弓形虫环介导等温扩增快速检测方法的建立

为了建立弓形虫的快速检测方法,本研究建立了一种灵敏、特异、快速的分子生物学检测方法——环介导等温扩增技术(LAMP)。用10倍系列稀释的DNA样品对该检测方法的灵敏度进行了测试,同时通过对扩增产物做内切酶验证;为证明LAMP技术的特异性,对新孢子虫、附红细胞体、瑟氏泰勒虫等病原体进行检测。结果显示:利用LAMP技术成功检测到弓形虫基因区,此方法的灵敏度可达到能检测5个拷贝DNA分子水平,酶切分析结果正确,并且特异性强,对新孢子虫、附红细胞体、瑟氏泰勒虫等病原体检测均为阴性。说明LAMP技术可应用于弓形虫的快速检测。     

吉林省2006--2010年羊传染性脓疱病的流行病学调查及分析

为了解近年来羊传染性脓疱病在吉林省的流行情况,本试验采用PCR方法对2006—2010年间采自吉林省9个不同市县85个养羊户疑似羊传染性脓疱病毒（ORFV）感染的628份痂皮样本（背部、乳房、四肢以及尾根等部位皮肤）进行羊传染性脓疱病毒核酸的检测,在各市县不同年龄不同品种羊的皮肤痂皮样本中均检测到一定程度的ORFV,平均阳性率为24．36％（153／628）,其中以1～6月龄羔羊的阳性检出率最高。应用间接ELISA方法对同期采集自上述地区羊群中的2160份血清样本进行ORFV抗体检测,其中有852份呈现0RFV抗体阳性反应,平均阳性率为39．44％（852／2160）。以上结果表明,ORFV在吉林省大部分地区羊群中的感染已经非常普遍。     

吉林省2006--2010年羊传染性脓疱病的流行病学调查及分析

为了解近年来羊传染性脓疱病在吉林省的流行情况,本试验采用PCR方法对2006—2010年间采自吉林省9个不同市县85个养羊户疑似羊传染性脓疱病毒（ORFV）感染的628份痂皮样本（背部、乳房、四肢以及尾根等部位皮肤）进行羊传染性脓疱病毒核酸的检测,在各市县不同年龄不同品种羊的皮肤痂皮样本中均检测到一定程度的ORFV,平均阳性率为24．36％（153／628）,其中以1～6月龄羔羊的阳性检出率最高。应用间接ELISA方法对同期采集自上述地区羊群中的2160份血清样本进行ORFV抗体检测,其中有852份呈现0RFV抗体阳性反应,平均阳性率为39．44％（852／2160）。以上结果表明,ORFV在吉林省大部分地区羊群中的感染已经非常普遍。     

快速检测大肠杆菌O157的LAMP方法的建立与评价

利用DNA环介导恒温扩增技术(Loop-mediated isothermal amplification,LAMP),以rfbE基因为靶序列,设计LAMP引物,建立LAMP反应体系与条件,进行灵敏度和特异性试验.结果显示,整个检测过程仅需1h即可通过肉眼直接目测试验结果.在灵敏度试验中,rfbE-LAMP对O157菌液和模拟阳性样品的最低检测限分别为1CFU/mL和1 CFU/g.在特异性试验中,以其他血清型大肠杆菌以及沙门杆菌基因组DNA为模板对rfbE基因进行检测,均没有发生非特异性扩增反应.试验建立的LAMP检测方法具有操作简单安全、检测灵敏度高、特异性高的特点,能够满足基层实验室、应急检测或现场监测等方面的使用需求.     

水疱性口炎病毒RT-LAMP快速检测方法的研究

根据逆转录环介导等温扩增(RT-LAMP)原理,针对水疱性口炎病毒(VSV)糖蛋白G基因序列中的6个区域设计内外各1对特异引物,建立扩增VSV糖蛋白(G)基因的RT-LAMP方法。扩增产物电泳呈特异的阶梯状条带分布,扩增产物加SYBR GREEN I染色呈特征性的黄绿色,肉眼可直接观察判定。同时,还建立了可以进行定量检测的实时RT-LAMP方法。特异性试验显示,本方法可快速检验鉴别VSV与口蹄疫病毒(FMDV)和猪水泡病病毒(SVDV)。敏感性试验显示,建立的实时RT-LAMP方法检测VSVRNA的最低检测量为0.01 PFU,比实时荧光RT-PCR显著提高。建立的LAMP方法可检测p-VSVNJ质粒DNA的最低量为6.36×10-3pg/μL(1.4×103copies/μL),比PCR也显著提高。综合表明,本研究建立RT-LAMP检测VSV的方法具有特异、敏感、快速、简便的特点,具有开发应用前景。     

Rapid diagnosis of duck plagues virus infection by loop-mediated isothermal amplification

Duck virus enteritis is a serious disease among farmed and free-living ducks (Anatidae) and a constant threat to the commercial duck industry in China. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed to rapidly detect and diagnose duck plague virus (DPV) in both farmed and wild waterfowl, and compared with polymerase chain reaction (PCR) method and real-time PCR method in accuracy, sensitivity and specificity. A set of four specific primers was successfully designed to recognize six distinct genomic sequences of UL6 protein from DPV, including one forward inner primer, one back inner primer and two outer primers. The optimum reaction temperature and time were verified to be 61.5 °C and 60 min, respectively. Comparative experiments showed that LAMP assay was a simple, rapid, accurate, sensitive and specific method for detecting DPV, and was superior to PCR assay in sensitivity and specificity for DNA amplification. In addition, challenge tests indicated the newly developed LAMP method was more sensitive for the diagnosis of DPV infection than virus isolation and PCR. LAMP assay would be a good alternative method for on-farm disease diagnosis.     

