猪繁殖与呼吸综合征检测与监测技术要点

近年来,mNGS作为新一代的高通量测序诊断工具,在多种病原混合感染检测和毒株在新发突发感染及混合感染等复杂病例诊断中具有极高的临床参考价值,在PRRS诊断和追踪猪群是否有新的PRRSV流行毒株、评估驯化效果等方面具有良好的应用前景。     

猪PRRSV和PCV2混合感染的诊断及全基因组遗传进化分析

为查明某猪场病猪的死亡原因，对该场的典型病死猪进行病理剖检，无菌采集肺脏、肾脏、脾脏和淋巴结病料，提取核酸，进行荧光定量PCR检测和高通量测序，最后确诊该猪场的发病原因为PRRSV和PCV2的混合感染。对PRRSV和PCV2全基因组进行遗传进化分析，发现检测到的PRRSV属于VR2332-like亚群，与国内外26株参考毒株的核苷酸同源性为61.7%～99.8%;PCV2属于PCV2d基因型，与国内外31株参考毒株的同源性为54.7%～99.7%。     

绿脓杆菌焦磷酸测序检测方法的建立

本试验旨在利用绿脓杆菌的序列信息分析和焦磷酸测序技术,建立一种快速、简单检测绿脓杆菌的方法。从培养的绿脓杆菌中提取DNA,PCR扩增目的基因片段,采用焦磷酸测序技术(pyrosequencing technology,PSQ)针对保守核苷酸区段的测序分析。通过焦磷酸测序后获得的序列信息与已知的目的基因的序列比对,能进一步确证毒株的序列信息为绿脓杆菌。利用焦磷酸测序能快速获取核酸的序列信息,可为毒株及早确证奠定基础。     

高通量测序分析1株H7N9流感病毒的全基因

为探索利用高通量测序开展H7N9亚型流感的监测、全基因分析和溯源研究的方法,使用Ion Torrent PGM测序仪对1株H7N9流感病毒进行了全基因测序,分析了此毒株的基因组特性和进化特征。结果显示,该毒株与2013年引起我国华东地区疫情的H7N9流感毒株的亲缘关系较近;纵观在不同年代和地点分离的H7N9流感病毒的6个内部基因,2013年疫情之前和之后的H7N9流感病毒存在明显差异。     

鹅副粘病毒F基因的克隆测序及核苷酸序列分析

以鹅副粘病毒 (GPV) SF0 2株的基因组 RNA为模板 ,通过 RT- PCR的方法扩增出 F基因片段 ,然后将其克隆至 T载体中。经 PCR鉴定后 ,对阳性克隆进行测序。测序后拼接得出 F基因的全序列。与 Gen Bank下载的 9株参考毒株比较 F基因编码区全核苷酸序列 ,发现所测 GPV- SF0 2毒株与参考毒株 L Z- NDV核苷酸序列同源性为 96 %,与F48E9同源性为 92 .5 %,与 L a Sota为 88%。     

应用锚定高通量测序技术对家畜粪便中线虫进行检测的初步探索

本研究对青海省内采集的牦牛和藏羊粪便样本中线虫进行了锚定高通量测序检测和PCR检测验证。结果表明,锚定高通量测序结果显示有效物种OTU共156条。物种数据库注释的有33种生物,包括已知动植物线虫(37.34%)、已知细菌(10.44%)和未知生物种(2.61%)。其中已知的动物线虫以钩口属为主,其次是血矛属。针对钩虫,PCR检测结果显示：海晏县阳性率最高(25%),其次是泽库县(8.82%)和河南县(6.9%),贵南县样本中钩虫阳性率为0。结果提示本研究中的锚定高通量测序方法的检测结果中尽管存在细菌,但客观上起到了缩小被检物种范围的作用,这为开展群体性高通量寄生虫检测的深入研究提供了参考资料。     

