5种常见犬腹泻病毒基因芯片检测方法的建立

为建立一种简便、快速、高效的可同时区分犬腺病毒1型（canine adenovirus type 1,CAV-1）、犬腺病毒2型（canine adenovirus type 2,CAV-2）、犬冠状病毒（canine coronavirus,CCV）、犬瘟热病毒（canine distemper virus,CDV）、犬细小病毒（canine parvovirus,CPV） 5种常见犬腹泻病毒的基因芯片诊断方法,本研究以CAV-1、CAV-2、CCV、CDV、CPV 5种犬腹泻病毒为靶病毒,根据NCBI上收录的病毒基因序列在其保守区域内设计引物,在此基础上根据变异区域设计针对每种病毒的探针2~3条,优化检测体系的各反应条件,确定该检测方法的特异性与敏感性,建立可同时区分5种病毒的基因芯片检测方法。结果显示,建立的基因芯片检测方法可同时检测以上述5种犬腹泻病毒,其中PCR的退火温度为55℃、延伸时间为1 min 15 s;探针与PCR产物的杂交温度40℃、杂交时间2.5 h时,该方法对CAV-1、CAV-2、CCV、CDV和CPV 5种病毒标准品的检测限分别为0.2 fg/μL、2 fg/μL、2 fg/μL、20 pg/μL和0.02 fg/μL,具有较高的灵敏性;同时对犬副流感病毒进行特异性试验,发现无阳性信号出现,具有较强的特异性;对14份临床腹泻样品检测结果显示,基因芯片方法对CAV-1、CAV-2、CCV、CDV和CPV 5种病毒的阳性检出率分别为28.57%、50.00%、64.28%、14.28%和85.71%,并且基因芯片检测方法的敏感性较PCR要高10~100倍。以上结果表明,本研究建立的基因芯片检测方法具有特异、敏感等特点,对临床中犬类混合感染病毒检测具有一定的诊断意义。     

Establishment and Application of TaqMan MGB Fluorescent-Quantitative PCR Assay for Detection of Canine Parvovirus

In order to establish a TaqMan MGB fluorescent-quantitative PCR (FQ-PCR) assay for detecting canine parvovirus (CPV) specifically, sensitively and rapidly, a highly sensitive and specific TaqMan MGB FQ-PCR assay was developed using the specific primers and TaqMan MGB probe designed basing on the conservative sequences of VP2 gene of CPV in GenBank. The sensitivity, specificity and repetition assay of FQ-PCR assay were tested, and 46 clinic suspicious CPV infected samples were detected by the FQ-PCR assay in contrast to the routine PCR method. The results indicated that the FQ-PCR was successfully established. The developed FQ-PCR assay was able to detect as little as 1×10¹copies/μL of recombinant pGEX-T/CPV plasmid DNA, and the sensitivity of which was 100 times more than that of the routine PCR. The specificity assay exhibited that positive signals could be obtained from recombinant pGEM-T/CPV plasmid, but not from the genomic DNA or total cDNA of the other 5 kinds of pathogenic microorganism acting as the controls. The repetition tests were carried out by detection repeated 3 times for 3 different concentrations of recombinant pGEX-T/CPV plasmid, and the results indicated that the FQ-PCR was reproducible. Twenty-three positive results from 46 clinic suspicious CPV infected samples were obtained, which showed the better sensitivity than that of the routine PCR, with 19 positive samples from the same 46 suspected samples. The study suggested that the CPV FQ-PCR method was successfully established, and suitable for clinic rapid diagnosing of CPV and early detection of latent infection.     

同时检测犬瘟热病毒和犬细小病毒双重PCR方法的建立

为建立可以同时检测犬瘟热病毒(CDV)和犬细小病毒(CPV)的双重PCR方法,本研究根据GenBank登录的CDV N蛋白序列和CPV NS基因保守序列,设计合成2对特异性引物。通过优化反应条件,对CDV阳性病毒株反转录后的cDNA模板和CPV的DNA模板进行双重PCR扩增,同时得到2条与试验设计相符的669 bp(CDV)和392 bp(CPV)特异性条带,建立了同时检测CDV和CPV的双重PCR方法。实验结果表明:在同一PCR反应体系中可以同时检测这2种病毒,而对犬腺病毒Ⅰ型、犬腺病毒Ⅱ型、狂犬病毒检测均为阴性;CDV和CPV的最低检出限分别为101.8TCID50和101.4TCID50。采用该方法对在黑龙江省不同地区所采集的30份犬病料样品进行检测,CDV阳性率为30%;CPV阳性率为23.33%,表明建立的PCR方法可以用于临床诊断。     

