猪圆环病毒3型TaqMan-MGB荧光定量PCR方法的建立及应用
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摘要
为建立猪圆环病毒3型(PCV3)荧光定量PCR检测方法,本研究根据GenBank中PCV3基因序列设计特异性引物和探针,经过反应体系和条件优化,建立了特异性检测PCV3的TaqMan-MGB荧光定量PCR方法。该检测方法在4.78×10~1拷贝/μL～4.78×10~9拷贝/μL质粒标准品范围内均有良好的线性关系;该方法特异性试验结果显示,其与多种常见猪病病毒均无交叉反应,特异性良好;本研究建立的方法敏感性是常规PCR方法的100倍,敏感性较高;批内批间重复性试验变异系数均小于2.3%,重复性良好。对临床样品的检测结果显示,该方法对PCV3的检出率高于常规PCR方法,并且PCV3阳性样品多存在混合感染情况。该方法的建立为PCV3的实验室诊断及流行病学调查提供了快速、准确的检测手段。
To established a sensitive detection method of porcine circovirus 3(PCV3), a TaqMan-MGB real-time PCR assay was established using specific primers and probe designed according to the genome sequence of PCV3. With the optimized reactions conditions, the established method was specific for PCV3 detection without cross-reaction with other common porcine viruses. Results showed that the method had linearity within the template ranges from 4.78×10~1 to 4.78×10~9 copies/μL and was100 times more sensitive than that of routine PCR assay. The coefficient of variation in the reproducible assays was less than 2.3%. The positive detection rate of PCV3 in clinical samples using the developed method was higher than that using the conventional PCR method and the PCV3 positive samples were mostly mixed infection. The establishment of the real-time PCR method provides a rapid and accurate detection means for the laboratory diagnosis and epidemiological investigation of PCV3.
To established a sensitive detection method of porcine circovirus 3(PCV3), a TaqMan-MGB real-time PCR assay was established using specific primers and probe designed according to the genome sequence of PCV3. With the optimized reactions conditions, the established method was specific for PCV3 detection without cross-reaction with other common porcine viruses. Results showed that the method had linearity within the template ranges from 4.78×10~1 to 4.78×10~9 copies/μL and was100 times more sensitive than that of routine PCR assay. The coefficient of variation in the reproducible assays was less than 2.3%. The positive detection rate of PCV3 in clinical samples using the developed method was higher than that using the conventional PCR method and the PCV3 positive samples were mostly mixed infection. The establishment of the real-time PCR method provides a rapid and accurate detection means for the laboratory diagnosis and epidemiological investigation of PCV3.
引文
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[7]曹玉琴,付丹,杨尧,等.PEDV-TGEV-RV多重RT-PCR鉴别检测方法的建立与应用[J].中国兽医科学,2015,45(09):881-887.
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[9]田青,郑浩,童武,等.伪狂犬病毒实时荧光定量PCR方法的建立[J].中国动物传染病学报,2015,23(3):1-6.
[10]郭慧娟,李秀丽,张国伟,等.猪圆环病毒2型TaqMan荧光定量PCR检测方法的建立[J].中国畜牧兽医,2014,41(5):39-44.
[11]徐朋丽,张鸿鑫,张宇,等.猪圆环病毒3型PCR检测方法的建立及应用[J].中国兽医学报,2017,39(9):727-730.
[12]Chen Gui-hua,Tang Xiao-yu,Sun Yuan,et al.Development of a SYBR green-based real-time quantitative PCR assay to detect PCV3 in pigs[J].J Virol Methods,2018,251(1):129-132.
[13]Wang Jian-chang,Zhang Yong-ning,Wang Jin-feng,et al.Development of a TaqMan-based real-time PCR assay for the specific detection of porcine circovirus 3[J].J Virol Methods,2017,248(10):177-180.
[14]Li Xiang-dong,Qiao Ming-ming,Sun Ming,et al.A duplex real-time PCR assay for the simultaneous detection of porcine circovirus 2 and circovirus 3[J].Virol Sin,2018,33(2):181-186.
[15]Ku X,Chen Fang-zhou,Li P,et al.Identification and genetic characterization of porcine circovirus type 3 in China[J].Transbound Emerg Dis,2017,64(3):703-708.
[2]Segalés J,Kekarainen T,Cortey M.The natural history of porcine circovirus type 2:from an inoffensive virus to a devastating swine disease?[J].Vet Microbiol,2013,165(1-2):13-20.
[3]Baekbo P,Kristensen C S,Larsen L E.Porcine circovirus diseases:A review of PMWS[J].Transbound Emerg Dis,2012,59(1):60-67.
[4]夏叶,郭家宏,廖学文,等.猪圆环病毒2d亚型病毒样颗粒的制备[J].上海农业学报,2017,33(6):43-48.
[5]Zheng Shu-xuan,Wu Xiao-yan,Zhang Li,et al.The occurrence of porcine circovirus 3 without clinical infection signs in Shandong Province[J].Transbound Emerg Dis,2017,64(5):1337-1341.
[6]李晓菲,陈婷,魏笑笑,等.猪圆环病毒3型PCR检测方法的建立与应用[J].黑龙江畜牧兽医,2018,11(22):77-80.
[7]曹玉琴,付丹,杨尧,等.PEDV-TGEV-RV多重RT-PCR鉴别检测方法的建立与应用[J].中国兽医科学,2015,45(09):881-887.
[8]王建华,陈小金,赵丹,等.非洲猪瘟病毒、猪瘟病毒和猪繁殖与呼吸综合征病毒多TaqMan荧光定量RT-PCR检测方法的建立[J].中国预防兽医学报,2017,39(11):907-911.
[9]田青,郑浩,童武,等.伪狂犬病毒实时荧光定量PCR方法的建立[J].中国动物传染病学报,2015,23(3):1-6.
[10]郭慧娟,李秀丽,张国伟,等.猪圆环病毒2型TaqMan荧光定量PCR检测方法的建立[J].中国畜牧兽医,2014,41(5):39-44.
[11]徐朋丽,张鸿鑫,张宇,等.猪圆环病毒3型PCR检测方法的建立及应用[J].中国兽医学报,2017,39(9):727-730.
[12]Chen Gui-hua,Tang Xiao-yu,Sun Yuan,et al.Development of a SYBR green-based real-time quantitative PCR assay to detect PCV3 in pigs[J].J Virol Methods,2018,251(1):129-132.
[13]Wang Jian-chang,Zhang Yong-ning,Wang Jin-feng,et al.Development of a TaqMan-based real-time PCR assay for the specific detection of porcine circovirus 3[J].J Virol Methods,2017,248(10):177-180.
[14]Li Xiang-dong,Qiao Ming-ming,Sun Ming,et al.A duplex real-time PCR assay for the simultaneous detection of porcine circovirus 2 and circovirus 3[J].Virol Sin,2018,33(2):181-186.
[15]Ku X,Chen Fang-zhou,Li P,et al.Identification and genetic characterization of porcine circovirus type 3 in China[J].Transbound Emerg Dis,2017,64(3):703-708.