套式RT—PCR方法检测临床样品中的猪流感病毒

猪流感是由猪流感病毒(Swine influenza vi rus, SIV)引起的一种呼吸道传染病,具有发病率高、死亡率低的特点,罹患流感的怀孕母猪还有并发流产的危险。猪流感已给我国养猪业带来严重危害。SIV属于正粘病毒科 A 型流感病毒属[1]。A型流感病毒可感染多种动物,一般认为,水禽是流感病毒的自然宿主和基因库,猪则是人流感病毒株与禽流感病毒株的中间宿主和混合器[1],通过基因重配产生抗原转移而导致新流感病毒株的出现。研究病毒两种受体[2],因此所有A型流感病毒都能感染猪,猪在流感病毒的生态分布中占有重要地位。目前猪流感的主要流行血…     

套式RT-PCR检测猪繁殖和呼吸综合征病毒的研究

参照ATCC VR-2332株及LV株保守区段设计了3条引物,以此建立了检测猪繁殖和呼吸综合征病毒的套式RT-PCR方法。利用其分别对ATCC VR-2332株、LV株及B13株进行套式RT-PCR,结果从3个不同地区分离的毒株中均能特异性的扩增出相应的片段,大小分别约为430bp(预期片段为430bp)、410bp(预期片段为413bp)及410bp(预期片段为413bP),而3个非PRRSV的病毒(猪瘟病毒、细小病毒及伪狂犬病毒)均未扩增出相应的片段。其敏感性可达到10^-2TCI     

西尼罗病毒的RT-PCR检测与鉴定

建立西尼罗病毒敏感、特异、快速的RT-PCR检测方法用于实验室诊断和流行病学监测。采用一步RT-PCR和套式PCR法对西尼罗病毒感染的乳鼠脑和细胞培养上清进行扩增,并对扩增产物进行序列测定。两种方法均可分别从两种组织中扩增出与预期大小相一致的片段,套式PCR法比一步RT-PCR法更为敏感,该扩增片段与西尼罗病毒埃及Eg101株相应序列的同源性为99％。     

应用套式RT-PCR检测SARS患者血液样品中SARS冠状病毒

建立了套式RT-PCR方法检测SARS患血液样品中SARS冠状病毒的方法,并检测血液样品257份。同时将RT-PCR与ELISA检测IgG、IgM的方法进行了比较。结果RT-PCR方法的总检出率为72％,IgG总检出率为68％,IgM总检出率为43％。     

单管半套式RT-PCR法检测贝类中轮状病毒的研究

轮状病毒(Rotavirus)是一种以贝类为载体的重要食源性病毒,目前来说,RT_PCR是贝类中轮状病毒最有效的检测方法,但通常由于病毒含量较低以及贝类中存在大量的PCR抑制物,致使将其运用于实际样本的检测时灵敏度仍较低。在实验室模拟自然环境,以人工播毒的方式使贝类富集轮状病毒,运用单管半套式RT_PCR法进行检测,并将单管半套式RT_PCR与ELISA、RT_PCR的检测灵敏度做了比较,表明单管半套式RT_PCR比ELISA的灵敏度高10 0 0倍,是传统RT_PCR的10倍,有时甚至10 0倍。另外,单管半套式RT_PCR法降低了实验过程中的内源性和外源性污染,使检测所需时间从6h缩短至4 5h ,大大改进和完善了食源性病毒检测方法。并同时以整只贝和仅以贝的消化道为样检测表明,以消化道为样时的病毒检出率和检测灵敏度较高。     

猪流感病毒分离鉴定

用非免疫鸡胚从我国上海、福建、湖北分离到3株猪流感病毒,分别命名为SSH1、SFJ1和SHB1.SIV分离株第5代病毒液对0.7%豚鼠红细胞的血凝活性分别1∶28、1∶27和1∶28,与H3亚型标准血清血凝抑制价1∶28、1∶27和1∶28,但不能被鸡新城疫阳性血清所抑制.纯化的病毒在电镜下观察,可见到猪流感典型的病毒粒子.病毒液接种于小白鼠,可表现出临床症状,剖杀后可观察到病毒性肺炎.猪体回归试验,可表现出临床症状和病理变化,从肺脏中可分离到病毒并且RT-PCR也可检测到病毒相应的基因片段.从纯化的病毒中提取RNA,进行RT-PCR,可扩增出预期的条带.     

