2 research outputs found
Completely Free from PAM Limitations: Asymmetric RPA with CRISPR/Cas12a for Nucleic Acid Assays
Experimentally, Cas12a can recognize multiple protospacer
adjacent
motif (PAM) sequences and is not restricted to the “TTTN”.
However, the application of the CRISPR/Cas12a system is still limited
by the PAM for double-stranded DNA (dsDNA). Here, we developed asymmetric
RPA (Asy-RPA) to completely break the limitations of PAM. Asy-RPA
not only achieved efficient amplification but also converted dsDNA
to single-stranded DNA (ssDNA) without complicated steps. The ssDNA
products activated the trans-cleavage activity of
Cas12a, outputting signals. The application of Asy-RPA completely
freed Cas12a from the PAM, which can be more widely used in nucleic
acid detection, such as lumpy skin disease virus, with an actual detection
limit as low as 1.21 × 101 copies·μL–1. More importantly, Cas12a was intolerant to mutations
on ssDNA. This provided technical support for the detection and identification
of wild-type Mycobacterium tuberculosis (WT-TB) and rifampin-resistant mutant-type M. tuberculosis (MT-TB). The detection limit was as low as 1 fM for 1% mixed samples.
The detection and availability of different treatment options for
treatment-resistant and WT-TB were significant for the elimination
of TB. In summary, the platform consisting of Asy-RPA and CRISPR/Cas12a
was suitable for the detection of various viruses and bacteria and
was a boon for the detection of dsDNA without recognizable PAM
Rapid and Ultrasensitive Approach for the Simultaneous Detection of Multilocus Mutations to Distinguish Rifampicin-Resistant Mycobacterium tuberculosis
The
untested empirical medications exacerbated the development
of multidrug-resistant Mycobacterium tuberculosis (MDR-TB). Here, we develop a rapid and specific method based on
loop-mediated isothermal amplification and duplex-specific nuclease
for distinguishing rifampicin-resistant M. tuberculosis. Three probes were designed for the codons 516, 526, and 531 on
the RNA polymerase β-subunit (rpoB) gene. These three sites
accounted for more than 90% of the total mutations of the ropB gene
in the rifampicin-resistant strain. The approach can perform simultaneous
and sensitive detection of three mutant sites with the actual detection
limit as 10 aM of DNA and 62.5 cfu·mL–1 of
bacteria in 67 min under isothermal conditions. Moreover, the positive
mode of the approach for MDR-TB can not only deal with the randomness
and diversity of mutations but also provide an easier way for medical
staff to read the results. Therefore, it is a particularly valuable
method to handle major and urgent MDR-TB diagnostics