猪博卡病毒G1基因群和猪流行性腹泻病毒双重TaqMan荧光定量PCR检测方法的建立及应用
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摘要
为建立同时快速定量检测猪博卡病毒(PBoV) G1基因群和猪流行性腹泻病毒(PEDV)的方法,本研究参照GenBank登录的PBoV的NP1基因和PEDV的M基因保守序列,设计了引物和探针,经优化反应条件后,建立了能够同时检测PBoV G1基因群和PEDV的双重TaqMan荧光定量PCR方法。特异性试验结果显示该方法与PCV2、PRV、PDCoV、PRoV和TGEV无交叉反应,敏感性试验结果显示该方法对PBoV、PEDV的质粒标准品检测下限分别为21.8拷贝/μL和31.7拷贝/μL;且组内、组间变异系数均小于4%,重复性好。应用本实验建立的方法对2017年5月～2018年8月,河北省部分地区采集的142份仔猪腹泻样品检测结果显示,PBoV G1阳性率为18.3%(26/142),PEDV阳性率为62.7%(89/142),其中PBoV G1与PEDV共感染率为10.6%(15/142),该方法优于常规PCR方法。本研究建立的双重TaqMan荧光定量PCR方法对PBoV和PEDV的准确检测、病原监测、流行病学调查等均具有重要意义。
In order to establish a rapid and quantitative method for porcine bocavirus(PBoV) genogroup G1 and porcine epidemic diarrhea virus(PEDV) detection, primers and probes were designed based on the conserved sequence of NP1 gene of PBoV and the M gene of PEDV. After optimizing the reaction conditions and reaction system, a duplex TaqMan real-time PCR method for simultaneous detection of PBoV genogroup G1 and PEDV was successfully established. The results showed the methods had no cross-react with PCV2, PRV, PDCoV, PRoV and TGEV, and had the ability to detect 21.8 copies/μL and 31.7 copies/μL for PBoV and PEDV, respectively, and the coefficient variation of intra-and inter-assay were less than 4%, indicating the established methods had high specificity, sensitivity and repeatability. The detection of 142 clinical samples collected from the piglets with diarrhea in Hebei province during May 2017 to August 2018 showed that the positive rate of PBoV G1 was 18.3%(26/142), and the positive rate of PEDV was 62.7%(89/142), and the co-infection rate of PBoV G1 and PEDV was 10.6%(15/142), which was superior to the detection result of conventional PCR. The established method could contribute to the accurate diagnosis, pathogenic surveillance and epidemiological investigation of PBoV and PEDV infections in pigs.
In order to establish a rapid and quantitative method for porcine bocavirus(PBoV) genogroup G1 and porcine epidemic diarrhea virus(PEDV) detection, primers and probes were designed based on the conserved sequence of NP1 gene of PBoV and the M gene of PEDV. After optimizing the reaction conditions and reaction system, a duplex TaqMan real-time PCR method for simultaneous detection of PBoV genogroup G1 and PEDV was successfully established. The results showed the methods had no cross-react with PCV2, PRV, PDCoV, PRoV and TGEV, and had the ability to detect 21.8 copies/μL and 31.7 copies/μL for PBoV and PEDV, respectively, and the coefficient variation of intra-and inter-assay were less than 4%, indicating the established methods had high specificity, sensitivity and repeatability. The detection of 142 clinical samples collected from the piglets with diarrhea in Hebei province during May 2017 to August 2018 showed that the positive rate of PBoV G1 was 18.3%(26/142), and the positive rate of PEDV was 62.7%(89/142), and the co-infection rate of PBoV G1 and PEDV was 10.6%(15/142), which was superior to the detection result of conventional PCR. The established method could contribute to the accurate diagnosis, pathogenic surveillance and epidemiological investigation of PBoV and PEDV infections in pigs.
引文
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[2]胡军勇,张倩,王丹丹,等.猪博卡病毒PCR检测方法的建立及其初步流行病学调查[J].中国预防兽医学报,2012,34(08):632-636.
[3]Gunn L,Collins P J,Fanning S,et al.Detection and characterisation of novel bocavirus(genus Bocaparvovirus)and gastroenteritis viruses from asymptomatic pigs in Ireland[J].Infect Ecol Epidemiol,2015,5:27270.
[4]覃绍敏,吴健敏,马琳,等.猪博卡病毒全基因组序列分析与基因分型研究[J].中国预防兽医学报,2014,36(02):150-153.
[5]周宇,李春萍,朱事康,等.猪博卡病毒PCR检测方法的建立及初步应用[J].中国预防兽医学报,2016,38(09):715-718.
[6]Zhang Bin,Tang Cheng,Yue Hua,et al.Viral metagenomics analysis demonstrates the diversity of viral flora in piglet diarrhoeic faeces in China[J].J Gen Virol,2014,95(Pt 7):1603-1611.
[7]Zhou Feng,Sun Hao-ting,Wang Yu-yan.Porcine bocavirus:Achievements in the past five years[J].Viruses,2014,6(12):4946-4960.
[8]翟少伦,陈胜男,魏文康.猪博卡病毒的研究进展[J].病毒学报,2012,28(02):190-193.
[9]Zhang Qian,Hu Rui-ming,Tang Xi-biao,et al.Occurrence and investigation of enteric viral infections in pigs with diarrhea in China[J].Arch Virol,2013,158(8):1631-1636.
[10]叶星宇,聂奎,聂福平.猪博卡病毒感染及检测方法研究进展[J].动物医学进展,2014,35(06):127-131.
[11]刘政龙,王金良,沈志强.猪流行性腹泻病毒研究进展[J].中国畜牧兽医,2015,42(09):2506-2511.
[12]Zhou Xin-rong,Zhang Tian-sheng,Song De-ping,et al.Comparison and evaluation of conventional RT-PCR,SYBR green I and Taq Man real-time RT-PCR assays for the detection of porcine epidemic diarrhea virus[J].Mol Cell Probes,2017,33:36-41.