Fujian Provincial Key Laboratory of Inspection and Quarantine Technology Research /Fujian Entry-Exit Inspection and Quarantine Bureau, Fuzhou 350001, China
Abstract:In the present study, we aimed to establish a specific and sensitive nested reverse transcription polymerase chain reaction (RT-PCR) assay for detection of Nipah virus. Two pairs of specific-primers were designed according to the sequence of N gene of Nipah virus, and the RNA positive controls of synthetic was used for the nested RT-PCR reaction system. The system was optimized and the specificity and sensitivity of this system were evaluated and used to detect the 100 clinical samples. The results showed that the homology of the outer primers and the inner primers were all 100%, comparing with the origin gene. As demonstrated by the specificity test, the outer and inner primers designed in this study could not be amplified from the Newcastle disease virus (NDV), Rinderpest virus (RPV) and Japanese encephalitis virus (JEV). The lowest sensitivity concentration of the nested RT-PCR as proved by the sensitivity test was 39 fg/μL RNA of the standard template, and 100 clinical samples were negative detected by the nested RT-PCR. These data suggest that a rapid, specific and sensitive nested RT-PCR technique for detection N gene of Nipah virus has been established primarily, and it will be used for animal disease surveillance, inspection and quarantine.
张体银,张志灯,郑腾,白泉阳,王武军,林素洁. 尼帕病毒巢式RT-PCR方法的建立和初步应用[J]. 中国人兽共患病学报, 2014, 30(6): 599-602.
ZHANG Ti-yin,ZHANG Zhi-deng,ZHENG Teng,BAI Quan-yang,WANG Wu-jun,LIN Su-jie. Development of nested RT-PCR for detection of Nipah virus. Chinese Journal of Zoonoses, 2014, 30(6): 599-602.
[1]Li Y, Wang JM, Hickey AC, et al. Antibodies to Nipah or Nipah-like viruses in bats, China[J]. Emerg Infect Dis, 2008, 14(12): 1974-1976. DOI: 10.3201/eid1412.080359 [2]Chen JM, Guo LX, San CY, et al. A stable and differentiable RNA positive control for reverse transcription-polymerase chain reaction[J].Biotechnol Lett,2006,28(22):1787-1792.DOI: 10.1007/S10529-006-9161-0 [3]Zeng ZL. Development of nucleic acid testing methods and epidemiological surveillance for three neuro tropic viruses[D]. Chongqing: Chongqing Medical University, 2008: 67. (in Chinese) 曾志磊.三种嗜神经病毒的核酸检测方法和初步流行病学研究方法进行[D]. 重庆:重庆医科大学, 2008. [4]Wang L, Harcourt BH, Yu M, et al. Molecular biology of Hendra and Nipah viruses[J]. Microbes Infect, 2001, 3: 279-287. DOI: 10.1016/S1286-4579(01)01381-8 [5]Wacharapluesadee S, Hemachudha T. Duplex nested RT-PCR for detection of Nipah virus RNA from urine specimens of bats[J]. J Virol Methods, 2007, 141: 97-101. DOI:10.1016/ j.jviromet.2006.11.023 [6]Ge JJ. Study on molecular epidemiology of Nipah virus in Guizhou Zunyi and its vicinal regtons[D]. Zunyi: Zunyi Medical University, 2009: 47. (in Chinese) 葛均江.遵义及周边地区Nipah 病毒感染分子流行病学研究[D].遵义:遵义医学院, 2009. [7]Zhang L, Jin G, Feng YX, et al. Detection of Nipah virus infection in brain tissue of donkeys in Ili, Xinjiang[J]. Chin J Microecol, 2010, 22(9): 793-797. (in Chinese) 张亮,金戈,冯裕星,等.新疆驴脑组织尼帕病毒N基因片段的检测[J].中国微生态学杂志,2010,22(9): 793-797. [8]Zhao ZY, Liu SY, Luo XD. Detection of exogenous Jaagsiekte Sheep Retrovirus by the nested RT-PCR technique[J]. Chin J Zoonoses, 2011, 27(6): 547-551. (in Chinese) 赵泽贇,刘淑英,罗学东.绵羊肺腺瘤病毒的巢式RT-PCR技术检测[J].中国人兽共患病学报,2011,27(6): 547-551. [9]Guillaume V,Lefeuwe A,Faure C,et al. Specific detection of Nipah virus using real-time RT-PCR(TaqMan)[J]. J Virol Methods, 2004, 120(2): 229-237. DOI: 10.1016/j.jviromet. 2004.05.018 [10]Xiao C, Liu YJ, Xu XR,et al. Preparation and characterization of monoclonal antibodies against recombinant nucleocapsid protein of Henipahvirus[J]. Chin J Vet Sci, 2007, 27(4): 433-436. DOI: 10.3969/j.issn.1005-4545.2007.04.001 (in Chinese) 肖昌,刘勇军,徐兴然,等.尼帕病毒和亨德拉病毒核蛋白单克隆抗体的制备与鉴定[J].中国兽医学报,2007, 27(4): 433-436. [11]Chua KB, Chua BH, Wang CW. Anthropogenic deforestation, El Nio and the emergence of Nipah virus in Malaysia[J]. Malays J Pathol, 2002, 24(1): 15-21.