环介导等温扩增技术快速检测猪流行性腹泻病毒

为建立一种快速、灵敏的检测猪流行性腹泻病毒(PEDV)的环介导等温扩增(LAMP)检测方法,本研究针对PEDV的NP基因设计LAMP引物,对反应体系优化并进行特异性及敏感性等试验.结果表明,该方法在65℃下恒温扩增60 min,经SYBR Green Ⅰ染色后仅肉眼观察即可判定结果.该方法特异性好,与传染性胃肠炎病毒、猪轮状病毒、猪瘟病毒及大肠杆菌等无交叉反应,对重组质粒最低检测限度为16拷贝/μL,灵敏度高于普通PCR.该LAMP检测方法特异性强,灵敏度高,操作方便,适于临床检测PEDV.     

猪巨细胞病毒环介导等温扩增方法的建立

根据GenBank已登录的猪巨细胞病毒(Porcine cytomegalovirus,PCMV)gB基因的DNA序列设计合成4对特异性引物,通过优化反应条件,以及敏感性、特异性检测,建立了猪巨细胞病毒的环介导等温扩增检测方法(Loop-mediated isothermal amplification,LAMP);用建立的检测方法对2013年来自四川省雅安、成都、绵阳、眉山、乐山、德阳、遂宁、资阳等市的136份病料进行猪巨细胞病毒检测,将检测结果与常规PCR方法检测结果进行比对。结果显示,本试验建立的方法检测灵敏度可达10拷贝/μL,是常规PCR的10倍;136份病料用LAMP检测,其中有89份样品为猪巨细胞病毒阳性,其阳性率与常规PCR方法检测的阳性相符率为100%。结果表明,本试验成功建立了PCMV的LAMP检测方法,可用于临床上PCMV感染猪的确诊,对PCMV的快速诊断、综合防控等具有重要意义。     

猪流行性腹泻病毒LAMP检测方法的建立及初步应用

猪流行性腹泻病毒是引起仔猪腹泻死亡的主要病原。为实现对猪流行性腹泻病毒(PEDV)的快速检测,利用PEDV S基因设计特异性引物,通过优化各种反应条件,建立了检测PEDV的环介导等温扩增快速检测方法(LAMP)。此方法特异性好,敏感度强,将反转录后的病毒c DNA稀释到108倍仍可检出,是PCR方法的100倍。本研究建立的LAMP方法操作简便、灵敏度高、特异性强,为临床PEDV的快速检测和预防奠定基础。     

猪圆环病毒2型环介导等温扩增检测方法的建立与应用

以猪圆环病毒2型为材料,设计了3对引物,构建了猪圆环病毒2型环介导等温扩增（LAMP）检测方法,并进行敏感性和特异性确定。建立的猪圆环病毒2型的LAMP检测方法,可以采用琼脂糖凝胶电泳、颜色变化、生成的沉淀等现象判定反应结果,为猪圆环病毒2型的现场快速检测提供了一种更加简便快速的方法,可以满足现场检疫的需要。     

猪细环病毒2型环介导等温扩增检测方法的建立

为了能快速、特异的检测猪细环病毒2型(TTSuV2),本研究针对TTSuV2全基因序列的非编码区域和第1个开放性阅读框前端设计了2对引物,建立了TTSuV2的环介导等温扩增(LAMP)检测方法并对反应成分和条件进行了梯度摸索。试验结果显示,该LAMP检测方法最佳反应条件为64 ℃恒温90 min,可特异性检测TTSuV2,与猪细环病毒1型、猪圆环病毒2型、猪瘟病毒、猪繁殖与呼吸综合征病毒和猪博卡病毒无交叉反应,病毒最低检出限为100拷贝/μL。结果表明,建立的LAMP方法具有快速、特异且灵敏的特点,可在TTSuV2快速检测方面提供一定的技术支持。     

