Sensitive and simultaneous detection of hygiene indicator bacteria using an enhanced CRISPR/Cas system in combination with a portable fluorescence detector

Shin, Jiye; Yoon, Taehwi; Park, Jeung Hun; Park, Sun-Hee

doi:10.1016/j.snb.2022.131871

Cited by 19 publications

(10 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…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

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Methodsmentioning

confidence: 99%

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

et al. 2022

Self Cite

View full text Add to dashboard Cite

show abstract

“…First, it requires a lot of energy to raise the temperature to 95 °C for the denaturation of double-stranded DNA (dsDNA) [ 8 ]. Second, repeated thermal cycling steps (e.g., 40 cycles) are required; accordingly, specialized equipment is necessary [ 8 , 9 ].…”

Section: Introductionmentioning

confidence: 99%

Low-Temperature Loop-Mediated Isothermal Amplification Operating at Physiological Temperature

Nam

Kim

et al. 2023

Biosensors

Self Cite

View full text Add to dashboard Cite

Loop-mediated isothermal amplification (LAMP) is one of the most widely used isothermal amplification technologies in molecular diagnostics. However, LAMP operates at a high temperature of 65 °C; thus, operating LAMP at a lower temperature is desirable to maximize its usefulness for on-site diagnosis. In this study, we propose a new version of LAMP, termed low-temperature LAMP, which operates at the physiological temperature of 37 °C. Low-temperature LAMP differs from conventional LAMP operating at 65 °C in terms of the concentrations of MgSO4 and deoxyribonucleoside triphosphates (dNTPs), as well as the lengths of DNA probes, which are crucial for the execution of low-temperature LAMP. Under the optimal conditions, the amplification efficiency of low-temperature LAMP is comparable to that of conventional LAMP. In addition, the ligation reaction at 37 °C, which is necessary to detect actual target nucleic acids, is combined without altering the temperature, enabling the identification of miR-21, a cancer-promoting oncogenic miRNA, with high sensitivity and selectivity. The method described in this paper does not require expensive DNA modifications or special additives and would facilitate the widespread application of LAMP in facility-limited or point-of-care settings, paving the way to improvements in other isothermal-amplification-based techniques.

show abstract

“…Recently, a new isothermal method, the split T7 promoter-based isothermal transcription amplification with light-up RNA aptamer (START), was developed, which completed detection within 30 min, however, the need for two types of DNA probes complicated the system. 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.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2023

View full text Add to dashboard Cite

show abstract

Sensitive and simultaneous detection of hygiene indicator bacteria using an enhanced CRISPR/Cas system in combination with a portable fluorescence detector

Cited by 19 publications

References 33 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

Low-Temperature Loop-Mediated Isothermal Amplification Operating at Physiological Temperature

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

Contact Info

Product

Resources

About