鉴别非洲猪瘟病毒和猪瘟病毒野毒株二重TaqMan MGB实时荧光定量PCR检测方法的建立及初步应用

旨在建立特异、敏感的实时荧光定量PCR（FQ-PCR）方法,用于非洲猪瘟病毒（ASFV）和猪瘟病毒（CSFV）野毒株的快速鉴别检测。针对ASFV的P72基因和CSFV野毒株的5'UTR非编码区序列的保守区域分别设计1对特异性引物和1条探针,经优化反应条件,建立一种基于TaqMan MGB探针技术的FQ-PCR方法,验证方法的敏感性、特异性和稳定性,对50份临床样品进行检测,并与猪瘟国标方法及OIE推荐的非洲猪瘟检测方法进行比较分析。结果显示：建立的鉴别ASFV和CSFV野毒株二重FQ-PCR检测方法在10⁰~10⁶拷贝·μL^-1模板范围内有良好的线性关系;对ASFV和CSFV基因出现阳性扩增信号,但对猪瘟病毒疫苗株、猪繁殖与呼吸综合征病毒、猪伪狂犬病病毒、猪圆环病毒2型、猪细小病毒、猪乙型脑炎病毒、副猪嗜血杆菌等病原对照未出现扩增;批内、批间试验变异系数在1.18%~2.08%,重复性良好;对ASFV和CSFV的最低检测模板浓度均为10拷贝·μL^-1;利用建立的二重FQ-PCR方法对50份临床样品进行检测,检测结果与猪瘟国标方法及OIE推荐的非洲猪瘟检测方法结果完全一致。本研究成功建立了鉴别ASFV和CSFV野毒株二重TaqMan MGB FQ-PCR方法,为ASFV和CSFV野毒株的鉴别诊断提供了快速、敏感、特异且能满足临床检测需求的检测方法。     

化脓隐秘杆菌TaqMan实时荧光定量PCR检测方法的建立

本试验旨在建立检测化脓隐秘杆菌(Arcanobacterium pyogenes,A.pyogenes)特异、灵敏的TaqMan实时荧光定量PCR检测方法。根据GenBank公布的化脓隐秘杆菌溶血素(pyolysin,PLO)基因高保守序列,设计特异性引物和探针建立检测体系,用于化脓隐秘杆菌的快速检测,并对该方法的特异性和灵敏度进行检测。结果显示,本试验建立的TaqMan实时荧光定量PCR方法仅对化脓隐秘杆菌的检测结果为阳性;该方法最低检测DNA浓度为77.6 fg,最低检测细菌浓度为63 CFU/mL。采用本研究建立的方法检测23份林麝临床病例样品,共鉴定出16株化脓隐秘杆菌,与API Coryne生化鉴定方法的结果相同。本研究为化脓隐秘杆菌的检测提供了一种灵敏、特异、快速的检测方法,其可用于化脓隐秘杆菌的诊断和流行病学调查。     

Establishment and Application of Real-time Quantitative PCR Assay for Detection of Classical Swine Fever Virus

A Real-time quantitative PCR assay for detection of classical swine fever virus (CSFV) was developed using the specific probe and primers designed basing on the E2 gene of CSFV. The Real-time quantitative PCR assay was established using the total RNA of CSFV as template. The specificity, sensitivity and repeatability of the assay were tested, and samples taken from clinic suspicious CSFV infected pigs had been testified by the established assay. The results indicated that the Real-time quantitative PCR assay was successfully established, and showed a good linear relationship at a template range of 10¹ to 10⁶ copies/μL with a coefficient correlation of 0.999; The specificity of the assay revealed that amplifications were showed on CSFV samples, but other pathogens had no amplifications; The sensitivity of the assay was 10 copies/μL nucleic acid and 1 TCID₅₀/mL virus; Meanwhile,19 positive samples were detected, which were consistent with results of CSFV detected by Nested RT-PCR, cloning and sequencing. The eatablished Real-time quantitative PCR assay was specific, sensitive rapid and suitable for early detection and epidemiological study of CSFV.     

Establishment of TaqMan Real-time PCR Assay for Detection of Arcanobacterium pyogenes

The study was aimed to establish a specific and sensitive TaqMan Real-time PCR assay for detection of Arcanobacterium pyogenes (A.pyogenes).Based on the conservative sequence of pyolysin (PLO) gene of A.pyogenes published in GenBank, specific primers and TaqMan probes were designed. The TaqMan Real-time PCR assay was established, and the specificity and sensitivity were tested.The specificity test results showed that only A.pyogenes exhibited typical curves.The detection sensitivity of this assay was 77.6 fg genomic DNA per 20 μL reaction, and 63 CFU/mL for pure cultures.16 out of 23 clinical samples were positive detected by the TaqMan Real-time PCR assay, which were consistent with API Coryne identification.The TaqMan Real-time PCR assay developed in this study was specific and sensitive for detection of A.pyogenes, and it could be used for identification and epidemiological investigation of A.pyogenes.     

