| Post-weaning multisystemic wasting syndrome is a kind of important disease caused by PCV2, mortality is up to more than 40% in the serious disease region and surviving pigs rarely recover. Because PCV2 severely damages the immune system of pigs,leading to a variety of poor vaccine response, immuning failure and induce other disease outbreaking. Therefore, the early diagnosis and elimination of the PCV2-infected pigs become the focus of prevention and treatment of PMWS. It is necessary to develop a qualitative and quantitative rapid detection method of PCV2-infected pigs for the prevention and control of PMWS. Porcine parvovirus disease is one of the major breeding pigs disorders caused by a porcine parvovirus, the first parity sow suffering from the disease will occur abortion, infertility, stillbirth, fetal malformation, mummified and weak aberdeen. In addition, PPV can enhance the weaning multisystemic wasting syndrome caused by porcine circovirus. PPV is distributed widely and pigs are infected by water, food, mating, placenta and other means. PPV can prolonged survival in the external environment, it is insensitive to most disinfectants and it is difficult to control, cause great damage to the pig industry. Therefore,it is very necessary to establish a kind of rapid and specific detection method fiting in the grass-roots promotion.This study is designed to establish PCV2 loop-mediated isothermal amplification and quantitative PCR methods and PPV loop-mediated isothermal amplification detection method, develop the live visualization LAMP rapid qualitative detection kit for PCV2 and PPV and the quantitative real-time PCR detection kit for PCV2, optimize the reaction system and the parameters,and research the preparation process. The main contents and results are as follows: 1. The establishion of the porcine parvovirus(PPV) LAMP visual rapid detection method and the development of the kit.The objective of this study was to develope effective diagnostic technique and diagnostic kit for porcine parvovirus(PPV), to establish a rapid, sensitive and specific detection of porcine parvovirus(PPV) loop-mediated isothermal amplification(LAMP) method,it provide an accurate and reliable tool for the diagnosis of porcine parvovirus. According the PPV sequence published in GenBank, LAMP primers were designed in its conserved sequence using PrimerExplorer V3, by optimizing conditions such as the proportion of primer, the temperature of the reaction, the time of the reaction, the components of the PCR, etc, the LAMP detection method for PPV virus-specific amplification for the DNA was established, the LAMP assay kit was developed, and the sensitivity, specificity, reproducibility and shelf life properties of the kit were evaluated. The specific amplification of high efficiency for the DNA of PPV was obtained at 60 ℃, 50 min.The clinical samples were tested by this test kit, the detection limit for PPV by the LAMP detection was 10 copies of PPV genome, whereas the detection limit by conventional PCR was 1000 copies of PPV genome, the analysis of clinical samples indicated that the LAMP method was highly sensitive. The detection did not cross-react with porcine circovirus type 2(PCV2), porcine reproductive and respiratory syndrome virus(PRRSV), porcine pseudo rabies virus(PRV)and classical swine fever virus(CSFV). When 1100 samples were tested using this test kit, 320 were detected as positive. The stability test showed that this kit can survive at least 40 times post-repeated freezed and thawed. The results indicated that the establishment of this method and the test kit are specific,sensitive and stable, and may provide a simple and rapid method for monitoring PPV and epidemiology surveys. 2. The establishion of the porcine circovirus type 2(PCV2)LAMP visual rapid tests and the development of the kit.The objective of this study was to establish effective diagnostic technique and diagnostic kit for porcine circovirus type 2(PCV2)and to establish a rapid, sensitive and specific detection of porcine circovirus type 2(PCV2)loop-mediated isothermal amplification(LAMP) method,it provide an accurate and reliable tool for the diagnosis of porcine circovirus type 2. According the PCV2 sequence published in GenBank, LAMP primers were designed in its conserved sequence using PrimerExplorer V3, by optimizing conditions such as the proportion of primer, the temperature of the reaction, the time of the reaction, the components of the PCR, etc, the LAMP detection method for PCV2 virus-specific amplification of the DNA was established, the LAMP assay kit was developed, and the sensitivity, specificity, reproducibility and shelf life properties of the kit were evaluated. The specific amplification of high efficiency for the DNA of PCV2 was obtained at 59 ℃, 45 min. The clinical samples were tested by this test kit, the detection limit for PCV2 by the LAMP detection was 10 copies of PCV2 genome, whereas the detection limit by conventional PCR was 1000 copies of PCV2 genome, the analysis of clinical samples indicated that the LAMP method was highly sensitive. The detection did not cross-react with classical swine fever virus(CSFV), porcine reproductive and respiratory syndrome virus(PRRSV) and porcine pseudo rabies virus(PRV).When 1100 samples were tested using this test kit, 950 were detected as positive. The stability test showed that this kit can survive at least 40 times post-repeated freezed and thawed. The specific detection of PCV2 LAMP method was established successfully, the kit has a good specificity, reproducibility and sensitivity, It can be used for rapid detection for clinical PCV2 infection and molecular epidemiology. 3. The establishion of the porcine circovirus type 2(PCV2)quantitative real-time PCR tests and the development of the kit.The objective of this study was to establish effective quantitative diagnostic technique and diagnostic kit for porcine circovirus type 2(PCV2). According the PCV2 sequence published in GenBank, specific primers and a probe were designed for the ORF2 gene of PCV2 using Primer 5, by optimizing system,10-fold serial dilutions of plasmid standards was established by recombinant plasmid, fluorescence quantitative PCR amplification was carried out and standard curve was made, the quantitative real-time PCR detection method for PCV2 virus-specific amplification of the DNA was established, the quantitative real-time PCR detective kit was developed, and the sensitivity, specificity, reproducibility and shelf life properties of the kit were evaluated. The cycle threshold(Ct) and standard DNA templates of the test kit has excellent linearity in the 101 to 107 copies / u L of DNA concentration in a range, the correlation coefficient is 0.9999. The study has good reproducibility,the coefficient of variation for Ct values varied from 0.59% to 1.05% in a batch experiment and from 1.9% to 4.2% in inter-assay test. The detection did not cross-react with classical swine fever virus(CSFV), porcine reproductive and respiratory syndrome virus(PRRSV) and porcine pseudo rabies virus(PRV),the limits of detection and quantitation were 10 and 100 copies of PCV2 genome, respectively. When 1000 samples were tested using this test kit, 710 were detected as positive. The results indicated that the establishment of this method and the detection kit are specific,sensitive and stable, early diagnosis and quantitative analysis of PCV2 infection are achieved, and it lays a foundation for the prevention and control of PCV2.
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