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A family-wide rt-pcr assay for detection of paramyxoviruses and application to a large-scale surveillance study

机译:用于检测副粘病毒的全家族性rt-pcr检测方法及其在大规模监测研究中的应用

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摘要

textabstractFamily-wide molecular diagnostic assays are valuable tools for initial identification of viruses during outbreaks and to limit costs of surveillance studies. Recent discoveries of paramyxoviruses have called for such assay that is able to detect all known and unknown paramyxoviruses in one round of PCR amplification. We have developed a RT-PCR assay consisting of a single degenerate primer set, able to detect all members of the Paramyxoviridae family including all virus genera within the subfamilies Paramyxovirinae and Pneumovirinae. Primers anneal to domain III of the polymerase gene, with the 3′ end of the reverse primer annealing to the conserved motif GDNQ, which is proposed to be the active site for nucleotide polymerization. The assay was fully optimized and was shown to indeed detect all available paramyxoviruses tested. Clinical specimens from hospitalized patients that tested positive for known paramyxoviruses in conventional assays were also detected with the novel family-wide test. A high-throughput fluorescence-based RT-PCR version of the assay was developed for screening large numbers of specimens. A large number of samples collected from wild birds was tested, resulting in the detection of avian paramyxoviruses type 1 in both barnacle and white-fronted geese, and type 8 in barnacle geese. Avian metapneumovirus type C was found for the first time in Europe in mallards, greylag geese and common gulls. The single round family-wide RT-PCR assay described here is a useful tool for the detection of known and unknown paramyxoviruses, and screening of large sample collections from humans and animals.
机译:全家庭范围的分子诊断测定是在暴发期间初步识别病毒并限制监视研究成本的宝贵工具。副粘病毒的最新发现要求进行这样的测定,其能够在一轮PCR扩增中检测所有已知和未知的副粘病毒。我们已经开发了一种由单个简并引物组组成的RT-PCR分析方法,能够检测副粘病毒科的所有成员,包括副粘病毒科和肺炎支原体亚科内的所有病毒属。引物退火至聚合酶基因的结构域III,反向引物的3'端退火至保守基序GDNQ,该基团被认为是核苷酸聚合的活性位点。该测定法经过充分优化,并显示出确实可以检测出所有可用的副粘病毒。新型全科试验还检测了住院患者的临床标本,这些患者在常规检测中对已知副粘病毒的检测呈阳性。开发了一种基于高通量荧光的RT-PCR版本的测定方法,用于筛选大量样品。对从野生鸟类收集的大量样品进行了测试,结果发现在藤壶和白额雁中都检出了禽副粘病毒1型,在藤壶鹅中检出了8型禽副粘病毒。在欧洲,首次在野鸭,灰雁和普通鸥中发现了C型禽肺炎病毒。此处描述的单轮全家族RT-PCR测定法是检测已知和未知副粘病毒以及筛选人和动物的大量样品的有用工具。

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