单核细胞增生李斯特氏菌实时荧光定量PCR快速检测方法的建立

为建立单核细胞增生李斯特菌(Listeria monocytogenes,LM)的快速检测方法,本研究以LM iap基因为靶基因设计合成引物及TaqMan探针,建立实时荧光定量PCR快速检测LM的方法。结果显示,对15株试验菌株进行实时荧光定量PCR检测,只有LM菌株检测为阳性,表明该检测方法特异性强;该方法的灵敏度为6.5 CFU/mL;稳定性和重复性试验结果表明,同一样品重复检测4次Ct值的变异系数均小于2%;利用该检测方法对采集的139份样品进行检测,共计检出3份LM阳性样品,与国标法(GB 478930-2010)检测结果一致。该检测方法灵敏度高、特异性强、重复性好,具有良好的实用性。     

Development of Real-time PCR Method for Rapid Detection of Vibrio parahaemolyticus

To establish a rapid assay for detection of Vibrio parahaemolyticus(VP), Real-time PCR method was developed targeting to toxR gene of Vibrio parahaemolyticus.The results showed that the test for 15 bacteria strains using the Real-time PCR method, only Vibrio parahaemolyticus test was positive, indicating that the method had high specificity.In addition, the sensitivity of Real-time PCR was 4.9 CFU/mL.Furthermore, a total of 3 positive samples for Vibrio parahaemolyticus were detected from 150 clinical samples by the Real-time method, which was in accordance with the testing result by GB 4789.7-2013 standard detection protocol.Therefore, the Real-time method provided a novel rapid and sensitive detection method with good practicality for Vibrio parahaemolyticus infection.     

副溶血弧菌实时荧光定量PCR快速检测方法的建立

为建立检测副溶血弧菌(Vibrio parahaemolyticus,VP)的快速检测方法,本研究以VP toxR基因为靶基因设计合成引物及TaqMan探针,建立了实时荧光定量PCR快速检测VP的方法。结果显示,对15株试验菌株进行实时荧光定量PCR检测,只有VP检测为阳性,表明该检测方法特异性强;该方法的灵敏度为4.9 CFU/mL,利用该检测方法对采集的150份样品进行检测,共计检出3份VP阳性样品,与国标法(GB 4789.7-2013)检测结果一致,显示了良好的实用性。该检测方法灵敏度高、特异性强,具有良好的实用性。     

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.     

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。     

鸭痘病毒TaqMan荧光定量PCR检测方法的建立

为建立检测鸭痘病毒的TaqMan荧光定量PCR方法,本试验克隆了鸭痘病毒P4b基因,构建重组质粒pMD-DPV-P4b,并将其作为标准阳性模板。参照GenBank收录的禽痘病毒P4b基因设计合成1对特异性引物及与该引物相匹配的特异探针。以定量的10倍系列稀释的质粒pMD-DPV-P4b为标准品,通过对反应条件进行优化,建立了一种检测鸭痘病毒的TaqMan荧光定量PCR方法。结果显示,该方法与禽流感病毒、鸭黄病毒、鸭肝炎病毒、新城疫病毒、鸭瘟病毒和小鹅瘟病毒等其他水禽病毒,以及山羊痘病毒和鸡痘病毒等其他痘病毒均无交叉反应,特异性好。该方法最低检测限为1.29×10²拷贝/μL,比普通PCR检测方法高100倍。组内和组间变异系数均小于2%。结果表明,本试验所建立方法具有灵敏、特异、安全、快速的特点,适用于鸭痘病毒的检测。     

Establishment of TaqMan Real-time PCR Method for Detection of Duck Poxvirus

To establishment a TaqMan Real-time PCR method for detection of duck poxvirus (DPV),we cloned the P4b gene of DPV.The specific primers and probe were designed according to the nucleotide sequence of avipoxvirus available in GenBank.Recombinant plasmid pMD-DPV-P4b was employed as positive standard template for Real-time PCR.By optimization of reaction conditions,a TaqMan Real-time PCR method for detection of DPV was established.The results of specificity test proved this method had no cross-react with other waterfowl vial agents and poxviruses including avian influenza virus,duck flavivirus,duck hepatitis virus,Newcastle disease virus,duck entertitis virus,goose parvovirus,goatpox virus and fowlpox virus.The detection limit of the assay was 1.29×10² copies/μL of viral DNA,which was 100 times higher than that of the routine PCR.Reproducibility test showed that the CVs of intra assay and inter assay were both less than 2%.Above results supported that the assay was suitable for the detection of DPV very well.     

