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Immunology and Infection

人类诺沃克病毒的表面上检测拭子采样方法

Published: February 6th, 2017

DOI:

10.3791/55205

1Division of Viral Diseases, Centers for Disease Control and Prevention

A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.

诺如病毒的人是流行性和世界各地的零星胃肠炎的主要原因。因为大多数感染或者直接经由人对人的路线通过环境表面或食物传播或间接地受污染的非生物媒介和无生命的表面是用于病毒的期间诺罗病毒爆发的传播重要的车辆。

我们开发和评估使用大泡拭子用于检测和从硬表面人类诺如病毒的打字的协议。纤维拭子或防静电抹布相比,大泡拭子允许从高达700 cm 2的马桶座面恢复病毒(范围1.2-33.6%)。该协议包括用于病毒从拭子提取和使用旋转柱的病毒RNA的进一步浓缩的步骤。总共127已经从在游船和长期护理设施的表面,其中诺罗病毒性胃肠炎已经收集到217拭子样品(58.5%)报道药检呈阳性被RT-qPCR的诺如病毒GII。这些29(22.8%)的可以成功基因分型。总之,检测使用我们开发可以帮助确定环境污染的暴发期间水平以及检测病毒的当临床样品是不可用的协议环境表面上诺罗病毒的;它也可能有利于整治策略的有效性进行监测。

诺如病毒的人是流行性和散发性急性胃肠炎全世界1,2,3的主要原因。该病毒是极具传染性并通过直接的人发生互动的人或间接通过受污染的食物,水或环境表面接触传播。诺如病毒可棚长时间和长期的环境表面上的病毒的生存已记载1,2,3。在高峰脱落,十亿病毒颗粒被每克释放粪便和呕吐物还含有足够数量的病毒颗粒中,以引起感染4,5,6,7,8,EF“> 9,10,另外,可以很容易地发生无生命的表面和人的皮肤之间的病毒传输2,1....

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1.棉签取样的场

  1. 穿一双干净的手套。
  2. 测量取样区域的大小,而无需使用卷尺或直尺接触到表面。尝试尽可能准确地估计该地区并填写报告表(补充表1)。
  3. 检查可能的泄漏和标签样本运输袋,包棉签拭子套件。
  4. 移动拭子穿过取样面积如下:在水平方向的行程,在垂直方向的一个行程,并在对角方向的一个笔划。不要擦拭表面面积超过700 平方厘米大.......

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图1呈现拭子采样协议的流程图。这个协议包括四个主要步骤; 1)样品的采集,2)样品存储和运输,3)病毒RNA纯化和浓缩和4),RT-qPCR分析和基因分型。

图1
图1:流量为诺如病毒的环境表面取样最终协议的图表

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诺如病毒有18和10 3病毒颗粒20之间的50%的人感染剂量。因此,即使表面的低水平的污染可能对公共健康危险。拭子采样协议的若干方面进行了评价,包括:1)不同拭子的材料,2)在运输过程中贮存条件拭子,3)病毒RNA的浓度,和4)噬菌体MS2作为内提取的控制。

直到最近,才由棉,涤纶,锦纶和抗静电擦拭)制成拭子的性能进行了评估,并提出适?.......

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The authors have no acknowledgements.

....

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NameCompanyCatalog NumberComments
Generic name for kits
Macrofoam swabPremoistened EnviroMax Swab kit Puritan2588060PFUW
 RNA Lysis buffer CDC UNEX bufferMicrobiologicsCat No MR0501
RNA extraction spin columnMidi columnOmega BiotekCat No R6664-02
RNA purification spin columnZymol RNA Clean and Concentrator kit Zymo ResearchCat No R1016
Real time RT-PCR kitAgPath kit One-Step RT-PCR KitLife TechnologiesCat No 4387391
Conventional RT-PCR kitQiagen one step RT-PCR kitQiagen kitCat No 210212
Gel extraction kitQiagen QIAquick gel extraction kitQiagen kitCat No 28704 or 28706
Coliphage MS2ATCCCat No 15597-B1
RNA run-off transcriptsBacteriophage MS2 (ATCC No. 15597-B1) can be cultivated using Escherichia coli (E.coli) Famp (ATCC No. 700891). 
Realtime PCR platformApplied BiosystemsModel ABI 7500GI and GII RNA run off transcripts were quantified spectrophotometrically at A260, diluted in diethyl pyrocarbonate-treated water to 1 × 106 copies/ μl, and stored at −80°C with 1.0 U /μl RNasin (Promega, Madison, WI). 
Optical 96-well reaction plateThermo ScientificCat No 4316813
MicroAmp Clear Adhesive Film Thermo ScientificCat No 4306311

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