CRISPR/Cas12a-Assisted Ligation-Initiated Loop-Mediated Isothermal Amplification (CAL-LAMP) for Highly Specific Detection of microRNAs

Abstract

Loop-mediated isothermal amplification (LAMP) has been increasingly applied in nucleic acid detection for clinical diagnosis and monitoring pathogenic microorganisms due to its isothermal nature and high sensitivity. However, the false-positive signal resulting from the non-specific amplification and the complexity of primer design are still technically challenging for wide applications. In this paper, we developed the CRISPR/ Cas12a-assisted sequence-specific detection of LAMP products to eliminate the effect of non-specific amplification from primer dimers and spurious amplicons. Moreover, by designing a pair of target-specific stem-loop DNA probes, we greatly simplified the primer design for LAMP. The DNA probes could be ligated to form a double-stem-loop DNA template by the detected target, which initiated LAMP reaction and achieved one-nucleotide resolution due to the highly specific ligase reaction. Using microRNAs (miRNAs) as the model targets, the CRISPR/Cas12a-assisted ligation-initiated loop-mediated isothermal amplification (CAL-LAMP) can sensitively detect as low as 0.1 fM miRNAs with high specificity.U p to now, the amplification-based assays are still the most practical methods for the detection of sequence-specific nucleic acids, which have been increasingly important for the detection of genetic biomarkers, clinical diagnosis and prognosis, and monitoring pathogenic microorganisms. 1,2 Generally, polymerase chain reaction (PCR) is primarily adopted to amplify and detect the specific nucleic acid targets. However, the requirement of precise thermal cycles makes PCR have its limitations. First, PCR is prone to false positive arising from the amplification artifacts (such as the primer dimers). Second, the precise thermal cycles necessitate specialized equipment, which is greatly challenging the wide applications in the source-poor setting. Finally, the 2 n amplification fashion of PCR leads to long analysis time, which is a limitation in the requirements of large-scale detection of nucleic acid.As alternatives to PCR, isothermal nucleic acid amplification (INAP) has been intensively investigated, including rolling circle amplification (RCA), 3−6 helicase-dependent amplification (HDA), 7,8 recombinase polymerase amplification ( R P A ) , 9 − 1 2 e x p o n e n t i a l a m p l i fi c a t i o n r e a c t i o n (EXPAR), 13−16 and loop-mediated isothermal amplification (LAMP). 17,18 Among these INAP techniques, LAMP is preferred owing to its high amplification efficiency and rapidness, which can generally amplify a few copies of DNA targets to detectable levels in less than an hour under isothermal conditions. Hence, LAMP has become a broadly

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

“…In this study, we optimized the nucleotide sequence and length of the DNA reporter to maximize the trans-cleavage activity of Cas12a. After a thorough screening process, a high-efficiency DNA reporter (5′-TTATT-CCCCC-3′; TTATT-5C) that exceeded the performance of the existing AT-rich DNA reporter (5′-TTATT-3′) [ 25 , 26 ] was discovered and successfully applied to the detection of the Salmonella enterotoxin ( stn ) gene. Consequently, the new DNA reporter described here was able to enhance the detection sensitivity by approximately tenfold compared to that achieved using the existing AT-rich DNA reporter.…”

Hybridization chain reaction circuit-based electrochemiluminescent biosensor for SARS-cov-2 RdRp gene assay

“…In recent years, isothermal nucleic acid amplification technology has developed rapidly, and several isothermal amplification methods are highly sensitive, and some have successfully moved to commercialization [ [9] , [10] , [11] , [12] ]. Depending on the reaction principle, isothermal amplification can be divided into loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), nucleic acid sequence-independent amplification, helicase-dependent amplification (HDA), strand displacement amplification (SDA), and cross-priming amplification (CPA) [ 7 , [13] , [14] , [15] ]. These techniques have advantages and disadvantages and have different applications in nucleic acid detection.…”

“…The detection of LAMP amplification products can be done by various methods, including agarose gel electrophoresis, turbidity detection, and color determination [ 13 ]. The coloring agents used in the color determination are divided into two categories: one is metal ion indicator, such as calcium xanthophyll, hydroxyl LAMP has the following advantages compared with previous nucleic acid amplification methods: A).…”

One-Pot Endonucleolytically Exponentiated Rolling Circle Amplification by CRISPR-Cas12a Affords Sensitive, Expedited Isothermal Detection of MicroRNAs

“…23,24 A variety of CRISPR assays have been reported for DNA and viral RNA detection which normally require an additional PCR or isothermal pre-amplification step to achieve desirable detection sensitivity. [25][26][27][28] Following the same strategy, sensitive CRISPR-based miRNA assays were also developed by incorporating pre-amplification of miRNA targets by various isothermal reactions, including RCA, [29][30][31] LAMP, 32 and cascade amplification. 33 However, these methods involving two separate pre-amplification and CRISPR-mediated readout steps require multi-step manual operations, which not only leads to complicated assay workflow and extended turnaround time, but also increases the risk of analytical variations and false results due to human error, enzymatic degradation, and crosscontaminations.…”