The authors would like to thank Sophie Castagnet and Nadia Doubli-Bounoua for their technical support. This work received financial support from the General Council of Calvados and the agreement of Region Basse-Normandie and French Government (CPER 2007-2013; project R25 p3). The authors would like to thank the experts of the AFNOR group and particularly Jean-Philippe Buffereau and Eric Dubois.

<ol>
	<li>Brault, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immune+response+of+foals+to+natural+infection+with+equid+herpesvirus-2+and+its+association+with+febrile+illness.">The immune response of foals to natural infection with equid herpesvirus-2 and its association with febrile illness.</a> Vet.Immunol.Immunopathol. 137 (1-2), 136-141 (2010).</li><li>Fortier, G., Van Erck, E., Pronost, S., Lekeux, P., Thiry, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Equine+gammaherpesviruses:+pathogenesis,+epidemiology+and+diagnosis.">Equine gammaherpesviruses: pathogenesis, epidemiology and diagnosis.</a> Vet.J. 186 (2), 148-156 (2010).</li><li>Hue, E. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+quantitation+of+equid+gammaherpesviruses+(EHV-2,+EHV-5)+in+nasal+swabs+using+an+accredited+standardised+quantitative+PCR+method.">Detection and quantitation of equid gammaherpesviruses (EHV-2, EHV-5) in nasal swabs using an accredited standardised quantitative PCR method.</a> J Virol.Methods. 198 (1), 18-25 (2014).</li><li>Diallo, I. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+real-time+PCR+for+the+detection+and+differentiation+of+equid+herpesvirus+1+(EHV-1)+and+equid+herpesvirus+4+(EHV-4).">Multiplex real-time PCR for the detection and differentiation of equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4).</a> Vet.Microbiol. 4 (1-3), 93-103 (2007).</li><li>Williams, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Equine+multinodular+pulmonary+fibrosis:+a+newly+recognized+herpesvirus-associated+fibrotic+lung+disease.">Equine multinodular pulmonary fibrosis: a newly recognized herpesvirus-associated fibrotic lung disease.</a> Vet.Pathol. 44 (6), 849-862 (2007).</li><li>Mullis, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+enzymatic+amplification+of+DNA+in+vitro:+the+polymerase+chain+reaction.">Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction.</a> Cold Spring Harb.Symp.Quant.Biol. 51 (1), 263-273 (1986).</li><li>EPA Office of Water (4607). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> EPA-815-B-04-001. Quality Assurance/Quality Control Guidance for Laboratories Performing PCR Analyses on Environmental Samples. , (2004).</li><li>Telford, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Equine+herpesviruses+2+and+5+are+gamma-herpesviruses.">Equine herpesviruses 2 and 5 are gamma-herpesviruses.</a> Virology. 195 (2), 492-499 (1993).</li><li>Fortier, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+equid+herpesvirus-5+in+respiratory+liquids:+A+retrospective+study+of+785+samples+taken+in+2006-2007.">Identification of equid herpesvirus-5 in respiratory liquids: A retrospective study of 785 samples taken in 2006-2007.</a> Vet.J. 182 (2), 346-348 (2009).</li><li>Brault, S. A., Bird, B. H., Balasuriya, U. B., MacLachlan, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+heterogeneity+and+variation+in+viral+load+during+equid+herpesvirus-2+infection+of+foals.">Genetic heterogeneity and variation in viral load during equid herpesvirus-2 infection of foals.</a> Vet.Microbiol. 147 (3-4), 253-261 (2011).</li><li>Association Francaise de Normalisation. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> NFU 47-600-1. Animal health analysis methods-PCR-Part 1: Requirements and recommandations for the implementation of veterinary PCR. , (2015).</li><li>Association Francaise de Normalisation. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> NFU 47-600-2. Animal health analysis methods-PCR-Part 2: Requirements and recommendations for the development and the validation of veterinary PCR. , (2015).</li><li>ISO. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> EN ISO-CEI 17025. General requirements for the competence of testing and calibration laboratories. , (2005).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manual of Diagnostic Tests and Vaccines for Terrestrial Animals 2010. Chapter1.1.1.4/ 5. Principles and Methods of Validation of Diagnostic Assay for Infectious Diseases. , (2009).</li><li>Apaza, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+genogrouping+of+noroviruses+from+children's+stools+by+Taqman+One-step+RT-PCR.">Detection and genogrouping of noroviruses from children's stools by Taqman One-step RT-PCR.</a> J Vis.Exp. (65), e3232 (2012).</li><li>Lorenz, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymerase+chain+reaction:+basic+protocol+plus+troubleshooting+and+optimization+strategies.">Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies.</a> J Vis.Exp. (63), e3998 (2012).</li><li>Sanger, F., Coulson, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+method+for+determining+sequences+in+DNA+by+primed+synthesis+with+DNA+polymerase.">A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase.</a> J Mol.Biol. 94 (3), 441-448 (1975).</li><li>Sanger, F., Nicklen, S., Coulson, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+sequencing+with+chain-terminating+inhibitors.">DNA sequencing with chain-terminating inhibitors.</a> Proc.Natl.Acad.Sci.U.S.A. 74 (12), 5463-5467 (1977).</li><li>NCBI. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> BLAST Homepage and Selected Search Pages. Introducing the BLAST homepage and form elements/functions of selected search pages. , (2015).</li><li>Greiner, M., Gardner, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+diagnostic+tests+in+veterinary+epidemiologic+studies.">Application of diagnostic tests in veterinary epidemiologic studies.</a> Prev.Vet Med. 45 (1-2), 43-59 (2000).</li><li>Blanchard, P., Regnault, J., Schurr, F., Dubois, E., Ribiere, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intra-laboratory+validation+of+chronic+bee+paralysis+virus+quantitation+using+an+accredited+standardised+real-time+quantitative+RT-PCR+method.">Intra-laboratory validation of chronic bee paralysis virus quantitation using an accredited standardised real-time quantitative RT-PCR method.</a> J Virol.Methods. 180 (1-2), 26-31 (2012).</li></ol>

