Triple-recognition strategy for one-pot detection of single nucleotide variants by aligner-mediated cleavage-triggered exponential amplification.

The detection of single nucleotide variants (SNVs) is important for the diagnosis and treatment of cancer. To date, researchers have devised several methods to detect SNVs, but most of them are complex and time-consuming. To improve SNVs detection specificity and sensitivity, we developed a triple-recognition strategy, which facilitates aligner-mediated cleavage-triggered exponential amplification (Trec-AMC-EXPAR) for the rapid, specific, and one-pot detection of SNV. Under optimized conditions, Trec-AMC-EXPAR detected two clinically significant SNVs, PIK3CAH1047R and EGFR L858R within 80 min, with a reliable detection of 0.1% SNV in the wide type, which is lower than that of allele-specific PCR (AS-PCR) for detecting SNV. Finally, by spiking into normal human serum samples, mutants mixed with the wild-type targets in different ratios were analyzed, resulting in the relative standard deviation (RSD) of recovery ratios <3%. The findings suggested the potential application of Trec-AMC-EXPAR in clinical disease diagnosis. In summary, the proposed Trec-AMC-EXPAR technique provides a novel fast and convenient method for one-pot detection of SNV with high sensitivity and specificity.

Surface-enhanced Raman spectroscopy integrated with aligner mediated cleavage strategy for ultrasensitive and selective detection of methamphetamine.

New drugs and illicit synthesized mixtures detection at crime scenes is a great challenge for detection method, which requires anti-interference and ultrasensitive methods to detect methamphetamine (METH) in seized street samples and biological fluids. Herein, we constructed a surface-enhanced Raman sensing method based on aligner mediated cleavage (AMC) of nucleic acid for quantitative detection of METH for the first time. This method we proposed relied on AMC to achieve programmable sequence-specific cleavage of METH aptamer linked by gold nanoparticles (METH aptamer-Au NPs), the cleavage product-Au NPs conjugates (cleavage aptamer-Au NPs) would hybridize with complementary DNA (cDNA)-Au NPs, resulting in the aggregation of the Au NPs and concomitant plasmonic coupling effect. Besides, due to the base number of METH aptamer-Au NPs was decreased, the interparticle distance of the Au NPs was shortened, which increased the electric field enhancement factor. Thus, under the irradiation of the laser, rhodamine 6G (R6G) adsorbed on Au NPs generated a strong Raman signal. The detection limit reached 7 pM, the linear range was from 10 pM to 10 nM, and this detection method also showed good anti-interference ability and reproducibility in serum.

Aligner-Mediated Cleavage-Based Isothermal Amplification for SARS-CoV-2 RNA Detection.

Rapid detection of SARS-CoV-2 RNA is critical for reducing the global transmission of COVID-19. Here, we report a simple and versatile assay for detection of SARS-CoV-2 RNA based on aligner-mediated cleavage-based strand displacement amplification (AMC-SDA). The entire amplification procedure takes less than 25 min without professional instruments or requirement of specific target sequences and can reach a limit of detection of attomolar RNA concentration. Using pseudovirus as mimicry of clinical SARS-CoV-2 positive samples, we achieved a diagnostic accuracy of 100% in 10 simulated samples (five positive and five negative). We anticipate that our method will provide a universal platform for rapid and accurate detection of emerging infectious diseases.

Aligner mediated cleavage of nucleic acids for site-specific detection of single base mismatch.

Single base mismatch can always connect with various gene-related diseases, whose determination has aroused widespread interest. So far, various methods have been developed to determine the common base mismatch. However most of them are complex, time-consuming. Herein, we report a novel method, which only need one conventional endonuclease (NEase) and achieve site-specific cleavage in a programmable way, to detect single base mismatch, termed aligner-mediated cleavage-based single base mismatch discrimination (AMCMD). The DNA aligner (DA) is in a stem-loop structure, consistent with an incomplete recognition site of NEase on its stem and a 5'-side arm complementary to the target sequence (TS). Once TS contains matched base and hybridizes with DA, the complete recognition site of NEase is formed, and the TS will be cleavaged with fast speed, while converse is not. Based on it, the method can clearly distinguish mismatched and complementary bases. Without sample pre-processing, we were able to obtain and verify all the test result in about 30 min through the polyacrylamide gel electrophoresis analysis. This endows the proposed method with a simpler advantage. Then we combined AMCMD and EXPAR to create a new method for single base mismatch discrimination, the short sequence obtained by AMCMD as a target to trigger EXPAR, with a detection limit at 1pM level. Another process with human serum underlines that AMCMD is compatible with the complex biological sample, thus it has the potentials for practical applications.

Aligner-mediated cleavage-triggered exponential amplification for sensitive detection of nucleic acids.

Exponential amplification reaction (EXPAR), as a simple and high sensitive method, holds great promise in nucleic acids detection. One major challenge in EXPAR is the generation of trigger DNA with a definite 3'-end, which now relies on fingerprinting technology. However, the requirement of different endonucleases for varying target sequences and two head-to-head recognition sites in double stranded DNA, as well as the confinement of trigger DNA's 3'-end to be near/within the recognition site, usually subject EXPAR to compromised universality and/or repeated matching of reaction conditions. Herein, we report a simple and universal method for high sensitive detection of nucleic acids, termed aligner-mediated cleavage-triggered exponential amplification (AMCEA). The aligner-mediated cleavage (AMC) needs only one nicking endonuclease and can make a break at any site of choice in a programmable way. Thus, the 3'-end of target DNA can be easily redefined as required, a key step for initiating the amplification reaction. This capability endows the proposed AMCEA with excellent universality and simplicity. Moreover, it is sensitive and specific, with a detection limit at amol level, a broad dynamic range of 5～6 orders of magnitude and the ability to distinguish single nucleotide mutation. Experiments performed with human serum indicate that AMCEA is compatible with the complex biological sample, and thus has the potentials for practical applications.