猪圆环2型病毒环介导等温扩增实时检测方法的建立与评价

以猪圆环2型病毒为材料,设计了2对引物,构建了猪圆环2型病毒实时环介导等温扩增（LAMP）检测方法,并进行敏感性和特异性确定。建立的猪圆环2型病毒实时LAMP检测方法,通过应用实时浊度仪可以对猪圆环2型病毒进行快速检测。     

非洲猪瘟病毒环介导等温扩增快速检测方法的建立

本试验利用环介导等温扩增技术（LAMP）建立了一种快速高效检测非洲猪瘟病毒的方法。整个反应在水浴锅中恒温进行,不需要其他昂贵仪器,30 min即可得到阳性结果,灵敏度是OIE标准PCR方法的100倍,并且与其他常见猪DNA病毒无交叉反应。该方法非常适合在野外现场或缺乏试验条件的边远地区检测非洲猪瘟病毒。     

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。     

鸭瘟病毒环介导等温扩增检测方法的建立及应用

为建立一种简便、快速、灵敏度高、特异性好的鸭瘟病毒(DPV)检测方法,根据GenBank中公布的DPV UL6基因组序列,设计了3对特异性引物,建立了LAMP快速检测方法。该方法的灵敏度可达0.1fg,对其他鸭常见病原体检测结果均为阴性,扩增反应只需在水浴锅中60min内即可完成,可通过肉眼观察颜色变化直接判定结果。结果表明,所建立的LAMP方法简便快捷,省时省力,是适用于现场和基层实验室快速、准确检测DPV的分子生物学方法。     

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.     

基于钙黄绿素显色的可视化LAMP检测猪流行性腹泻病毒的研究

针对猪流行性腹泻病毒(PEDV)的NP基因设计1套环介导等温扩增(LAMP)引物,在反应体系中添加钙黄绿素/氯化锰指示剂代替传统的反应后添加SYBR Green Ⅰ染料,建立了基于钙黄绿素的可视化LAMP检测PEDV的方法。该方法能在65 ℃ 1 h内特异性扩增PEDV,与猪瘟病毒(CSFV)、猪传染性胃肠炎病毒(TGEV)、猪轮状病毒(PoRV)及大肠杆菌等均无交叉反应,检测下限为3.73 pg/μL,其结果可用肉眼判断,快捷方便。用该方法对龙岩学院动物医学研究所接诊的93份腹泻病例进行检测,结果表明,LAMP方法检测PEDV的阳性率为43.01%(40/93),高于普通PCR的检出率(38.7%,36/93)。本试验建立的可视化LAMP检测方法能用于PED诊断。     

Establishment of Real-time Recombinase Polymerase Amplification for Detection of Porcine Epidemic Diarrhoea Virus

To develop a precise and rapid diagnosis method for detecting porcine epidemic diarrhoea virus (PEDV), a series of recombinase polymerase amplification (RPA) primers and exo-probes were established based on the highly conserved M gene of PEDV. Then a Real-time RPA assay was developed to detect PEDV using pUC57 plasmid carrying M gene fragment of PEDV as template, and the membrane or nucleotide capsid proteins from TGEV, PRRSV, PCV2 and CSFV were utilized as control. Then the sensitivity and specificity of this Real-time RPA assay was evaluated. The results showed that the Real-time reaction could detect PEDV specifically at 39℃ within 20 min with the detection limit of 10 copies/μL of plasmid DNA, and there was no cross-reaction with other control viral pathogens. Besides, the established Real-time PRA method could successfully detecte the PEDV M gene in the plasma and plasma protein power. The Real-time established in this study was simple, rapid and sensitive, which could be a novel and reliable method for diagnosing and control of PED.     

