Establishment And Application Of A Nucleic Acid Detection Method For Porcine Circovirus Type 2 Based On RAA-CRISPR/Cas12a

Porcine circovirus disease(PCVD),which is caused by porcine circovirus bring about Porcine Reproductive Dysfunction Syndrome(SMEDI),Weanling Multisystem Failure Syndrome(PMWS),Dermatitis Nephrotic Syndrome(PNDS),Piglet Congenital Tremor(CT),Porcine Respiratory Syndrome(PRDS)and Enteritis.World-spread of PCV2 has caused significant economic losses in pig industry.Accurate and time-saving detection of PCV2 is nassary to reduced the potential economic losses.Previous study reported many useful examining methods for PCV2 detection,most of which showed high sensitivity and specificity.Specific instruments and professional operrators needed limited its clinical application.Therefore,establishment a time-saving and simply detection method is of great value for the prevention and control of PCV2.Development of CRISPR/Cas system provide a train of thought that usinng in nucleic detection.Previous study reported CRISPR/Cas12 a system exhibiting its exonucleolytic activity to non-specific ss DNA while recognizing the target sequence.According to the characteristic of the CRISPR/Cas12 a protein,a fluorescent quenching group-modified ss DNA molecule were designed and added into detection system.Potential ss DNA fluorescence molecule were designed with a Fluorescence quenching group in its 3?-stream and the fluorescence signal detected only when the target sequence is detected,this fluorescence signal also can be observed by the naked eye.The ss DNA modified molecule were also interpreted with a test strip with the same principle.RAA(Recombinase-mediated isothermal nucleic acid amplification)is a method using recombinase,single-stranded binding protein and DNA polymerase to amplify DNA under constant temperature conditions(such as 37°C),which is not require instruments and is esaily to operate.At present,the isothermal amplification technology has been combined with the CRISPR/Cas system to detect PCV3,ASFV,PRRSV.But it has not been used for the detection of PCV2.In this study,a rapid detection of PCV2 method was established by combination of RAA isothermal amplificion and CRISPR/Cas12 a system.The results of this study are as follows:(1)As Cas12 a protein was successfully expressed and purified and exhibited its exonucleolytic activity.As Cas12 a protein was successfully expressed after induction for 2.5 h under the conditions of IPTG concentration of 0.5 m M,temperature of 30 °C,and 180 r/min,and the As Cas12 a protein was purified by Ni-NTA resin and MBP tag purification column.(2)Optimal g RNA was designed and selected.After analyzing the conserved sequence of PCV2,the Protospacer sequence was found according to the PAM sequence recognized by As Cas12 a,and the g RNA was designed.The test results proved that the designed g RNA could be used for PCV2 detection.(3)RAA primers for PCV2 examining were successfully designed and selected.In this study,four pairs of primers were designed and the best one was selected by isothermal amplification and PCR assays.This primer can amplify the sequence containing detection target sequence of PCV2 and its amplification efficiency is high.(4)PCV2 detection method based on RAA-CRISPR/Cas12 a was successfully established and optimized.In this study,one-tube reaction and two-tube one were evaluated and the results showed one-tube reaction was better than two-tube one.Orthogonal experiments were operated and the results showed the optimal concentrations of As Cas12 a protein and g RNA was 100 n M and 200 n M respectively.Changes of fluorescence signal values in different reaction time and the optimal strip test time were operated and the best time checking time within 30 minutes.(5)Tests specificity,sensitivity and repeatability were operated and evaluated.Among the positive plasmids of PCV2,PCV3,PRV,PRRSV,CSFV,ASFV that were detected in this method,the only positive one is PCV2,indicating this method has high specificity.The sensibility tests show PCV2 could be detected with a lower dose of 10 copies/?L.The inter-assay coefficient of variation was less than 10%,which proved that the detection method had good repeatability.(6)Detection assays proved that the method can be used for the clinical bio-simple detection of PCV2.Bio-samples were detected by this methods and PCR assays,sepatately.The results showed that among the 52 samples,14 cases were positive and 38 cases were negative by this method,13 cases were positive and 39 cases were negative by PCR assays.The consistency was reach to 98%,which indicated this method could be used for PCV2 detection.In conclusion,this study established a RAA-CRISPR/Cas12a-based method for PCV2 detection,which provide a time-saving and simple method for PCV2 checking.This tests can be detected by blue light irradiation or test strips.The method has good specificity and high sensitivity,also with simple operation and time-saving feactures,which provides a new technical method for PCV2 detection.