A rapid and high-throughput Helicobacter pylori RPA-CRISPR/Cas12a-based nucleic acid detection system

Liu, Hua; Wang, Jinbin; Hu, Xiuwen; Tang, Xueming; Zhang, Chao

doi:10.1016/j.cca.2022.12.013

Cited by 23 publications

(3 citation statements)

References 45 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Our optimized method, which involves adjusting the reporter type, reporter length, and Cas12a/crRNA ratio, led to a nearly 50-fold increase in transcleavage activity and more than a three-order-of-magnitude improvement in detection sensitivity. Compared with other optimization methods that require complex changes to the underlying logic, our approach is simple and easily transferable to other optimization characters, such as engineered crRNA 50 and toehold-activated Cas12a, 51 or pre-amplification reactions, such as RPA, 52 LAMP, 53 and RCA. 51 Our findings have not only practical implications for CRISPR-based measurement science but also shed new light on the trans-cleavage mechanism of Cas12a.…”

Section: ■ Conclusionmentioning

confidence: 99%

Unlocking the Full Potential of Cas12a: Exploring the Effects of Substrate and Reaction Conditions on Trans-Cleavage Activity

Liu

Zhang

et al. 2023

Anal. Chem.

View full text Add to dashboard Cite

Section: ■ Conclusionmentioning

confidence: 99%

Unlocking the Full Potential of Cas12a: Exploring the Effects of Substrate and Reaction Conditions on Trans-Cleavage Activity

Liu

Zhang

et al. 2023

Anal. Chem.

View full text Add to dashboard Cite

“…The relative fluorescence intensity was measured using the multi-purpose microplate reader (Tecan, Mannedorf, Switzerland), with excitation and emission wavelengths of 530 nm and 560 nm, respectively. The detection process was described in our previous report [30].…”

Section: Lamp Rpa-crispr/cas12a-ics and The Lamp Rpa-crispr/cas12a-fl...mentioning

confidence: 99%

Isothermal Amplification and CRISPR/Cas12a-System-Based Assay for Rapid, Sensitive and Visual Detection of Staphylococcus aureus

Xu,

Zeng,

et al. 2023

Foods

Self Cite

View full text Add to dashboard Cite

Staphylococcus aureus exists widely in the natural environment and is one of the main food-borne pathogenic microorganisms causing human bacteremia. For safe food management, a rapid, high-specificity, sensitive method for the detection of S. aureus should be developed. In this study, a platform for detecting S. aureus (nuc gene) based on isothermal amplification (loop-mediated isothermal amplification—LAMP, recombinase polymerase amplification—RPA) and the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas12a) proteins system (LAMP, RPA-CRISPR/Cas12a) was proposed. In this study, the LAMP, RPA-CRISPR/Cas12a detection platform and immunochromatographic test strip (ICS) were combined to achieve a low-cost, simple and visualized detection of S. aureus. The limit of visual detection was 57.8 fg/µL of nuc DNA and 6.7 × 102 CFU/mL of bacteria. Moreover, the platform could be combined with fluorescence detection, namely LAMP, RPA-CRISPR/Cas12a-flu, to establish a rapid and highly sensitive method for the detection of S. aureus. The limit of fluorescence detection was 5.78 fg/µL of genomic DNA and 67 CFU/mL of S. aureus. In addition, this detection platform can detect S. aureus in dairy products, and the detection time was ~40 min. Consequently, the isothermal amplification CRISPR/Cas12a platform is a useful tool for the rapid and sensitive detection of S. aureus in food.

show abstract

“…PCR-based methods have been widely used for H. pylori detection in clinical circumstances, however, it has its limitations in sensitivity, and requires well-equipped laboratory [10]. Combining CRISPR/Cas with isothermal amplification has recently been demonstrated for nucleic acid detection with aM-level sensitivity [12]. However, the majority of reported systems still requires elevated incubation temperature, which limited its applications for better Point-of-Care diagnostics.…”

Section: Introductionmentioning

confidence: 99%

Integrated RPA-CRISPR/Cas12a system towards Point-of-Care H. pylori detection

Li,

Deng,

Zhang

et al. 2023

2023 45th Annual International Conference of the IEEE Engineering in Medicine &Amp; Biology Society (EMBC)

View full text Add to dashboard Cite

The rapidly advanced CRISPR/Cas biosensing technology provides unprecedent potential for the development of novel biosensing systems. It provides a new approach for realizing rapid, sensitivity and highly specific pathogen nucleic acid detection, with the capability to combine other technologies, including Polymerase Chain Reaction or isothermal amplifications. The detection of Helicobacter pylori (H. pylori), one of the most common human pathogens to cause various gastroduodenal diseases, has also been explored with the assistance of CRISPR/Cas systems. However, gaps still remain for the development of end-user friendly sensing systems.In this study, a combined RPA-CRISPR/Cas12a biosensing system has been established. It shown the capability to quantitively detect the presence of H. pylori genome DNA with 4 orders of magnitude linear range, and sensitivity of 1.4 copies/µL. The overall reaction can be done within 45 mins at room temperature, which eliminates the needs for heating instrumentation. In addition, with the addition of pullulan as a protective reagent, the potential of storing CRISPR/Cas12a system reagents by using a freeze-dry approach has also been demonstrated.Clinical Relevance -This study represents a novel exploration to applying CRISPR/Cas12a-based biosensing technology to the detection of pathogen DNA with improved potential for the development of Point-of-Care diagnostics. This critical aspect of our technology will contribute to address the newly emerged pathogenic threats and support public health systems.

show abstract

A rapid and high-throughput Helicobacter pylori RPA-CRISPR/Cas12a-based nucleic acid detection system

Cited by 23 publications

References 45 publications

Unlocking the Full Potential of Cas12a: Exploring the Effects of Substrate and Reaction Conditions on Trans-Cleavage Activity

Unlocking the Full Potential of Cas12a: Exploring the Effects of Substrate and Reaction Conditions on Trans-Cleavage Activity

Isothermal Amplification and CRISPR/Cas12a-System-Based Assay for Rapid, Sensitive and Visual Detection of Staphylococcus aureus

Integrated RPA-CRISPR/Cas12a system towards Point-of-Care H. pylori detection

Contact Info

Product

Resources

About