人博卡病毒2基因组扩增及序列分析

为获得人博卡病毒2(Human bocavirus 2,HBoV2)基因组序列,以2010年HBoV2单阳粪便标本为材料,PCR方法扩增HBoV2基因组不同区域,经序列拼接后得到5444bp的全基因组序列(HBoV2-NC)。系统进化分析显示,HBoV2-NC与兰州株HBoV2亲缘关系最近;DINAMelt末端回文结构预测证实,HBoV2-NC 5′末端存在反向互补序列,具有典型的细小病毒末端的茎环样结构;接头法PCR扩增得到HBoV2-NC部分末端侧翼序列。     

双重荧光RPA扩增方法快速鉴别两种血清型水泡性口炎病毒

本研究利用重组酶聚合酶扩增(RPA)技术,根据水泡性口炎病毒印第安纳型(VSV-IND)和新泽西型(VSVNJ)相对保守的L基因为靶序列设计并筛选出两套特异性的引物和探针,建立了快速鉴别检测水泡性口炎病毒两种血清型的双重荧光RT-RPA方法。试验结果表明,该方法特异性强,与猪水泡病病毒(SVDV)、口蹄疫病毒(FMDV)、蓝舌病病毒(BTV)、猪繁殖与呼吸综合征病毒(PRRSV)、牛病毒性腹泻病毒(BVDV)、猪瘟病毒(CSFV)等灭活抗原核酸无交叉反应;灵敏度高,最低可检测核酸浓度为12.7fg/μL;简便快速,反应时间短(15min),且重复性好。利用所建立方法对124份临床样品进行检测,结果与双重荧光RT-PCR一致。本文为VSV-IND和VSV-NJ的快速鉴别检测提供一种新方法,尤其适合现场检疫或基层实验室的快速检测。     

用SARS冠状病毒全基因组芯片杂交方法分析SARS-CoV

为从临床样品中检测和分析SARSCoV病毒打基础,并为分析SARSCoV病毒的复制和转录等机理提供一种有效方法。以SARS冠状病毒TOR2株序列作为标准设计和制备一种覆盖SARS冠状病毒全基因组的寡聚核苷酸芯片,探针长度为70nt,每相邻的探针序列重复25nt,共660条。用该芯片分析了细胞培养的SARSCoV病毒总RNA、7个SARSCoV病毒的基因克隆片段。对RNA样品用随机引物进行反转录PCR获得cDNA。对DNA用随机引物扩增和dUTPcy3标记。结果用这种芯片杂交检测SARSCoV病毒RNA可见阳性信号呈全基因组分布,并且有多处连续的阳性信号点;用正常人的白细胞RNA为对照,杂交未出现明显阳性信号。检测7个SARSCoV病毒基因克隆片段,在该片段相应的探针区段出现连续阳性信号点。这种方法可有效地检测和分析样品中SARS冠状病毒全基因组的信息。     

鹅副黏病毒环介导等温扩增快速检测方法的建立及应用

参照GenBank中登录的鹅副黏病毒(gallinacean paramyxovirus,GPMV)序列(AY325797),针对GPMV的F基因保守区域设计了4条引物,通过优化反应条件,建立了检测GPMV的逆转录环介导等温扩增技术(RT-LAMP),并进行了特异性试验、敏感性试验及临床样本检测.结果显示,建立的RT-LAMP方法对GPMV阳性样品扩增产物的电泳呈特征性梯状条带,而鸭瘟病毒(DPV)、鹅细小病毒(GPV)、番鸭细小病毒(MDPV)、鸭肝炎病毒(DHV)、鸭疫里默氏杆菌(Riemerella anatipestifer)、鸭源沙门氏菌(Salmonella anatamu)、鸡马立克病毒(MDV)、鸡传染性支气管炎病毒(IBV)的扩增产物电泳后均无扩增条带.建立的RT-LAMP方法对GPMV的最低检出量为9.5 fg的cDNA模板,敏感性比常规RT-PCR方法高1 000倍.表明建立的RT-LAMP方法特异性强、敏感性高.对6份临床样本的检出率为100％ (6/6),与常规RT-PCR的符合率为100％.     

