Validation of a redesigned pan-poliovirus assay and real-time PCR platforms for the global poliovirus laboratory network.
Validation of a redesigned pan-poliovirus assay and real-time PCR platforms for the global poliovirus laboratory network.
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DOI：
10.1371/journal.pone.0255795
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发表时间：
2021
期刊：
影响因子：
3.7
通讯作者：
Vega E
中科院分区：
文献类型：
--
作者：
Sun H;Harrington C;Gerloff N;Mandelbaum M;Jeffries-Miles S;Apostol LNG;Valencia MAD;Shaukat S;Angez M;Sharma DK;Nalavade UP;Pawar SD;Pukuta Simbu E;Andriamamonjy S;Razafindratsimandresy R;Vega E
脊灰病毒（PV）的监测和检测对于监测根除进展仍然至关重要。使用实时RT-PCR试剂盒进行细胞内分化（ITD）是监测工作流程的关键，其中在细胞培养分离后筛选病毒，然后通过测序验证子集。ITD试剂盒是一系列实时RT-PCR检测试剂盒，可使用标准WHO病毒分离方法筛选致细胞病变效应（CPE）阳性细胞培养物。由于ITD筛查是脊髓灰质炎病毒鉴定工作流程中的关键程序，因此实时PCR平台的性能验证是使用ITD试剂盒检测脊髓灰质炎病毒的核心要求。此外，需要不断更新和改进ITD测定以简化所有平台的判读，以确保所有实时机器能够检测阳性实时信号。使用ITD试剂盒和重新设计的脊髓灰质炎病毒探针验证了四个平台（ABI 7500实时系统、Bio-Rad CFX 96、Stratagene MX3000 P和Qiagen Rotor-Gene Q）。实时RT-PCR泛脊髓灰质炎病毒（PanPV）检测中的脊髓灰质炎病毒探针用双淬灭剂（Zen™）重新设计，以减少背景荧光和潜在的假阴性。使用由184种脊髓灰质炎病毒和非脊髓灰质炎肠道病毒组成的组对更新的PanPV探针进行了评价。为了进一步验证更新的PanPV探针，新的检测方法在五个全球脊髓灰质炎实验室网络（GPLN）实验室（马达加斯加、印度、菲律宾、巴基斯坦和刚果民主共和国）进行了试点测试。与标准PanPV探针相比，更新的PanPV探针性能显示出减少背景荧光和减少假阳性的数量。
Surveillance and detection of polioviruses (PV) remain crucial to monitoring eradication progress. Intratypic differentiation (ITD) using the real-time RT-PCR kit is key to the surveillance workflow, where viruses are screened after cell culture isolation before a subset are verified by sequencing. The ITD kit is a series of real-time RT-PCR assays that screens cytopathic effect (CPE)-positive cell cultures using the standard WHO method for virus isolation. Because ITD screening is a critical procedure in the poliovirus identification workflow, validation of performance of real-time PCR platforms is a core requirement for the detection of poliovirus using the ITD kit. In addition, the continual update and improvement of the ITD assays to simplify interpretation in all platforms is necessary to ensure that all real-time machines are capable of detecting positive real-time signals. Four platforms (ABI7500 real-time systems, Bio-Rad CFX96, Stratagene MX3000P, and the Qiagen Rotor-Gene Q) were validated with the ITD kit and a redesigned poliovirus probe. The poliovirus probe in the real-time RT-PCR pan-poliovirus (PanPV) assay was re-designed with a double-quencher (Zen™) to reduce background fluorescence and potential false negatives. The updated PanPV probe was evaluated with a panel consisting of 184 polioviruses and non-polio enteroviruses. To further validate the updated PanPV probe, the new assay was pilot tested in five Global Polio Laboratory Network (GPLN) laboratories (Madagascar, India, Philippines, Pakistan, and Democratic Republic of Congo). The updated PanPV probe performance was shown to reduce background fluorescence and decrease the number of false positives compared to the standard PanPV probe.
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影响因子：
168.9
作者：
HULL, HF;WARD, NA;DEQUADROS, C
通讯作者：
DEQUADROS, C
影响因子：
9.4
作者：
Kilpatrick, DR;Nottay, B;Kew, OM
通讯作者：
Kew, OM
影响因子：
5.4
作者：
Burns, CC;Shaw, J;Kew, O
通讯作者：
Kew, O
影响因子：
5
作者：
YANG, CF;DE, LN;KEW, OM
通讯作者：
KEW, OM
DOI：
10.1080/00365510701666031
发表时间：
2008-01-01
影响因子：
2.1
作者：
Trajman, A.;Luiz, R. R.
通讯作者：
Luiz, R. R.