RPA-Cas12a Method Was Established And Initial Application For Detection Of Actinobacillus Pleuropneumoniae

Actinobacillus pleuropneumoniae(APP)is one of the main pathogenic bacteria which can cause respiratory diseases in the modern intensive breeding process of pigs,it can cause exudative,fibrous,necrotizing,hemorrhagic pneumonia and pleurisy,that have brought huge economic losses to the global pig industry.Under the policy of Chinese promotion for no antibiotics feeding,it was bring unprecedented challenges to the prevention and control of bacterial diseases.Since this disease is usually spread by pigs which were sub-clinically infected,the rapid identification is extremely important for the prevention and control of APP.However,the current diagnostic methods for APP are unable to be widely used in the field,due to complicated operating procedures,high requirements for personnel and equipment,and cost.Therefore,a method which is simple operation,flexible application,convenient interpretation,sensitive and rapid,will has important value for the prevention and control of APP.The CRISPR/Cas system is an acquired immune system of bacteria and archaea,which can recognize and cut specific DNA and RNA target sequences under the guidance of specific gRNA.Some CRISPR/Cas systems show incidental non-specific cleavage activity while cutting the target sequence,that is,non-specific single-stranded DNA or RNA is cut indiscriminately.For example,activated Cas12 a has non-specific cleavage activity on single-stranded DNA.Therefore,the presence of target genes in the system can be determined by adding labeled single-stranded DNA molecules to the detection system and detecting whether the single-stranded DNA molecules are cleaved.This method is simpler and more intuitive than general traditional diagnostic methods.It has been developed for nucleic acid detection of a variety of pathogens.In order to establish a rapid detection method for APP in this study,we combine RPA amplification with Cas12 a.According to the conservative sequence of specific gene(apxⅣA)of APP,CRISPR/Cas12a-specific probes(gRNA)were designed and synthesized in vitro,and then the recombinase polymerase amplification(RPA)primers were designed and synthesized according to the gRNA target,a quickly method was established for the detection of APP,The specificity,sensitivity and repeatability of the method were evaluated afterwards.And the lungs sample of mouses disease material was detected later.The results show that this method has high specificity and it is negative for other common pathogens such as Haemophilus parasuis(HPS),Streptococcus suis(SS),Salmonella enteritidis(SE),porcine circovirus(PCV),porcine pseudorabies virus(PRV)and Porcine epidemic diarrhea virus(PEDV).The test results of sensitivity show that the limit of detection for recombinant plasmid can reach 10 copies,and for the gradient diluted bacteria samples can reach 10 CFU,which is nearly 1000 times more sensitive than normal PCR and has good repeatability.This method shows high detective rate for samples of lung isolated from diseased mouse.In summary,above results show that we establish a detection method in this research can be used for APP,it can be performed at 37°C,and the detection results can be displayed in a variety of ways(direct observation with the naked eyes、dynamic detection by a microplate reader or dipstick).It has good sensitivity and specificity,and lots of metrits,for example,it is simple operation and no need for expensive equipment,and can be used in diverse scenarios.So,it will provide a practical technical means for the rapid detection and epidemiological investigation of APP.