猪圆环病毒的分离鉴定及检测方法的建立
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摘要
猪圆环病毒(PCV)是近年受到广泛关注的危害世界养猪业的重要病源之一。PCV具有PCV1和PCV2两种基因型。根据致病性、抗原性、核苷酸序列,将从PK15细胞系分离的圆环病毒列为圆环病毒1型(PCVl),把与断奶仔猪多系统衰竭综合征(PMWS)等密切相关的圆环病毒列为2型(PCV2)。本研究主要是从河北保定地区某猪场疑似PMWS的发病猪病料中分离到一株病毒,经PCR鉴定、生物学特性的鉴定,进而建立快速准确的临床诊断方法。
无菌采集疑似PMWS的病猪病料,将其腹股沟淋巴结接种PK-15细胞。由于猪圆环病毒在PK-15细胞上不产生细胞病变,接种72h后收毒,盲传至四代。根据GenBank上已发表的PCV2基因序列,设计并合成了一对扩增ORF2基因序列的特异性引物,对细胞毒提取DNA,经PCR扩增,得到485bp特异性的核酸片段。
本研究采用过氧化物酶单层实验(IPMA)测定PCV2分离毒的TCID50,并基于此方法对理化特性进行研究。实验结果表明:该毒株病毒液在PH3和PH12作用1h后TCID50均没有显著变化,毒株表现出较强的耐酸碱性;对胰蛋白酶不敏感;经乙醚、氯仿处理后TCID50的差值均小于2,说明该病毒没有囊膜。试验结果均符合PCV2生物学特性。
根据Genbank上已发表的猪细小病毒(PPV)和猪伪狂犬病毒(PRV)的基因序列,应用Primer Premier 5.0软件进行分析,设计并合成扩增PPV VP2基因片段和PRV UL39基因片段的特异性引物,并建立多重PCR的方法,在同一个反应体系中可同时扩增PCV2、PPV和PRV的特异性片段。通过对引物用量、反应温度、模板用量等反应条件的优化,达到较为理想的扩增效果,并且特异性和重复性良好。之后对PCV2的PCR产物用XbaI单酶切,获得了267bp和228bp的两个片段,并构建质粒pMD18T-PPV和pMD18T-PPV,分别用BamHI和HindIII双酶切,获得PPV 158 bp和PRV 219 bp的目的片段,之后通过序列测序,分别证实了扩增的条带的准确性。
本研究建立了用于检测PCV2的双向乳胶凝集实验的诊断方法,通过对乳胶最佳致敏浓度、最佳致敏温度和最佳致敏时间的测定,确定致敏乳胶的最佳条件,并对致敏乳胶的稳定性进行检验,包括保存的温度和保存时间的检查。乳胶凝集抗原质量的鉴定包括用PBS、BBS、生理盐水进行的自凝性检测和用PRV阳性血清、猪瘟阳性血清、PCV阴性血清和犊牛血清进行的特异性检测。结果表明,本研究所建立的PCV2双向乳胶凝集实验准确快速、简便特异,适用于基层单位用于血清学诊断。
Porcine circovirus(PCV)is one of infectious disease sources in the world which was widesread focus on.There are two genotypes,PCV1 and PCV2. According to pathogenicity,antigenicity and nucleotide sequence,the porcine circovirus, which was gained from PK-15 cell line,was called PCV1,and the porcine circovirus,which was related with postweaning multisystemic wasting syndrome(PMWS),was called PCV2.In this study,a PCV2 strain was gained from swines,which was suspect to PMWS and from HeBei Baoding area.This strain was characterized by PCR and physicochemical affection,and a clinical diagnosis method was established,which was fast and precise.
The PCV2 strain was getted from PK-15 cell line which was inoculated by lymphoglandulae inguinales, The organism was from swine which was suspect to PMWS.For cytopathic effect doesn’t form in PK-15 cell line when porcine circovirus was inoculated,the PCV2 was harvested after inoculating for 72 hours and blind passaged for 4 generations.According to PCV2 gene order which were published in GenBank,specific primers were designed and synthesized,and ORF2 gene order of PCV2 was amplified. After DNA extracting and PCR amplification,a 485bp specific nucleinic acid fragment was getted.
The TCID50 of PCV2 strain was determined by Immunoperoxidase Monolayer Assay(IPMA),physicochemical characteristics also be determined by this method.The result shows that the TCID50 of PCV2 strain has not remarkble changed after deal with 1 hour in PH3 or PH12,the strain has characteristics of strong resist acid and alkali,insensitivity for trypsinase,the difference of TCID50 are not reach to 2 after dealing with ether or chloroform.The result shows that PCV2 strain haven’t peplos which was consistent with the bionomics of PCV2.
Two pairs of specific primers were designed according to the sequences of PPV(VP2) and PRV(UL39),which were published in Genbank and Primer Premier 5.0 was used in the designe.PCV,PRV and PPV DNA could be amplified by the multipx PCR using these three sets of primers,yielding three specific fragments. By improving reactive condition of primer dosis,temperature and template dosis,better result getted ans specificity and reproducibility were fine.The PCR production of PCV2 was determined by XbaI,267bp and 228bp fragments were obtained,the pMD18T-PPV and pMD18T-PPV were determined by BamHI and HindIII, PPV 158 bp and PRV 219 bp fragments were getted.The sequencing result verifed the accuratissime of the fragments.