作者宣称，他们没有竞争的经济利益。

自2000年代，实时PCR已在越来越多的实验室取代金标准技术（细胞培养物和细菌培养方法）。该技术的实现是比较容易的。然而实验方法验证了分子检测和病原体量化必须确保准确，可重复的和可靠的数据。 由于提取步骤是生物材料的损失的主要来源，它可被认为是一个协议和另一个之间的量化误差的主要来源。因此，定量RT-PCR过程中的DNA质粒的标准曲线，主要文献报道的建立，表明病毒基因组的负载，但不考虑提取步骤。 在AFNOR标准NF U47-600-2整个方法验证过程从头策略的说明表示在这方面显著的进展。如在所示本文的EHV-2在马，或由他人在蜜蜂21，这就需要开发步骤，并与整个方法中PCR的表征和鉴定验证步骤之间明显不同。在这个有趣的方法的一个限制是，在协议的任何改变将导致重新验证的完整过程可能是非常昂贵的义务。这种限制也被事实量化的范围取决于从该病毒被提取（ 例如，呼吸液，器官，血液或尿液）的源上突出显示。事实上，每个矩阵呈现不同特异性在其物理化学特征和独立地定义用于通过qRT-PCR的病毒检测和​​定量的每个不同的矩阵是重要的。因此，每个生物样品的病毒基因组的负载可以更精确地从提取定量。表征还考虑到热循环模埃尔并且当使用预先充分表征的方法（ 例如，本文描述的EHV-2定量PCR法）就必须在父实验室或其他实验室新类型的机器，必须确认仪器的性能。一个qPCR分析的性能的确认是一个先决条件为所有测试带来到实验室。这通常是通过分析与已知特性的参考样品来实现的。这种检查是一个先决条件，并认为强制由NF 47-600-1 AFNOR标准为了验证的定量PCR（LOD，LOQ效率）的性能和整个方法（LOD，LOQ）的鲁棒性要求。不仅在开发和表征步骤，但在研究或诊断目的时，也，风险因素可以识别和很好的控制，以确保该协议的标准化。特别值得关注的是足够的员工培训，高素质的人才，耗材的质量控制使用和储存，立即环境条件和计量条件的认识，可能会影响参与试验的科学仪器的性能控制。对于实验室间比使用的参考样本也可以帮助控制的不确定性。在这种方式下，实验室之间的数据的比较可以容易。事实上，实验室间的水平测试是必要的，以评估和确认方法的再现性。 这是在分析生物基质的国际单位（IU）表达的病毒基因组的负载的结果（IU:拷贝/ ml用于流体或副本组织/ g）的更易于使用，以比较不同实验室之间的结果。所有LOQ上方的结果表示为拷贝/ ml和LOD和LOQ之间的结果被作为不可计量的阳性结果。以这种方式呈现所述基因组的定量数据符合更精确到PROCES分析第（基因组的扩增）。实际上，在细胞培养实验中，通过TCID 50的病毒载量的表达（中值组织培养感染剂量） 依赖于细胞和病毒株的性质。每一株行拥有其独特的感染动力学和一些病毒如超高压-2可能需要数天前的第一个细胞病变效应是明显的。 总之，定量RT-PCR表征的新方法应促进实验室之间的数据演示和解释的协调统一。这将是在将来像建立截止值的对的疾病状态的声明，而不是仅仅是病原体的存在或不存在下定量RT-PCR检测的潜在的新的应用中是非常有用的。

马科动物疱疹病毒-2（EHV-2）参与了呼吸综合征，具有潜在的临床表现，如鼻分泌物，咽炎和淋巴结肿大1-3。这种病毒也怀疑与马的差性能，这可能导致对赛马业2显著和负面经济影响有关。 到现在为止，黄金标准伽玛超高压（γ-超高压）的检测是细胞培养法。这个过程的第一不便之处是不存在的EHV-2和其它区分γ-超高压的（ 例如，EHV-5）。第二不便是细胞病变的过程，从12至28天采取体现4,5的发展缓慢。 一验证和标准化定量实时聚合酶链反应的发展（QRT-PCR）方法将有助于快速检测病毒，EHV-2和EHV-区分5，并研究了病毒基因组负荷和疾病由于量化方面之间的关系。 聚合酶链反应（PCR）是首次于1986年6穆利斯描述即将成为大多数生物诊断（人类，环境和兽医）等领域的新的黄金标准。该方法中，这是基于病原体的基因组的一部分的放大，具有许多优点:特异性，灵敏度和快速性。此外，扩增子污染的风险，因为定量RT-PCR检测的出现和质量保证7退去。然而，PCR方法作为新的黄金标准方法识别必要不仅仅是改进的性能数据，而且在整个方法的开发和验证步骤的控制的无性能随时间的退化的示范多。 用于检测的第一分子工​​具EHV-2分别为时刻c接着测序8与巢式PCR onsuming和参与非特异性扩增。疱疹病毒的靶基因是脱氧核糖核酸（DNA）聚合酶和DNA包装9。然而，嵌套式PCR显示按扩增子污染的高风险。自那时以来，常规PCR试 ​​验已设计用于扩增白细胞介素10样基因或糖蛋白B的基因，在2009年2审查。最近，为EHV-2 10的定量进行了描述实时PCR特性，但没有数据可用关于整个方法包括提取过程的验证。 在这个协议中，开发和验证程序为根据协会法国标准化中马呼吸流体超高压-2 DNA的检测和定量的定量PCR方法描述（AFNOR）NF标准U47-600 -3,11,12-，这是法国代表国际标准化委员会。此规范详细介绍11,12了"动物卫生分析方法兽医PCR的实施，开发和验证要求和建议"，根据NF EN ISO / CEI 17025 2005 13和OIE（世界动物卫生组织） 。建议2010年14超高压-2实时定量RT-PCR验证协议包括三部分过程:（一）实时定量RT-PCR检测方法的建立（二）实时荧光定量PCR检测的单独表征及（c）整体表征分析方法（从生物样品的PCR分析的核酸提取）。 的定量RT-PCR检测的，整个分析方法的特征包括的两个限制的定义:检测限（LOD）的限制和定量限（LOQ）的限制。检测限的95％的PCR表示最低数目的，可以在所有CA的95％待检测的每单位体积的核酸拷贝上课。定量限95％的PCR表示的，可以被确定考虑到不确定性核酸拷贝的最低量。 这种定量RT-PCR方法允许精确的检测和呼吸流体超高压-2快速定量。此外，该方法可以在其他实验室应用，以确保其他新的qRT-PCR测定的开发的标准处理程序和通用模板。

