检测基孔肯雅病毒SYBR Green I real-time PCR方法的建立及应用

目的建立一种基孔肯雅病毒的快速诊断方法。方法采用基孔肯雅病毒E1基因的重组质粒作为阳性标准品。优化反应条件,建立针对基孔肯雅病毒的SYBR Green I real-time PCR检测方法,并对其特异性、灵敏性和重复性进行评价。结果建立的SYBR Green I real-time PCR检测方法的Ct值与阳性标准品在6.94×10~0～6.94×10~8 copies/μl范围内呈良好的线性关系,相关性为0.997,斜率为-3.571;其检测下限为0.694 copies/μl,且对ZIKV、DENV和JEV等均无特异性扩增;所有稀释倍数的标准品在84.0℃出现特异性熔解峰;组内和组间变异系数均小于2%。采用该方法对93份蚊虫样品进行检测,基孔肯雅病毒核酸阳性率为2.15%,普通PCR核酸阳性率为1.07%。结论成功建立了针对基孔肯雅病毒E1基因的SYBR Green I real-time PCR检测方法。该方法灵敏、特异,可用于基孔肯雅病毒感染的快速诊断。     

口蹄疫病毒一步法TaqMan-MGB荧光定量RT-PCR检测方法的建立

摘要：为了快速高效的检测所有血清型口蹄疫病毒（FMDV）,根据FMDV聚合酶3D基因序列比对结果,设计特异性引物和MGB探针,通过反应条件的优化,建立了一步法荧光定量RT-PCR检测方法,并进行了特异性、敏感性、重复性试验。结果表明,该方法能特异性检测A型、O型、Asia I型FMDV,而对猪瘟、猪蓝耳病、猪圆环病毒、猪细小病毒、猪狂犬病毒、猪乙型脑炎等病原检测结果均为阴性;检测质粒的敏感性可达83.4copies/μL,检测病毒RNA的敏感性可达7.1fg/μL,比多重RT-PCR敏感性高10倍;对4份样品进行5次批内和批间重复检测,检测结果变异系数均小于2%。该方法特异性强、敏感性高、重复性好,可用于口蹄疫临床样品诊断、流行病学调查和畜产品安全监测。     

基于逆转录-酶促重组等温扩增原理的西尼罗病毒基因检测方法的建立

目的 建立一种可用于检测西尼罗病毒特异性基因片段的逆转录-酶促重组等温扩增(RT-ERA)方法。方法 以西尼罗病毒NS5基因的高度保守序列作为靶序列，根据ERA反应原理设计、合成引物及荧光探针，建立荧光RT-ERA反应体系。分别以不同拷贝数(10~4、10~3、10~2、10、1拷贝/μl)的含120 bp靶基因片段的重组质粒及不同浓度(10、1、10^-1、10^-2、10^-3 ng/μl)西尼罗病毒RNA为模板进行荧光RT-ERA法扩增，评价该方法的敏感度；分别以森林脑炎病毒、基孔肯雅病毒、Ⅰ型登革病毒、西尼罗病毒、乙型脑炎病毒、黄热病毒和甲型流感病毒H2N1的RNA为模板进行荧光RT-ERA法检测，评价该方法的特异性。结果 建立的荧光RT-ERA法可在39℃、20 min内特异性扩增西尼罗病毒RNA。敏感度检测结果显示，以重组质粒为模板，荧光RT-ERA法最低可检出的质粒拷贝数为1拷贝/μl；以RNA为模板，荧光RT-ERA法最低可检测浓度为10^-3 ng/μl。特异性检测结果显示，以森林脑炎病毒...     

