Applications of the CRISPR-Cas system for infectious disease diagnostics

Li, Peipei; Wang, Li; Yang, Junning; Di, Lijun; Li, Jingjing

doi:10.1080/14737159.2021.1922080

Cited by 16 publications

(10 citation statements)

References 108 publications

(112 reference statements)

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“…In recent years, the discovery of CRISPR-Cas system has revolutionized biology, enabling accurate editing of almost any DNA or RNA molecule ( 15 – 18 ). Some Cas proteins have been found to have accessory cleavage activity, such as Cas12a and Cas13a.…”

Section: Discussionmentioning

confidence: 99%

Based on CRISPR-Cas13a system, to establish a rapid visual detection method for avian influenza viruses

Zhang,

Wang,

Chen

et al. 2024

Front. Vet. Sci.

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To rapidly, specifically, and sensitively detect avian influenza virus (AIV), this research established a visual detection method of recombinase-aided amplification (RAA) based on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated proteins 13a (Cas13a) system. In this study, specific primers and CRISPR RNA (crRNA) were designed according to the conservative sequence of AIV Nucleprotein (NP) gene. RAA technology was used to amplify the target sequence, and the amplification products were visually detected by lateral flow dipstick (LFD). The specificity, sensitivity, and reproducibility of RAA-CRISPR-Cas13a-LFD were evaluated. At the same time, this method and polymerase chain reaction (PCR)-agarose electrophoresis method were used to detect clinical samples, and the coincidence rate of the two detection methods was calculated. The results showed that the RAA-CRISPR-Cas13a-LFD method could achieve specific amplification of the target gene fragments, and the detection results could be visually observed through the LFD. Meanwhile, there was no cross-reaction with infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), and Newcastle disease virus (NDV). The sensitivity reached 100 copies/μL, which was 1,000-fold higher than that of PCR-agarose electrophoresis method. The coincidence rate of clinical tests was 98.75 %, and the total reaction time was ~1 h. The RAA-CRISPR-Cas13a-LFD method established in this study had the advantages of rapid, simple, strong specificity, and high sensitivity, which provided a new visual method for AIV detection.

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Section: Discussionmentioning

confidence: 99%

Based on CRISPR-Cas13a system, to establish a rapid visual detection method for avian influenza viruses

Zhang,

Wang,

Chen

et al. 2024

Front. Vet. Sci.

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“…Infectious diseases pose serious threats on public health, causing 17 million deaths every year (Khan et al 2022 ). The mortality of infectious diseases account for 15% of global deaths (Li et al 2021 ). COVID-19 is a global pandemic that has placed enormous burdens on medical resources and social welfare.…”

Section: Discussionmentioning

confidence: 99%

Four thermostatic steps: A novel CRISPR-Cas12-based system for the rapid at-home detection of respiratory pathogens

Fang

Liu

Cheng

et al. 2023

Appl Microbiol Biotechnol

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The outbreak of coronavirus disease 2019 (COVID-19) in 2019 has severely damaged the world's economy and public health and made people pay more attention to respiratory infectious diseases. However, traditional quantitative real-time polymerase chain reaction (qRT-PCR) nucleic acid detection kits require RNA extraction, reverse transcription, and amplification, as well as the support of large-scale equipment to enrich and purify nucleic acids and precise temperature control. Therefore, novel, fast, convenient, sensitive and specific detection methods are urgently being developed and moving to proof of concept test. In this study, we developed a new nucleic acid detection system, referred to as 4 Thermostatic steps (4TS), which innovatively allows all the detection processes to be completed in a constant temperature device, which performs extraction, amplification, cutting of targets, and detection within 40 min. The assay can specifically and sensitively detect five respiratory pathogens, namely SARS-CoV-2, Mycoplasma felis (MF), Chlamydia felis (CF), Feline calicivirus (FCV), and Feline herpes virus (FHV). In addition, a cost-effective and practical small-scale reaction device was designed and developed to maintain stable reaction conditions. The results of the detection of the five viruses show that the sensitivity of the system is greater than 94%, and specificity is 100%. The 4TS system does not require complex equipment, which makes it convenient and fast to operate, and allows immediate testing for suspected infectious agents at home or in small clinics. Therefore, the assay system has diagnostic value and significant potential for further reducing the cost of early screening of infectious diseases and expanding its application. Key points • The 4TS system enables the accurate and specific detection of nucleic acid of pathogens at 37 °C in four simple steps , and the whole process only takes 40 min. •A simple alkali solution can be used to extract nucleic acid. • A small portable device simple to operate is developed for home diagnosis and detection of respiratory pathogens. Supplementary Information The online version contains supplementary material available at 10.1007/s00253-023-12568-3.

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“…Thus, for the highly sensitive detection of Escherichia coli O157:H7, the RPA–CRISPR/Cas12 system was developed [ 20 ], and LAMP–CRISPR/Cas [ 21 ]; for Salmonella spp.–RPA–CRISPR/Cas13 [ 22 ] and for Campylobacter jejuni –RPA–CRISPR/Cas12a [ 23 ]. Thus, biosensor systems based on isothermal amplifications and CRISPR/Cas are promising candidates for ultrasensitive detection of bacterial pathogens, which is confirmed by several review articles [ 24 , 25 ], and for the development of non-laboratory biosensor systems based on them [ 26 , 27 ].…”

Section: Introductionmentioning

confidence: 87%

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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Applications of the CRISPR-Cas system for infectious disease diagnostics

Cited by 16 publications

References 108 publications

Based on CRISPR-Cas13a system, to establish a rapid visual detection method for avian influenza viruses

Based on CRISPR-Cas13a system, to establish a rapid visual detection method for avian influenza viruses

Four thermostatic steps: A novel CRISPR-Cas12-based system for the rapid at-home detection of respiratory pathogens

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

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