Development of Real-time Quantitative PCR Method for Rapid Detection of Enterohemorrhagic Escherichia coli O157∶H7

A Real-time quantitative PCR method was developed for the rapid detection of enterohemorrhagic Escherichia coli （EHEC） O157∶H7 based on the primers and TaqMan probes which were designed for the conservative domain of rfbE gene of EHEC O157∶H7.The results showed that the sensitivity of Real-time PCR was 7.3 CFU/mL.20 from 310 samples of meat,egg,milk and its products,animal diarrhea materials and artificial contamination samples were positive detected by Real-time PCR assay,which was in accordance with the testing result according to AOAC standard.The results indicated that Real-time PCR assay was a sensitive,rapid and simple tool.     

猪繁殖与呼吸综合征病毒TaqMan-MGB实时荧光定量RT-PCR方法的建立及应用

为了快速、准确地检测猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome,PRRSV),本研究根据PRRSV基因序列设计特异性引物和探针,建立了一种可同时检测PRRSV经典毒株、高致病性变异毒株以及近几年中国新出现的NADC30-like毒株的TaqMan-MGB实时荧光定量方法,并对该方法的特异性、敏感性和重复性进行验证,同时用建立的实时荧光定量方法与常规PCR方法对临床收集的120份疑似PRRSV样品进行检测。结果表明,该方法特异性良好,对PRRSV的经典毒株、高致病性变异毒株及NADC30-like毒株均有良好的扩增,但对猪瘟病毒(CSFV)、猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)、猪轮状病毒(RV)、猪伪狂犬病病毒(PRV)、猪细小病毒(PPV)和猪圆环病毒2型(PCV2)的检测结果均为阴性,无交叉反应;模板浓度在101～108拷贝/μL范围内具有良好的线性关系,标准曲线结果显示其扩增的相关系数为0.9999,扩增效率为93%;敏感性高,约是常规PCR方法的100倍,最低可以检测到101拷贝/μL的模板;重复性好,批内和批间重复性试验变异系数分别为0.17%～0.90%和0.65%～2.34%;用本研究所建立的实时荧光定量检测方法对120份临床样品进行检测,PRRSV阳性检出率为59.2%(71/120),而常规PCR方法的PRRSV阳性检出率为44.2%(53/120)。该方法的建立为PRRSV的实验室诊断、流行病学调查,以及预防和控制中国PRRSV的流行提供了快速、准确的检测手段。     

检测产单核细胞李氏杆菌溶血素的胶体金试纸条制备及初步应用

产单核细胞李氏杆菌为重要的食源性病原菌,本研究旨在建立快速、灵敏、特异的检测方法,提高检测效率和检测通量。李氏杆菌溶血素(LLO)蛋白经原核表达后, 制备单克隆抗体, 测定单克隆抗体的亚型、效价及亲和力。柠檬酸钠法研制LLO胶体金试纸条,并对试纸条的特异性、敏感性、重复性、稳定性及模拟样品进行检测,并初步应用于实际海产品的检测。结果发现,制备的LLO单克隆抗体特异性强、稳定性高、重复性佳、灵敏度较高,亲和力好,亲和力常数为4.25×10⁸ L·mol^－1。人工污染样品试验表明灵敏度与纯细菌培养物基本一致,为3.0×10⁵CFU·mL^-1。将LLO胶体金试纸条应用于500份海产品的实际检测中,产单核细胞李氏杆菌的阳性检出率为2.20% (11/500),略低于国标检测方法(GB4789.30—2010)的2.40%(12/500)和PCR检测方法的2.40%(12/500),三者之间符合率为91.67%。另外缩短检测时间至15 min,检测效率大幅度提高。本研究建立的LLO胶体金检测方法可用于产单核细胞李氏杆菌的快速检测。     