<table><tbody><tr><td>AB-1900 natural color ABgene 96 well plate&amp;nbsp;</td><td>Dutsher</td><td>16924</td><td></td></tr><tr><td>Adhesive film qPCR Absolute&amp;nbsp;</td><td>Dutsher</td><td>16629</td><td>Adhesive film used for sealing the plate prior to the qRT-PCR run</td></tr><tr><td>0.5 ml microtubes, skirted, caps</td><td>Dutsher</td><td>039258</td><td></td></tr><tr><td>Ethanol 98%</td><td>Sodipro</td><td>SAF322941000</td><td></td></tr><tr><td>Primers</td><td>Eurofins</td><td>Custom order</td><td></td></tr><tr><td>Probe</td><td>Life Technologies</td><td>Custom order</td><td></td></tr><tr><td>Plasmid</td><td>Eurofins</td><td>Custom order</td><td></td></tr><tr><td>QIAamp&amp;nbsp; RNA viral Mini Kit&lt;br/&gt; (containing: QIAamp Mini column, AVL&amp;nbsp; buffer,&amp;nbsp; AW1 buffer, AW2 buffer, AVE buffer, collection tubes)&amp;nbsp;</td><td>Qiagen</td><td>52906</td><td>AVL buffer: pre-warm 5 min at 72 &amp;deg;C</td></tr><tr><td>Sequencing by Sanger method</td><td>Eurofins</td><td>Custom order</td><td></td></tr><tr><td>Taqman Universal PCR Master Mix</td><td>Life Technologies</td><td>4364340</td><td></td></tr><tr><td>Tris-EDTA buffer solution</td><td>Santa Cruz</td><td>sc-296653A</td><td></td></tr><tr><td>NanoDrop 2000c Spectrophotometer</td><td>Thermoscientific</td><td>ND-2000C</td><td></td></tr><tr><td>StepOnePlus Real-Time PCR systems</td><td>Life Technologies</td><td>4376600</td><td>pre-warm 15 min&amp;nbsp;</td></tr></tbody></table>

development and validation of a quantitative pcr method for equid herpesvirus-2 diagnostics in respiratory fluids

The protocol describes a quantitative RT-PCR method for the detection and quantification of EHV-2 in equine respiratory fluids according to the NF U47-600 norm. After the development and first validation step, two distinct characterization steps were performed according to the AFNOR norm: (a) characterization of the qRT-PCR assay alone and (b) characterization of the whole analytical method. The validation of the whole analytical method included the portrayal of all steps between the extraction of nucleic acids and the final PCR analysis. 
Validation of the whole method is very important for virus detection by qRT-PCR in order to get an accurate determination of the viral genome load. Since the extraction step is the primary source of loss of biological material, it may be considered the main source of error of quantification between one protocol and another. For this reason, the AFNOR norm NF-U-47-600 recommends including the range of plasmid dilution before the extraction step. In addition, the limits of quantification depend on the source from which the virus is extracted. Viral genome load results, which are expressed in international units (IU), are easier to use in order to compare results between different laboratories. 
This new method of characterization of qRT-PCR should facilitate the harmonization of data presentation and interpretation between laboratories.

