保存型與去活化型病毒檢體運送培養基的效能評估

病毒檢體運送培養基（transport media）的主要功能是保存檢體中病原體的活性、抗原及核酸，其可避免檢體在運送過程中受到汙染。由於新冠病毒（SARS-CoV-2）肺炎疫情（COVID-19）的爆發，其診斷係以拭子（swab）採集呼吸道檢體進行分子檢測及／或養殖細胞接種。啟新生物科技有限公司於2020年上市兩種通用型病毒檢體運送培養裝置，一為保存型CMP^(TM) viral transport medium w/oropharyngeal and/or nasopharyngeal flocked swab （VTM），可供P2+或P3等級檢驗室分離病毒及進行分子檢測；另一為去活化型CMP^(TM) inactivated viral transport medium w/oropharyngeal and/or nasopharyngeal flocked swab（I-VTM），僅可供檢驗室進行病毒之分子檢測。為了驗證此兩種病毒檢體運送培養基的效能，吾等根據CLSI（Clinical and Laboratory Standards Institute）之病毒運送系統之規範M40-A2（2014），選取與SARS-CoV-2同屬RNA病毒的腸病毒71型（Enteroviruses 71, EV 71）進行效能評估。測試時分別將10^3 PFU（plaque forming unit）病毒量接種至VTM或I-VTM中，並模擬病毒檢體的運送及保存條件，分別將兩種裝置的運送培養基置於4℃、-80℃及室溫，並分別貯存不同的指定時間點（在4℃保存6、24、48、72及96小時，而在-80℃及室溫保存48、72及96小時。此外，I-VTM在各測試溫度額外保存10分鐘、30分鐘、1、2、3及4小時）後將VTM或I-VTM內的EV 71分別移種於人類橫紋肌肉瘤細胞（human rhabdomyosarcoma cell line, RD cell）中，接著在5% CO_2培養箱中，培養48－72小時後觀察RD cell 的細胞病變效應（cytopathic effects, CPE）以及利用反轉錄－即時聚合酶鏈反應（quantitative reverse transcription PCR, qRT-PCR）偵測EV 71核酸（RNA）。本研究亦利用SARS-CoV-2檢測所用的三對基因片段（E gene、RdRp gene及N gene），以如同EV 71在VTM/I-VTM的測試條件下進行測試，結果顯示：（i）VTM在4℃、-80℃及室溫的貯存指定時間點皆能完整保存EV 71之活性，而在RD cell產生CPE；（ii）I-VTM在與EV 71接觸2小時後可將10^6 PFU的病毒完全裂解而將病毒去活化（滅活）；（iii）VTM及I-VTM在各種測試條件下皆能保存EV 71核酸及SARS-CoV-2之核酸而分別可被qRT-PCR方法檢出。基於上述的發現，吾等認為VTM及I-VTM適合用於病毒檢體的運送，個別檢驗室可根據病毒的檢驗方法、檢驗室設施的安全等級、檢驗標的病毒的風險等級（risk group）等選擇保存型或去活化型病毒檢體運送裝置輔助各種病原的檢測。

關鍵字

腸病毒71型；新冠病毒；病毒檢體運送培養裝置；細胞病變效應；反轉錄－即時聚合酶鏈反應

並列摘要

The main function of virus specimen transport media is to preserve the viability, antigen, and nucleic acid of viruses and to prevent the specimen from being contaminated during transportation. As diagnosis of the new pneumonia-causing coronavirus (SARS-CoV-2, COVID-19 virus) is based on swab collection of respiratory specimens for molecular testing and/or cell culture, the Creative Lifesciences Co., Ltd. (啟新生物科技有限公司) has launched two kinds of universal virus specimen transporting devices in 2020. One is CMP^(TM) preserved viral transport medium w/ oropharyngeal and/or nasopharyngeal flocked swab (VTM); it can be used in P2+ or P3 level laboratory for virus isolation and molecular detection. The other is CMP^(TM) inactivated viral transport medium w/ oropharyngeal and/or nasopharyngeal flocked swab (I-VTM); it can be used in P2 laboratory for virus molecular detection. To assess the effectiveness of these two virus specimen transport media, we tested them for transport of enteroviruses 71 (EV 71), which is also an RNA virus like the COVID-19 virus, according to the CLSI virus transport system specification M40-A2 (2014). During the test, both VTM and I-VTM were inoculated with 10^3 PFU of EV 71 placed at 4°C, -80°C and room temperature, and then examined at 6, 24, 48, 72, and 96 hours for those stored at 4°C and at 48, 72, and 96 hours for those stored at -80°C and room temperature. EV 71 inoculated I-VTMs were also examined at 10 min, 30 min, 1h, 2h, 3h, and 4h. The inoculated VTMs and I-VTMs were used to infect cells of the human rhabdomyosarcoma cell line (RD cell). The infected cells were incubated in a 5% CO_2 incubator for 48-72 hours and then examined for cytopathic effect (CPE) and proliferation of EV 71, and analyzed for viral nucleic acid by the reverse transcription real-time polymerase chain reaction (quantitative reverse transcription PCR, qRT-PCR). In this study, both VTM and I-VTM were also inoculated with three different sets of primers coded for E, RdRP, and N genes of SARS-CoV-2, and placed at 4, -80, and room temperature and analyzed for viral nucleic acid by the qRT-PCR at 24 and 96 hours. Results showed that: (i) VTM can effectively preserve EV 71 viability at 4°C, -80°C, and room temperature ; (ii) I-VTM can completely inactivate and lyse 10^6 PFU of viruses in 2 hours; (iii) VTM/I-VTM can effectively preserve EV 71 and SARS-CoV-2 nucleic acids for qRT-PCR analysis. Based on results of this study, we conclude that VTM or I-VTM is suitable for transport of virus specimens and suggest that Virology laboratories can select VTM or I-VTM based on their virus detection methods, safety level of the laboratory, and risk group of the target virus.

並列關鍵字

Enteroviruses 71 ； SARS-CoV-2 ； virus transport system ； cytopathic effects ； qRT-PCR

國際替代計量

保存型與去活化型病毒檢體運送培養基的效能評估

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