常规RT-PCR与荧光定量RT-PCR检测GⅠ、GⅡ型诺如病毒方法的建立及其应用北大核心CSCD

目的建立GI、GII型诺如病毒的常规RT-PCR和荧光定量RT-PCR检测方法,并对两种方法进行应用。方法优化筛选出最佳PCR反应体系与反应条件,并从灵敏性、特异性、临床样品检测等方面对建立的方法进行比较与评价。结果该两种方法特异性强,与札幌病毒、轮状病毒、星状病毒、腺病毒同时检测无交叉反应,同一体系内GI、GII型诺如病毒相互之间没有干扰;常规RT-PCR最低检测限为103copies/μL,荧光定量RT-PCR最低检测限为102copies/μL;对180份临床粪便样品进行检测,常规RT-PCR则检测率为5.56%(10/180),符合率达97.22%,荧光定量RT-PCR检测率为8.33%(15/180),符合率达100%;对15份阳性样品测序分析,证实均为诺如病毒。结论建立的常规RT-PCR与荧光定量RT-PCR均可用于诺如病毒的快速检测,荧光定量RT-PCR更为灵敏。     

常规RT-PCR与荧光定量RT-PCR检测GⅠ、GⅡ型诺如病毒方法的建立及其应用

目的建立GI、GII型诺如病毒的常规RT-PCR和荧光定量RT-PCR检测方法,并对两种方法进行应用。方法优化筛选出最佳PCR反应体系与反应条件,并从灵敏性、特异性、临床样品检测等方面对建立的方法进行比较与评价。结果该两种方法特异性强,与札幌病毒、轮状病毒、星状病毒、腺病毒同时检测无交叉反应,同一体系内GI、GII型诺如病毒相互之间没有干扰;常规RT-PCR最低检测限为103copies/μL,荧光定量RT-PCR最低检测限为102copies/μL;对180份临床粪便样品进行检测,常规RT-PCR则检测率为5.56%(10/180),符合率达97.22%,荧光定量RT-PCR检测率为8.33%(15/180),符合率达100%;对15份阳性样品测序分析,证实均为诺如病毒。结论建立的常规RT-PCR与荧光定量RT-PCR均可用于诺如病毒的快速检测,荧光定量RT-PCR更为灵敏。     

检测蚊虫感染黄病毒属病毒Real-time PCR方法的建立

目的 建立SYBR Green Real-time PCR检测和筛选黄病毒属病毒方法. 方法 参照文献报道的通用引物,以JEV cDNA 和DEN cDNA为模板,建立RT-PCR和SYBR Green Real-time PCR,检测和筛选黄病毒属病毒,并比较两者的敏感性. 结果 此黄病毒属引物适合两种反应体系,SYBR Green Real-time PCR方法的敏感性是RT-PCR方法的100倍,最低检出病毒浓度为0.5×10-2 PFU/ml. 结论 建立的两种方法均可用于黄病毒属病毒检测,以SYBR Green Real-time PCR方法具有更高的敏感性,对于黄病毒属病毒初筛检测具有应用价值.     

智能等温扩增方法建立乙肝病毒核酸检测体系及评估

目的:利用智能等温扩增方法(SMAP)建立乙型肝炎病毒(HBV)核酸检测体系。方法:以pGEMX-T-X重组质粒为标准品,应用SMAP、实时荧光定量PCR法(Real-time PCR)对标准品的连续梯度稀释样品和其他几种病毒核酸提取物进行对照试验,测定SMAP对HBV核酸检测的特异性与灵敏度。结果:HBV病毒SMAP检测体系,反应最短时间15min,最优反应时间30min,无特殊仪器要求,特异性好,灵敏度10copies/μl;HBV病毒Real-time PCR检测体系,反应时间约2h,需要实时荧光定量PCR仪,特异性好,灵敏度100copies/μl。结论:SMAP方法检测速度快,无特殊仪器要求,能够提高检查效率,降低诊疗成本,值得推广应用。     

鹦鹉热嗜衣原体实时定量PCR检测方法的建立

目的建立一种简便、快速、特异的荧光定量PCR检测方法,用于鹦鹉热嗜衣原体的快速检测。方法以主要外膜蛋白(MOMP)基因为靶序列设计特异性引物,采用SYBR GreenⅠ随机渗入法建立实时定量PCR检测方法。结果循环阈值(Ct)与标准DNA模板在1.0×102-1.0×107拷贝/μl浓度范围内呈良好的线性关系,相关系数为0.989。该方法用于鹦鹉热嗜衣原体的检测具有很高的特异性,其敏感性与常规PCR相比可以提高100倍。结论本研究建立的检测鹦鹉热嗜衣原体的实时定量PCR检测方法具有很高的特异性和敏感性,可以用于鹦鹉热嗜衣原体的检测。     