定量RT-PCR法中，如上所述，被实施，以检测和呼吸液体量化马科动物疱疹病毒-2。 图1示出了根据AFNOR标准NF的定量RT-PCR法的开发和验证的示意流程图表U47-600。引物和探针的特异性一步一步发展PCR的过程中进行了验证。仅EHV-2菌株在本系统中扩增。随后，定量RT-PCR检测的性能必须表征。 首先，估计检测限的PCR，进行6十倍系列稀释液，以建立该减弱区（ 图2）。在这个例子中，6十倍系列稀释液10 -5和10 -10之间进行估算LOD PCR（26,000 0.26拷贝/ 2.5微升样品之间）。的减排区间l的10 -9和10 -10稀释之间IES（2.6和0.26个拷贝/ 2.5微升样品之间）。为了确定这种情况下的LOD PCR值，质粒的6两倍连续稀释在此消减区作了5.2和0.16个拷贝/ 2.5微升样品之间。该LOD PCR值2.6张/ 2.5微升样品。 为了确定线性范围和LOQ 的PCR，所述的LOD PCR值被用来启动的6十倍系列稀释液的范围内，2.6（LOD PCR）和26万拷贝/ 2.5微升样品之间。 图3示出了用于EHV2线性回归定量RT-PCR从一个试验。线性回归（ 图4）的性能在使用表3所述的计算一式四份进行了验证。执行的计算根据标准来定义的线性范围绝对偏差我 VALUE≤0.25日志10，质粒负荷的任何水平的我 。在这种情况下，线性范围铺设2.6和26万之间拷贝/ 2.5微升样品。该LOQ PCR是在线性范围的最低浓度（ 即 2.6拷贝/在这种情况下，2.5微升样品）。 üLIN被确定为0.12日志10范围2.6-260,000拷贝/ 2.5微升的DNA。 发展（ 图1，蓝色）和定量RT-PCR（ 图1，黄色）表征之后，AFNOR NF U47-600规范建议从DNA提取到QRT-PCR（ 图1，橙色）全分析方法表征。如表4中描述的方法计算出的诊断的敏感性和特异性。在定量RT-PCR全分析方法的定量性能进行评价和验证，精确度轮廓（ <strong&gt;的图5）。 该协议，它使用先进设备，最先进的分子生物学技术，使我们能够检测和量化，从与呼吸系统疾病和/或感染的临床怀疑马获得172鼻拭子样品超高压-2病毒基因组的负载。超高压-2场（生物）样品的发生率在这一人群中为50％（172分之86）。的定量分析表明，EHV-2的病毒基因组的负载在年轻马匹显著更高和病毒基因组的负载的重新分配与年龄（ 图6）降低。在本研究中，在马驹检测到最高EHV-2病毒基因组的负载（1.9×10 11拷贝/ ml）（ 图6）。 <img alt="图1" src="/files/ftp_upload/53672/53672fig1.jpg" /> 图1:用于开发（蓝色）的工作流图中，定量RT-PCR的表征根据AFNOR标准NF U47-600-2（黄色）和从DNA提取到定量RT-PCR（橙色），整个分析方法的特征。该工作流程图恢复为发展的不同步骤中，定量RT的表征-PCR和DNA提取到定量RT-PCR全分析方法的表征。对于每一个步骤，工作流图表显示运行所需的数量，稀释被执行和所需分析师的数量。 <a href="https://www.jove.com/files/ftp_upload/53672/53672fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53672/53672fig2.jpg" /> 图2:来自用质粒的6十倍系列稀释液得到的实时PCR曲线的代表性结果消减区的测定 。为了估计减排区，6个十倍串行稀释液10 -5之间进行（26000拷贝/ 2.5微升样品）和10 -10（0.26份/ 2.5微升样品）。减排区位于10 -9稀释之间（2.6张/ 2.5微升样品）和10 -10（0.26份/ 2.5微升样品）。在这种情况下，质粒的6两倍连续稀释在这个减排区分别以确定LOD 95％PCR，5.2和0.16拷贝/ 2.5微升样品之间。 <a href="https://www.jove.com/files/ftp_upload/53672/53672fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53672/53672fig3.jpg" /> 图3:线性回归为EHV2定量RT-PCR定量检测的线性是产生结果成比例的存在于一个特定范围内的目标的浓度的能力。这可以通过进行建模线性回归的仪器响应（循环阈值或CT）与目标量的对数之间（Y = AX + B）（目标拷贝/ 2.5微升样品数）。 <a href="https://www.jove.com/files/ftp_upload/53672/53672fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53672/53672fig4.jpg" /> 图4:EHV-2的qPCR的线性回归的性能平均偏压代表所测量的质粒数量之间的平均差（ <img alt="fig4-1" src="/files/ftp_upload/53672/fig4-1.jpg" /> ），并为每个质粒水平的理论质粒量（X&#39;i）中 。竖线代表由下式给出的线性不确定性（U·利尼 ） <img alt="4-2所示" src="/files/ftp_upload/53672/fig4-2.jpg" /> 其中，SD&#39;i为标准偏差Ø<html> F量度质粒数量。 <a href="https://www.jove.com/files/ftp_upload/53672/53672fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/53672/53672fig5.jpg" /> 图5:基于所述EHV-2定量RT-PCR法的验证结果的准确度轮廓的绿线（圆）表示数据的真实性（系统误差或偏差）。的可接受限制由实验室（虚线）在±0.75日志10限定。在较低和较高的精度限制，确定从平均偏差±可靠性数据（红色线）的两倍标准差的每个质粒的负荷水平。 <a href="https://www.jove.com/files/ftp_upload/53672/53672fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 1"&gt; <img alt="图6" src="/files/ftp_upload/53672/53672fig6.jpg" /> 图6:根据年龄EHV-2的病毒基因组的负载的定量 EHV-2鼻拭子样品中检测的病毒基因组的载荷分布被表示为不同的年龄组。水平线代表标准偏差（M =个月）内的中间值。 *通过ANOVA显着不同与纽曼-两比较事后检验（P &lt;0.05）。 <a href="https://www.jove.com/files/ftp_upload/53672/53672fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 靶基因 </td><td colspan="3"> 引物，探针和plasmide序列（5&#39;-3&#39;） </td><td> 核苷酸位点 </td><td> 产品尺寸（核苷酸） </td><td colspan="2"><strong&gt;的热循环条件</td><td> 参考 </td></tr><tr><td rowspan="4"> EHV2的gB （HQ247755.1） </td><td colspan="3">前锋:GTGGCCAGCGGGGTGTTC </td><td> 2113至30年</td><td rowspan="4"> 78 </td><td> 95℃5分钟</td><td></td><td rowspan="4"> 11 </td></tr><tr><td colspan="3">反向:CCCCCAAAGGGATTYTTGAA </td><td> 2189年至2170年</td><td> 95℃15秒</td><td rowspan="2"> 45个循环</td></tr><tr><td colspan="3">探头:FAM-CCCTCTTTGGGAGCATAGTCTCGGGG-MGB </td><td> 2132年至2157年</td><td> 60℃1分钟</td></tr><tr><td colspan="3">质粒: ACCTGGGCACCATAGGCAAGGTGGTGGTCA ATGTGGCCAGCGGGGTGTTCTCCCTCTTTG GGAGCATAGTCTCGGGGGTGATAAGCTTTTT CAAAAATCCCTTTGGGGGCATGCTGCTCATA GTCCTCATCATAGCCGGGGTAGTGGTGGTG TACCTGTTTATGACCAGGTCCAGGAGCATAT ACTCTGCCCCCATTAGAATGCTCTACCCCGG GGTGGAGAGGGCGGCCCAGGAGCCGGGCG CGCACCCGGTGTCAGAAGACCAAATCAGGA ACATCCTGATGGGAATGCACCAATTTCAG </td><td> 2081年至2381年</td><td></td><td></td></tr></tbody></table> 表1:在该协议中使用的引物，探针和阳性合成DNA对照序列的质粒的序列（正合成DNA）对应核苷酸位置EHV2gB序列（HQ247755.1）的2081至​​2381年。