| African swine fever（ASF）is an acute,severe and highly contagious infectious disease caused by African swine fever virus（ASFV）.It can infect domestic pigs and wild boars of different varieties and ages,and the mortality of ASF is as high as 100%,which has caused more than 10 billion dollars economic losses in pig industry.As there is no effective treatment and commercial vaccine for ASF,rapid diagnosis,culling and other prevention to control the spread of the disease is important.At present,although the detection methods for ASFV have their own advantages,most of which still rely on large-scale laboratory instruments and professional operators.This increased the difficults to meet the needs of on-site rapid detection.Therefore,development a sensitive,fast,simple,time-saving and convenient methods for ASFV detection is of great significance to its prevention and control.Recently study exhibited the defense mechanism CRISPR/Cas system existing in most bacteria and all Archaea has gradually come into people’s vision.CRISPR/Cas12 a has become a research hotspot in pathogenic nucleic acid detection because of its exonuclease activity（cutting target ds DNA and other non-specific ss DNA）.Recombinase mediated nucleic acid amplification（RAA）is a method of amplification of DNA under constant temperature（such as 37°C）by using recombinase,single strand binding protein and DNA polymerase.It provide fast amplification and simple operation for nucleic sequences amplication under constant temperature conditions.Based on the two detection technologies,this study established an ASFV detection method without expensive instruments and professional operators by combining RAA and CRISPR/Cas12 a system.The specific research results are as follows:1.AsCas12 a protein was successfully expressed and purified,and its cleavage activity was verified.After the plasmid p MBP-AsCas12 a was transferred into E.coli BL21（DE3）,the required protein was successfully induced under the induction conditions of IPTG concentration of 0.5m M,temperature of 30 °C and 180r/min for 2.5h,and the AsCas12 a protein was purified by nickel column and MBP tag purification column.2.The positive standard plasmid was successfully constructed and the g RNA for ASFV and cas12 a was designed.PCR primers were designed according to the conserved B646 Lgene of ASFV published on Gen Bank,and the PMD-19T-p72 standard positive plasmid was constructed with the synthetic complete B646 L sequence as the template;In the highly conserved B646 L gene sequence of ASFV,g RNA was designed according to the PAM sequence recognized by Cas12 a.The feasibility of g RNA was verified by detection.3.RAA primers were successfully designed according to the corresponding targets.The results of isothermal amplification showed that the primers could amplify the target sequence and be used for detection.4.The method of detecting ASFV based on CRISPR/Cas12 a was successfully established and the system was optimized.The results showed that the optimal protein and g RNA concentration is 150 n M and 200 n M separately,reliable results can be obtained after 30 minutes of fluorescence detection reaction and 20 minutes of test strip detection reaction followed by incubation for 4 minutes.5.The specificity,sensitivity and repeatability of the method were evaluated.The results showed that the method have good specificity,no cross reaction with PCV2,PCV3,PRRSV,PRV and CSFV positive samples,and the lower dose of fluorescence detection and test strip detection was 10 copies/ μL.At the same time,the method has good repeatability.After multiple batches of positive samples with different copy numbers are tested,the intra batch coefficient of variation is less than 5% and the inter batch coefficient of variation is less than10%.To sum up,this study established a fast ASFV detection method,which can be completed under constant temperature（37 °C）,and results can be read directly by blue light irradiation or test strips,with good sensitivity and specificity,simple operation,no need for expensive experimental instruments and equipment and suitable for clinical detection,which provides a practical method for rapid diagnosis and epidemiological investigation of ASFV. |