禽流感病毒H1、H3、H5、N2亚型多重RT-PCR检测方法的初步研究

目的根据禽流感病毒H1、H3、H5亚型的HA基因和N2型NA基因的保守序列,设计出四对RT-PCR引物,建立一步法多重RT-PCR对禽流感H1、H3、H5、N2四亚型进行快速检测。方法利用所设计的四对引物,通过对该方法扩增条件的优化,成功建立快速检测禽流感病毒H1、H3、H5、N2四亚型的一步法多重RT-PCR。利用禽流感H1、H3、H5、N2四亚型毒株和其它相关标准毒株进行敏感性和特异性试验。结果与结论所建立的一步法多重RT-PCR具有较高的特异性和敏感性,与禽流感其它亚型和NDV、IBV、ARV?IBDV的核酸均无交叉反应。用该方法检测现场样品395份(4省20多个地区),结果与经典检测方法一致。     

尼帕病毒巢式RT-PCR方法的建立和初步应用

目的 建立灵敏、特异的检测尼帕病毒的巢式RT-PCR方法。方法 根据GenBank公布的尼帕病毒N基因序列,设计2对特异性引物(外引物和内引物),建立巢式RT-PCR方法,优化反应体系,测定特异性和灵敏度,并进行临床样品的检测。结果 该方法扩增的片段序列与源序列同源性均为100%。特异性试验结果表明本试验设计的内外侧引物不能从新城疫病毒、牛瘟病毒和日本乙型脑炎病毒中扩增出条带。敏感性试验结果表明,该方法最低能检测出的标准模板RNA浓度为39 fg/μL。100份临床样品检测结果全部为阴性。结论 初步建立了快速、灵敏、特异的检测尼帕病毒 N基因的巢式RT-PCR方法,可用于动物疫病监测和检验检疫等领域。     

Seronegative naturally acquired West Nile virus encephalitis in a renal and pancreas transplant recipient

S.A. Koepsell, A.G. Freifeld, A.R. Sambol, R.D. McComb, S.A. Kazmi. Seronegative naturally acquired West Nile virus encephalitis in a renal and pancreas transplant recipient
Transpl Infect Dis 2010: 12: 459–464. All rights reserved Abstract: West Nile virus (WNV), a single‐stranded RNA flavivirus, has spread across the United States since arriving in 1999. While asymptomatic or self‐limited in a majority of patients, WNV can cause a severe neuroinvasive disease, which occurs more often in transplant recipients with chronic immunosuppression. Diagnosis of acute WNV infection usually relies on serologic identification of immunoglobulin M (IgM) specific for the virus. We report a fatal case of naturally acquired WNV encephalitis in a renal and pancreas transplant recipient who was seronegative for WNV‐specific IgM but had detectable WNV RNA by nucleic acid amplification testing (NAAT) several weeks after the onset of symptoms. This case demonstrates the importance of using both serologic assays and NAAT for WNV in transplant recipients with the clinical suspicion of encephalitis.     

Induction of sterilizing immunity against West Nile Virus (WNV), by immunization with WNV-like particles produced in insect cells

No specific vaccine for West Nile virus (WNV) is currently available for human use. In the present study, we describe the generation of WNV-like particles (WNV-LPs) in insect cells by use of recombinant baculoviruses expressing the WNV structural proteins prME or CprME. BALB/c mice immunized with purified WNV-LPs developed WNV-specific antibodies that had potent neutralizing activities. Mice immunized with prME-like particles (prME-LPs) showed no morbidity or mortality after challenge with WNV. Immunization with prME-LPs can induce sterilizing immunity without producing any evidence of viremia or viral RNA in the spleen or brain. These results suggest that WNV-LPs hold promise as a vaccine candidate for WNV infection.     