通过使用特定的软件获得的在该协议中使用的引物和探针的设计。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td rowspan="2"> 病原体 </td><td rowspan="2"> 参考（来源） </td><td rowspan="2"> 菌株数 </td><td> 结果 </td></tr><tr><td> 超高压-2 </td></tr><tr><td rowspan="2"> 超高压-2 </td><td> VR701（ATCC） </td><td row跨度="2"&gt; 20 </td><td rowspan="2">正面的</td></tr><tr><td> 20个样品（FDL集合） </td></tr><tr><td rowspan="2"> 超高压-5 </td><td> KD05（GERC） </td><td rowspan="2"> 20 </td><td rowspan="2">负</td></tr><tr><td> 20个样品（FDL集合） </td></tr><tr><td rowspan="2"> 超高压-3 </td><td> VR352（ATCC） </td><td rowspan="2"> 2 </td><td rowspan="2">负</td></tr><tr><td> T934 WSV（GERC） </td></tr><tr><td rowspan="2"> 超高压-1 </td><td>肯塔基州肯塔基州株A（ATCC） </td><td rowspan="2"> 3 </td><td rowspan="2">负</td></tr><tr><td> 2个样本（FDL集合） </td></tr><tr><td> 超高压-4 </td><td> VR2230（ATCC） </td><td> 1 </td><td>负</td></tr><tr><td> 愚蠢的疱疹病毒AHV5 </td><td> FDL系列</td><td> 1 </td><td>负</td></tr><tr><td rowspan="2"> 马流感病毒</td><td> A /马/ Jouars / 4/2006的（H3N8） </td><td rowspan="2"> 1 </td><td rowspan="2">负</td></tr><tr><td> （登记号JX091752） </td></tr><tr><td> 马动脉炎病毒 </td><td> VR796（ATCC） </td><td> 2 </td><td>负</td></tr><tr><td> 马红球菌 </td><td> FDL系列</td><td> 1 </td><td>负</td></tr><tr><td> 马链球菌亚种。兽疫 </td><td> FDL系列</td><td> 1 </td><td>负</td></tr><tr><td> 马链球菌亚种。球菌 </td><td> FDL系列</td><td> 1 </td><td>负</td></tr><tr><td> 贝氏柯克斯体 </td><td> ADI-142-100（Adiagene） </td><td> 1 </td><td>负</td></tr><tr><td> 流产衣原体 </td><td> ADI-211-50（Adiagene） </td><td> 1 </td><td>负</td></tr><tr><td> 克雷伯菌PNEUmoniae </td><td> FDL系列</td><td> 1 </td><td>负</td></tr></tbody></table> 表2:用于EHV-2定量RT-PCR的分析的特异性。 <img alt="表3" src="/files/ftp_upload/53672/53672table3.jpg" /> 表3:偏置和线性度的不确定性（改编自NF U47-600-2 12） 的计算 。对于每一个试验中，线性回归的性能（Y = AX + b）的使用，其中y是所得到的循环阈值表的验证; a是斜率得到; x是质粒水平和b是截距i是质粒级别（ⅰ从1变化至K的水平）;在叔k是使用质粒电平的数目（ 例如 ，k = 6 的他的表）; j是试验（J从1到I期试验）; I是试验的次数，包括（在该表例如 I = 4）3和6试验间X I是每个所估计的质粒数量。 我质粒的水平。X&#39; 我是方程x获得的理论质粒量"每个质粒我 i级= 10日志（X I）。在每个Ĵ试验中，对于每个i中得到的循环阈值 质粒级别与线性回归计算 Y I，J = 第j X I，J + B 学家 <img alt="表3-1" src="/files/ftp_upload/53672/table3-1.jpg" />是审判Ĵ期间测得的质粒数量。 我偏 </<html>子&gt;是测量质粒数量和用于每个试验的理论质粒数量和各质粒水平之间观察到的差异。 <img alt="表3-2" src="/files/ftp_upload/53672/table3-2.jpg" />是的平均值<img alt="表3-3" src="/files/ftp_upload/53672/table3-3.jpg" />由每个i质粒水平; SD&#39;i是测量的量的标准偏差<img alt="table3-4" src="/files/ftp_upload/53672/table3-4.jpg" />对于每个i 质粒水平; 平均偏差是偏向我的均值;ü 利尼是每个确定的线性不确定我从质粒计算SD&#39;i水平和平均偏差 。 <a href="https://www.jove.com/files/ftp_upload/53672/53672table3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 1"FO:保together.within页="1"FO:保持与 - next.within页="总是"&gt; <tbody><tr><td></td><td></td><td colspan="2">样品的真实身份</td></tr><tr><td></td><td></td><td>正面的</td><td>负</td></tr><tr><td rowspan="3">用全法得到的结果</td><td>正面的</td><td> RP（真阳） </td><td> FP（假阳性） </td></tr><tr><td>负</td><td> FN（假阴性） </td><td> RN（实际负） </td></tr><tr><td>总</td><td> RP + FN </td><td> FP + RN </td></tr><tr><td></td><td></td><td> SE = RP /（RP + FN） </td><td> SP = RN /（RN + FP） </td></tr></tbody></table> 表4: 整个方法的 诊断敏感性计算（SE） 和特异性（SP）所用的施瓦茨表计算confid在如NF U47-600-2描述的整个方法的敏感性和特异性的95％EnCE的间隔。

