塞内卡病毒荧光定量RT-PCR检测方法的建立及初步应用
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摘要
为建立塞内卡病毒(SVA)荧光定量RT-PCR (FQ-PCR)检测方法,本研究根据GenBank中SVA 3D基因保守区域序列设计了一对特异性引物和一条特异性探针,以SVA cDNA为模板,经优化反应条件,建立了SVA FQ-PCR检测方法,并对其进行了特异性、敏感性、重复性试验;利用所建立的方法对28份临床疑似SVA感染样品进行了检测,并与本实验室建立的SVA RT-PCR方法检测结果及测序结果比较分析。结果显示,该方法仅对SVA出现阳性扩增信号,对BHK-21正常细胞对照和口蹄疫病毒、猪繁殖与呼吸障碍综合征病毒、猪流行性腹泻病毒、猪圆环病毒Ⅱ型、猪伪狂犬病毒等5种病原均未出现扩增,特异性较强;该方法最低检测限为10.4拷贝/μL质粒标准品,比常规RT-PCR敏感性高10倍,敏感性较高;该方法批内及批间变异系数均小于4%,重复性好。利用该方法对28份疑似SVA感染样品检测显示,检出9份阳性样品,与测序结果一致,而常规RT-PCR仅检出6份阳性样品,其敏感性高于常规RT-PCR方法,两种方法的符合率为89.3%。本研究为SVA的早期检测提供了特异、敏感、快速的方法。
Fluorescence quantitative PCR(FQ-PCR) assay for detection of Senecavirus A(SVA) was developed. using the reverse transcribed total RNA from SVA cDNA as templates, and the nucleic acid sequence of SVA 3 D gene as specific primers.The specificity, sensitivity and repeatability of SVA FQ-PCR were tested. Twenty-eight suspected SVA positive pig samples was tested by the established FQ-PCR assay. The results showed the assay has a good linear relationship at a template range from1.04×101 copies/μL to 1.04×106 copies/μL, with a coefficient correlation of 0.998; the lower limit of detection was 10.4 copies/μL;the specificity of FQ-PCR assay showed no cross reactions to FMDV, PRRSV, PEDV, PCV2 and PRV; the coefficient of variation for intra-assay and inter-assay were both less than 4%. Meanwhile, 9 out of 28 samples were identified as positive by our assay,which was consistent with the sequencing results, while 6 samples were detected as positive by conventional RT-PCR assay. The coincidence rate of the two methods is 89.3%. We propose that SVA FQ-PCR can be a specific, sensitive, fast assay for the early detection of SVA.
Fluorescence quantitative PCR(FQ-PCR) assay for detection of Senecavirus A(SVA) was developed. using the reverse transcribed total RNA from SVA cDNA as templates, and the nucleic acid sequence of SVA 3 D gene as specific primers.The specificity, sensitivity and repeatability of SVA FQ-PCR were tested. Twenty-eight suspected SVA positive pig samples was tested by the established FQ-PCR assay. The results showed the assay has a good linear relationship at a template range from1.04×101 copies/μL to 1.04×106 copies/μL, with a coefficient correlation of 0.998; the lower limit of detection was 10.4 copies/μL;the specificity of FQ-PCR assay showed no cross reactions to FMDV, PRRSV, PEDV, PCV2 and PRV; the coefficient of variation for intra-assay and inter-assay were both less than 4%. Meanwhile, 9 out of 28 samples were identified as positive by our assay,which was consistent with the sequencing results, while 6 samples were detected as positive by conventional RT-PCR assay. The coincidence rate of the two methods is 89.3%. We propose that SVA FQ-PCR can be a specific, sensitive, fast assay for the early detection of SVA.
引文
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[7]林彦星,曹琛福,花群俊,等.塞内卡谷病毒荧光RT-RPA快速检测法的建立[J].中国兽医科学,2018,48(12):1497-1502.
[8] Kim W I, Kim J J, Cha S H, et al. Significance of genetic variation of PRRSV ORF5 in virus neutralization and molecular determinants corresponding to cross neutralization among PRRS viruses[J]. Vet Microbiol, 2013, 162(1):10-22.
[9] Adams M J,Lefkowitz E J,King A M Q,et al. Ratification vote on taxonomic proposals to the International committee on taxonomy of viruses(2015)[J]. Arch Virol,2015,160(7):1837-1850.
[10] Jamnikar C U, Grom J, ToplakI, et al. Real-time RT-PCR assay for rapid and specific detection of classical swine fever virus:Comparison of SYBR green and TaqMan MGB detection methods using novel MGB probes[J]. J Virol Methods, 2008, 147(2):257-264.
[11] Kutyavin I V,Afonina I A,Mills A,et al. 3'-minor groove binder-DNA probes increase sequence specificity at PCR extension temperature[J]. Nucleic Acids Res, 2000, 28(2):655-661.
[2] Venkataraman S,Reddy S P,Loo J, et al. Structure of Seneca Valley Virus-001:an oncolyticpicomavirus representing a new genus[J]. Structurce,2008,16(10):1555-1561.
[3] Bracht A J, O'Hearn E S. Real-time reverse transcription pcr assay for detection of senecavirus a in swine vesicular diagnostic specimens[J]. PLoS One, 2016, 11(1):e0146211.
[4] Resende T P, Marthaler D G, Vannucci F A. A novel RNAbased in situ hybridization to detect seneca valley virus in neonatal piglets and sows affected with vesicular disease[J]. PLoS One, 2017, 12(4):e0173190.
[5] Feronato C, Leme R A, Diniz J A, et al. Development and evaluation of a nested-PCR assay for senecavirus A diagnosis[J].Tropl Anim Health Prod, 2018, 50(2):337-344.
[6]樊晓旭,赵永刚,迟田英,等.塞尼卡谷病毒TagMan荧光定量PCR检测方法的建立[J].中国预防兽医学报,2016,38(12):959-962.
[7]林彦星,曹琛福,花群俊,等.塞内卡谷病毒荧光RT-RPA快速检测法的建立[J].中国兽医科学,2018,48(12):1497-1502.
[8] Kim W I, Kim J J, Cha S H, et al. Significance of genetic variation of PRRSV ORF5 in virus neutralization and molecular determinants corresponding to cross neutralization among PRRS viruses[J]. Vet Microbiol, 2013, 162(1):10-22.
[9] Adams M J,Lefkowitz E J,King A M Q,et al. Ratification vote on taxonomic proposals to the International committee on taxonomy of viruses(2015)[J]. Arch Virol,2015,160(7):1837-1850.
[10] Jamnikar C U, Grom J, ToplakI, et al. Real-time RT-PCR assay for rapid and specific detection of classical swine fever virus:Comparison of SYBR green and TaqMan MGB detection methods using novel MGB probes[J]. J Virol Methods, 2008, 147(2):257-264.
[11] Kutyavin I V,Afonina I A,Mills A,et al. 3'-minor groove binder-DNA probes increase sequence specificity at PCR extension temperature[J]. Nucleic Acids Res, 2000, 28(2):655-661.