A sensitive visual method for onsite detection of quarantine pathogenic bacteria from horticultural crops using an LbCas12a variant system

Jiao, Jian; Yang, Mengjie; Zhang, Tengfei; Zhang, Yingli; Yang, Mei; Li, Ming; Liu, Chonghuai; Song, Shangwei; Bai, Tuanhui; Song, Chunhui; Wang, Miaomiao; Pang, Hongguang; Feng, Jiancan; Zheng, Xianbo

doi:10.1016/j.jhazmat.2021.128038

Cited by 17 publications

(19 citation statements)

References 43 publications

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“…Only one article describes the scheme using RPA for the detection of E. amylovora -RPA-CRISPR/Cas with a FAM-BHQ probe [ 51 ]. The authors used phosphoribosyl transferase as the target gene and showed a detection limit of 10 3 CFU/mL.…”

Section: Resultsmentioning

confidence: 99%

“…However, in the article comparing LAMP and PCR [ 52 ], LAMP-based primers from [ 53 ] and qPCR assays showed high specificity for E. amylovora and were able to detect up to ×10 3 CFU/mL. Regarding immunoassays [ 41 , 51 ], all test systems based on isothermal amplifications turned out to be expectedly more sensitive, by 2–4 orders of magnitude depending on the type of test system.…”

Section: Resultsmentioning

confidence: 99%

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Comparison of Biosensing Methods Based on Different Isothermal Amplification Strategies: A Case Study with Erwinia amylovora

et al. 2022

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Isothermal amplifications allow for the highly sensitive detection of nucleic acids, bypassing the use of instrumental thermal cycling. This work aimed to carry out an experimental comparison of the four most promising techniques: recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP) coupled with lateral flow test or coupled with additional amplification based on CRISPR/Cas12a resulting from the fluorescence of the Cas12a-cleaved probe. To compare the four amplification techniques, we chose the bacterial phytopathogen Erwinia amylovora (causative agent of fire blight), which has a quarantine significance in many countries and possesses a serious threat to agriculture. Three genes were chosen as the targets and primers were selected for each one (two for RPA and six for LAMP). They were functionalized by labels (biotin, fluorescein) at the 5′ ends for amplicons recognition by LFT. As a result, we developed LAMP-LFT, LAMP-CRISPR/Cas, RPA-LFT, and RPA-CRISPR/Cas for E. amylovora detection. The detection limit was 104 CFU/mL for LAMP-LFT, 103 CFU/mL for LAMP-CRISPR/Cas, and 102 CFU/mL for RPA-LFT and RPA-CRISPR/Cas. The results of four developed test systems were verified by qPCR on a panel of real samples. The developed assays based on RPA, LAMP, CRISPR/Cas12a, and LFT are rapid (30–55 min), user-friendly, and highly sensitive for E. amylovora detection. All proposed detection methods can be applied to fire blight diagnosis and effective management of this disease.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Comparison of Biosensing Methods Based on Different Isothermal Amplification Strategies: A Case Study with Erwinia amylovora

et al. 2022

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“…Nevertheless, current research on non-nucleic acid targets is also of considerable interest. Many detection systems have developed, especially in the food and environmental fields, to detect pathogenic small molecules or parasites (Lei et al, 2022;You et al, 2022), or pathogens in agriculture (Jiao et al, 2022;Zhu et al, 2022).…”

Section: Summary and Future Directionsmentioning

confidence: 99%

Research progress of CRISPR-based biosensors and bioassays for molecular diagnosis

Chen

Shen

Wang

et al. 2022

Front. Bioeng. Biotechnol.

