ABSTRACT
To establish a method for simultaneous detection of porcine circovirus type 2 (PCV2) and porcine circovirus type 3 (PCV3), specific primers and TaqMan probes were designed after sequence alignment according to the specific sequences of PCV2 Cap gene and PCV3 Cap gene on GenBank. By optimizing the reaction conditions, a duplex fluorescence quantitative PCR detection method for simultaneous detection of porcine circovirus type 2 and 3 was established, and the specificity, sensitivity, and reproducibility were tested. Specificity test results showed that in addition to the positive test results for PCV2 and PCV3, tests for PRRSV, CSFV, PPV, PRV, PEDV, and TGEV were all negative with no cross-reaction, indicating its good specificity. Sensitivity test results showed that the minimum detection limit for detection of PCV2 and PCV3 can both reach 10 copies.L-1, indicating its high sensitivity. The coefficient of variation within and between groups of this method was less than 2%, indicating its good stability. A total of 181 pork and whole blood samples collected from Zhejiang Province were tested using the detection method established in this article and the standard common fluorescent PCR detection method. The results showed that the positive rate of PCV2 was 50.83% (92/181), the positive rate of PCV3 was 37.57% (68/181), and the co-infection rate of PCV2 and PCV3 was 12.15% (22/181). The above detection results of ordinary fluorescent PCR were 50.28% (91/181), 36.46% (66/181), and the co-infection rate was 11.60% (21/181). The coincidence rates of the two methods for PCV2 and PCV3 can reach 98.91% and 97.06%, and the coincidence rate for PCV2 and PCV3 mixed infection were 95.45%. In summary, the duplex fluorescence quantitative PCR detection method established in this experiment can distinguish PCV2 and PCV3 rapidly, which can be used for pathogen detection and epidemiological investigation.
ABSTRACT
Objective: To elucidate the mechanism of interferon gene stimulating factor (STING) in the anti-pathogenic microbial infection of pigs, so as to further provide a reference for the scientific prevention and control of viral diseases such as porcine transmissible gastroenteritis, epidemic diarrhea and porcine pseudorabies. Method: High-scored targets were found in exons 4 and 8 of STING gene and corresponding sgRNA sequences were designed based on CRISPR/ Cas9 technology. The annealed sgRNAs were linked with the enzyme digested LentiCRISPRV2 carrier with T4 DNA ligase to obtain LentiCRISPRV2-STING-sgRNA lentivirus carrier(STING-sgRNA);Different combinations of STING sgRNA lentivirus carriers, packaging plasmid psPAX2 and envelope plasmid pMD2.G were transfected into 293T cells to obtain lentivirus containing sgRNA and then transduced into 3D4/21 cells. Monoclonal cell lines were obtained by puromycin screening and limited dilution method. The knockout efficiencies of the STING gene were identified by PCR amplification, Sequencing and Western blotting;The effect of STING gene knockout on the expression of type I interferon was verified by real-time fluorescent quantitative PCR. Result: When 293T cells were transfected with different combinations of STING-sgRNA lentivirus carrier and HA-STING over expression vector, the editing effect of STING eukaryotic expression carrier could be detected in cells, and the combination of STING-sgRNA(1+5)lentivirus carrier showed the supreme editing efficiency. Thus, the STING-sgRNA(1+5)lentivirus carrier combined with the packaging plasmid psPAX2 and the envelope plasmid pMD2.G were transfected 293T cells to package lentivirus, and then infected 3D4/21 cells with lentivirus. The results showed that a 3D4/21 cell line with a large deletion of the STING gene(4989 bp)was obtained. The STING protein was not observed by Western blotting, indicating that the STING gene knockout 3D4/21 cells(3D4/ 21-STING-/-)were successfully constructed. The transcription level of IFN-beta in 3D4/21-STING-/- cells decreased significantly (P<0.05) compared with parental cells when stimulated by transfection of Haemophilusparasuis DNA. Conclusion : By applying CRISPR/Cas9 technology, STING gene is successfully knock out in 3D4/21 cells, resulting in loss of function of STING gene;STING knockout leads to the transcription disorder of type I interferon when cells are stimulated by DNA, which also suggests that STING gene may be a key factor in the anti-pathogenic microbial infection of pigs.
ABSTRACT
In order to build a specific, sensitive and rapid detection method for PAstV3 detection, the PAstVB gene sequences in Genbank were used and the conserved region in ORFlb was selected to design specific primers and TaqMan probe. Clinical stool samples were collected and preliminary detected by this newly established real-time RT-PCR method after reaction systems and conditions optimization. This detection method established in this study has a good linear relationship with the standard curve, with R2 value up to 0.9971. The sensitivity is 100 times higher than conventional PCR method, The variation co-efficient of in-batch and inter-batch repeatability test is less than 2.0%, indicating good repeatability. The detection results of Clinical samples showed that the positive rate of this method is higher than conventional PCR method. The establishment of this method provides a rapid detection means for PAstV3 laboratory diagnosis and epidemiological investigation.
ABSTRACT
To establish a J2-KD (knockdown) cell line stably expressing interfered IFITM1 and study the effect of interference with IFITMI gene on the infection of PCV2, PRV and TGEV. Gene cloning tech- niques were used to constructed pLKO. l-EGFP-Puro-IFITMI recombinant vector, which was co-transfected into 293 FT cells with lentiviral packaging plasmids psPAXZ and pMDZ. G to produce green fluorescent protein labeled lentiviruses expression IFITMlshRNA, the viral supernatant was collected at 48 hours after post transfection. J2 cells were infected with the harvested lentiviruses, screened by puromycin and cloned via cell limited dilution. Real-time PCR identify that the cell lines with stable interference with IFITMl gene were obtained, and via MTT method verify that interference with IFITMI expression had no effect on the growth of J2 cells, the successfully constructed J2 stable cell line interfere with IFITMl expression was named as JZ-KD. PRV, PCV2 and TGEV infected J2-KD cells, respectively. Using real-time fluorescence quantitative PCR detect virus replication. The results showed that J2-KD cell line was successfully generated with interfered IFITMl expression;the copy number of PCV2 and TGEV were in- creased, while PRV was decreased in J'Z-KD cell. Indicating that the interference of IFITMI gene expression markedly inhibited the replication of PRV while promoted that. of TGEV and PCV2, providing a basis for further study on the function of porcine IFITMI protein and elucidates its antiviral mechanism.