伪狂犬病病毒野毒株与gE基因缺失疫苗株微滴数字PCR鉴别方法的建立
|
摘要
为建立伪狂犬病病毒(PRV)野毒株与gE基因缺失疫苗株的鉴别诊断和准确定量的微滴数字PCR(ddPCR)方法,本研究以PRV gD和gE基因为靶基因,设计并合成了2套特异性引物和Taq Man探针,建立了分别针对PRV gD和gE基因的dd PCR方法。结果显示,本研究建立的dd PCR方法能够特异性地鉴别检测PRV野毒株和疫苗株,与圆环病毒2型、猪瘟病毒、猪流感病毒、猪繁殖与呼吸综合征病毒等均无交叉反应;该方法针对PRV gD、gE基因的检测限分别为3.9拷贝/μL、2.3拷贝/μL,分别比相应的荧光定量PCR (qPCR)方法敏感性高10倍和5倍;针对PRV gD、gE基因的批内和批间重复性试验结果变异系数(C.V)均小于4%;通过对45份临床样品的检测,结果显示,与病毒分离方法相比,dd PCR方法敏感性为91.7%,特异性为97.2%,符合率为97.8%;与dd PCR方法相比,q PCR的敏感性为83.3%,特异性为94.3%,符合率为95.6%。本研究建立的dd PCR方法在检测低拷贝临床样品时敏感性更高、特异性强、重复性好,可以作为PRV野毒株与疫苗株的鉴别、准确定量的有效检测手段。
In this study,a set of 2 novel assays for quantitative detection and differentiation of wild-type pseudorabies virus(PRV)and gE-deleted PRV vaccine strain were developed by using droplet digital PCR(ddPCR)based on the two sets of primers and Taq Man probes according to the conservative region of PRV gD and gE genes.The ddPCRs were highly specific for PRV,without cross-reaction with other common swine viral pathogens,such as porcine circovirus type 2,classical swine fever virus,swine influenza virus,porcine reproductive and respiratory syndrome virus.The detection limit for the gD and gE genes of the ddPCRs were 3.9 copies/μL and 2.3 copies/μL respectively,which showed that the ddPCRs provided 10-fold and 5-fold sensitivity improvement in contrast of the real time PCR(qPCR)previously established in our laboratory,respectively.The coefficient of variation of the intra-batch and inter-batch reproducibility tests of the ddPCRs were 2.06%,3.20% and 3.03%,3.88%,respectively. A total of 45 clinical sample detection results showed that the sensitivity, specificity and the coincidence rate of the ddPCR assays were 91.7%, 97.2% and 97.8%, respectively, compared with the virus isolation detections; and the sensitivity,specificity and the coincidence rate of the qPCR were 83.3%, 94.3% and 95.6%, respectively, compared with the ddPCR using the same primers and probes. The ddPCR assays were more sensitive than qPCR for the detection of low concentrations of PRV in clinical samples. Thus, the established ddPCR assays provide an appropriate detection platform for absolute quantitative detection and differentiation of wild-type PRVs from gE-deleted vaccine strain of PRV.
In this study,a set of 2 novel assays for quantitative detection and differentiation of wild-type pseudorabies virus(PRV)and gE-deleted PRV vaccine strain were developed by using droplet digital PCR(ddPCR)based on the two sets of primers and Taq Man probes according to the conservative region of PRV gD and gE genes.The ddPCRs were highly specific for PRV,without cross-reaction with other common swine viral pathogens,such as porcine circovirus type 2,classical swine fever virus,swine influenza virus,porcine reproductive and respiratory syndrome virus.The detection limit for the gD and gE genes of the ddPCRs were 3.9 copies/μL and 2.3 copies/μL respectively,which showed that the ddPCRs provided 10-fold and 5-fold sensitivity improvement in contrast of the real time PCR(qPCR)previously established in our laboratory,respectively.The coefficient of variation of the intra-batch and inter-batch reproducibility tests of the ddPCRs were 2.06%,3.20% and 3.03%,3.88%,respectively. A total of 45 clinical sample detection results showed that the sensitivity, specificity and the coincidence rate of the ddPCR assays were 91.7%, 97.2% and 97.8%, respectively, compared with the virus isolation detections; and the sensitivity,specificity and the coincidence rate of the qPCR were 83.3%, 94.3% and 95.6%, respectively, compared with the ddPCR using the same primers and probes. The ddPCR assays were more sensitive than qPCR for the detection of low concentrations of PRV in clinical samples. Thus, the established ddPCR assays provide an appropriate detection platform for absolute quantitative detection and differentiation of wild-type PRVs from gE-deleted vaccine strain of PRV.
引文
[1] An Tong-qin, Peng Jin-mei, Tian Zhi-jun, et al. Pseudorabies virus variant in Bartha-K61-vaccinated pigs, China, 2012[J].Emerg Infec Dis, 2013, 19(11):1749-1755.
[2] Michael J R, Kelli L H, Brian H K, et al. Quantification of zoonotic bacterial pathogens within commercial poultry processing water samples using droplet digital PCR[J]. Adv Microbiol,2013, 3:403-411.
[3]陈亚娜,王静,訾占超,等.伪狂犬病病毒微滴数字PCR定量检测方法的建立[J].畜牧兽医学报,2017, 48(9):1705-1710.
[4]原霖,訾占超,亢文华,等.猪圆环病毒1型微滴数字PCR定量检测方法的建立[J].中国兽医学报,2017,37(11):2043-2047.
[5] Bidshahri R, Attali D, Fakhfakh K, et al. Quantitative detection and resolution of BRAF V600 status in colorectal cancer using droplet digital PCR and a novel wild-type negative assay[J]. J Mol Diagn, 2016, 18(2):190-204.