猪瘟病毒实时荧光定量PCR检测方法的建立与应用

为建立一种快速、敏感、特异的猪瘟病毒（classical swine fever virus,CSFV）实时荧光定量PCR检测方法,本研究根据GenBank中CSFV E2基因保守区域序列,设计了一对特异性引物和一条特异性探针,以CSFV总RNA为反转录模板,经优化反应条件,建立CSFV实时荧光定量PCR检测方法,并对其进行了特异性、敏感性、重复性试验;利用所建立的方法对35份临床疑似CSFV感染样品进行了检测。结果表明,本研究建立的CSFV实时荧光定量PCR检测方法在10¹~10⁶拷贝/μL范围内有很好的线性关系,相关系数为0.999;CSFV细胞培养物出现阳性扩增信号,但ST正常细胞对照和其他8种病原对照未出现扩增,特异性良好;该方法重复性好、敏感性高,最低检测模板浓度为10拷贝/μL,并且CSFV的最低检测限为1 TCID₅₀/mL;自35份疑似CSFV感染样品中检出19份阳性样品,与本课题组建立的CSFV Nested RT-PCR检测结果和克隆测序结果一致。本研究成功建立了CSFV实时荧光定量PCR检测方法,可用于CSFV的快速检测。     

成都地区宠物犬感染犬瘟热病毒和犬呼吸道冠状病毒的分子流行病学调查

为了解成都地区宠物犬犬瘟热病毒（canine distemper virus,CDV）和犬呼吸道冠状病毒（canine respiratory coronavirus,CRCoV）的感染情况,本试验应用RT-PCR对采自成都地区8家动物医院共计420份出现呼吸道症状的宠物犬鼻腔棉拭子样本进行分子检测。结果发现,从420份样本中,检出213份CDV阳性,检出率为50.71%;检出247份CRCoV阳性,检出率为58.81%;CDV和CRCoV混合感染的检出率为41.19%。表明成都地区宠物犬感染CDV和CRCoV较为严重,且二者混合感染率较高。宠物犬CDV和CRCoV的检出率与年龄、性别、品种、季节和免疫状况等因素的关系存在不同程度的差异。其中,1~3月龄幼犬检出率最高,分别为74.40%和79.20%;纯种犬的检出率较其他犬种高,分别为59.37%和62.22%;春季CDV的检出率较高,为56.19%,而冬季CRCoV的检出率较高,为67.59%;未免疫犬的检出率较高,分别为64.42%和63.46%。该研究丰富了成都地区宠物犬CDV和CRCoV的流行病学资料,为该地区宠物犬CDV和CRCoV的诊断及防控提供了基本数据。     

犬源牛犬细小病毒和犬圆环病毒二联PCR检测方法的建立及应用

本研究旨在建立可同时检测犬源牛犬细小病毒(CBoV)和犬圆环病毒(CCV)的二联PCR检测方法,并对两种病毒病当前的流行情况进行监测和调查.分别将已发表的CBoV和CCV基因组序列进行同源性比对,选择高同源区段,应用Primer Primier 5计算机软件设计并合成了2对特异性扩增引物,目的片段大小分别为170 bp...     

环介导等温扩增技术快速检测犬细小病毒方法的建立

旨在建立一种利用环介导等温扩增技术(LAMP)检测犬细小病毒感染的新方法。根据Gen-Bank中犬细小病毒(CPV)VP2基因序列,设计4条LAMP特异性引物,对反应条件、特异性、敏感性、可视化效果和应用效果进行研究。结果显示,在65℃等温条件下、1h内可完成LAMP扩增过程;病毒的最低检出限量为10-2个TCID50/mL;特异性和可视效果良好;对36份临床标本进行检测,阳性检出率为80.5%(29/36),检出率高于普通PCR 72.2%(26/36)。建立的LAMP检测方法,显示了较高的特异性和敏感性,而且兼具高效、快捷、可视化的优势,为临床检测犬细小病毒感染提供了一种快速简便的新方法。     

