RT-LAMP CRISPR-Cas12/13-Based SARS-CoV-2 Detection Methods

Selvam, Kasturi; Najib, Mohamad Ahmad; Khalid, Muhammad Fazli; Mohamad, Suharni; Palaz, Fahreddin; Ozsoz, Mehmet; Ismail, Asma

doi:10.3390/diagnostics11091646

Cited by 35 publications

(25 citation statements)

References 43 publications

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“…There is a broad range of combination possibilities of LAMP with downstream analytical systems for the detection of amplification products. They include standard gel electrophoresis 17 and quantitative amplification (qLAMP) 18 over different colorimetric reagents (phenol red 19 , leuco crystal violet 20 , SYBR green 21 , malachite green 22 ) to lateral flow assays (LFA) 23 or combinations with CRISPR-Cas Systems 24 . The most commonly used are LFA and colorimetric assays but the major limitation of the mentioned techniques is that they are not suitable for the distinction of false positive results.…”

Section: Introductionmentioning

confidence: 99%

Investigation and validation of labelling loop mediated isothermal amplification (LAMP) products with different nucleotide modifications for various downstream analysis

Warmt

Yaslanmaz

Henkel

2022

Sci Rep

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Loop mediated isothermal amplification (LAMP) is one of the best known and most popular isothermal amplification methods. It's simplicity and speed make the method particularly suitable for point-of-care diagnostics. Nevertheless, false positive results remain a major drawback. Many (downstream) applications are known for the detection of LAMP amplicons like colorimetric assays, in-situ LAMP or CRISPR-Cas systems. Often, modifications of the LAMP products are necessary for different detection applications such as lateral flow assays. This is usually achieved with pre-modified primer. The aim of this study is to evaluate amplicon labelling with different modified nucleotides such as Cy5-dUTP, biotin-dUTP and aminoallyl-dUTP as an alternative to pre-labelled primers. To realise this, the effects on amplification and labelling efficiency were studied as a function of molecule size and nucleotide amount as well as target concentration. This research shows that diverse labelling of LAMP amplicons can be achieved using different, modified nucleotides during LAMP and that these samples can be analysed by a wide range of downstream applications such as fluorescence spectroscopy, gel electrophoresis, microarrays and lateral flow systems. Furthermore, microarray-based detection and the ability to identify and distinguish false positives were demonstrated as proof of concept.

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Section: Introductionmentioning

confidence: 99%

Investigation and validation of labelling loop mediated isothermal amplification (LAMP) products with different nucleotide modifications for various downstream analysis

Warmt

Yaslanmaz

Henkel

2022

Sci Rep

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“…The COVID‐19 pandemic has pushed researchers to develop innovative approaches for on‐field virus detection to contain the virus spread effectively. Therefore, since the inception of the COVID‐19 pandemic, many groups have reported development of accurate, easy to use and field‐deployable diagnostic assays for SARS‐CoV‐2 based on CRISPR system (Javalkote et al, 2020; Selvam et al, 2021). Many reports improvised or added some novel aspect to the previously published CRISPR‐based diagnostics.…”

Section: Discussionmentioning

confidence: 99%

An improved, simple and field-deployable CRISPR-Cas12a assay for the detection of SARS-CoV-2

Misra

Rangu

Phulsundar

et al. 2022

Journal of Applied Microbiology

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Aims The RT‐PCR is the most popular confirmatory test for SARS‐CoV‐2. It is sensitive, but high instrumentation cost makes it difficult for use outside routine clinical setup. This has necessitated the development of alternative methods such as CRISPR‐based DETECTR method which uses lateral flow technology. Although accurate and sensitive, this method is limited by complex steps and recurrent cost of high‐quality lateral flow strips. The main goal of this study was to improve the Cas12a‐based SARS‐CoV‐2 DETECTR method and develop a portable and field‐deployable system to reduce the recurring consumable cost. Methods and results Specific regions of N and E genes from SARS‐CoV‐2 virus and human RNase P (internal control) were reverse transcribed (RT) and amplified by loop‐mediated isothermal amplification (LAMP). The amplified products were detected by a Cas12a‐based trans‐cleavage reaction that generated a fluorescent signal which could be easily visualized by naked eye. Detection of internal control, RNase P gene was improved and optimized by redesigning RT‐LAMP primers. A number of steps were reduced by combining the reagents related to the detection of Cas12a trans‐cleavage reaction into a single ready‐to‐use mix. A portable, cost‐effective battery‐operated instrument, CRISPR‐CUBE was developed to run the assay and visualize the outcome. The method and instrument were validated using both contrived and patient samples. Conclusions The simplified CRISPR‐based SARS‐CoV‐2 detection and instrument developed in this study, along with improved design for internal control detection allows for easier, more definitive viral detection requiring only reagents, consumables and the battery operable CRISPR‐CUBE. Significance and impact of study Significant improvement in Cas12 method, coupled with simple visualization of end point makes the method and instrument deployable at the point‐of‐care (POC) for SARS‐CoV‐2 detection, without any recurrent cost for the lateral flow strips which is used in other POC methods.

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“…However, qPCR needs expensive equipment and skilled workers [21]. Recently, CRISPR-Cas12 mediated trans-collateral activity was widely applied to nucleic acid detection [11,22,23]. The CRISPR-Cas12 detection system is accurate and specific, and it can also combine various readout and amplification technologies [24][25][26].…”

Section: Discussionmentioning

confidence: 99%

Detection of Klebsiella pneumoniae DNA by PCR-Based CRISPR-lbCas12a System

Wang¹,

Tang²,

Wei³

et al. 2021

Preprint

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Klebsiella pneumonia (K. pneumoniae) is a Gram-negative bacterium that causes nosocomial infections in the lung, bloodstream, and urinary tract. Therefore, detecting K. pneumoniae in early time is important in preventing severe infections. However, clinical detection of K. pneumoniae requires a long time of agar plate culture. Nucleic acid detection like qPCR is precise but requires expensive equipment. Recent research reveals that collateral cleavage activity of CRISPR-LbCas12a has been applied in nucleic acid detection. In this study, PCR combined with CRISPR-LbCas12a targeting the K. pneumoniae system was established. This system showed excellent detection specificity and sensitivity in both bench work and clinical samples. Due to its advantages, its application can meet different detection requirements in health centers where qPCR is not accessible.

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RT-LAMP CRISPR-Cas12/13-Based SARS-CoV-2 Detection Methods

Cited by 35 publications

References 43 publications

Investigation and validation of labelling loop mediated isothermal amplification (LAMP) products with different nucleotide modifications for various downstream analysis

Investigation and validation of labelling loop mediated isothermal amplification (LAMP) products with different nucleotide modifications for various downstream analysis

An improved, simple and field-deployable CRISPR-Cas12a assay for the detection of SARS-CoV-2

Detection of Klebsiella pneumoniae DNA by PCR-Based CRISPR-lbCas12a System

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