Abstract
False-positive results cause a major problem in nucleic acid amplification, and require external blank/negative controls for every test. However, external controls usually have a simpler and lower background compared to the test sample, resulting in underestimation of false-positive risks. Internal negative controls, performed simultaneously with amplification to monitor the background level in real-time, are therefore appealing in both research and clinic. Herein, we describe a nonspecific product-activated single-stranded DNA-cutting approach based on CRISPR (clustered regularly interspaced short palindromic repeats) Cas12a (Cpf1) nuclease. The proposed approach, termed Cas12a-based internal referential indicator (CIRI), can indicate the onset of nonspecific amplification in an exponential rolling circle amplification strategy here combined with an optomagnetic readout. The capability of CIRI as an internal negative control can potentially be extended to other amplification strategies and sensors, improving the performance of nucleic acid amplification-based methodologies.
Highlights
Nucleic acid amplification plays a vital and basic role in both research and clinic, its accuracy depends highly on the awareness of risk factors and the control of falsepositive and false-negative results [1,2,3]
The proposed approach, termed Cas12abased internal referential indicator (CIRI), can indicate the onset of nonspecific amplification in an exponential rolling circle amplification strategy here combined with an optomagnetic readout
In strategies that recycle amplicons as primers, nonspecific background products (NBPs) are nearly identical to specific products, which means that the quality of the results depends highly on the performance of external negative controls
Summary
Nucleic acid amplification plays a vital and basic role in both research and clinic, its accuracy depends highly on the awareness of risk factors and the control of falsepositive and false-negative results [1,2,3]. The most sensitive nucleic acid amplification strategies are those employing exponential amplification formats in which amplicons (amplification products) are recycled as primers or templates [4]. In strategies that recycle amplicons as primers (e.g. primer generation-rolling circle amplification [PG-RCA]), NBPs are nearly identical to specific products, which means that the quality of the results depends highly on the performance of external negative controls. We report a CRISPR-Cas12a-based NBPactivated molecular strategy that digests all single-stranded DNAs (ssDNA) including the amplicons, and suppresses the output signal upon NBP generation This approach, named Cas12a-based internal referential indicator (CIRI), is performed together with PG-RCA [10] to indicate the level of nonspecific amplification for the tested samples. In addition to PG-RCA, CIRI can be implemented in (i) exponential amplification schemes that recycle amplicons as primers, e.g., bidirectional strand-displacement amplification [11] and exponential amplification reaction (EXPAR) [12]; (ii) cascade amplification schemes containing a step utilizing amplicons of the previous step as primers or triggers, e.g., hairpin-mediated quadratic enzymatic amplification [13]; and (iii) cascade amplification schemes containing an RCA step, e.g., circle-to-circle amplification [14]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