鹅副粘病毒HN基因的克隆与序列分析

以鹅副粘病毒GPV-SF02毒株的基因组RNA为模板，通过RT-PCR方法扩增出HN基因片段，然后将其克隆至T载体中。经PCR鉴定后，对阳性克隆进行测序；测序后拼接得出HN基因的全序列。经分析HN基因编码区核苷酸序列，所测GPV-SF02毒株与参考毒株NL-96核苷酸序列的同源性为86%。     

云南牟定狂犬病病毒株N基因和G基因序列分析

自英国引进3株荧光素标记的针对狂犬病毒核蛋白的单克隆抗体,建立狂犬病直接免疫荧光诊断技术。根据已知狂犬病毒核蛋白和糖蛋白基因序列,参照已发表文献,设计合成4对PCR引物和2对克隆引物,以弱毒疫苗毒株为阳性对照,建立狂犬病RT-PCR及套式PCR诊断技术。自牟定采集发病犬脑组织样品15份,免疫荧光及PCR诊断结果均为阳性。对病毒N基因和G基因全序列经RT-PCR扩增,克隆至pMD18-T载体进行测序（登录号分别为：EU095330、EU253477）,并与已知代表毒株对应序列进行比对及系统发育分析,结果表明：云南牟定狂犬病毒属于基因Ⅰ型和血清Ⅰ型毒株,与近年从广西、浙江、江苏、湖北等省分离的毒株遗传关系密切,在系统发育树中形成同一小分支并均属于亚组群Ⅱ毒株。     

羊传染性脓疱口炎病毒株的分离鉴定

利用牛肾(Madin-Darby bovine kidney,MDBK)细胞对上海市崇明区某羊场的痂皮病例进行病毒分离,获得一株羊口疮病毒(orf virus,ORFV),命名为ORFVSH-01。参考NCBI数据库中ORFV的保守基因B2L的序列,设计一对特异性引物,进行PCR扩增并测序。结果表明：接种病料悬液的MDBK细胞出现细胞病变,成功分离到毒株;经PCR扩增和测序,分离的毒株为ORFV,命名为ORFVSH-01。     

Mhp强弱毒株P97基因的克隆与序列测定

利用聚合酶链式反应(PCR)技术扩增出猪肺炎支原体弱毒株R659及强毒株F19的P97基因，PCR产物经纯化后，克隆至载体pGEM-Teasyvector，转化DH5a感受态细胞，筛选阳性克隆，提取质粒，对目的片段测序，结果表明：R659的P97全基因大小为3416bp，F19的P97全基因大小为3329bp，强弱毒株间核苷酸同源性为96．9％，弱毒株R659与标准株232A核苷酸同源性为95．6％，强毒株F19与232A株核苷酸同源性为95．1％。     

石河子地区宠物犬蜱的种类鉴定及其携带布鲁氏菌的检测

为了解新疆石河子地区宠物犬体表寄生蜱的种类及蜱携带布鲁氏菌情况,本试验在形态学分类的基础上,基于线粒体基因12S rRNA和细胞色素氧化酶Ⅰ（COⅠ）对宠物犬体表采集寄生蜱进行分子生物学检测,使用DNAMAN软件比对分析序列同源性,并应用序列分析软件Mega 7.0邻接法构建蜱种系统发育进化树,分析蜱种的遗传进化关系;基于布鲁氏菌外膜蛋白Omp22基因对宠物犬体表寄生蜱进行布鲁氏菌PCR检测,确定宠物犬蜱布鲁氏菌携带情况。结果显示,宠物犬体表寄生的436只蜱的形态学与线粒体基因（12S rRNA和COⅠ）分子生物学鉴定结果一致,均为新疆优势蜱种之一——图兰扇头蜱（Rhipicephalus turanicus）。基于12S rRNA基因构建的蜱种系统发育树显示,本试验所得宠物犬体表寄生图兰扇头蜱序列与GenBank中已知图兰扇头蜱的序列聚为一大支。基于布鲁氏菌Omp22基因的巢式PCR扩增结果显示,宠物犬体表寄生图兰扇头蜱携带布鲁氏菌的阳性率为4.82%（21/436）,且同源性为100%。BLAST比对显示,本试验所得宠物犬图兰扇头蜱携带的布鲁氏菌与新疆流行株YC31（GenBank登录号：MK201679.1）、QH5（GenBank登录号：MK201678.1）、EM3（GenBank登录号：MK201677.1）和ML9（GenBank登录号：MK201676.1）的同源性均为100%。本研究通过形态学及分子生物学探讨了新疆石河子地区宠物犬体表寄生蜱的种类及携带布鲁氏菌基本情况,为宠物犬体表寄生蜱的种类及蜱传疾病的监测和控制等研究工作提供了基础资料。     

