Rapid Detection Of Microsporum Canis By Recombinase Aided Amplification

Object Microsporum canis is one of the most important pathogens of fungal dermatosis in clinic.It is widely distributed in the world,causing harm to human health and animal production.Traditional detection methods are time-consuming and low accuracy.Although molecular biology method shortens the detection time,it needs not only professional personnel to operate,but also depends heavily on laboratory instruments.RAA?Recombinase Aided Amplification?can produce a large number of target gene amplification within 20⁴0 minutes at about 39?.In this study,two RAA methods for detection of microsporidium canis were established,and new methods for rapid detection of microsporidium canis in clinic was expected.Method Referring to the U4/U6 sn RNA-associated-splicing factor PRP24 sequence?MCYG₀8382?of Microsporum canis,primers and probes for RAA reaction were designed.To prepare DNA positive standard of Microsporum canis.RAA method and LF-RAA method were established.The reaction temperature,reaction time,specificity and sensitivity of the two methods were evaluated.At the same time,78 clinical samples were detected by PCR,RAA and LF-RAA.The results were compared to evaluate the clinical application value of the two RAA detection methods.Result RAA method established in this study has the best reaction temperature is 24?³9?,the lowest reaction time is 10 minutes,good specificity,the minimum detection sensitivity can reach 10 orders of magnitude,and the total coincidence rate of clinical detection is 91.03%.LF-RAA method established in this study has the best reaction temperature is 24?³9?,the lowest reaction time is 20 minutes,good specificity,the lowest detection sensitivity can reach 103copies/?L,and the total coincidence rate of clinical detection is 87.18%.Conclusion The two RAA detection methods for Microsporidium canis established in this study have wide reaction temperature range,short reaction time,good specificity and sensitivity,high coincidence rate.Because these two methods have low requirements for equipment and low technical requirements for experimenters,they can be used as new methods for clinical detection of microsporidium canis in the future.