犬瘟热病毒RT-LAMP检测方法的建立及初步应用

环介导等温基因扩增技术(loop-mediated isothermal amplification,LAMP)是一种特异、灵敏、快速的检测方法。为提高犬瘟热病毒(CDV)检测效率、降低检测成本,针对犬瘟热病毒N基因保守区的8个位点设计了6条引物进行RT-LAMP一步法扩增。对反应体系及条件优化,检测特异性、敏感性,扩增产物通过凝胶电泳、显色反应和浑浊度比较进行判定。试验结果表明,该方法特异性强、敏感度高,操作简便、快速、便于观察,适用于门诊和现场检测。     

犬瘟热—犬冠状病毒双重PCR诊断方法的建立及初步应用

Two pairs of PCR primers were designed according to sequence of canine distemper virus （CDV） and canine coronavirus （CCV） from GenBank, respectively, which could amplify 550 bp fragment for CDV and 225 bp fragment for CCV. The products of PCR were cloned to pMD18-T for sequencing, which proved to be specific. Positive plasma were developed for standard DNA, and the sensitivity result of duplex PCR showed that the method could amplify 0.1 ng/μL nucleic acid for both of viruses. The result of rudimentary application showed that the method was specific, sensitive, efficient, and was a new detecting method for the mixed infection of CDV and CCV.     

犬瘟热病毒单克隆抗体的研制及胶体金试纸条检测方法的建立

本研究利用原核表达、纯化的犬瘟热病毒(Canine distemper virus,CDV)N 蛋白免疫BALB/C小鼠,成功筛选到两株分泌CDV N 蛋白单克隆抗体的杂交瘤细胞株,分别命名为D3、D6。2株杂交瘤细胞分泌单克隆抗体敏感性、特异性良好,能识别不同来源的犬瘟热病毒株,单抗亚类均为IgG1。以D3作为金标抗体,D6作为检测抗体,兔抗鼠IgG作为质控线抗体,制备犬瘟热病毒胶体金检测试纸条,检测犬瘟热病毒敏感性为1000TCID50,能有效检测区分犬瘟热病毒、犬细小病毒(Canine parvovirus,CPV)、犬腺病毒2型(Canine adenovirus type 2,CAV2)、狂犬病病毒(Rabies virus,RV)。综上,本研究建立的胶体金试纸条检测方法,灵敏度高、特异性强,适用于犬瘟热病毒临床快速诊断。     

用半套式PCR检测犬瘟热病毒的研究

根据ＧｅｎＢａｎＫ中Ｂａｒｒｅｔｔ报道的犬瘟热病毒Ｏｎｄｅｒｓｔｅｐｏｏｒｔ弱毒株的附着或血凝蛋白基因序列,设计合成了能扩增７６０ｂｐ基因片段的半套式引物。用异硫氰酸胍－－酚－－仿一步抽取法提取细胞总ＲＡＮ进行反转录,再以此产物进行半套式ＰＣＲ扩增,并筛选出最佳ＰＣＲ扩增。结果经半套式ＰＣＲ扩增能得到与设计片段大小相同的产物,并且不扩增犬细小病毒、犬腺病毒、犬冠状病毒、狂犬病病毒的核酸。敏感性试验     

荧光定量RT-PCR法对犬瘟热病毒PS株感染犬血清和粪便中病毒的检测

为研究犬人工感染犬瘟热病毒(CDV)后病毒在血清和粪便中的含量及变化,本实验根据CDV核衣壳蛋白(NP)基因序列设计一对特异引物,扩增NP基因.并以NP基因重组质粒作为阳性标准品,建立检测CDV的SYBR GreenⅠ荧光定量RT-PCR方法.结果表明,该方法102拷贝～109拷贝范围内具有良好的线性关系,相关系数为R2=0.998,扩增效率为E=98.3％,敏感度高,最低检测限为6.1拷贝/μL,重复性检测变异系数低于2.62％.该方法与常规RT-PCR方法比较,其敏感性提高102倍.两只人工感染CDV PS强毒株的犬,分别于接种后17d、19d发病死亡,采用荧光定量RT-PCR方法对人工感染犬的血液和拭子液中的病毒核酸栽量进行定量检测以及对收集的22份临床样本拭子液进行检测,均检测到CDV.本研究结果表明,该方法可以用于CDV核酸定量检测,为该病毒的致病机理及病毒传播途径的研究提供了一种快速和敏感的检测手段.     