新城疫液相芯片快速检测方法的建立

目的：建立可检测新城疫病毒（Newcastle disease virus,NDV）的液相芯片快速检测技术。方法：用DNAStar软件对GEN-BANK中NDV的NP基因进行序列分析设计NDV特异性探针并标记生物素,利用该探针与荧光编码微球偶联后与抽提的NDV病毒RNA的RT-PCR产物杂交反应,用液相芯片检测仪（Liquichip 200）检测荧光信号建立了NDV快速液相芯片检测方法。结果：检测结果显示,该法具有较好的特异性,不与H5AIV和H9AIV反应;检测灵敏度达到150个EID50;该法与鸡胚病毒分离法检出NDV的符合率达到97.1%。结论：初步建立了检测NDV的液相芯片技术,为进一步搭建NDV全新快速高通量检测平台奠定了基础,也为其他同类病毒的快速高通量检测提供了借鉴和经验。     

利用单基因组扩增法对马传染性贫血病毒疫苗株异质性的分析

前期研究发现,马传染性贫血病毒（Equine infectious anemia virus EIAV）中国弱毒疫苗株并非单一病毒,而是由多种准种（quasispecies）组成的种群。阐明该疫苗株的具体构成,对于确定优势疫苗株和分析其在体内的进化具有重要意义。本研究比较了传统RNA病毒测序法（即bulk PCR）和单基因组扩增法（Single-genome Amplification, SGA）在扩增EIAV疫苗株囊膜表面蛋白gp90基因 V3~V5区序列上的差异。结果发现,利用SGA法和bulk PCR法获得的序列在组内差异率分别为1.84%和1.88%。进一步序列比较发现,SGA法扩增的序列中除了含有与bulk PCR法中同源性较高的序列外,还存在bulk PCR法未检出的含强毒株LN40特异性位点,以及单个氨基酸缺失的序列。上述序列的存在为该疫苗株“多克隆构成”假说提供了佐证。此外,在对抽样偏差分析中发现,由于疫苗株中各种病毒准种在量上的差异,使得传统bulk PCR法不能有效的扩增组成比例较低的病毒准种,而导致测得的序列组成不能完全代表实际情况。SGA法通过对单基因组分的扩增和测序,可避免bulk PCR法的以上缺陷,在分析以准种形式存在的RNA病毒序列方面具有独特的优势。     

应用等温环介导扩增方法检测蜜蜂残翅病毒的研究

本文的目的在于建立用于临床检测残翅病病毒(Deformed wing virus,DWV)的等温环介导扩增技术(Loopmediated isothermal amplification,LAMP),为该疾病的预防和控制提供理论依据。在DWV基因保守序列设计4条引物,探究LAMP扩增的最优条件,并与常规的PCR(polymerase chain reaction)检测方法进行比较。建立的LAMP方法检测下限为0.89 pg,灵敏度比PCR高100倍而且特异性好。临床检测显示建立的LAMP方法可行、准确、方便、灵敏。针对DWV的LAMP建立的检测方法为养蜂生产第一线检测和预防DWV提供了技术支持,有一定的应用价值。     

9种呼吸道病毒微流体芯片检测体系构建与应用

近年来,越来越多新的致病性呼吸道病毒被发现,给口岸检验检疫机构的预防和控制造了成极大困扰,建立甲型流感病毒(Influenza A virus,FluA)、乙型流感病毒(Influenza B virus,FluB)、副流感病毒(Human Parainfluenza virus,HPIV)、冠状病毒(Coronavirus,Cov)、呼吸道合胞病毒(Respiratory syncytial virus,RSV)、人偏肺病毒(Humanmetapneumovirus,HMPV)、人博卡病毒(Human bocavirus,Hbov)、腺病毒(Adenovirus,Adv)和鼻病毒(Rhinovirus,Rhv)9种呼吸道病原体微流体芯片检测体系。选择人β-actin基因作为靶基因设计内标引物探针,根据9种呼吸道病原体基因组的保守区设计相应的引物探针;在Ultra Fast Labchip Real-time PCR V280平台上,筛选合适的RT-PCR Master Mix;对扩增体系中引物探针浓度进行优化,用优化后的扩增体系进行灵敏度和特异性验证;用100例临床标本进行回顾性检测...     