犬流感病毒RT-LAMP快速检测方法的建立

为了建立一种便捷、灵敏的犬流感病毒(CIV)检测技术,本研究根据CIV相对保守的M片段设计3对特异性引物,建立CIV的RT-LAMP检测方法,对反应体系中的MgSO4、Betaine、Bst DNA PoLymerase、dNTP、引物浓度等分别进行了优化,并进行敏感性和特异性试验。结果表明:所建立的反应体系在恒温水浴锅中作用45min即可得到其特有的阶梯状条带,而且对犬细小病毒、犬瘟病毒和犬副流感病毒的扩增结果均为阴性。该方法对CIV RNA的最小检测限为0.1pg,灵敏性高于一步RT-PCR方法。RT-LAMP和普通RT-PCR方法检测临床样品的符合率为93.02%。该方法为犬流感病毒的临床检测提供了一种简便、实用的方法,为基层检疫提供了方便。     

快速诊断伪狂犬病毒的LAMP方法的建立

采用LAMP技术建立了一种针对伪狂犬病毒的可视、快速、灵敏、特异的检测方法。应用PrimerExplorerV4软件,根据伪狂犬gE基因的保守序列设计了4条引物,优化了反应体系与反应时间,检测了特异性与灵敏性。发现该方法只需要40 min就能检测出结果,具有良好的特异性,灵敏度为普通PCR的100倍,最低可检测出质量浓度为6.2×10-9mg/L的病毒DNA。该方法的建立为伪狂犬病的快速诊断提供了新的思路。     

Establishment and Application of Loop-mediated Isothermal Amplification for Rapid Detection of Porcine Epidemic Diarrhea Virus

This study was aimed to establish a loop-mediated isothermal amplification (LAMP) assay for detection of porcine epidemic diarrhea virus (PEDV), and provide a simple, sensitive, accurate and reliable tool for diagnosis of PEDV.The conservative PEDV N gene (GenBank accession number: KT799997) of PEDV was selected as a target to design six specific primers.The reaction system and temperature of LAMP were optimized, and the LAMP method for specific amplification of PEDV was established. Results showed that the PEDV LAMP detection method was established successfully, and it could detect PEDV specifically at 60℃ for 60 min,and the detection limit was 91 copies/μL, which was one hundred-fold higher than conventional RT-PCR method.75 clinical samples were detected by LAMP and PCR, respectively, the coincidence of LAMP and PCR was about 97.3%. All the data suggested that the LAMP assay had strong specificity, high sensitivity, simple operation, low equipment requirement, and was suitable for rapid detection of PEDV clinical samples.     

猪流行性腹泻病毒环介导等温扩增检测方法的建立及应用

试验旨在建立一种快速检测猪流行性腹泻病毒（PEDV）的环介导等温扩增方法（LAMP）,为诊断PEDV提供简便、敏感、准确可靠的工具。参考GenBank中PEDV基因序列（登录号：KT799997）,针对PEDVN基因设计了6条引物,对所建立的LAMP反应体系、反应温度进行优化,建立可特异性扩增PEDV的LAMP方法。结果显示,本试验成功建立了PEDV LAMP检测方法,在60℃恒温下反应60 min,能特异性地检测PEDV,检测限量为91拷贝/μL,比常规PCR方法的敏感性高100倍。对比75份临床样本的LAMP和常规RT-PCR法检测结果,显示两种方法符合率为97.3%。综上所述,本试验建立的LAMP方法具有特异性强、敏感性高,操作简单,设备要求低的特点,适用于PEDV临床样本的快速检测。     

新城疫病毒逆转录环介导等温扩增检测方法的建立

为建立一种新城疫病毒(NDV)的简便、快速、高灵敏度的检测方法,本研究根据NDV融合蛋白基因(F基因)设计合成6条特异性引物,在恒温水浴中进行核酸扩增反应,对反应温度、反应时间以及反应液中Mg2+浓度进行优化,获得最佳反应条件,结果经凝胶电泳分析并在可见光下直接观察.整个RT-LAMP检测用时0.5 h,检测敏感度比RT-PCR高100倍,对禽流感病毒、鸡传染性支气管炎病毒和鸡传染性喉气管炎病毒无交叉反应.本研究建立的NDV RT-LAMP检测方法速度快、不需要高精密的仪器,而且具有灵敏度高、特异性强,操作简单等特点,有望在核酸扩增检测方法中取代RT-PCR技术.     

