10 copies/μL, EV-A71, it 1 copy/μL. Clinical sample assays demonstrated that, compared qPCR, accuracy HNB-LAMP-CRISPR 95.7 100%, respectively. Significance: In summary, this offers reliable user-friendly Also worth mentioning is that provided method has beneficial effects on rapid visualized detection.
Language: Английский
Citations
0
Microchemical Journal, Journal Year: 2025, Volume and Issue: unknown, P. 113480 - 113480
Published: March 1, 2025
Language: Английский
Citations
0
Microbial Biotechnology, Journal Year: 2025, Volume and Issue: 18(4)
Published: April 1, 2025
ABSTRACT We developed a sensitive and specific method based on recombinase‐aided amplification (RAA) clustered regularly interspaced short palindromic repeats (CRISPR)‐CRISPR‐associated protein 13a (Cas13a). This method, named CRISPR‐based Rapid Efficient Test (CRISPRET), is designed for the early diagnosis of Influenza B (FluB) with aim shortening its transmission chain. identified conserved regions in Virus (IBV) NS gene forward reverse primers along crRNAs. then established optimised reaction system, Nucleic Acid Positive Reference Materials IBV were used to evaluate detection limit (DL) CRISPRET. Additionally, we collected 257 clinical samples, comprising 127 samples from patients infection 130 healthy individuals, subjected them dual using CRISPRET qPCR positive predictive value (PPV), negative (NPV), sensitivity specificity one primer, two primers, crRNAs establish optimise CRISPR ET. The demonstrated DL 500 copies·μL −1 when assisted by appropriate equipment. Despite requiring auxiliary equipment 30‐min reaction, ET enables nucleic acid within approximately first 5 min, achieving high (100%), (97.69%), PPV (97.69%) NPV concordance rate 98.83% qPCR. offers simple, field‐applicable, one‐step rapid IBV. It has strong potential field‐testing applications intelligent integration into existing diagnostic systems.
Language: Английский
Citations
0
Microbiology Spectrum, Journal Year: 2025, Volume and Issue: unknown
Published: April 25, 2025
ABSTRACT Influenza B virus (Flu B) is a prevalent respiratory pathogen responsible for seasonal influenza epidemics. Despite its clinical significance, there remains lack of rapid and accurate diagnostic methods Flu detection. In this study, we developed novel detection system, named Fast-Flu, by integrating reverse transcription recombinase polymerase amplification (RT-RPA) with the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system (CRISPR/Cas). Through optimization reaction temperature adjustment Cas12a concentrations, successfully balanced RPA CRISPR/Cas12a trans-cleavage activity, enabling establishment one-step system. The Fast-Flu demonstrated ability to specifically identify within 45 min, limit 58 copies per test. It eliminates need uncapping operations minimizes risk cross-contamination, without cross-reactivity other pathogens. When evaluated using 101 throat swab samples, achieved sensitivity 56.25% specificity 100% compared PCR-based method, an overall concordance rate 93.06% (94/101). development RT-RPA-CRISPR/Cas12a represents significant advancement in rapid, convenient, B, highlighting potential diagnosis. Furthermore, future technical improvements enhance sensitivity, RT-RPA-CRISPR assay holds promise as versatile tool nucleic acid RNA viruses. IMPORTANCE global health concern, diagnosis crucial effective prevention control. integration isothermal (CRISPR)/CRISPR-associated has high Although CRISPR/Cas-based systems have been detection, existing platforms require transfer amplified products into through operations, which increases cross-contamination. amplification-CRISPR/Cas12a method one-pot By optimizing concentration, streamlined contamination-free workflow. This innovative approach not only improves but also serves valuable reference constructing CRISPR/Cas pathogens targets, paving way broader applications molecular diagnostics.
Language: Английский
Citations
0
Biosensors, Journal Year: 2024, Volume and Issue: 14(7), P. 348 - 348
Published: July 17, 2024
The COVID-19 pandemic has highlighted the urgent need for rapid and accurate diagnostic methods various infectious diseases, including SARS-CoV-2. Traditional RT-PCR methods, while highly sensitive specific, require complex equipment skilled personnel. In response, we developed an integrated RT-LAMP-MS assay, which combines reverse transcription loop-mediated isothermal amplification (RT-LAMP) with microscanning (MS) technology detecting assay uses magnesium pyrophosphate formed during LAMP as a visual marker, allowing direct observation via microscopy without additional chemical indicators or probes. For SARS-CoV-2/IC sample-LAMP reagent mixture was added to microchip SARS-CoV-2 primers internal controls, then incubated at 62 °C 30 min in heat block, followed by analysis using microscanner. clinical tests, showed 99% sensitivity 100% specificity, is identical RT-LAMP results comparable commercial Allplex
Language: Английский
Citations
3
Infectious Diseases of Poverty, Journal Year: 2024, Volume and Issue: 13(1)
Published: Dec. 9, 2024
Abstract Background Toxoplasma gondii oocysts, excreted in cat feces, pose a significant health risk to humans through contaminated soil and water. Rapid accurate detection of T. environmental samples is essential for public protection. Methods We developed novel, single-tube method that integrates loop-mediated isothermal amplification (LAMP), the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12b system, lateral flow immunoassay strips rapid, visual identification . This targets B1 gene , initially amplifies it with LAMP, directed by single-guide RNA (sgRNA). It then recognizes amplified target activates trans-cleavage, cutting nearby single-stranded DNA (ssDNA) reporters. Fluorescence was performed using 6-Carboxyfluorescein (FAM)-12N-Black Hole Quencher-1 (BHQ1) reporter, while Fluorescein Isothiocyanate (FITC)-12N-Biotin enabled on strips. The tested its ability detect various genotypes related parasites, assessing specificity broad-spectrum applicability. further applied real-world evaluate practicality. Results LAMP-CRISPR/Cas12b exhibited high capability, successfully identifying nine distinguishing them from 11 other parasitic species. Sensitivity testing at both molecular (plasmid) practical (oocyst) levels showed limits 10 copies/μL 0.1 oocyst, respectively. When 112 (soil, water, feces), demonstrated 100% sensitivity, accurately reflecting known infection rates. Conclusions offers robust, innovative approach monitoring zoonotic samples, implications surveillance. Graphical
Language: Английский
Citations
3
Diagnostics, Journal Year: 2024, Volume and Issue: 14(9), P. 967 - 967
Published: May 6, 2024
Influenza viruses cause highly contagious respiratory diseases that millions of deaths worldwide. Rapid detection influenza is essential for accurate diagnosis and the initiation appropriate treatment. We developed a loop-mediated isothermal amplification lateral flow assay (LAMP-LFA) capable simultaneously detecting A B. Primer sets B were designed to target conserved regions segment 7 nucleoprotein gene, respectively. Optimized through various primer set ratios, operated at 62 °C 30 min. For total 243 (85 positive, 58 positive 100 negative) nasopharyngeal swab samples, performance A/B multiplex LAMP-LFA was compared with commercial AllplexTM Respiratory Panel 1 (Seegene, Seoul, Korea). The demonstrated specificity 98% non-infected clinical along sensitivities 94.1% samples 96.6% showed high sensitivity specificity, indicating it reliable use in low-resource environment.
Language: Английский
Citations
1
Journal of Virological Methods, Journal Year: 2024, Volume and Issue: unknown, P. 115035 - 115035
Published: Sept. 1, 2024
Language: Английский
Citations
0