Loop-mediated isothermal amplification-based nucleic acid lateral flow assay for the specific and multiplex detection of genetic markers

Kim, Seokhwan; Kim, Jung Ho; Kim, Seokjoon; Park, Jung Soo; Seok, Byung; Lee, Eun Sung; Han, Jiqing; Shin, Jiye; Jang, Young Jun; Park, Ki Soo

doi:10.1016/j.aca.2022.339781

Cited by 28 publications

(11 citation statements)

References 22 publications

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“…All bacterial strains ( Salmonella enterica , Staphylococcus aureus , Klebsiella pneumoniae , Pseudomonas aeruginosa , Enterobacter cloacae ) were cultured in Luria–Bertani (LB) medium (BD, Franklin Lakes, NJ, USA) with shaking (150 rpm) at 37 °C for 24 h. After the cultures were centrifuged at 5000 g for 10 min to obtain cell pellets, the gDNA was then isolated using a Total DNA Extraction S&V kit (Bionics, Seoul, Korea) according to the manufacturer’s instructions. The concentrations of gDNA were evaluated using a Nanodrop instrument (Spectramax iD5 multi-mode microplate reader, Molecular Devices, San José, CA, USA), and the gDNA was stored at − 20 °C until use [ 28 , 29 ].…”

Section: Methodsmentioning

confidence: 99%

Highly Efficient DNA Reporter for CRISPR/Cas12a-Based Specific and Sensitive Biosensor

et al. 2022

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In addition to cis-cleavage activity that recognizes and cleaves nucleic acid sequences, a trans-cleavage activity that indiscriminately and non-specifically cleaves single-stranded DNA or RNA has been discovered in some Cas proteins, including Cas12a and Cas13a. Various detection methods using this activity have been widely reported. Herein, we describe a new highly efficient DNA reporter (5′-TTATT-CCCCC-3′; TTATT-5C) that outperformed the existing AT-rich DNA reporter (5′-TTATT-3′) used in most Cas12a-based target nucleic detection assays. By systematically investigating the effect of DNA reporter length and sequence on the trans-cleavage activity of Cas12a, we achieved up to a 100-fold increase in fluorescence signal intensity derived from the trans-cleavage activity of Cas12a compared to that achieved using the existing AT-rich DNA reporter. The new DNA reporter was also applied, along with the existing AT-rich DNA reporter, for the detection of the Salmonella enterotoxin ( stn ) gene. Importantly, both detection speed and limit were significantly enhanced with the new DNA reporter. In addition, polymerase chain reaction (PCR) was adopted to the CRISR/Cas-Based system of the new DNA reporter, thereby confirming its practical applicability. The high-efficiency DNA reporter described herein can pave the way for further improving the trans-cleavage activity of other Cas proteins, as well as the sensitivity of CRISPR/Cas-Based systems. Supplementary Information The online version contains supplementary material available at 10.1007/s13206-022-00081-0.

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Section: Methodsmentioning

confidence: 99%

Highly Efficient DNA Reporter for CRISPR/Cas12a-Based Specific and Sensitive Biosensor

et al. 2022

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“…21 A promising new method based on the CRISPR/Cas system for the simultaneous detection of hygiene-related bacteria was developed, 22 but it was more time-consuming (2 h) and too complex when compared with our ASEA system. Although recently developed isothermal amplification methods combined with nucleic acid lateral flow assay (NALFA) obviated the need for real-time fluorescent PCR instruments, 23 the low cost of ASEA (only $0.24 per reaction), 24 makes it more competitive in price. During the COVID-19 pandemic, fluorescent PCR instruments have been made available in most remote regions and they are no longer the limiting factors for food safety inspections.…”

Section: Introductionmentioning

confidence: 99%

Rapid, specific and sensitive detection of Vibrio parahaemolyticus in seafood by accelerated strand exchange amplification

et al. 2023

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“…However, the relatively poor detection sensitivities and false‐positive detection results of the above methods have limited their applications in food safety monitoring (Kumar et al., 2008; Uyttendaele et al., 2003). Nucleic acid amplification technologies, represented by polymerase chain reaction (PCR) and isothermal amplification techniques (S. Kim et al., 2022), have rapidly emerged as useful approaches for Salmonella detection (T. H. Kim et al., 2019). Nevertheless, amplification‐based technologies require professional operations and skilled workers unsuited for resource‐limited areas.…”

Section: Introductionmentioning

confidence: 99%

CRISPR Cas12a‐based “sweet” biosensor coupled with personal glucose meter readout for the point‐of‐care testing of Salmonella

Zhou

Huang

Wang

et al. 2022

Journal of Food Science

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Salmonella is a pathogen that comes from different animal-originated foods and poses a significant threat to human health. The present detection methods for Salmonella are time-consuming and labor-intensive and requires skilled workers and specialized instruments. In this study, the conservative invA was selected as the target gene, and a quantitative detection method for Salmonella with wide availability and user-friendliness was established based on CRISPR Cas12a and a personal glucose meter (PGM). The indirect signal transformation from the original target DNA to the final glucose signal was achieved through RAA, CRISPR Cas12a reaction, enzymic reaction, and glucose signal reading by a PGM (accu-chek type from Roche). This PGMs-CRISPR assay showed a detection sensitivity of Salmonella as low as 5 colony-forming units (CFU)/reaction in either pure culture or artificially contaminated food samples and exhibited specificity between Salmonella isolates and non-Salmonella isolates. Furthermore, quantitative detection of Salmonella in spiked milk samples was also achieved within the range from 1 to 1 × 10 3 CFU/reaction. Subsequently, the correlation and consistency between the PGMs-CRISPR assay and quantitative polymerase chain reaction (qPCR) in detection of Salmonella in spiked milk samples were achieved. Therefore, a highly sensitive, portable, quantitative, and user-friendly detection method based on CRISPR Cas12a and PGMs was developed in this study for Salmonella detection and identification.

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Loop-mediated isothermal amplification-based nucleic acid lateral flow assay for the specific and multiplex detection of genetic markers

Cited by 28 publications

References 22 publications

Highly Efficient DNA Reporter for CRISPR/Cas12a-Based Specific and Sensitive Biosensor

Highly Efficient DNA Reporter for CRISPR/Cas12a-Based Specific and Sensitive Biosensor

Rapid, specific and sensitive detection of Vibrio parahaemolyticus in seafood by accelerated strand exchange amplification

CRISPR Cas12a‐based “sweet” biosensor coupled with personal glucose meter readout for the point‐of‐care testing of Salmonella

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