| Coronavirus Disease 2019 caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)is a continuous pandemic that poses a severe challenge to public health and global economy.Staphylococcus aureus(S.aureus)is a common foodborne pathogen,causing food poisoning and resulting in a serious threat to human health.The conventional detection methods for the above-mentioned pathogenic microorganisms have shown their advantages.However,they rely on large-scale equipment and professional operators which greatly limit the application range,and are difficult to popularize in regions with relative backward conditions and scarce experimental resources.In recent years,the CRISPR/Cas system composed of clustered regularly interspace short palindromic repeat(CRISPR)and CRISPR associated proteins(Cas)has attracted extensive attention by scientists,among which CRISPR/Cas12a has become a hot topic in the in vitro diagnosis field due to its crRNA-guided non-specific ssDNA cleavage activity triggered by target DNA.Recombinase aided amplification(RAA)breaks through the limitation of precise temperature control equipment,and achieves the exponential amplification of target nucleic acid under constant and mild conditions.In addition,combining CRISPR/Cas12a technology with portable lateral flow assay is expected to overcome the shortcomings of the conventional detection techniques for pathogenic microorganisms and achieve more sensitive,specific,rapid,and simple detection.Therefore,this research intends to combine the CRISPR/Cas12a system,RAA isothermal amplification technology,fluorescence or lateral flow strip signal output methods to construct a point-of-care testing(POCT)platform for SARS-CoV-2 and S.aureus.First,a SARS-CoV-2 detection platform based on CRISPR/Cas12a system and fluorescence analysis technology was established in this paper.N gene was selected as the detection target,and the human RNaseP gene fragment was used as the internal reference.After optimizing the reaction conditions,the sensitivity of the technology reached 1×100 copies/?L,and it showed good accuracy and specificity when detecting clinical samples.The entire detection process took only 60 minutes.Therefore,this detection platform showed the characteristics of high sensitivity,strong specificity,efficiency,and had a good development potential,which was of great significance for the early diagnosis,prevention,and control of COVID-19.Besides,a detection platform for S.aureus was constructed based on the above principles,and the nuc gene of S.aureus and 16S rDNA of Staphylococcus genus were selected as detection targets.In combination with the RAA technology,the detection sensitivity of the platform was 1×100 CFU/reaction,and the detection sensitivity of the artificially contaminated milk sample was 2×101 CFU/mL.The platform also showed good accuracy in the detection of other food samples.The whole detection process required only 70 min,which could meet the actual detection demand of S.aureus in food.To better meet the demand of POCT detection,lateral flow strip assay was introduced in this paper based on the CRISPR fluorescence detection system of S.aureus,and a CRISPR-lateral flow strip(CRISPR-LF)detection platform was established.The detection platform could be observed by naked-eye observation or measured by a strip reader.The detection parameters were equivalent to the above-mentioned fluorescence detection system and the whole detection process was shortened to 60 min.Since the platform not only achieved rapid,sensitive,accurate,and specific detection of target bacteria,but also improved the portability and user-friendliness of the detection,it was more suitable for use in areas with backward economy and poor experimental resources.Therefore,the CRISPR-LF detection platform had a good development prospect in the field of POCT detection of pathogenic bacteria and was of great significance for food safety supervision,prevention,and diagnosis of foodborne diseases. |
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