国内部分省市肉兔及蜱虫携带土拉杆菌的流行病学调查

本调查旨在探明土拉杆菌在国内部分省份的流行情况。利用本实验室建立的土拉杆菌通用及亚种PCR检测研究方法，对国内肉兔饲养密度比较大的省份和牛羊存栏的部分省市开展了肉兔和蜱虫中土拉杆菌的检测，并对阳性样本进行了土拉杆菌亚种的鉴定和基因测序验证。研究结果表明，肉兔肝脏组织和牛羊携带蜱虫中土拉杆菌DNA检测阳性，其中218份肉兔样本中12份阳性（阳性率5.5%），490份蜱虫样本中15份阳性（阳性率3.1%）；从地区分布来看，山东、河南、四川肉兔病料检测阳性，云南、山东蜱虫阳性率高；PCR检测显示其亚种为F.h，为土拉杆菌毒力较强的亚种。本次调查显示土拉杆菌在肉兔、蜱虫中存在，山东、云南地区存在土拉杆菌病公共卫生安全风险，应引起有关科研院校、医疗单位和政府部门的高度重视。     

Epidemiological Investigation of Francisella tularensis Transmitted by Rabbits and Ticks in Some Provinces of China

The aim of this investigation was to find out the prevalence of Francesella tularensis in some provinces of China. Using the general and subspecies PCR detection method for Francesella tularensis established in our laboratory, PCR detection of Francesella tularensis in rabbits and ticks was carried out in provinces with high-density rabbits feeding and some provinces and cities with sheep and cattle stock. The results showed that DNA detection of Francesella tularensis from rabbit tissues and ticks carried by cattle and sheep was positive. 12 out of 218 rabbit samples were positive (5.5% positive rate), and 15 tick samples were positive (3.1%) of 490 tick samples. In terms of geographical distribution, most of the positive rabbit samples were from Shandong, Henan and Sichuan province, however, ticks collected from Yunnan and Shandong province showed a higher positive rate. PCR detection showed that Francesella tularensis subspecies in this investigation was F.h, which was a subspecies with strong toxicity. This investigation revealed that Francesella tularensis presented in rabbits and ticks, public health and safety risks of Francesella tularensis existed in Shandong and Yunnan province, which should be paid more attention by the relevant scientific research institutions, medical institutions and government departments.     

Determination of an efficient and reliable method for DNA extraction from ticks

Molecular detection of pathogenic microorganisms in ticks is based on DNA amplification of the target pathogen; therefore, extraction of DNA from the tick is a major step. In this study, we compared three different tick DNA extraction protocols based on an enzymatic digestion by proteinase K followed by DNA extraction by a commercial kit (method 1), or on mortar crushing, proteinase K digestion and phenol/chloroform DNA extraction (method 2) and fine crushing with a beads beater, proteinase K digestion and DNA extraction using a commercial kit (method 3). The absence of PCR inhibitors and the DNA quality were evaluated by PCR amplification of the tick mitochondrial 16S rRNA gene using tick-specific primers. With method 1, 23/30 (77%) of the samples were extracted; with method 2, 30/31 (97%) of the samples were extracted and with method 3, 30/30 (100%) of the samples were extracted. DNA extraction efficiency using method 3 is significantly higher than DNA extraction efficiency using method 1 (100% versus 77%, P < 0.05). There was no significant difference between methods 2 and 3. Method 3 was however more adapted to cohort studies than method 2. This technique was validated for cohort tick DNA extraction and applicable to the treatment of small samples such as nymphs and soft ticks with 100% efficiency.     