应用套式PCR检测猪细小病毒

从猪细小病毒基因组的ＶＰ２基因上,选择两对引物进行套式聚合酶链反应体外扩增,产物经２％ａｇａｒｏｓｅ凝胶电泳和生物素标记的探针鉴定。证实具有特异性,敏感性实验证明,大套式ＰＣＲ能检测到１ ＴＣｉＤ５０的病毒量,在临床上应用方法能从不同的组织样品和粪便中检测出猪细小病毒,具有很高的敏感性。     

西部马脑炎病毒基因组序列的半套式RT-PCR检测

为进一步提高RT－PCR检测西部马脑炎病毒（WEE）病毒基因组方法的敏感性,采用半套式PCR扩增病毒基因组特异序列,首先采用逆转录法将病毒基因组RNA逆转录为cDNA,然后以此cDNA为模板,进行扩增。对扩增后电泳检查无可见DNA条带的产物进行半套式PCR;与此同时对扩增的循环数、Mg^ 浓度和退火温度等条件进行了优化,以进一步提高扩增的特异性。结果第一轮PCR未扩出特异笥片段的WEE病毒稀释度,其半套式扩增出特定大小的DNA产物;同时优化的条件提高了扩增产物的特异性。扩增产物约为190bp的单一DNA片段,其大小与预期的相一致,结果表明采用半套式RT－PCR方法检测WEE病毒的基因组序列的敏感性可提高100倍以上。     

用套式RT-PCR检测兰州地区肺炎患儿下呼吸道分泌物中的RSV和HPIV-3

为了调查兰州地区肺炎患儿中RSV和HPIV-3的感染状况,分别在RSV的G蛋白基因的保守序列和HPIV-3的HN基因的保守序列处,设计了套式引物,采用套式RT-PCR的方法检测了60份兰州地区肺炎患儿下呼吸道分泌物样本,结果显示,60份样本中RSV阳性为14例(23%),HPIV-3阳性为12例(20%)。并分别随机对其中的5份样本的扩增产物进行了基因序列测定,结果5份RSV阳性样本与参考株序列的同源性均大于97%,5份HPIV-3阳性样本与参考株序列的同源性大于97%。表明兰州地区下呼吸道感染患儿中,RSV和HPIV-3是常见的感染病原体。     

猪流感病毒分离鉴定

用非免疫鸡胚从我国上海、福建、湖北分离到3株猪流感病毒,分别命名为SSH1、SFJ1和SHB1。SIV分离株第5代病毒液对0．7％豚鼠红细胞的血凝活性分别1：2^8、1：2^7和1：2^8,与H3亚型标准血清血凝抑制价1：2^8、1：2^7和1：2^8,但不能被鸡新城疫阳性血清所抑制。纯化的病毒在电镜下观察,可见到猪流感典型的病毒粒子。病毒液接种于小白鼠,可表现出临床症状,剖杀后可观察到病毒性肺炎。猪体回归试验,可表现出临床症状和病理变化,从肺脏中可分离到病毒并且RT-PCR也可检测到病毒相应的基因片段。从纯化的病毒中提取RNA,进行RT-PCR,可扩增出预期的条带。     

Airborne Detection and Quantification of Swine Influenza A Virus in Air Samples Collected Inside,Outside and Downwind from Swine Barns

Airborne transmission of influenza A virus (IAV) in swine is speculated to be an important route of virus dissemination, but data are scarce. This study attempted to detect and quantify airborne IAV by virus isolation and RRT-PCR in air samples collected under field conditions. This was accomplished by collecting air samples from four acutely infected pig farms and locating air samplers inside the barns, at the external exhaust fans and downwind from the farms at distances up to 2.1 km. IAV was detected in air samples collected in 3 out of 4 farms included in the study. Isolation of IAV was possible from air samples collected inside the barn at two of the farms and in one farm from the exhausted air. Between 13% and 100% of samples collected inside the barns tested RRT-PCR positive with an average viral load of 3.20E+05 IAV RNA copies/m³ of air. Percentage of exhaust positive air samples also ranged between 13% and 100% with an average viral load of 1.79E+04 RNA copies/m³ of air. Influenza virus RNA was detected in air samples collected between 1.5 and 2.1 Km away from the farms with viral levels significantly lower at 4.65E+03 RNA copies/m³. H1N1, H1N2 and H3N2 subtypes were detected in the air samples and the hemagglutinin gene sequences identified in the swine samples matched those in aerosols providing evidence that the viruses detected in the aerosols originated from the pigs in the farms under study. Overall our results indicate that pigs can be a source of IAV infectious aerosols and that these aerosols can be exhausted from pig barns and be transported downwind. The results from this study provide evidence of the risk of aerosol transmission in pigs under field conditions.     

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.     