猪流行性腹泻病毒实时荧光RPA等温检测方法的建立

为建立一种简单、快速的猪流行性腹泻病毒（PEDV）分子检测方法,本研究基于PEDV病毒M基因保守序列,设计一系列扩增引物及其荧光探针,以包含PEDV病毒M基因片段的pUC57质粒为模板,同时以包含猪传染性胃肠炎病毒（TGEV）、猪繁殖与呼吸综合征病毒（PRRSV）、猪圆环病毒2型（PCV2）及古典猪瘟病毒（CSFV）等病毒膜蛋白或核衣壳蛋白基因序列的质粒作为对照,建立了一种PEDV实时荧光重组酶聚合酶扩增（RPA）等温检测方法,测定了方法的特异性与敏感性。结果表明,该实时荧光RPA方法可在39℃恒温反应20 min特异性扩增PEDV病毒M基因,与TGEV、PRRSV等对照病毒基因无交叉反应,其检测限为10拷贝/μL。应用该实时荧光RPA方法可有效检测出血浆及血浆蛋白粉中的PEDV核酸。本研究建立的实时荧光RPA检测方法简单、快速、灵敏度高,可为PEDV的检测防控提供一种新的、可靠的技术支持。     

Establishment of Indirect ELISA Detection Method for Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea (PED) is an acute, highly contagious enteric disease of pigs. Porcine epidemic diarrhea virus (PEDV) is the causative agent of PED. PED has caused significant economic losses to the pig industry. In this study,the purified PEDV as the coating antigen, by optimizing the ELISA reaction conditions,the indirect ELISA antibody detection method was established. The optimized reaction conditions were as follows: Antigen working concentration was 20 μg/mL; Serum sample dilution was 1:500;It was coated at 4℃ overnight; The plates were blocked by 5% calf serum incubated at 37℃ for 1 h; The secondary antibody was diluted at 1:10 000,incubated at 37℃ for 1 h. It was judged as positive when the cutoff value D_{450 nm}≥0.289,as negative when D_{450 nm}≤0.236,and as suspicious between 0.289 and 0.236.It could not react with the positive sear of other six viruses such as porcine respiratory and reproductive syndrome virus,porcine circovirus 2, classical swine fever virus, porcine parvovirus,pseudo rabies virus and foot-and-mouth disease virus. The variation coefficient of repeated test was less than 10%.74 pig serum samples from Jiangsu, Jiangxi, Fujian and Guangdong were detected,and the positive rate was 84%.It indicated that this method could be used for PEDV epidemiological surveys and diagnosis in the future.     

猪流行性腹泻病毒间接ELISA检测方法的建立

猪流行性腹泻（porcine epidemic diarrhea,PED）是由猪流行性腹泻病毒（porcine epidemic diarrhea virus,PEDV）引起的猪的一种急性、高度传染性肠道疾病,给养猪业造成了严重的经济损失。本研究以纯化的PEDV 为包被抗原,通过优化 ELISA 反应条件,建立了间接 ELISA 抗体检测方法,其反应条件为：抗原最佳包被浓度为 20 μg/mL,血清标准品最佳稀释度为 1：500,包被时间为4℃过夜,5%小牛血清37℃封闭1 h,二抗 1：10 000稀释,37℃作用1 h,抗体临界值为 D_{450 nm}≥ 0.289判为阳性,D_{450 nm}≤ 0.236判为阴性,介于二者之间为可疑。检测猪繁殖与呼吸综合征病毒、猪圆环病毒2型、猪瘟病毒、伪狂犬病毒、细小病毒和口蹄疫病毒标准血清抗体均为阴性,重复试验变异系数均小于10%,对江苏、江西、福建、广东地区74份猪血清样品进行检测,抗体阳性率达84%,表明该方法具有较好敏感性、特异性和重复性,可用于 PEDV 抗体检测和流行病学调查。     

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.     

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.     

猪流行性腹泻病毒RT-PCR检测方法的建立

试验根据GenBank上发表的猪流行性腹泻病毒(PEDV)核衣壳(N)蛋白基因序列,设计合成了一对寡聚核苷酸引物,扩增大小为245 bp的目的片段,建立起PEDV N基因的RT-PCR检测方法。试验结果表明,该方法具有很强特异性和很高的敏感性,可作为猪流行性腹泻临床快速诊断的一种方法。     

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。