酵母全基因组芯片的应用

酵母作为最常用的模式生物,其全基因组测序最先完成。利用已知的酵母基因组信息,结合基因芯片技术,可进一步系统研究酵母的功能基因组表达。基因芯片技术是上世纪末发展起来的一项集分子生物学、生物信息学和电子学等科目的生物高新技术。酵母全基因组芯片,可以用以从基因表达水平,研究环境、物理、化学因子、毒理和药物作用的机制,在最终阐明酵母基因组功能的同时,为生物学研究提供更优化的模式生物模型。     

Evaluation of methods for single hair DNA amplification

Because of the low amount of DNA in single hairs, it may be difficult to obtain reliable genotypes for forensic and conservation genetics studies. We therefore compared different methods for reliably genotyping single hair samples. Our results indicate that preliminary whole genome amplification can increase the likelihood of successfully genotyping a single hair compared to other commonly used protocols. The difference between the methods is small for single locus comparisons, but it becomes more important in multi-locus comparisons. The economic and time costs of the whole genome amplification may prevent its large-scale use in non-invasive monitoring programs. Nevertheless, it may be a very useful approach for the analysis of especially valuable samples.     

不同单细胞全基因组扩增方法的比较及MALBAC在辅助生殖中的应用

单细胞全基因组扩增(whole genome amplification, WGA)是指在单细胞水平对全基因组进行扩增的新技术,其原理是将分离的单个细胞的微量全基因组DNA进行扩增,获得高覆盖率的完整的基因组后进行高通量测序,用于揭示细胞异质性。目前,WGA方法主要包括引物延伸预扩增(primer extension preamplification PCR, PEP-PCR)、简并寡核苷酸引物PCR (degenerate oligonucleotide primed PCR, DOP-PCR)、多重置换扩增(multiple displacement amplification, MDA)、多次退火环状循环扩增(multiple annealing and looping-based amplification cycles, MALBAC)等。本文对不同的单细胞WGA方法的原理及应用情况分别进行了阐述,并对其扩增效率进行评价和比较,包括基因组覆盖度、均一性、重现性、SNV (single-nucleotide variants)和CNV (copy number variants)检测力等。综合对比不同单细胞WGA方法后发现,MALBAC的扩增均一性最高、等位基因脱扣率最低、重现性最好,且对于CNV和SNV的检测效果最好。本文还阐述了MALBAC技术在人类单精子减数重组、非整倍体分析以及人类卵细胞基因组研究中的应用。     

Whole genome amplification of the obligate intracellular pathogen Coxiella burnetii using multiple displacement amplification

This study demonstrates that whole genome multiple displacement amplification (MDA) is a promising technique for downstream genomic analysis of fastidious obligate intracellular pathogens such as Coxiella burnetii. The MDA technology can help in obtaining sufficient genetic material from highly infectious agent and thus minimizing repeated culturing and associated biohazard.     

Isothermal amplification detection of nucleic acids by a double-nicked beacon

Isothermal and rapid amplification detection of nucleic acids is an important technology in environmental monitoring, foodborne pathogen detection, and point-of-care clinical diagnostics. Here we have developed a novel method of isothermal signal amplification for single-stranded DNA (ssDNA) detection. The ssDNA target could be used as an initiator, coupled with a double-nicked molecular beacon, to originate amplification cycles, achieving cascade signal amplification. In addition, the method showed good specificity and strong anti-jamming capability. Overall, it is a one-pot and isothermal strand displacement amplification method without the requirement of a stepwise procedure, which greatly simplifies the experimental procedure and decreases the probability of contamination of samples. With its advantages, the method would be very useful to detect nucleic acids in point-of-care or field use.     

一种快速检测微囊藻毒素mcyG基因的新技术——环介导恒温扩增

利用环介导恒温扩增(LAMP)技术,以微囊藻毒素(Microcystins,MCs)合成基因簇中的mcyG基因为靶序列,设计了1 套LAMP引物,建立了LAMP反应体系并进行灵敏度和特异性实验。结果表明mcyG基因的最低检测限为:24 cfu/mL,远低于常规PCR(Polymerase Chain Reaction)。整个检测过程仅需40 min,且可直接目测结果。特异性实验中, 13 株淡水常见水华蓝藻分属:色球藻属(Chroococcus)、念珠藻属(Nostoc)、鱼腥藻属(Anabaena)、束丝藻属(Aphanizomenon)、微囊藻属(Microcystis),其中10 株呈阳性反应, 3 株为阴性。在野外样品检测中,来自太湖与黄庆苗池塘的水样PCR检测显示阴性反应,而LAMP检测均呈阳性反应,提示此两处水样中可能含有产毒微囊藻,显示出了LAMP检测方法良好的野外检测和预警能力。综合上述,LAMP检测方法能够快速检测产微囊藻毒素的关键基因,且结果可视化。该方法简便、快捷、不依赖特殊检测设备,极具推广前景。     