猪性控精子纯度实时荧光定量PCR检测方法的建立

研究旨在利用实时荧光定量PCR法检测杜洛克猪性控精子，以期建立一种快速、准确且经济高效的性控精子纯度检测方法。选取位于猪Y染色体上的Y染色体性别决定区（sex-determining region Y，SRY）基因和位于X染色体上的A-激酶锚定蛋白4（A-kinase anchoring protein 4，AKAP4）基因，以猪耳组织样提取的基因组DNA为模板进行PCR扩增验证引物的特异性，利用胶回收试剂盒进行回收并质粒小提，将获得的两种质粒经检测后稀释至相同浓度（20 ng/μL），混合构建含有不同比例SRY和AKAP4基因的质粒模板，用于绘制检测精子纯度所用标准曲线的反应模板。分别使用SRY和AKAP4基因特异引物检测3个混合的X精子（P.x_gro1、P.x_gro2、P.x_gro3）和3个混合的Y精子（P.y_gro1、P.y_gro2、P.y_gro3）的纯度。结果显示，用SRY基因特异性引物检测P.x_gro1、P.x_gro2、P.x_gro3精子的纯度分别为91.44%、91.93%、88.99%，P.y_gro1、P.y_gro2、P.y_gro3精子的纯度分别为89.91%、87.31%、88.71%；用AKAP4基因特异性引物检测P.x_gro1、P.x_gro2、P.x_gro3精子的纯度分别为91.44%、91.93%、88.99%，P.y_gro1、P.y_gro2、P.y_gro3精子的纯度分别为89.91%、87.31%、88.71%；卡方适合性检验结果显示，两次检测结果之间差异不显著，表明使用这种方法进行精子纯度检测所得结果准确。本试验通过实时荧光定量PCR法准确检测了经流式细胞仪分选后的精子的纯度，建立了一种利用实时荧光定量PCR法检测猪精液中X精子和Y精子比例的新方法。     

Establishment of QuantStudioTM 3D Digital PCR Method for Detection of Equine Herpesvirus Type 1

The purpose of this study was to establish a highly sensitive 3D digital PCR (3D-dPCR) method for the detection of equine herpesvirus 1 (EHV-1),which could accurately and quantitatively detect the samples with low EHV-1 content and realize the early diagnosis and prevention of equine rhinopneumonia.According to the conserved region of EHV-1 glycoprotein B gene,we designed specific primers and probes,optimized the concentration and annealing temperature of primers in the 3D-dPCR reaction system,analyzed the sensitivity,specificity and repeatability of this method,and established the 3D-dPCR method of EHV-1.In this study,the best concentration of primer and probe of 3D-dPCR was 0.4 and 0.4 μmol/L respectively,the best annealing temperature was 60 ℃,R² of the absolute quantitative curve of the method was 0.998,the linear relationship was good,the sensitivity was about 10 times higher than that of Real-time PCR,and the minimum detection limit was 5.83 copies/μL.There was no cross reaction with EHV-4,Theileria equi and the nucleic acid of equine arteritis.The results showed that the positive rate of 3D-dPCR was 66.7%,which was higher than that of Real-time PCR for EHV-1 in OIE (64.2%).The results of 3D-dPCR were consistent with those of Real-time PCR,and the sensitivity of 3D-dPCR to the samples with low virus content was higher,which could effectively detect suspicious samples.The results showed that the established 3D-dPCR method was more sensitive,specific and reproducible for the detection of clinical samples with low copy number,and could be used for the accurate and quantitative detection of EHV-1.     

猪支原体肺炎LAMP-LFD快速检测方法的建立及初步应用

旨在采用环介导等温扩增（LAMP）和横向流动试纸条（LFD）相结合的方法，建立一种快速、特异，过程可视化的猪肺炎支原体（Mycoplasma hyopneumoniae，Mhp）检测方法。针对猪肺炎支原体（Mhp）P36基因设计5套特异性引物和1条异硫氰酸荧光素（FITC）标记的探针，进行LAMP扩增反应。将生物素标记的LAMP产物与FITC标记的探针进行特异性杂交，并使用横向流动试纸条完成扩增产物的检测。经优化，LAMP最佳反应条件为65℃，反应15 min，从基因组DNA提取到LFD结果判断只需40 min左右，比常规PCR技术缩短近2 h。LAMP-LFD可特异性地检出猪肺炎支原体（Mhp），对猪鼻支原体（Mhr）及猪圆环病毒（PCV）等常见猪病病原的检测结果为阴性。灵敏性试验表明，LAMP-LFD对Mhp的检测灵敏度为1×10⁰个拷贝DNA，是普通PCR的1 000倍。利用本方法可从采集的88份临床疑似病料样品中检测到64份阳性，国标PCR方法可检测到56份阳性，符合率可达90.9%。综上，本研究建立的LAMP-LFD方法可特异、准确地应用于Mhp的检测，而且灵敏度高、操作简单、仪器设备依赖性低、检测成本低、耗时短，适合基层实验室、应急检测或现场监测等使用，具有较高的推广价值，有望发展成为Mhp快速检测的有效手段。     