Watch this Scientific Journal Video about Development and Validation of a Quantitative PCR Method for Equid Herpesvirus-2 Diagnostics in Respiratory Fluids at JoVE.com

Development and Validation of a Quantitative PCR Method for Equid Herpesvirus-2 Diagnostics in Respiratory Fluids

Here, we present a protocol for the development and validation of a quantitative PCR method used for the detection and quantification of EHV-2 DNA in equine respiratory fluids. The EHV-2 qRT-PCR validation protocol involves a three-part procedure: development, characterization of qRT-PCR assay alone, and characterization of the whole analytical method.

为马科动物疱疹病毒2诊断的呼吸道液体定量PCR检测方法的开发和验证

The protocol describes a quantitative RT-PCR method for the detection and quantification of EHV-2 in equine respiratory fluids according to the NF U47-600 ...

development-validation-quantitative-pcr-method-for-equid-herpesvirus

Research

JoVE Journal

Immunology and Infection

28.2K 次观看.  LABÉO Frank Duncombe. Here, we present a protocol for the development and validation of a quantitative PCR method used for the detection and quantification of EHV-2 DNA in equine respiratory fluids. The EHV-2 qRT-PCR validation protocol involves a three-part procedure: development, characterization of qRT-PCR assay alone, and characterization of the whole analytical method.

视频: Development and Validation of a Quantitative PCR Method for Equid Herpesvirus-2 Diagnostics in Respiratory Fluids

Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant limitations, viruses are more resistant to many inactivation processes and standard fecal indicators do not inform on the source of contamination. The development of cost-effective methods for the concentration of viruses from water and molecular assays facilitates the applicability of viruses as indicators of fecal contamination and as microbial source tracking (MST) tools. Adenoviruses and polyomaviruses are DNA viruses infecting specific vertebrate species including humans and are persistently excreted in feces and/or urine in all geographical areas studied. In previous studies, we suggested the quantification of human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) by quantitative PCR (qPCR) as an index of human fecal contamination. Recently, we have developed qPCR assays for the specific quantification of porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as animal fecal markers of contamination with sensitivities of 1-10 genome copies per test tube. In this study, we present the procedure to be followed to identify the source of contamination in water samples using these tools. As example of representative results, analysis of viruses in ground water presenting high levels of nitrates is shown.
Detection of viruses in low or moderately polluted waters requires the concentration of the viruses from at least several liters of water into a much smaller volume, a procedure that usually includes two concentration steps in series. This somewhat cumbersome procedure and the variability observed in viral recoveries significantly hamper the simultaneous processing of a large number of water samples.
In order to eliminate the bottleneck caused by the two-step procedures we have applied a one-step protocol developed in previous studies and applicable to a diversity of water matrices. The procedure includes: acidification of ten-liter water samples, flocculation by skimmed milk, gravity sedimentation of the flocculated materials, collection of the precipitate and centrifugation, resuspension of the precipitate in 10 ml phosphate buffer. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyomaviruses of interest are quantified by qPCR. High number of samples may be simultaneously analyzed using this low-cost concentration method.
The procedure has been applied to the analysis of bathing waters, seawater and river water and in this study, we present results analyzing groundwater samples. This high-throughput quantitative method is reliable, straightforward, and cost-effective.

The study describes a cost-effective method for the identification of the source of fecal/urine contamination or contamination by nitrates in water using qPCR for the specific quantification of human/porcine/bovine DNA viruses, adenoviruses and polyomaviruses, proposed as MST tools.

微生物源使用特定的人类和动物病毒的追踪成本效益的方法

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant ...

科研

免疫与感染

Rapid Diagnosis of Avian Influenza Virus in Wild Birds: Use of a Portable rRT-PCR and Freeze-dried Reagents in the Field

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory flyways. Sampling of wild birds for avian influenza virus (AIV) is often conducted in remote regions, but results are often delayed because of the need to transport samples to a laboratory equipped for molecular testing. Real-time reverse transcriptase polymerase chain reaction (rRT-PCR) is a molecular technique that offers one of the most accurate and sensitive methods for diagnosis of AIV. The previously strict lab protocols needed for rRT-PCR are now being adapted for the field. Development of freeze-dried (lyophilized) reagents that do not require cold chain, with sensitivity at the level of wet reagents has brought on-site remote testing to a practical goal.
Here we present a method for the rapid diagnosis of AIV in wild birds using an rRT-PCR unit (Ruggedized Advanced Pathogen Identification Device or RAPID, Idaho Technologies, Salt Lake City, UT) that employs lyophilized reagents (Influenza A Target 1 Taqman; ASAY-ASY-0109, Idaho Technologies). The reagents contain all of the necessary components for testing at appropriate concentrations in a single tube: primers, probes, enzymes, buffers and internal positive controls, eliminating errors associated with improper storage or handling of wet reagents. The portable unit performs a screen for Influenza A by targeting the matrix gene and yields results in 2-3 hours. Genetic subtyping is also possible with H5 and H7 primer sets that target the hemagglutinin gene.
The system is suitable for use on cloacal and oropharyngeal samples collected from wild birds, as demonstrated here on the migratory shorebird species, the western sandpiper (Calidrus mauri) captured in Northern California. Animal handling followed protocols approved by the Animal Care and Use Committee of the U.S. Geological Survey Western Ecological Research Center and permits of the U.S. Geological Survey Bird Banding Laboratory. The primary advantage of this technique is to expedite diagnosis of wild birds, increasing the chances of containing an outbreak in a remote location. On-site diagnosis would also prove useful for identifying and studying infected individuals in wild populations. The opportunity to collect information on host biology (immunological and physiological response to infection) and spatial ecology (migratory performance of infected birds) will provide insights into the extent to which wild birds can act as vectors for AIV over long distances.