基于环介导恒温扩增技术的大肠杆菌O157:H7快速检测试剂盒的评价

【目的】评价基于环介导恒温扩增技术(LAMP)的大肠杆菌O157:H7(Escherichia coli O157:H7)快速检测试剂盒的实效性。【方法】测定快速检测试剂盒的特异性、灵敏度、重复性、保质期以及运输稳定性,并与传统方法对比检测实际样品。【结果】大肠杆菌O157:H7标准菌株样品均检测为阳性,非大肠杆菌O157:H7标准菌株样品均检测为阴性,未发现有交叉反应;试剂盒最低检验限为29 CFU;该试剂盒的特异性、灵敏度及准确度与传统方法相比具有较高的一致性;试剂盒对高菌量目标菌和阴性菌样品的检测重复率均为100%,对低菌量目标菌样品的批间检测重复率为94%。试剂盒可在4°C保存9个月以上,并且可进行变温储存72 h以上。【结论】该试剂盒特异性好,灵敏度高,重复性好,储存方便,检测结果稳定、可靠,适用于对食品中大肠杆菌O157:H7的检测需求。     

Loop‐mediated isothermal amplification for the rapid detection of Gardnerella vaginalis

The aim of this study was to develop a method for the rapid detection of Gardnerella vaginalis, which is proposed to play a key role in the pathogenesis of bacterial vaginosis. Specific loop‐mediated isothermal amplification (LAMP) primers were designed and used to detect target DNA within 45 min under isothermal conditions. Comparative screening indicated that the LAMP assay is superior to PCR in terms of rapidity, and is equivalent in sensitivity and specificity. This LAMP assay can be used for rapid screening and detection of G. vaginalis in vaginal samples; the limit of detection is 10 fg DNA.

     

Development and evaluation of a loop‐mediated isothermal amplification assay for rapid detection of chicken anaemia virus

Aim: Chicken anaemia virus (CAV) causes an economically important viral disease in chickens worldwide. The main aim of this study was to establish a rapid, sensitive and specific loop‐mediated isothermal amplification (LAMP) assay for detecting CAV infection. Methods and Results: A set of four specific LAMP primers were designed based on the nucleotide sequence of the CAV VP2 gene, which encodes a nonstructural protein. These were used for the amplification of a specific target region of the VP2 gene. LAMP amplicons were successfully amplified and detected by DNA electrophoresis and by direct naked eye SYBR Green I visualization. A sensitivity test systematically demonstrated that the LAMP assay was superior to a conventional PCR assay with a minimum concentration limit of 100 fg compared to 10 ng for the conventional PCR. The specificity of the LAMP assay for CAV detection is consistent with conventional PCR. Using this established LAMP assay, infected and uninfected clinical samples obtained from an experimental farm were fully verified. Conclusions: A novel nucleic acid‐based approach of LAMP assay was successfully developed for detecting CAV infection. Significance and Impact of the Study: In this study, these results indicate that the developed LAMP assay herein for CAV detection is a time‐effective, simple, sensitive and specific test that can be used as an alternative approach in the future for large‐scaled diagnosis on the farm of CAV infection.     

Evaluation of a rapid microbial detection method via phage lytic amplification assay coupled with Live/Dead fluorochromic stains

AIMS: To develop a method for rapid detection of bacteria via bacteriophage amplification coupled with exogenous fluorochromic stains. METHODS AND RESULTS: A method for the rapid detection of bacteria was developed which consisted of exposing the sample suspected to contain target cells to host-specific phage. After at least one infection cycle, bacteria known to be infected by the phage (helper cells) were added and the number of nascent phage particles was estimated using the Live/Dead BacLight Bacterial Viability kit. Using Pseudomonas aeruginosa, it was shown that the dead helper cell population following phage infection was proportional to the initial number of target cells present in the original sample. Approximately 1 x 10(1) CFU per ml of P. aeruginosa could be detected within 4 h without the need for enrichment. CONCLUSIONS: The phage lytic amplification assay coupled with exogenous fluorochromic stains was able to detect approx. 1 x 10(1) CFU per ml of the target bacterium within 4 h. SIGNIFICANCE AND IMPACT OF THE STUDY: A method to detect low number of bacterial cells in a sample within 4 h without the need for enrichment was developed.