A Signal-off Cas14a1-based Platform For Detection Of Methicillin-resistant Staphylococcus Aureus

In recent years,diseases in aquaculture have caused great economic losses to aquatic products,polluted water environment and seriously impacted the quality of aquatic products.The use of antibiotics has been becoming an effective method for the treatment of aquatic animal diseases,but the drug-resistant bacteria caused by the nonstandard use of antibiotics have brought new problems to the prevention and control of aquatic animal diseases.Methicillin-resistant Staphylococcus aureus(MRSA)is a common kind of zoonotic multi-drug resistant bacteria,which can be transmitted to humans through aquaculture environments and pose a major threat to public health and food safety.Therefore,early and accurate detection of MRSA is the key to guide the correct use of antibiotics and control the spread of infectious super bacteria.In recent years,biosensors based on clustered regularly spaced short palindromic repeat-related proteins(CRISPR/Cas)have attracted much attention due to their high specificity and sensitivity.The specific binding of target nucleic acid and guiding RNA(sg RNA)is the mechanism of CRISPR/Cas biosensor.By simply changing the sequence of sg RNA spacer,they can be used to detect multiple targets.Recently,the newly discovered small Cas protein Cas14a1(61.52 KDa)was found to have cis/trans ss DNA cleavage activity.At present,the reported biosensors based on Cas14a1 use the trans-ss DNA cleavage ability of Cas14a1 to enhance the fluorescence signal by using the ss DNA labeled with fluorescent groups and quenching agent(FQ).There is no report that the detection is carried out by inhibiting the fluorescence signal.Therefore,we proposed a biosensor based on Cas14a1 signal closure.In this study,a biosensor based on Cas14a1 signal closure was established to identify MRSA.In this biosensor system,the designed single-stranded DNA could not only activate the trans-cutting ability of dual Cas14a1-sg RNA complex,but also serve as a primer for the amplification of methicillin-resistant gene(mec A).With mec A genome as the target,combined with PCR amplification technology,primers can be transformed into ds DNA products without PAM sites in the presence of MRSA,resulting in the decrease of trans-cutting activity of Cas14a1.After optimizing the reaction conditions,the detection sensitivity of MRSA could reach 3.08 ng/m L and had high detection specificity with six model bacteria such as sensitive Staphylococcus aureus(MSSA).This detection platform was applied to the detection of tilapia infection in actual samples.The tilapia was injected with MRSA,and the control group was injected with sterile PBS buffer.After 10 days of normal culture,the whole blood samples of tilapia were extracted.After the extraction of genomic DNA,Cas14a1 was detected,and q PCR was used as the control.The positive sample detection rate of this detection system was 100%.This study provides a new idea for the design of Cas14a-based biosensor platform,and expands the application of CRISPR/Cas14a1 system in the diagnosis of resistant genes.