[6] Zhang Yan-ni,Tang En-tu, Du Zhi-qiang. Detection of MET gene copy number in cancer samples using the droplet digital PCR method[J]. PLoS One, 2016, 11(1):e0146784.
[7] Dalmira F U, Melina P U, Jos Ubenigno V T, et al. Development, optimization, and evaluation of a duplex droplet digital PCR assay to quantify the T-nos/hmg copy number ratio in genetically modified maize[J]. Anal Chem, 2016, 88(1):812-819.
[8]冯兆民,赵翔,邹晓辉,等.基于微滴式数字PCR技术的甲型流感病毒绝对定量方法的建立及应用[J].病毒学报,2017, 33(1):1-5.
[9] Wang Qiu-shi, Yang Xin, He Yong, et al. Droplet digital PCR for absolute quantification of EML4-ALK gene rearrangement in lung adenocarcinoma[J]. J Mol Diagin, 2015, 17(5):515-520.
[10]唐连飞,禹思宇,周宇,等.数字RT-PCR技术检测H3N2亚型猪流感病毒方法的建立[J].中国畜牧兽医,2017,44(6):1847-1853.
[11] Alikian M, Ellery P, Forbes M, et al. Next-generation sequencing-assisted DNA-based digital PCR for a personalized approach to the detection and quantification of residual disease in chronic myeloid leukemia patients[J]. J Mol Diagp, 2016, 18(2):176-189.
[12]兰德松,顾贵波,侯振中.伪狂犬病病毒野毒株与gE基因缺失疫苗株TaqMan实时荧光定量PCR鉴别方法的建立[J].中国畜牧兽医,2018,45(7):1804-1812.
[13]刘存,陈书民,刘砚涵,等.伪狂犬病病毒Qihe547分离株主要保护性抗原基因序列的变异分析[J].中国预防兽医学报,2016, 38(4):331-334.
[14]朱小甫,吴旭锦.鉴别伪狂犬病毒gE基因缺失疫苗毒与野毒套式PCR方法的建立[J].中国动物传染病学报,2016, 24(4):26-30.
[15] Ma Wen-jun, Kelly M L, Juergen A R, et al. Development of real-time polymerase chain reaction assays for rapid detection and differentiation of wild-type pseudorabies and gene-deleted vaccine viruses[J]. J Vet Diagn Invest, 2008, 20:440-447.
[2] Michael J R, Kelli L H, Brian H K, et al. Quantification of zoonotic bacterial pathogens within commercial poultry processing water samples using droplet digital PCR[J]. Adv Microbiol,2013, 3:403-411.
[3]陈亚娜,王静,訾占超,等.伪狂犬病病毒微滴数字PCR定量检测方法的建立[J].畜牧兽医学报,2017, 48(9):1705-1710.
[4]原霖,訾占超,亢文华,等.猪圆环病毒1型微滴数字PCR定量检测方法的建立[J].中国兽医学报,2017,37(11):2043-2047.
[5] Bidshahri R, Attali D, Fakhfakh K, et al. Quantitative detection and resolution of BRAF V600 status in colorectal cancer using droplet digital PCR and a novel wild-type negative assay[J]. J Mol Diagn, 2016, 18(2):190-204.
[6] Zhang Yan-ni,Tang En-tu, Du Zhi-qiang. Detection of MET gene copy number in cancer samples using the droplet digital PCR method[J]. PLoS One, 2016, 11(1):e0146784.
[7] Dalmira F U, Melina P U, Jos Ubenigno V T, et al. Development, optimization, and evaluation of a duplex droplet digital PCR assay to quantify the T-nos/hmg copy number ratio in genetically modified maize[J]. Anal Chem, 2016, 88(1):812-819.
[8]冯兆民,赵翔,邹晓辉,等.基于微滴式数字PCR技术的甲型流感病毒绝对定量方法的建立及应用[J].病毒学报,2017, 33(1):1-5.
[9] Wang Qiu-shi, Yang Xin, He Yong, et al. Droplet digital PCR for absolute quantification of EML4-ALK gene rearrangement in lung adenocarcinoma[J]. J Mol Diagin, 2015, 17(5):515-520.
[10]唐连飞,禹思宇,周宇,等.数字RT-PCR技术检测H3N2亚型猪流感病毒方法的建立[J].中国畜牧兽医,2017,44(6):1847-1853.
[11] Alikian M, Ellery P, Forbes M, et al. Next-generation sequencing-assisted DNA-based digital PCR for a personalized approach to the detection and quantification of residual disease in chronic myeloid leukemia patients[J]. J Mol Diagp, 2016, 18(2):176-189.
[12]兰德松,顾贵波,侯振中.伪狂犬病病毒野毒株与gE基因缺失疫苗株TaqMan实时荧光定量PCR鉴别方法的建立[J].中国畜牧兽医,2018,45(7):1804-1812.
[13]刘存,陈书民,刘砚涵,等.伪狂犬病病毒Qihe547分离株主要保护性抗原基因序列的变异分析[J].中国预防兽医学报,2016, 38(4):331-334.
[14]朱小甫,吴旭锦.鉴别伪狂犬病毒gE基因缺失疫苗毒与野毒套式PCR方法的建立[J].中国动物传染病学报,2016, 24(4):26-30.
[15] Ma Wen-jun, Kelly M L, Juergen A R, et al. Development of real-time polymerase chain reaction assays for rapid detection and differentiation of wild-type pseudorabies and gene-deleted vaccine viruses[J]. J Vet Diagn Invest, 2008, 20:440-447.