| Point-of-care testing(POCT)of nucleic acids plays an increasingly important role in several fields,such as the prevention and control of infectious diseases,clinical diagnosis of genetic disease,inspection and quarantine,and environmental monitoring.CRISPR effector proteins represented by Cas12 and Cas13 nuclease achieved signal amplification through their novel trans-cleavage activities,which provided a new development direction for the POCT of nucleic acids.However,nucleic acid detection technologies based on CRISPR still face many challenges in replacing existing biological molecular methods.Traditional CRISPR systems are challenging to meet the practical needs of portable and rapid detection due to their apparent defects,such as relying on professional operators and large equipment,complex reaction process,and considerable time consumption.Given the current situation and development trend of POCT,this paper focused on portable signal readout and integrated detection systems based on CRISPR for nucleic acid detection.On the one hand,based on the transduction and output of detection signals as the key point,we constructed a series of biosensors combining cascade enzyme reactions,paper-based microfluidic chips,functional magnetic beads,and other platforms with noble metal nanoparticles,DNA smart hydrogel,and other biological materials.In this case,the fluorescence signal in traditional CRISPR platforms was replaced with a series of simple and easy-to-read signals,in which the glucose signal can be obtained with a personal glucose meter,and the color or distance signals can be read directly with the naked eyes.With the help of these three novel signal readout methods mentioned above,CRISPR Cas12 systems were established to achieve the POCT of SARS-COV-2,genetically modified crops,and invasive fungi,respectively.These three detection methods not only have comparable or higher sensitivity and specificity to traditional quantitative PCR,but also eliminate the reliance on professional personnel and large equipment,improving the user-friendliness of the detection platform and having better practical application scenarios.On the other hand,nucleic acid detection platforms based on CRISPR are usually composed of three parts:nucleic acid amplification system,CRISPR recognition system,and signal transduction and readout system.Among them,nucleic acid amplification is a key component of the entire detection system,but its design is relatively complex and the reaction time is longer.Therefore,in this paper,the initial reaction based on CRISPR Cas13a was combined with the cyclic amplification based on polydopamine nanospheres biosensors to replace the traditional nucleic acid amplification reactions,which eliminated the dependence of the conventional CRISPR Cas 13a system on NAA technologies,and established the portable "one-pot detection"of nucleic acids.Consequently,sensitive POCT of miRNA was achieved with the help of this integrated detection platform,which is expected to create a practical tool for the clinical diagnosis of nucleic acid markers related to diseases. |
