Abstract：In order to identify the Torque Teno virus (TT virus), a PCR-DHPLC assay was performed in this study. Primers specific were selected according to the characteristics of TT virus nucleic acid sequence to conduct PCR, and PCR products assayed by DHPLC. We analyzed the sensitivity, specificity, repeatability of PCR-DHPLC and applied it preliminarily on clinical detection. The specific testing was performed with TTV, HBV, HCV and HEV, no cross reaction were found, and the PCR-DHPLC assays we developed had good specification and nice repeatability. Sensitivity analysis showed that the developed PCR-DHPLC assays could detect 1.0×101 copy/μL. Then we detected 32 serum samples by this method, real-time PCR and normal PCR at same time. The results showed that 17 TTV positives results could be observed by PCR-DHPLC for 32 samples, it is consistent with real-time PCR test results and 15 positive by normal RT-PCR. PCR-DHPLC assays showed nice specification, sensitivity, repeatability, and could be used in epidemiological investigation.
杨春华, 廖芸, 罗秋红, 孙思扬, 夏彪, 祝建新. 输血传播病毒PCR-DHPLC检测技术的建立及初步应用[J]. 中国人兽共患病学报, 2017, 33(11): 979-983.
YANG Chun-hua, LIAO Yun, LUO Qiu-hong, SUN Si-yang, XIA Biao, ZHU Jian-xin. Development and preliminary application of PCR-DHPLC assays for detection of the Torque Teno virus. Chinese Journal of Zoonoses, 2017, 33(11): 979-983.
[1] Nishizawa T, Okamoto H, Konishi K, et al. A novel DNA virus (TTV) associated with elevated transaminase levels in posttransfusion hepatitis of unknown etiology[J]. Biochem Biophys Res Communi, 1997, 241: 92-97. doi:10.1006/bbrc.1997.7765 [2] Zhuang H. Research status and prospect of TT virus [J].Chin J Inter Med,1999,38(11):727-728.(in Chinese) 庄辉.TT病毒的研究现状及展望 [J].中华内科杂志,1999,38(11):727-728. [3] Okamoto H, Takahashi M, Nishizawa T, et al. Genomic characterization of TT viruses (TTVs) in pigs, cats and dogs and their relatedness with species-specific TTVs in primates and tupaias[J]. J Genomic Virol, 2002, 83: 1291-1297. doi:10.1099/0022-1317-83-6-1291 [4] Zheng H, Ye L, Fang X, et al. Torque teno virus (SANBAN isolate) ORF2 protein suppresses NF-kappa B pathways via interaction with IkappaB kinases[J]. J Virol, 2007, 81: 1917-11924. doi:10.1128/JVI.01101-07 [5] Biaqini P. Classification of TTV and related viruses (Anelloviruses)[J]. Curr Topics Microbiol Immunol, 2009, 331: 21-33 [6] Okamoto H, Nishizawa T, Ukita M. A novel unenveloped DNA virus (TT virus) associated with acute and chronic non-A to G hepatitis.[J]. Intervirology,1999, 42(2-3):196-204. doi:24961 [7] Li Y, Xiao J. Research progress of TTV detection methods[J]. Chongqing Med, 2006, 35(18):1718-1720.(in Chinese) 李阳,肖洁. TTV 检测方法研究进展 [J].重庆医学, 2006, 35(18):1718-1720. [8] Yang CH, Sun SY,Sun J, et al.Development and application of real-time PCR for detection of the Torque teno sus virus[J].Chin J Vet Sci,2017, 37(6): 999-1006. (in Chinese) 杨春华,孙思扬,孙洁,等.猪细环病毒实时荧光PCR检测技术的建立及应用 [J]. 中国兽医科学, 2017, 37(6): 999-1006. [9] Kekarainen T, Lopezsoria S, Segale J. Detection of swine Torque teno virus genogroups 1 and 2 in boar sera and semen[J]. Theriogenology, 2007, 68: 966-971. doi:10.1016/j.theriogenology.2007.07.010 [10] Wang MM, Zhou YJ, Chen ZY, et al. Identification and survey of torque teno virus in pigs in china[J]. Chin J Prevent Vet Med, 2009, 31 (10): 751-755.(in Chinese) 王礞礞, 周艳君, 陈宗艳,等. 我国猪群中TTV 的鉴定及其分子流行病学分析[J]. 中国预防兽医学报, 2009, 31 (10): 751-755. [11] Brassard J, Gagen M J, Houde A, et al. Development of a real-time TaqMan PCR assay for the detection of porcine and bovine Torque teno virus[J]. J Appl Microbiol, 2010, 108: 2191-2198. doi:10.1111/j.1365-2672.2009.04624.x [12] Okamoto H, Takahashi M, Nishizawa T, et al. Marked genomic heterogeneity and frequent mixed infection of TT virus demonstrated by PCR with primers from coding and noncoding regions[J]. Virology, 1999, 259(2): 428-436. doi:10.1006/viro.1999.9770 [13] Ott C, Duret L, Chemin I, et al. Use of a TT virus ORF1 recombinant protein to detect anti-TT virus antibodies in human sera[J]. J General Virol, 2000, 81: 2949-2953. doi:10.1099/0022-1317-81-12-2949 [14] Vignolini T, Macera L, Antonelli G, et al. Investigation on torquetenovirus (TTV) microRNA transcriptome in vivo [J]. Virus Res, 2016, 124: 3457-3463. doi:10.1016/j.virusres.2016.03.003 [15] Focosi D, Antonelli G, Pistello M, et al. Torquetenovirus: the human virome from bench to bedside[J]. Clin Microbiol Infect, 2016, 22(7): 589-593. doi:10.1016/j.cmi.2016.04.007 [16] Haloschan M, Bettesch R, Görzer I, et al. TTV DNA plasma load and its association with age, gender, and HCMV IgG serostatus in healthy adults[J]. Age (Dordr),2014, 36(5):9716-9723. doi:10.1007/s11357-014-9716-2 [17] Yang CH,Zhu JX, Zhong Y, et al.Development and application of PCR-DHPLC assays for detection of the porcine reproductive and respiratory syndrome virus[J].Chin J Vet Sci,2012,32(2):167-171. (in Chinese) 杨春华, 祝建新, 钟毅, 等. 猪繁殖与呼吸综合征病毒PCR-DHPLC检测新技术的建立及应用[J].中国兽医学报,2012, 32(2):167-171. [18] Yang CH, Cao JJ, Zhong Y, et al.Construction of identification of Klebsiella pneumoniae in milk and diary product with PCR-DHPLC[J]. Microbiol Chin, 2010,37(12):1805-1810. (in Chinese) 杨春华, 曹际娟, 钟毅, 等. 乳及乳制品中肺炎克雷伯氏菌PCR-DHPLC检测新技术的建立[J]. 微生物学通报, 2010,37(12):1805-1810. [19] Yang CH, Cao JJ, Gui JX, et al. Construction of identification of Enterobacter sakazakii in milk and diary product with PCR-DHPLC[J]. Microbiol Chin, 2008, 35 (11): 1845-1849. (in Chinese) 杨春华, 曹际娟, 桂家祥, 等. 乳及乳制品中阪崎肠杆菌PCR-DHPLC检测新技术的建立[J]. 微生物学通报, 2008, 35 (11): 1845-1849.
2017, Vol. 33 