This study describes diagnosis of avian influenza in wild birds using a portable rRT-PCR system. The method takes advantage of freeze-dried reagents to screen wild birds in a non-laboratory setting, typical of an outbreak scenario. Use of molecular tools provides accurate and sensitive alternatives for rapid diagnosis.

在野生鸟类禽流感病毒快速诊断：使用便携式的RRT - PCR和在外地的冻干试剂

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory ...

Evaluation of a Universal Nested Reverse Transcription Polymerase Chain Reaction for the Detection of Lyssaviruses

To detect rabies virus and other member species of the genus Lyssavirus within the family Rhabdoviridae, the pan-lyssavirus nested reverse transcription polymerase chain reaction (nested RT-PCR) was developed to detect the conserved region of the nucleoprotein (N) gene of lyssaviruses. The method applies reverse transcription (RT) using viral RNA as template and oligo (dT)15 and random hexamers as primers to synthesize the viral complementary DNA (cDNA). Then, the viral cDNA is used as a template to amplify an 845 bp N gene fragment in first-round PCR using outer primers, followed by second-round nested PCR to amplify the final 371 bp fragment using inner primers. This method can detect different genetic clades of rabies viruses (RABV). The validation, using 9,624 brain specimens from eight domestic animal species in 10 years of clinical rabies diagnoses and surveillance in China, showed that the method has 100% sensitivity and 99.97% specificity in comparison with the direct fluorescent antibody test (FAT), the gold standard method recommended by the World Health Organization (WHO) and the World Organization for Animal Health (OIE). In addition, the method could also specifically amplify the targeted N gene fragment of 15 other approved and two novel lyssavirus species in the 10th Report of the International Committee on Taxonomy of Viruses (ICTV) as evaluated by a mimic detection of synthesized N gene plasmids of all lyssaviruses. The method provides a convenient alternative to FAT for rabies diagnosis and has been approved as a National Standard (GB/T36789-2018) of China.

A pan-lyssavirus nested reverse transcription polymerase chain reaction has been developed to detect specifically all known lyssaviruses. Validation using rabies brain samples of different animal species showed that this method has a sensitivity and specificity equivalent to the gold standard fluorescent antibody test and could be used for routine rabies diagnosis.

一种通用嵌套逆转录酶链反应检测裂解病毒的评价

To detect rabies virus and other member species of the genus Lyssavirus within the family Rhabdoviridae, the pan-lyssavirus nested reverse transcription ...

遗传学

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

在动物标本呼吸道病原体的高通量检测通过纳米PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

Field Postmortem Rabies Rapid Immunochromatographic Diagnostic Test for Resource-Limited Settings with Further Molecular Applications

Functional rabies surveillance systems are crucial to provide reliable data and increase the political commitment necessary for disease control. To date, animals suspected as rabies-positive must be submitted to a postmortem confirmation using classical or molecular laboratory methods. However, most endemic areas are in low- and middle-income countries where animal rabies diagnosis is restricted to central veterinary laboratories. Poor availability of surveillance infrastructure leads to serious disease underreporting from remote areas. Several diagnostic protocols requiring low technical expertise have been recently developed, providing opportunity to establish rabies diagnosis in decentralized laboratories. We present here a complete protocol for field postmortem diagnosis of animal rabies using a rapid immunochromatographic diagnostic test (RIDT), from brain biopsy sampling to the final interpretation. We complete the protocol by describing a further use of the device for molecular analysis and viral genotyping. RIDT easily detects rabies virus and other lyssaviruses in brain samples. The principle of such tests is simple: brain material is applied on a test strip where gold conjugated antibodies bind specifically to rabies antigens. The antigen-antibody complexes bind further to fixed antibodies on the test line, resulting in a clearly visible purple line. The virus is inactivated in the test strip, but viral RNA can be subsequently extracted. This allows the test strip, rather than the infectious brain sample, to be safely and easily sent to an equipped laboratory for confirmation and molecular typing. Based on a modification of the manufacturer&#39;s protocol, we found increased test sensitivity, reaching 98% compared to the gold standard reference method, the direct immunofluorescence antibody test. The advantages of the test are numerous: rapid, easy-to-use, low cost and no requirement for laboratory infrastructure, such as microscopy or cold-chain compliance. RIDTs represent a useful alternative for areas where reference diagnostic methods are not available.

We present a complete protocol for postmortem diagnosis of animal rabies under field conditions using a rapid immunochromatographic diagnostic test (RIDT), from brain biopsy sampling to final interpretation. We also describe further applications using the device for molecular analysis and viral genotyping.

现场验尸狂犬病快速免疫性诊断测试资源有限设置与进一步分子应用

Functional rabies surveillance systems are crucial to provide reliable data and increase the political commitment necessary for disease control. To date, ...

