CRISPR/Cas12a-based method coupled with isothermal amplification to identify Alternaria spp. isolated from wheat grain samples DOI Creative Commons
Aisha Shaizadinova, Meruyert Amanzholova, И.В. Рукавицина

et al.

Frontiers in Microbiology, Journal Year: 2025, Volume and Issue: 15

Published: Jan. 15, 2025

Alternaria fungal species are considered major plant pathogens, infecting various crops and resulting in significant agricultural losses. Additionally, these can contaminate grain with multiple mycotoxins that harmful to humans animals. Efficient pest management relies on timely detection identification of phytopathogens samples, facilitating prompt selection a crop protection strategy. Conventional tools, such as morphological characterization based polymerase chain reaction (PCR)-based methods, time-consuming laboratory-bound, limiting their implementation for on-site diagnostics essential the industry. Isothermal amplification including nucleic acid sequence-based (NASBA), loop-mediated isothermal (LAMP), recombinase (RPA), enable at constant temperatures, making them ideal point-of-care without need thermal cycling equipment. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a)-based identification, coupled represents an emerging acid-based technology detecting pathogens high accuracy sensitivity. This study aimed develop CRISPR/Cas12a-based method integrated RPA specific spp. isolated from wheat samples. The developed targeted β-tubulin gene was successfully identified strains within 20-min followed by 30-min CRISPR/Cas12a visualization results. Specificity test included pathogenic commonly hosted grain, Fusarium Bipolaris sorokiniana, Nigrospora oryzae revealed specificity species. Furthermore, exhibited sensitivity, DNA down 100 copies, validated real-time fluorescence readout. A assay employed visualize results reaction, demonstrating substantial potential In conclusion, we present potentially sustainable approach rapid, precise, nucleic-acid-based

Language: Английский

Exploration of Isothermal Nucleic Acid Amplification Techniques in the Biomedical Field DOI Creative Commons

Sukanya Kundu,

Rajat Varshney, Sourabh Sulabh

et al.

Gene and Genome Editing, Journal Year: 2024, Volume and Issue: 7, P. 100032 - 100032

Published: Jan. 21, 2024

Nucleic acid amplification techniques (NATs) are frequently used in the molecular biology arena. Polymerase chain reaction (PCR) and its variants one of most popular NATs. The requirement a sophisticated thermocycler skilled technician for PCR limits use resource-limited laboratories fields. Alternatively, isothermal technique can also deliver proficiency, simplicity, sensitivity, fidelity without need thermocycler. Several methods have been devised newer concepts emerging. Some them loop-mediated amplification, whole genome rolling circle nucleic sequence-based polymerase spiral reaction. These cost-effective, practicable, easy-to-perform diagnostic assays current trends to identify pathogens, tumors, embryo sex, genetically modified organisms. Isothermal be microfluidic devices point-of-need diagnostics. This review focuses on development common processes, their characteristics, acceptance over PCR.

Language: Английский

Citations

6
Nanopore sequencing of infectious fluid is a promising supplement for gold-standard culture in real-world clinical scenario DOI Creative Commons

Manna Zhao,

Yongyang Zhang,

Li Chen

et al.

Frontiers in Cellular and Infection Microbiology, Journal Year: 2024, Volume and Issue: 14

Published: Jan. 30, 2024

Introduction Infectious diseases are major causes of morbidity and mortality worldwide, necessitating the rapid identification accurate diagnosis pathogens. While unbiased metagenomic next-generation sequencing (mNGS) has been extensively utilized in clinical pathogen scientific microbiome detection, there is limited research about application nanopore platform-based mNGS diagnostic performance various infectious fluid samples. Methods In this study, we collected 297 suspected fluids from 10 centers detected them with conventional microbiology culture platform–based mNGS. The objective was to assess detective nanopore-sequencing technology (NST) real-world scenarios. Results Combined gold-standard adjudication, demonstrated nearly 100% positive predictive agreements microbial-colonized sites, such as respiratory urinary tracts. For samples initially sterile body microorganisms were highly pathogens, negative relatively higher than those particularly abscess 95.7% cerebrospinal fluid. Furthermore, consistent also observed antimicrobial resistance genes drug susceptibility testing pathogenic strains Escherichia coli , Staphylococcus aureus Acinetobacter baumannii . Discussion Rapid NST a promising tool supplement culture, it potential improve patient prognosis facilitate treatment diseases.

Language: Английский

Citations

6
Detection of monkeypox virus based on a convenient and sensitive single-step RPA-CRISPR/Cas12a strategy DOI Creative Commons

Tao Yu,

Zhen Rong,

Zhixia Gu

et al.

RSC Advances, Journal Year: 2024, Volume and Issue: 14(21), P. 14775 - 14783

Published: Jan. 1, 2024

The global outbreak of monkeypox virus (MPXV) has highlighted the need for rapid molecular diagnostics techniques. In this study, a single-step recombinase polymerase amplification (RPA)-CRISPR/Cas12a system was developed and sensitive detection MPXV. limit assay 1 copy per μL extracted nucleic acids. A heating lysis method integrated to further simplify sample processing workflow shorten time 40 min from result. reaction mixture can be lyophilized improve its accessibility in resource-limited settings. analysis results proposed RPA-CRISPR/Cas12a clinical MPXV positive negative samples were 100% consistent with standard PCR assay. These demonstrate feasibility efficiency accurate real-world settings, showcasing potential utility urgent practical

Language: Английский

Citations

6
RPA-CRISPR-Cas-Mediated Dual Lateral Flow Assay for the Point-of-Care Testing of HPV16 and HPV18 DOI

K. Zhang,

Qingmei Li, Kun Wang

et al.