鸭腺病毒A型TaqMan实时荧光定量PCR检测方法的建立

试验旨在建立鸭腺病毒A型（duck adenovirus A,DAdV-A）TaqMan实时荧光定量PCR检测方法。根据DAdV-A Hexon基因序列设计特异性引物和探针,建立了基于TaqMan探针检测DAdV-A的实时荧光定量PCR检测方法,对其特异性、灵敏性、重复性进行检测,用建立的TaqMan实时荧光定量PCR检测方法和常规PCR方法同时对福建地区临床收集的85份番鸭源病料进行DAdV-A感染的检测,比较其符合率。结果表明,试验成功建立了检测DAdV-A的实时荧光定量PCR检测方法,其扩增相关系数为0.996,扩增效率为99.9%;特异性强,对鸭常见病原（如鸭瘟病毒、鹅细小病毒、番鸭细小病毒、鸭圆环病毒、鸭源大肠杆菌、鸭疫里默氏杆菌和鸭源禽多杀性巴氏杆菌）检测均为阴性;灵敏度高,最低检测限为8.37拷贝/μL;重复性好,组内变异系数和组间变异系数分别为0.54%~1.28%和0.61%~2.39%。对临床送检的85份病料,TaqMan实时荧光定量PCR方法的阳性率为7.06%（6/85）,PCR方法的阳性率为5.88%（5/85）,且PCR检测的阳性样品经TaqMan实时荧光定量PCR方法检测均为阳性,符合率为100%。本研究建立了基于TaqMan探针检测DAdV-A的实时荧光定量PCR检测方法,为鸭群中开展DAdV-A的分子流行病学研究提供了有效技术手段。     

Establishment of a Duplex TaqMan Real-time PCR Method for Detection of Bovine Viral Diarrhea Virus Types 1 and 2

【Objective】 This study was aimed to establish a duplex TaqMan Real-time PCR method for rapid detection of Bovine viral diarrhea virus types 1(BVDV1) and 2(BVDV2).【Method】 Specific primers were designed based on the 5'-non-coding region of 95 strains of BVDV1 and BVDV2 in GenBank.The positive plasmids containing the target fragments of BVDV1 and BVDV2 were constructed.The reaction conditions were optimized, the standard curve was constructed, the specificity, sensitivity and reproducibility of the duplex TaqMan Real-time PCR method were tested, and the established duplex TaqMan Real-time PCR assay was used to detect the clinical samples collected from Jilin province.【Result】 The results showed that the optimal annealing temperature of the duplex TaqMan Real-time PCR was 57.0 ℃, the optimum primer concentration was 0.5 μmol/L, and the optimum probe concentration was 0.3 μmol/L.The standard curves of BVDV1 and BVDV2 were Y=-3.54X+37.36 (R²=0.990) and Y=-3.18X+35.95 (R²=0.997), respectively.There was no specific amplification of Infectious bovine rhinotracheitis virus (IBRV), Bovine respiratory syncytial virus (BRSV) and Bovine parainfluenza virus type 3 (BPIV3), with intra- and inter-batch CV less than 3%, and the lower limit of detection was 10 copies/μL.The results of clinical samples showed that the overall positive rate was 23.1% (36/156), of which 17.9% (28/156) were positive for BVDV1 and 5.1% (8/156) were positive for BVDV2.【Conclusion】 In this study, a duplex TaqMan Real-time PCR method was established, which could identify BVDV types 1 and 2 simultaneously, quickly and accurately, and provided technical support for the prevention, control and purification of BVDV.     

犬细小病毒昆明流行株的分离鉴定与VP2基因序列分析

为了解昆明市犬细小病毒(canine parvovirus,CPV)流行及抗原变异情况,有针对性地筛选疫苗及研发新疫苗,本研究从昆明市部分宠物医院收集6份疑似CPV粪便样品进行病毒分离培养,对获得的疑似病毒液进行PCR、免疫荧光试验(IFA)、血凝效价测定、TCID50测定及VP2基因序列分析。结果显示,分离的1株病毒能使F81猫肾细胞产生明显的细胞病变(CPE),PCR扩增产物大小为164bp,IFA鉴定感染的F81细胞出现绿色荧光,血凝效价为1∶512,病毒TCID50为10-4.375/0.1mL,VP2基因PCR扩增条带大小为1 755bp。对分离株VP2结构蛋白进行测序分析,判定该分离株为CPV-2a亚型,与标准毒株存在5个变异氨基酸位点,属于变异株。将测序结果与商品化疫苗株及国内参考株序列进行比对分析,结果显示与弱毒苗Intervet/vaccine/06和Pfizer/vaccine/06核苷酸同源性均为98.7%;与国内参考毒株序列同源性为98.2%～99.5%,与ANTU-1和WH02/06株同源性最高;与ANTU-1株、WH02/06株及BJ01株亲缘关系最近,位于进化树的同一簇。本研究对监测CPV的遗传变异趋势及疫苗的研制具有重要意义。