Antibody Test Strip Of Porcine Parvovirus And Preparation Of Establishment Of LAMP-LFD Method For Detection

Porcine parvovirus(PPV)is a major causative agent in a syndrome of reproductive failure in swine.The main clinical characteristic lesion of this disease is the sow’s abortion and the death of the fetus,the weak,absorbed and like mummy.PPV is mainly infected with a relatively low viral and long-term continuous infection,infected boars and sows are primary infection.The virus can be discharged from the semen after PPV infection,and it is transmitted to the sow by mating and artificial junction.Then PPV breaks through the sow placental barrier to spread to the fetus,Causes sow abortion,fetal death,and surviving piglets contain the virus for life.Studies have shown that 30% to 50% PPV antibody positive pigs are PPV virus carriers,which makes great difficulties in the development of epidemic prevention measures and disease purification of pigs.Loop-mediated isothermal amplification(LAMP)is a constant temperature amplification technology of nucleic acid under the action of strand displacement DNA polymerase.It has the advantages of simple operation and high sensitivity.However,when it is used for PPV nucleic acid detection,the results show that the link has the defects of being unable to determine the specific amplification and easy to misjudge when the dose is small.In view of the above two difficulties to be solved,this study developed a rapid antibody test strip to distinguish PPV wild virus infection from vaccine virus.A detection method combining LAMP and FITC nucleic acid detection Lateral flow dipstick(LFD)has been established for PPV antigen detection.The biotin tag can specifically indicate whether the target gene has been amplified,The visualization result judgment standard of the test strip is more accurate and intuitive.1.This study the non-structural protein NS1 gene sequence of 32 PPV strains registered by Gen Bank was analyzed,and its homology ranged from 98.42% to 99.95%.A pair of specific primers for the NS1 gene sequence were designed with the Chinese strain(China,AY5883318)as the research object,F:5′-CATATGACAAGGTGAAAAGGCTGTA-3′,R:5′-CTCGAGAGTAAGGTGCCTTTGACATAATT-3′.By optimizing the PCR reaction system,the full-length 1989 kb gene fragment of the NS1 protein was successfully amplified,and the NS1 gene fragment was connected to the p ET-28 a vector to construct the p ET28aNS1 recombinant vector.The p ET28a-NS1 recombinant plasmid was transformed into E.coli DH5α competent cells,and the expression was induced for 12 h at 26°C and 0.1 m M IPTG.The results of SDS-PAGE electrophoresis showed that there was a clear band at the relative molecular weight of 75.5 KDa,Same size as expected.The bacterial cells were lysed by sonication,and the solubility of the expressed protein was analyzed.The results showed that the expressed protein mainly existed in the form of inclusion bodies.The bacteria were purified by nickel column,then denatured and renatured,and identified by Western-blot and ELISA with PPV-positive serum,the renatured protein has good biological activity.It lays the foundation for the preparation of PPV antibody detection test strip and the establishment of LAMP-LFD detection method.2.A Conjugate pad was prepared with 1 m L of colloidal gold-labeled NS1 protein at a working concentration of 27.5 μg;HF1350425 nitrocellulose membrane(NC membrane)was selected,and SPA and PPV polyclonal serum Ig G with a concentration of 0.3 mg/m L were sprayed on the NC membrane at a distance of 0.5 cm,they are used as the detection line(T line)and the quality control line(C line)of the test strip respectively.Assemble the PPV antibody detection test strip.The performance indicators of the test strip were measured,and the test result is the detection limit of this test strip of PPV antibody test strip for positive serum was 1:6400,which was equivalent to ELISA method;which was comparable to PRV,PCV-2,PRRSV,JEV,CSFV positive sera have no cross-reaction;the repeated test results of three different batches of test strips are completely consistent;the test strips are sampled every 3 months,there is no change in appearance within 12 months,and the sensitivity and specificity are good;110 clinical samples were tested.The results of pig serum samples were compared with commercial ELISA kits,and the coincidence rate between the test strips and the kits was 95.45%.The above results show that the PPV antibody detection test strip has high sensitivity,strong specificity,good repeatability and stability.3.According to the principle of LAMP technology,Primer Explorer V5 software was used to design four specific primers for the target gene sequence of NS1 protein,a biotin tag was added to the 5’ end of the inner primer FIB,and a FITC probe was designed between the gene fragments B1 c and B2 c to The constructed p ET28a-NS1 recombinant plasmid was used as a template to establish a PPV-LAMP detection system,and the optimal reaction temperature of the PPV-LAMP system was determined to be 64℃ and the optimal reaction time was 50 min.Labeled with 1 m L of colloidal gold and 27.5 μg of FITC-m Ab,avidin with a concentration of 0.5 mg/ml and 1 mg/ml of goat anti-mouse Ig G were used as the detection line and the quality control line of the test strip,respectively,according to the test strip preparation procedures Assemble FITC-LFD.After the PPV-LAMP reaction was completed,1 μL of FITC probe was added to terminate the reaction,and FITC-LFD was used for detection.A PPV-LAMP-LFD detection method was established,and its sensitivity,specificity,and clinical application were determined.The results showed that the lowest detection limit of LAMP-LFD for p ET28a-NS1 template was 1×10 copies/μL,and its sensitivity was 10 times higher than that of agarose gel and real-time quantitative PCR methods.No cross-reaction,with good specificity;early monitoring of PPV infection can detect PPV virus positive in the 4th hour after challenge;150 clinical sera were tested,and the results were compared with the fluorescence quantitative PCR kit.The compliance rate is 99.33%.The LAMP-LFD detection method provides a research basis for the early diagnosis of PPV infection,epidemic prevention and epidemiological investigation.