Bioconjugate Chemistry, Journal Year: 2024, Volume and Issue: 35(11), P. 1797 - 1804

Published: Oct. 15, 2024

The incidence of cervical cancer caused by human papillomavirus (HPV) infection has increased in recent years. More than half all cases are due to HPV16 and HPV18 infection, so testing is essential prevent cancer. HPV mainly carried out hospitals, but it subject time specialized medical facilities. On the other hand, home self-testing using simple diagnostics would present an attractive alternative privacy flexibility with regard place, provided sufficient sensitivity specificity can be achieved. In this work, a dual lateral flow assay based on RPA-CRISPR-Cas12a/13a (named RC-LFA) for detection was described. Taking advantage cleavage Cas12a Cas13a, CRISPR-Cas12a/Cas13a system designed detect HPV18. strip two test lines suit CRISPR-Cas12a/Cas13 system. RC-LFA achieves rapid simultaneous high (10 copies/μL) about 40 min from extraction nucleic acid instrument-free readout. user-friendly instrument-free, making promising method self-tests at home.

Language: Английский

Citations

6
Phosphoramidate Azole Oligonucleotides for Single Nucleotide Polymorphism Detection by PCR DOI Open Access
Alexey S. Chubarov, Elizaveta E. Baranovskaya, Igor P. Oscorbin

et al.

International Journal of Molecular Sciences, Journal Year: 2024, Volume and Issue: 25(1), P. 617 - 617

Published: Jan. 3, 2024

Detection of the Kirsten rat sarcoma gene (KRAS) mutational status is an important factor for treatment various malignancies. The most common KRAS-activating mutations are caused by single-nucleotide mutations, which usually determined using PCR, allele-specific DNA primers. Oligonucleotide primers with uncharged or partially charged internucleotide phosphate modification have proved their ability to increase sensitivity and specificity single nucleotide mutation detection. To enhance detection, novel oligonucleotides four types phosphates modification, phosphoramide benzoazole (PABA) (PABAO), was used prove concept on KRAS model. molecular effects different site-specific PABA in a primer template synthesis full-length elongation product PCR efficiency were evaluated. (AS-PCR) plasmid templates showed significant analysis without changes Cq values compared unmodified primer. universal mismatch-like disturbance, can be polymorphism discrimination applications. insights affect structural features PABAO connection AS-PCR results, improvements support further design platforms biological targets testing.

Language: Английский

Citations

5
Amplifying mutational profiling of extracellular vesicle mRNA with SCOPE DOI

Jayeon Song,

Mi Hyeon Cho, Hayoung Cho

et al.

Nature Biotechnology, Journal Year: 2024, Volume and Issue: unknown

Published: Oct. 7, 2024

Language: Английский

Citations

5
CRISPR-Cas based diagnostic tools: Bringing diagnosis out of labs DOI
Abu Sufiyan Chhipa,

Ekta Radadiya,

Snehal S. Patel

et al.

Diagnostic Microbiology and Infectious Disease, Journal Year: 2024, Volume and Issue: 109(2), P. 116252 - 116252

Published: March 8, 2024

Language: Английский

Citations

4
Development of a duplex recombinase polymerase amplification-lateral flow strip assay with 1 min of DNA extraction for simultaneous identification of pork and chicken ingredients DOI

Jinchao Feng,

Hangzhen Lan,

Daodong Pan

et al.

Journal of Food Composition and Analysis, Journal Year: 2024, Volume and Issue: 132, P. 106253 - 106253

Published: April 23, 2024

Language: Английский

Citations

4
Recent Advances in and Application of Fluorescent Microspheres for Multiple Nucleic Acid Detection DOI Creative Commons
Zhu Chen,

Gaoming Luo,

Jie Ren

et al.

Biosensors, Journal Year: 2024, Volume and Issue: 14(6), P. 265 - 265

Published: May 22, 2024

Traditional single nucleic acid assays can only detect one target while multiple targets simultaneously, providing comprehensive and accurate information. Fluorescent microspheres in multiplexed detection offer high sensitivity, specificity, multiplexing, flexibility, scalability advantages, enabling precise, real-time results supporting clinical diagnosis research. However, face challenges like complexity, costs, sample handling issues. The review explores the recent advancements applications of fluorescent detection. It discusses versatility various fields, such as disease diagnosis, drug screening, personalized medicine. highlights possibility adjusting performance by modifying concentrations carrier forms, allowing for tailored applications. emphasizes potential microsphere technology revolutionizing advancing health, treatment, medical

Language: Английский

Citations

4
Towards Pathogen-Free Coconut Germplasm Exchange DOI Creative Commons
Chongxi Yang, Van Anh Nguyen,

Naga Prafulla Chandrika Nulu

et al.

Plants, Journal Year: 2024, Volume and Issue: 13(13), P. 1809 - 1809

Published: June 30, 2024

Coconut (

Language: Английский

Citations

4