In this study,double orientation latex agglutination text was established which was used to detect PCV2.The optimum condition of allergizing latex was definited by determining the density,temperature and time.The stability of the allergized latex was tested,which was contained preservative temperature and preservative time.The qualitative appreciation of latex agglutination antigen contains self-cohere detection ,which used PBS,BBS and sodium chloride,and specificity detection,which used PRV,swine plague positive serum,PCV negative serum and calf serum.It is revealed that the double orientation latex agglutination text is of high speciality and simplicity and makes the basis of serological diagnosis of PCV2.
无菌采集疑似PMWS的病猪病料,将其腹股沟淋巴结接种PK-15细胞。由于猪圆环病毒在PK-15细胞上不产生细胞病变,接种72h后收毒,盲传至四代。根据GenBank上已发表的PCV2基因序列,设计并合成了一对扩增ORF2基因序列的特异性引物,对细胞毒提取DNA,经PCR扩增,得到485bp特异性的核酸片段。
本研究采用过氧化物酶单层实验(IPMA)测定PCV2分离毒的TCID50,并基于此方法对理化特性进行研究。实验结果表明:该毒株病毒液在PH3和PH12作用1h后TCID50均没有显著变化,毒株表现出较强的耐酸碱性;对胰蛋白酶不敏感;经乙醚、氯仿处理后TCID50的差值均小于2,说明该病毒没有囊膜。试验结果均符合PCV2生物学特性。
根据Genbank上已发表的猪细小病毒(PPV)和猪伪狂犬病毒(PRV)的基因序列,应用Primer Premier 5.0软件进行分析,设计并合成扩增PPV VP2基因片段和PRV UL39基因片段的特异性引物,并建立多重PCR的方法,在同一个反应体系中可同时扩增PCV2、PPV和PRV的特异性片段。通过对引物用量、反应温度、模板用量等反应条件的优化,达到较为理想的扩增效果,并且特异性和重复性良好。之后对PCV2的PCR产物用XbaI单酶切,获得了267bp和228bp的两个片段,并构建质粒pMD18T-PPV和pMD18T-PPV,分别用BamHI和HindIII双酶切,获得PPV 158 bp和PRV 219 bp的目的片段,之后通过序列测序,分别证实了扩增的条带的准确性。
本研究建立了用于检测PCV2的双向乳胶凝集实验的诊断方法,通过对乳胶最佳致敏浓度、最佳致敏温度和最佳致敏时间的测定,确定致敏乳胶的最佳条件,并对致敏乳胶的稳定性进行检验,包括保存的温度和保存时间的检查。乳胶凝集抗原质量的鉴定包括用PBS、BBS、生理盐水进行的自凝性检测和用PRV阳性血清、猪瘟阳性血清、PCV阴性血清和犊牛血清进行的特异性检测。结果表明,本研究所建立的PCV2双向乳胶凝集实验准确快速、简便特异,适用于基层单位用于血清学诊断。
Porcine circovirus(PCV)is one of infectious disease sources in the world which was widesread focus on.There are two genotypes,PCV1 and PCV2. According to pathogenicity,antigenicity and nucleotide sequence,the porcine circovirus, which was gained from PK-15 cell line,was called PCV1,and the porcine circovirus,which was related with postweaning multisystemic wasting syndrome(PMWS),was called PCV2.In this study,a PCV2 strain was gained from swines,which was suspect to PMWS and from HeBei Baoding area.This strain was characterized by PCR and physicochemical affection,and a clinical diagnosis method was established,which was fast and precise.
The PCV2 strain was getted from PK-15 cell line which was inoculated by lymphoglandulae inguinales, The organism was from swine which was suspect to PMWS.For cytopathic effect doesn’t form in PK-15 cell line when porcine circovirus was inoculated,the PCV2 was harvested after inoculating for 72 hours and blind passaged for 4 generations.According to PCV2 gene order which were published in GenBank,specific primers were designed and synthesized,and ORF2 gene order of PCV2 was amplified. After DNA extracting and PCR amplification,a 485bp specific nucleinic acid fragment was getted.
The TCID50 of PCV2 strain was determined by Immunoperoxidase Monolayer Assay(IPMA),physicochemical characteristics also be determined by this method.The result shows that the TCID50 of PCV2 strain has not remarkble changed after deal with 1 hour in PH3 or PH12,the strain has characteristics of strong resist acid and alkali,insensitivity for trypsinase,the difference of TCID50 are not reach to 2 after dealing with ether or chloroform.The result shows that PCV2 strain haven’t peplos which was consistent with the bionomics of PCV2.
Two pairs of specific primers were designed according to the sequences of PPV(VP2) and PRV(UL39),which were published in Genbank and Primer Premier 5.0 was used in the designe.PCV,PRV and PPV DNA could be amplified by the multipx PCR using these three sets of primers,yielding three specific fragments. By improving reactive condition of primer dosis,temperature and template dosis,better result getted ans specificity and reproducibility were fine.The PCR production of PCV2 was determined by XbaI,267bp and 228bp fragments were obtained,the pMD18T-PPV and pMD18T-PPV were determined by BamHI and HindIII, PPV 158 bp and PRV 219 bp fragments were getted.The sequencing result verifed the accuratissime of the fragments.
In this study,double orientation latex agglutination text was established which was used to detect PCV2.The optimum condition of allergizing latex was definited by determining the density,temperature and time.The stability of the allergized latex was tested,which was contained preservative temperature and preservative time.The qualitative appreciation of latex agglutination antigen contains self-cohere detection ,which used PBS,BBS and sodium chloride,and specificity detection,which used PRV,swine plague positive serum,PCV negative serum and calf serum.It is revealed that the double orientation latex agglutination text is of high speciality and simplicity and makes the basis of serological diagnosis of PCV2.
引文
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