Rapid and visual RPA-CRISPR/Cas12a detection for Staphylococcus pseudintermedius and methicillin-resistant S.pseudintermedius in clinical samples of dogs and cats  

作　　者：Pingping Gao Shuobo Shi Di Zhang Yueping Zhang 

机构地区：[1]National Key Laboratory of Veterinary Public Health and Safety,College of Veterinary Medicine,China Agricultural University,Beijing 100193,China [2]Beijing Advanced Innovation Center for Soft Matter Science and Engineering,College of Life Science and Technology,Beijing University of Chemical Technology,Beijing 100029,China [3]Beijing Zhongnongda Veterinary Hospital Co.,Ltd,Beijing 100193,China.

出　　处：《One Health Advances》2023年第1期149-161,共13页全健康进展（英文）

基　　金：funded by the National Natural Science Foundation of China(32100156);China Agricultural University Teaching Animal Hospital Talent Funding.

摘　　要：Staphylococcus pseudintermedius can cause severe infections of the skin,ear and other tissues in dogs and cats.Methi-cillin-resistant S.pseudintermedius(MRSP)has recently become more prevalent,posing a severe threat to companion animals and public health.Therefore,rapid and accurate diagnosis of S.pseudintermedius and MRSP infections in dogs and cats is essential for timely controlling infections.The development of CRISPR/Cas technology offers an innovative solution for rapid diagnosis.Here,we established an assay combining recombinant polymerase amplification(RPA)and CRISPR/Cas12a.By separately detecting the spsJ gene,the specific gene of S.pseudintermedius,and the mecA gene,the methicillin resistance gene,this method allows for the direct detection of methicillin-susceptible S.pseud-intermedius(MSSP)and MRSP in clinical samples at 37℃for a total of 40 min,The results can be directly visualized by the naked eye under blue light.The limits of detection of the RPA-CRISPR/Cas12a assay were 103 copies per reaction for the spsJ gene and 104 copies per reaction for the mecA gene.The RPA-CRISPR/Cas12a detection successfully detected and differentiated clinical isolates of MSSP and MRSP without cross-reactivity with other tested bacteria species.The evaluation of the detection performance of RPA-CRISPR/Cas12a with 47 clinical samples(without culture)from dogs and cats showed that the results of detection were 100%consistent with those of clinical culture and colony sequencing,which was more sensitive than PCR.RPA-CRISPR/Cas12a assay can quickly and sensitively detect S.pseudintermedius and MRSP in clinical samples without expensive instruments,making it suitable for small veterinary clinics.

关 键 词：CLINICAL METHICILLIN RESISTANT 

分 类 号：S85[农业科学—兽医学]