Ⅰ型脊髓灰质炎野病毒实时荧光RT-PCR快速检测方法的评价

Study on the Fast Testing Strategy for identifying the wild poliovirus Ⅰ

目的评价和探讨Ⅰ型脊髓灰质炎（脊灰）野病毒快速检测策略。方法采集671名来自脊灰野病毒流行疫区的在京学生粪便标本，采用一组实时荧光RT-PCR代替标准方法分别进行肠道病毒通用型检测、脊灰病毒分型检测、Ⅰ型脊灰疫苗株和野毒株鉴别检测。并利用Ⅰ、Ⅱ和Ⅲ型脊灰病毒标准株（Sabin株）和Ⅰ型野毒株对实时荧光RT-PCR和标准方法的灵敏度进行比较和评价。结果（1）新脊灰病毒检测流程（肠道病毒通用型检测＋脊灰病毒分型检测＋脊灰野毒鉴别检测）检出非脊灰肠道病毒33例；脊灰病毒疫苗株16例，其中Ⅰ型5例、Ⅱ型1例、Ⅲ型3例、Ⅰ＋Ⅱ型1例、Ⅰ＋Ⅲ型4例、Ⅰ＋Ⅱ＋Ⅲ型2例；脊灰病毒Ⅰ型野毒株3例。（2）3种实时荧光RT-PCR检测脊灰病毒比标准方法敏感1～100倍。其中肠道通用型检测和脊灰病毒分型检测2种方法对Ⅱ型脊灰病毒疫苗株的检测，比标准方法敏感100倍；脊灰病毒分型检测能准确区分Ⅰ、Ⅱ和Ⅲ型脊灰疫苗株，脊灰野毒株鉴别检测对Ⅰ型疫苗株的检测比标准方法敏感10倍；3种实时荧光RT-PCR均能有效检测出Ⅰ型脊灰野毒株，比标准方法敏感10倍。结论新的脊灰病毒检测流程，可大幅缩短检测时间，提高检测通量，且灵敏度高于标准方法，适合于脊灰应急检测应对突发疫情。

Objective To explore the Fast Testing Sstrategy （FTS） for wild poliovirus Ⅰ （WP1）. Methods Epidemiological investigations were carried out on 671 students from WP1 epidemic areas in China. A set of real time RT-PCR assays, including panenterovirus testings （PE） assay, poliovirus serotypings （PS） assay and the assay distinguishing wild strain from vaccine strain of poliovirus I （DWV） were introduced into the screening program for WPV1 to replace the conventional RT-PCR, recommended by the China National Polio Laboratory （GNPL）. Additionally, sensitivities of all the assays were assessed by poliovirus type I to HI （Sabin stain） and the isolated WPV1. Results （ 1 ） 33 non-poliovirus enterovirus （NPEV） cases were detected, with 16 polio vaccine- related cases including 5 polio Ⅰ, 1 polio Ⅱ, 3 poliom, 1 polio Ⅰ ＋11,4 polio Ⅰ＋ Ⅲ and 2 polio Ⅰ ＋ Ⅱ＋Ⅲ. Three WPV1 cases were also detected in this study and confirmed by GNPL. （2）For polio virus vaccine strain, sensitivities of the set of real time RT-PCR assays ranged from 1 to 100 times than that of the in-house RT-PCR assay. The sensitivities of PE and PS assays for the detection of polio Ⅱ were 100 times than that of the RT-PCR assay and the sensitivity of DWV assay used for the detection of polio I were 10 times than that of the RT-PCR assay. For WPV1, the sensitivity of three real time RT-PCR was 10 times higher than that of the RT-PCR assay. Conclusion The novel FTS for WPV1 suggested by this study would include PE, PS and DWV. It not only could greatly shorten the testing time but also more sensitive than the RT-PCR and suited for emergency detection for WPV1.