铁蛋白1基因在边缘革蜱各发育龄期、吸血期及不同器官中的表达分析

试验旨在了解铁蛋白1（ferritin 1,Fer1）基因在边缘革蜱各发育龄期、吸血期及不同器官中的表达情况。用实时定量荧光PCR法分析了Fer1基因在边缘革蜱各发育阶段、吸血期和不同器官中的相对表达情况,以饥饿雌蜱为基准（1 FC）,用2^-△△Ct法计算结果。结果显示,雌性蜱在开始吸血后第72 h Fer1基因的相对表达量最高,在刚刚孵化的饥饿成蜱（雌、雄）、卵及幼蜱中相对表达量较低;在若蜱中相对表达量较高,其中,幼蜱和若蜱在饥饿状态下比同时期饱血蜱的相对表达量高。在雌蜱的不同器官中,以饱血蜱唾液腺为基准（1 FC）,半饱血蜱中肠的表达量最高,在卵巢和马氏管不表达;在饱血蜱唾液腺、中肠、卵巢和马氏管中均表达,其中,在卵巢和马氏管中的表达量较高,中肠次之,唾液腺中的表达较低。试验结果表明,边缘革蜱体内Fer1基因的相对表达量随着蜱吸血过程变化而变化,这与蜱体内消化细胞的完善及消化酶在该过程中的变化相关,可为该类蜱铁代谢通路中关键功能基因的研究提供参考。     

Prevalence of tick-borne encephalitis virus in ticks from southern Korea

The prevalence of tick-borne encephalitis virus (TBEV) in southern Korea was determined by collecting ticks using tick drags. A total of 4,077 of 6,788 ticks collected were pooled (649 pools) according to collection site, species, and developmental stage and assayed for TBEV. The TBEV protein E and NS5 gene fragments were detected using RT-nested PCR in six pools of nymphs collected from Jeju Island (2,491 ticks). The minimum field detection rates for TBEV were 0.17% and 0.14% for Haemaphysalis longicornis and Haemayphysalis flava nymphs, respectively. The 252 bp NS5 and 477 bp protein E gene amplicons were sequenced. Phylogenetic analysis showed that the NS5 and protein E genes of the Jeju strain were clustered with Western subtype (98.0% and 99.4% identity, respectively). The Western subtype of TBEV is endemic in Korea, including Jeju Island. The study of vector and zoonotic host susceptibility to TBEV is required to better understand its potential impact on public health.     

PCR-based detection of Theileria ovis in Rhipicephalus bursa adult ticks

Tick-borne diseases in ruminants are common in tropical and subtropical regions and lead to meat and milk production losses. In this study, polymerase chain reaction (PCR) was used to assess the presence of Theileria ovis in Rhipicephalus bursa ticks. We have demonstrated that the PCR enabled detection of T. ovis in field isolates of R. bursa collected from naturally infested sheep and goats in eastern Turkey. The sampling was done in spring season (between May and June 2004). A total of 420 R. bursa were collected and randomly selected 192 number of them (97 female and 95 male) were dissected. Primers specific for 520 bp fragments small subunit ribosomal RNA (ssu rRNA) gene of T. ovis amplified products from 37 of the 192 (19.27%) samples. The parasite was detected in 17 (17.52%) female and in 20 (21.05%) male ticks. Two T. ovis amplicons from the tick samples were purified and sequenced. The resulting sequences were identical to the nucleotide sequence of the Turkish sheep strain of T. ovis. These results showed that R. bursa might play an important role in the field as a natural vector of T. ovis.     