Detection of Anaplasma platys in dogs using real-time loop-mediated isothermal amplification

Anaplasma platys is a parasite of canine platelets that causes infectious cyclic thrombocytopenia. In this study, a novel real-time loop-mediated isothermal amplification (RT-LAMP) method was developed to detect A. platys. RT-LAMP primer sets were designed using a citrate synthase gene sequence and the assay was performed at 63 °C for 30 min. No cross-reactivity was observed with other Anaplasma or Ehrlichia spp. and the method exhibited a similar level of sensitivity in detecting the organism in 58 canine blood samples to that of a nested PCR. This RT-LAMP is a rapid and potentially cost-effective method of diagnosing A. platys infection in dogs.     

Application of the colorimetric loop-mediated isothermal amplification (LAMP) technique for genotyping Cre-driver mice

The Cre-loxP system is widely used to investigate the cell-type specific roles of genes of interest. Cre-driver mice are required for cell-type specific knockout during the Cre-loxP reaction. To maintain Cre-driver mouse strains, Polymerase chain reaction (PCR)-oriented genotyping targeting the Cre gene cassette is usually conducted. In this study, we instead applied a colorimetric loop-mediated isothermal amplification (LAMP) method for Cre-genotyping. Among four sets of primers designed by the in silico program, one set effectively amplified the Cre cassette of three Cre-driver strains, but not of C57BL/6 mice. This LAMP-oriented method reduces assay time by less than half compared to the PCR-based method, and can be carried out using a conventional isothermal incubator. Applying this LAMP method may accelerate genotyping of Cre-driver mice.     

快速检测双芽巴贝斯虫LAMP方法的建立

为提高双芽巴贝斯虫(Babesia bigemina)检出率,本研究采用环介导等温扩增技术(LAMP)建立一种快速、灵敏、特异的B.bigemina检测方法。根据GenBank上公布的Babesia bigemina细胞色素b(Cytochrome b,cyt b)基因序列,设计4条特异地识别B.bigemina的cyt b基因6个特殊区域的LAMP引物,优化反应体系和条件,在Bst DNA聚合酶的作用下,65 ℃反应60 min,加入SYBR Green Ⅰ后观察。结果表明,该LAMP检测方法特异性强,与牛巴贝斯虫(Babesia bovis)等DNA不发生交叉反应;敏感性高,对B.bigemina的cyt b基因最小检测值为0.085 fg/μL,是一般PCR方法的1000倍。该方法具有简单、快速、低成本的特点,可用于B.bigemina的基层现场快速检测。     

LAMP方法及其在病原微生物检测中的应用

环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)是一门新兴的分子生物学技术,该技术具有操作简单、不需要大型仪器设备、成本低廉、反应时间短、结果判定简单等优点,且具有比常规分子生物学方法更高的灵敏度和特异性.目前该技术在细菌、病毒及支原体等病原微生物的快速检测和早期诊断中已广泛应用.通过对国内外LAMP研究进展及该技术在细菌、病毒、支原体等致病菌中的诊断应用进行综述,以期介绍该技术的优缺点和应用现状,从而为今后的广泛应用提供参考依据.     

环介导等温扩增技术在病原检测中的应用

文章介绍了环介导等温扩增技术的的基本原理、特点及其在传染性疾病中检测和诊断的应用。环介导等温扩增技术具有特异性强、等温灵敏、操作简单、产物易检测等优点。环介导等温扩增技术已经被用来快速诊断动物的传染病。目前可检测圆环病毒2型、鹅细小病毒、伪狂犬病病毒、高致病性禽流感病毒、新城疫病毒、副猪嗜血杆菌、单核细胞增生性李斯特氏菌、猪肺炎支原体及弓形虫基因等。     

Loop-mediated Isothermal Amplification Method and Its Application in Pathogen Detection

As a new molecular biology technique,the loop-mediated isothermal amplification (LAMP) has the advantages of easy to operate,no need of specialized devices,low cost,short reaction time and so on.It has higher sensitivity and specificity than the common molecular biology techniques.It has been widely used in rapid and early infection by variety of pathogens including bacteria,virus and mycoplasmas.We reviewed the research progress on LAMP techniques and the diagnostic applications in the bacteria,viruses,Mycoplasma and other pathogens at home and abroad,so as to understand the advantages and disadvantages of LAMP and application situation,and provide a reference for its widespread application in the future.     

鸡传染性法氏囊病毒反转录环媒恒温检测方法的初步建立

本研究利用Primer Explorer V4软件设计了针对鸡传染性法氏囊病毒（IBDV）VP1基因保守区6个特异性部位的4条引物,建立了IBDV的反转录环媒恒温检测方法。该方法反应体系在恒温水浴锅中作用1 h即可得到其特有的阶梯状条带,加入荧光素后肉眼可直接观察结果。本方法特异性高,对IBDV的最低检出量为10-6稀释倍数,敏感性是普通RT-PCR的100倍。反转录环媒恒温检测方法和普通RT-PCR方法检测临床样品的符合率为93.8%。因此该方法可以作为IBDV的综合防治和疾病诊断的实用方法。