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CRISPR/Cas technology originated from the immune mechanism of archaea and bacteria and was awarded the Nobel Prize in Chemistry in 2020 for its success in gene editing. Molecular diagnostics is highly valued globally for its development as a new generation of diagnostic technology. An increasing number of studies have shown that CRISPR/Cas technology can be integrated with biosensors and bioassays for molecular diagnostics. CRISPR-based detection has attracted much attention as highly specific and sensitive sensors with easily programmable and device-independent capabilities. The nucleic acid-based detection approach is one of the most sensitive and specific diagnostic methods. With further research, it holds promise for detecting other biomarkers such as small molecules and proteins. Therefore, it is worthwhile to explore the prospects of CRISPR technology in biosensing and summarize its application strategies in molecular diagnostics. This review provides a synopsis of CRISPR biosensing strategies and recent advances from nucleic acids to other non-nucleic small molecules or analytes such as proteins and presents the challenges and perspectives of CRISPR biosensors and bioassays.

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“…A clustered regularly interspaced short palindromic repeat (CRISPR)-associated (CRISPR/Cas) system has been proven powerful in developing assays for nucleic acid biomarkers. The RNA-guided recognition enables the CRISPR tools to be highly specific, and the multiple turnover of collateral cleavage provides signal amplification that contributes to sensitivity improvement. − CRISPR-based nucleic acid detection has been widely applied in monitoring environmental and food safety, mainly targeting pathogens and genetically modified organisms . To enhance the sensitivity, the CRISPR technologies are generally combined with isothermal amplification strategies, such as recombinase polymerase amplification and loop-mediated isothermal amplification .…”

Section: Introductionmentioning

confidence: 99%

“…The RNA-guided recognition enables the CRISPR tools to be highly specific, and the multiple turnover of collateral cleavage provides signal amplification that contributes to sensitivity improvement. − CRISPR-based nucleic acid detection has been widely applied in monitoring environmental and food safety, mainly targeting pathogens and genetically modified organisms . To enhance the sensitivity, the CRISPR technologies are generally combined with isothermal amplification strategies, such as recombinase polymerase amplification and loop-mediated isothermal amplification . Using CRISPR tools to detect non-nucleic acid biomarkers, however, requires compatible biorecognition and signal transduction elements.…”

Section: Introductionmentioning

confidence: 99%

Csm6-DNAzyme Tandem Assay for One-Pot and Sensitive Analysis of Lead Pollution and Bioaccumulation in Mice

Yang

Xue

et al. 2022

Anal. Chem.

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Lead contamination in the environment tends to enter the food chain and further into the human body, causing serious health issues. Herein, we proposed a Csm6-DNAzyme tandem assay (termed cDNAzyme) using CRISPR/Cas III-A Csm6 and GR-5 DNAzyme, enabling one-pot and sensitive detection of lead contamination. We found that Pb2+-activated GR-5 DNAzyme produced cleaved substrates that can serve as the activator of Csm6, and the Csm6-DNAzyme tandem improved the sensitivity for detecting Pb2+ by 6.1 times compared to the original GR-5 DNAzyme. Due to the high specificity of DNAzyme, the cDNAzyme assay can discriminate Pb2+ from other bivalent and trivalent interfering ions and allowed precise detection of Pb2+ in water and food samples. Particularly, the assay can achieve one-step, mix-and-read detection of Pb2+ at room temperature. We used the cDNAzyme assay to investigate the accumulation of lead in mice, and found that lead accumulated at higher levels in the colon and kidney compared to the liver, and most of the lead was excreted. The cDNAzyme assay is promising to serve as analytical tools for lead-associated environmental and biosafety issues.

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A sensitive visual method for onsite detection of quarantine pathogenic bacteria from horticultural crops using an LbCas12a variant system

Cited by 17 publications

References 43 publications

Comparison of Biosensing Methods Based on Different Isothermal Amplification Strategies: A Case Study with Erwinia amylovora

Comparison of Biosensing Methods Based on Different Isothermal Amplification Strategies: A Case Study with Erwinia amylovora

Research progress of CRISPR-based biosensors and bioassays for molecular diagnosis

Csm6-DNAzyme Tandem Assay for One-Pot and Sensitive Analysis of Lead Pollution and Bioaccumulation in Mice

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