Detection of rickettsial DNA in ixodid ticks recovered from dogs and cats in Japan

DNA from ticks recovered from 1137 dogs and 133 cats from all over Japan were examined for Rickettsia infection by citrate synthase gene (gltA)-based PCR and partial nucleotide sequencing. A total of 91 dog tick samples and 18 cat tick samples showed a single band of the appropriate size in the nested PCR. Sequence analysis was successfully performed on 102 samples. DNA of Rickettsia japonica or closely related Rickettsia spp. strains were detected from 38 ticks in 16 prefectures mainly in western Japan. The other 33, detected from 13 prefectures including Hokkaido and Okinawa, were found to be Rickettsia helvetica or closely related strains. A total of 29 DNA that showed highest homology with Rickettsia akari or closely related strains were detected in 19 prefectures, widespread throughout Japan. Rickettsia canada-like DNA was detected from Haemaphysalis sp. removed from a dog in Fukuoka, and ;Candidatus Rickettsia tarasevichiae'-like DNA was from Ixodes sp. removed from a dog in Hokkaido.     

荧光定量PCR检测硬蜱体内莱姆病螺旋体的研究

为建立一个荧光定量PCR检测硬蜱体内莱姆病螺旋体的方法,根据GenBank登录的莱姆病螺旋体鞭毛蛋白FlaB序列,应用生物学软件进行序列比对,在保守的C段区设计与筛选特异引物和TaqMan探针。对荧光定量PCR反应体系与条件进行优化,验证方法的特异性、敏感性,并通过对感染螺旋体的蜱样本的检测,评价该方法的实用价值。结果显示,自然感染莱姆病螺旋体的35份蜱标本检测阳性符合率100%,正常蜱20份标本的检测结果均为阴性。该方法对牛巴贝斯原虫、泰勒原虫、边缘无浆体、金龟子绿僵菌、大肠杆菌等蜱体常见病原微生物所抽提的DNA的检测均呈阴性。荧光定量PCR方法检测质粒的灵敏度可达1×102拷贝/μL。TaqMan荧光定量PCR方法检测硬蜱体内莱姆病螺旋体具有较好的敏感性和特异性,可适于莱姆病的流行病学调查和监控。     

Identification of the etiologic agent of epizootic bovine abortion in field-collected Ornithodoros coriaceus Koch ticks

Epizootic bovine abortion (EBA), or foothill abortion as it has often been termed, is a tick-borne disease of pregnant cattle recognized in California, Nevada and Oregon. The primary objective of this study was to better define the relationship of a novel deltaproteobacterium, the putative etiological agent of EBA (aoEBA), with the Pajaroello tick (Ornithodoros coriaceus Koch), the recognized vector of EBA. Three developmental stages of O. coriaceus (larva, nymph, and adult) were collected from five locations in California, Nevada and Oregon. A polymerase chain reaction (PCR), developed for detection of aoEBA, was applied to DNA extracted from ticks. Southern blotting of the PCR products increased the number of ticks determined to be carrying the bacteria by seven-fold, suggesting the majority of infected ticks carry relatively low numbers of the pathogen. An effort was made to determine if an artificial blood meal would stimulate replication of the bacterial pathogen, thereby increasing the frequency in which aoEBA could be identified; no statistically significant effect was evident. The number of ticks determined to be carrying aoEBA varied with geographic location and ranged from 5 to 20%. aoEBA was found in both adults (12% of the males and 12% of the females) and nymphs (13%) but not larvae. Comparative analysis of dissected ticks provided strong evidence that the salivary gland was the most common location of aoEBA in field-collected ticks. No significant correlations were identified between the frequency of infection and tick weight, suggesting that increasing tick age and increased number of blood meals did not increase infectivity.