Detection and Isolation of Swine Influenza A Virus in Spiked Oral Fluid and Samples from Individually Housed,Experimentally Infected Pigs: Potential Role of Porcine Oral Fluid in Active Influenza A Virus Surveillance in Swine

Background

The lack of seasonality of swine influenza A virus (swIAV) in combination with the capacity of swine to harbor a large number of co-circulating IAV lineages, resulting in the risk for the emergence of influenza viruses with pandemic potential, stress the importance of swIAV surveillance. To date, active surveillance of swIAV worldwide is barely done because of the short detection period in nasal swab samples. Therefore, more sensitive diagnostic methods to monitor circulating virus strains are requisite.

Methods

qRT-PCR and virus isolations were performed on oral fluid and nasal swabs collected from individually housed pigs that were infected sequentially with H1N1 and H3N2 swIAV strains. The same methods were also applied to oral fluid samples spiked with H1N1 to study the influence of conservation time and temperature on swIAV infectivity and detectability in porcine oral fluid.

Results

All swIAV infected animals were found qRT-PCR positive in both nasal swabs and oral fluid. However, swIAV could be detected for a longer period in oral fluid than in nasal swabs. Despite the high detectability of swIAV in oral fluid, virus isolation from oral fluid collected from infected pigs was rare. These results are supported by laboratory studies showing that the PCR detectability of swIAV remains unaltered during a 24 h incubation period in oral fluid, while swIAV infectivity drops dramatically immediately upon contact with oral fluid (3 log titer reduction) and gets lost after 24 h conservation in oral fluid at ambient temperature.

Conclusions

Our data indicate that porcine oral fluid has the potential to replace nasal swabs for molecular diagnostic purposes. The difficulty to isolate swIAV from oral fluid could pose a drawback for its use in active surveillance programs.     

基于杂交链式(HCR)反应的甲型流感病毒检测技术研究

甲型流感病毒的现场快速检测对于流感的及时有效防控具有重要意义．本研究基于杂交链式(HCR)反应,利用GO对荧光基团的猝灭作用及共同实现了对甲型流感病毒的快速检测．当目标序列存在时,可引发HCR反应,使短链DNA形成长链,保护FAM荧光基团不被猝灭,从而实现目标物的检测．实验结果表明,该方法在10～40 nmol/L范围内荧光强度与目标检测物浓度表现出了良好的线性关系,检测范围为5～100 nmol/L．这种HCR等温扩增检测技术具有较好的样本检测能力,具有等温、无酶、反应体系简单、操作步骤简便等优点,表现出良好的现场检测应用前景．     

H13亚型禽流感病毒实时荧光定量PCR检测方法的建立及其应用

H13亚型禽流感病毒以往只在海鸥、鸭等水禽中被发现,2018年,我们实验室报道了蛋鸡感染H13亚型禽流感病毒的证据,表明H13亚型禽流感病毒存在从水禽中溢出感染陆禽的风险,因而有必要加强对H13亚型禽流感病毒的监测。实时荧光定量PCR由于具有特异性强、灵敏度高、检测时间短等优点,在病原监测工作中得到了广泛应用。本研究以H13亚型禽流感病毒的HA基因作为靶基因,设计了一对特异性荧光定量PCR检测引物,通过特异性试验、敏感性试验等,建立了H13亚型禽流感病毒实时荧光定量PCR检测方法,并应用建立的检测方法对海鸥粪便样品进行核酸检测。实验结果发现,该检测方法的灵敏度为1.60×10⁰拷贝/μL;该检测方法对H1、H3、H5、H6、H7和H9等6个亚型的禽流感病毒无交叉反应;该检测方法的敏感性是常规PCR的10倍;海鸥粪便样品中H13亚型禽流感病毒的核酸检测阳性率为18.8%。我们建立的H13亚型禽流感病毒实时荧光定量PCR检测方法具有灵敏度高、特异性强等特点,该检测方法可用于大量临床样品的快速检测,为H13亚型禽流感病毒监测工作的顺利开展提供了技术支撑。     

Epidemic Status of Swine Influenza Virus in China

As one of the most significant swine diseases, in recent years, swine influenza (SI) has had an immense impact on public health and has raised extensive public concerns in China. Swine are predisposed to both avian and human influenza virus infections, between that and/or swine influenza viruses, genetic reassortment could occur. This analysis aims at introducing the history of swine influenza virus, the serological epidemiology of swine influenza virus infection, the clinical details of swine influenza, the development of vaccines against swine influenza and controlling the situation of swine influenza in China. Considering the elaborate nature of swine influenza, a more methodical surveillance should be further implemented.