West Nile virus: a primer for infection control professionals

In 1999, an outbreak of human West Nile encephalitis occurred in New York City. During the outbreak, 62 cases of human West Nile virus (WNV) infection were diagnosed, with 7 deaths. This was the first time that human WNV infections were detected in the Western Hemisphere. By 2002, the total number of human cases of WNV that year alone reached 4156, with 284 fatalities. In addition, investigations have shown that WNV can be acquired through organ transplantation, blood transfusion, breast milk, transplacental transmission, and occupational exposure. This article provides an overview of the 2002 WNV epidemic in the United States and reviews the epidemiology of WNV, the clinical presentation of human WNV infection, and the prevention and control of this emerging pathogen.     

West nile virus encephalitis

West Nile virus (WNV) is a small RNA virus. It was first isolated in the blood of a febrile woman in the West Nile district of Uganda in 1937. Although WNV has caused human disease in Africa and Europe since its identification, the first documented human infections occurred in the United States in 1999. Wild birds are the reservoir for WNV, and most transmission to humans occurs after the bite of an infected mosquito. In humans, 80% of infections are asymptomatic and nearly 20% cause a mild self-limiting illness called WNV fever. Less than 1% will develop central nervous system (CNS) infection, which manifests as meningitis, encephalitis, or acute flaccid paralysis. The case fatality rate for CNS infection is approximately 15%. Human vaccine is not available. Personal mosquito protection remains the best prevention, and treatment is supportive.     

First serological evidence of West Nile virus activity in horses in Serbia

West Nile virus (WNV), the most widely distributed flavivirus worldwide, has lately reemerged in Europe, causing worrisome outbreaks in humans and horses. Serological analysis by enzyme-linked immunoassay and plaque reduction neutralization test showed for the first time in Serbia that 12% of 349 horses presented specific neutralizing WNV antibodies, which in one case also cross-neutralized Usutu virus (USUV). This is the first time that anti-USUV high neutralizing antibody titers are reported in horses. All these data indicate that WNV and USUV are circulating in the region and advise on the convenience of implementing surveillance programs.     

Detection of antibodies to West Nile virus in horses, Costa Rica, 2004

We conducted a serosurvey for West Nile virus (WNV) infection in equines in Costa Rica in 2004. Antibodies to WNV were detected in 28% of the horses using an epitope blocking ELISA that is specific for WNV. WNV infection was confirmed for a subset of these sera by plaque reduction neutralization tests and Western blot. This is the first evidence of WNV activity in Costa Rica.     

In Vitro and in Vivo Evaluation of Mutations in the NS Region of Lineage 2 West Nile Virus Associated with Neuroinvasiveness in a Mammalian Model

West Nile virus (WNV) strains may differ significantly in neuroinvasiveness in vertebrate hosts. In contrast to genetic lineage 1 WNVs, molecular determinants of pathogenic lineage 2 strains have not been experimentally confirmed so far. A full-length infectious clone of a neurovirulent WNV lineage 2 strain (578/10; Central Europe) was generated and amino acid substitutions that have been shown to attenuate lineage 1 WNVs were introduced into the nonstructural proteins (NS1 (P250L), NS2A (A30P), NS3 (P249H) NS4B (P38G, C102S, E249G)). The mouse neuroinvasive phenotype of each mutant virus was examined following intraperitoneal inoculation of C57BL/6 mice. Only the NS1-P250L mutation was associated with a significant attenuation of virulence in mice compared to the wild-type. Multiplication kinetics in cell culture revealed significantly lower infectious virus titres for the NS1 mutant compared to the wild-type, as well as significantly lower amounts of positive and negative stranded RNA.     

West Nile virus antibody prevalence in red-winged blackbirds (Agelaius phoeniceus) from North Dakota, USA (2003-2004)

This study was designed to explore the role that red-winged blackbirds (Agelaius phoeniceus) may have played in disseminating West Nile virus (WNV) across the United States. Using enzyme-linked immunosorbent assays designed to detect WNV antibodies in avian species we were able to determine the WNV antibody prevalence in a cohort of red-winged blackbirds in central North Dakota in 2003 and 2004. The peak WNV antibody prevalence was 22.0% in August of 2003 and 18.3% in July of 2004. The results of this study suggest that red-winged blackbird migratory populations may be an important viral dispersal mechanism with the ability to spread arboviruses such as WNV across the United States.