猪链球菌自溶素的原核表达及其抗体间接ELISA检测方法的建立

【目的】 建立快速、准确检测猪链球菌自溶素（atl）抗体的间接ELISA方法。【方法】 根据GenBank登录的猪链球菌atl基因序列设计1对引物,采用PCR方法从猪源链球菌中获得atl基因序列;构建pET-32a-atl和pGEX-4T-1-atl重组质粒,并将重组质粒转化大肠杆菌BL21感受态细胞中进行诱导表达;以表达纯化后的atl-His融合蛋白为免疫原,免疫新西兰大白兔,制备多克隆抗体;以兔多克隆抗体为一抗,用Western blotting方法检测atl-GST融合蛋白的反应原性;以纯化后的atl-GST融合蛋白为包被抗原,猪链球菌感染阳性血清作为标准血清,建立猪链球菌自溶素抗体的间接ELISA检测方法。利用本试验建立的ELISA方法与商用猪链球菌2型ELISA抗体检测试剂盒同时对184份血清样品进行检测并用本试验建立的方法进行临床样品检测。【结果】 从猪链球菌中成功扩增出atl基因;构建的重组质粒,经诱导表达和纯化,获得大小为40 ku的atl-His和48 ku的atl-GST融合蛋白;经Western blotting验证,兔抗atl-His抗血清与atl-GST融合蛋白具有良好的反应原性;间接ELISA结果显示,该检测方法的阴阳临界值为0.318,阳性血清稀释至1：1 280检测仍为阳性;批内批间变异系数均<10%,表明该方法具有良好的重复性及敏感性。本试验所建立的ELISA方法检测出96份阳性样品,商用猪链球菌2型ELISA抗体检测试剂盒检测出66份阳性样品,符合率为71.7%。应用建立的方法检测458份不同饲养阶段的猪血清样品,其中检出277份阳性样品,阳性率为60.4%。【结论】 本试验成功建立了检测猪链球菌自溶素抗体的间接ELISA方法并进行了初步应用,为猪链球菌病血清流行病学调查奠定了基础。     

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.     

羊源多杀性巴氏杆菌重组酶聚合酶扩增诊断方法的建立

为建立适用于羊源多杀性巴氏杆菌的重组酶聚合酶扩增（RPA）诊断方法,本研究依据前期测序鉴定的羊源多杀性巴氏杆菌KMT1基因序列构建pET-28a （＋）-KMT1质粒标准品。设计基于RPA技术的特异性引物及RPA荧光探针,建立实时荧光RPA方法的最适反应体系。采用10倍梯度连续稀释的质粒标准品检测该RPA方法的敏感性并绘制相关性曲线;以10种不同菌株的基因组DNA为模板验证方法的特异性;用感染多杀性巴氏杆菌的山羊及小鼠组织样品对方法的可靠性进行验证。结果显示,本试验建立的实时荧光RPA方法最适反应温度为39 ℃,最佳引物为KMT1-Fe1,灵敏度达100拷贝/μL,检测下限为10拷贝/μL。与大肠杆菌、金黄色葡萄球菌、副伤寒沙门氏菌、副溶血弧菌、绿脓杆菌、产酸克雷伯菌、布鲁氏菌S2株和鲍曼不动杆菌均无交叉反应。对13个组织样本进行检测,阳性率为76.9%,与实时荧光定量PCR检测结果的符合率达92.3%。综上所述,本研究建立的羊源多杀性巴氏杆菌实时荧光RPA方法具有特异性强、灵敏度高、可靠、快速便捷等特点,适用于多杀性巴氏杆菌的临床分子诊断。     

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.     

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。     

7种食源性致病菌GeXP多重PCR检测方法的建立及其应用

本研究旨在建立一种能够同时鉴别包括大肠杆菌O157：H7、沙门菌、空肠弯曲菌、单核细胞增生李斯特菌、霍乱弧菌、副溶血弧菌和金黄色葡萄球菌7种常见食源性致病菌的GeXP多重PCR检测方法。根据这些致病菌在GenBank上公布的保守基因序列,设计合成了7对特异性GeXP引物。用单一或混合细菌样品的DNA模板优化反应条件,设置对照组,构建重组质粒,随机组合不同浓度的样品,验证所建立的GeXP方法的特异性、敏感性和准确性。最后用该方法检测120份临床样品,进一步验证所建立的GeXP检测方法的准确性和可靠性。结果显示,单一或混合模板的GeXP检测均能特异性出现相应清晰峰值,可在10³拷贝·μL^-1水平上同时特异地检测出7种细菌病原体,不同浓度模板混合时,本试验所建立的方法依然可检测出对应病原体。检测120份临床样品,GeXP多重PCR阳性率为2.50%（3/120）~15.83%（19/120）,普通PCR阳性率为2.50%（3/120）~15.00%（18/120）,GeXP多重PCR多检出8份阳性,表明GeXP方法更为敏感与准确。本研究建立的同时鉴别7种食源性致病菌的GeXP多重PCR检测方法,具有高通量、特异性强和敏感性高的特点,为食源性常见致病菌感染或混合感染提供了快速分子诊断方法。