Contact-Free Co-Culture Model for the Study of Innate Immune Cell Activation During Respiratory Virus Infection

The early interactions between the nasal epithelial layer and the innate immune cells during viral infections remains an under-explored area. The significance of innate immunity signaling in viral infections has increased substantially as patients with respiratory infections who exhibit high innate T cell activation show a better disease outcome. Hence, dissecting these early innate immune interactions allows the elucidation of the processes that govern them and may facilitate the development of potential therapeutic targets and strategies for dampening or even preventing early progression of viral infections. This protocol details a versatile model that can be used to study early crosstalk, interactions, and activation of innate immune cells from factors secreted by virally infected airway epithelial cells. Using an H3N2 influenza virus (A/Aichi/2/1968) as the representative virus model, innate cell activation of co-cultured peripheral blood mononuclear cells (PBMCs) has been analyzed using flow cytometry to investigate the subsets of cells that are activated by the soluble factors released from the epithelium in response to the viral infection. The results demonstrate the gating strategy for differentiating the subsets of cells and reveal the clear differences between the activated populations of PBMCs and their crosstalk with the control and infected epithelium. The activated subsets can then be further analyzed to determine their functions as well as molecular changes specific to the cells. Findings from such a crosstalk investigation may uncover factors that are important for the activation of vital innate cell populations, which are beneficial in controlling and suppressing the progression of viral infection. Furthermore, these factors can be universally applied to different viral diseases, especially to newly emerging viruses, to dampen the impact of such viruses when they first circulate in na&#239;ve human populations.

This protocol details an investigation of the early interactions between virally infected nasal epithelial cells and innate cell activation. Individual subsets of immune cells can be distinguished based on their activation in response to viral infections. They can then be further investigated to determine their effects on early antiviral responses.

用于研究呼吸道病毒感染期间先天免疫细胞活化的非接触式共培养模型

The early interactions between the nasal epithelial layer and the innate immune cells during viral infections remains an under-explored area. The ...

An Improved and High Throughput Respiratory Syncytial Virus (RSV) Micro-neutralization Assay

Respiratory syncytial virus-specific neutralizing antibodies (RSV NAbs) are an important marker of protection against RSV. A number of different assay formats are currently in use worldwide so there is a need for an accurate and high-throughput method for measuring RSV NAbs. We describe here an imaging-based micro-neutralization assay that has been tested on RSV subgroup A and can also be adapted for RSV subgroup B and different sample types. This method is highly reproducible, with inter-assay variations for the reference antiserum being less than 10%. We believe this assay can be readily established in many laboratories worldwide at relatively low cost. Development of an improved, high-throughput assay that measures RSV NAbs represents a significant step forward for the standardization of this method internationally as well as being critical for the evaluation of novel RSV vaccine candidates in the future.

An Improved and High Throughput Respiratory Syncytial Virus RSV Micro-neutralization Assay

This study describes a high throughput, imaging-based micro-neutralization assay to determine the titer of neutralizing antibodies specific for respiratory syncytial virus (RSV). This assay format has been tested on different sample types.

一种改进的高通量呼吸道合胞病毒 (rsv) 微中和检测方法

Respiratory syncytial virus-specific neutralizing antibodies (RSV NAbs) are an important marker of protection against RSV. A number of different assay ...

医学

Efficient SARS-CoV-2 Quantitative Reverse Transcriptase PCR Saliva Diagnostic Strategy utilizing Open-Source Pipetting Robots

The emergence of the recent SARS-CoV-2 global health crisis introduced key challenges for epidemiological research and clinical testing. Characterized by a high rate of transmission and low mortality, the COVID-19 pandemic necessitated accurate and efficient diagnostic testing, particularly in closed populations such as residential universities. Initial availability of nucleic acid testing, like nasopharyngeal swabs, was limited due to supply chain pressure which also delayed reporting of test results. Saliva-based reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) testing has shown to be comparable in sensitivity and specificity to other testing methods, and saliva collection is less physically invasive to participants. Consequently, we developed a multiplex RT-qPCR diagnostic assay for population surveillance of Clemson University and the surrounding community. The assay utilized open-source liquid handling robots and thermocyclers instead of complex clinical automation systems to optimize workflow and system flexibility. Automation of saliva-based RT-qPCR enables rapid and accurate detection of a wide range of viral RNA concentrations for both large- and small-scale testing demands. The average turnaround for the automated system was &#60; 9 h for 95% of samples and &#60; 24 h for 99% of samples. The cost for a single test was $2.80 when all reagents were purchased in bulk quantities.

The protocol describes a SARS-CoV-2 diagnostic method that utilizes open-source automation to perform RT-qPCR molecular testing of saliva samples. This scalable approach can be applied to clinical public health surveillance as well as to increase the capacity of smaller university laboratories.

利用开源移液机器人的高效SARS-CoV-2定量逆转录酶PCR唾液诊断策略

The emergence of the recent SARS-CoV-2 global health crisis introduced key challenges for epidemiological research and clinical testing. Characterized by ...

使用 ddPCR 定量眼泪中的病毒载体进行生物脱落研究

A Validatable Droplet Digital Polymerase Chain Reaction Assay for the Detection of Adeno-Associated Viral Vectors in Bioshedding Studies of Tears

The use of viral vectors to treat genetic diseases has increased substantially in recent years, with over 2,000 studies registered to date. Adeno-associated viral (AAV) vectors have found particular success in the treatment of eye related diseases, as exemplified by the approval of voretigene neparvovec-rzyl. To bring new therapies to market, regulatory agencies typically request qualified or validated bioshedding studies to evaluate release of the vector into the environment. However, no official guidelines for the development of molecular based assays to support such shedding studies have been released by the United States Food and Drug Administration, leaving developers to determine best practices for themselves. The purpose of this protocol is to present a validatable protocol for the detection of AAV vectors in human tears by droplet digital polymerase chain reaction (ddPCR) in support of clinical bioshedding studies. This manuscript discusses current industry approaches to molecular assay validation and demonstrates that the method exceeds the target assay acceptance criteria currently proposed in white papers. Finally, steps critical in the performance of any ddPCR assay, regardless of application, are discussed.

作者聚焦:通过定量复杂基质中的病毒载体来解决分子研究中的监管差距 

Here, we present a protocol for the development and validation of good laboratory practices in the compliant detection of adeno-associated viral vectors in human tears by droplet digital polymerase chain reaction in support of clinical development of gene therapy vectors.