通过聚合酶链反应进行可靠的高危型人乳头瘤病毒DNA检测：方法间和方法内比较
Reliable high risk HPV DNA testing by polymerase chain reaction: an intermethod and intramethod comparison.
作者信息
Jacobs M V, Snijders P J, Voorhorst F J, Dillner J, Forslund O, Johansson B, von Knebel Doeberitz M, Meijer C J, Meyer T, Nindl I, Pfister H, Stockfleth E, Strand A, Wadell G, Walboomers J M
机构信息
Department of Pathology, University Hospital Vrije Universiteit, Amsterdam, The Netherlands.
出版信息
J Clin Pathol. 1999 Jul;52(7):498-503. doi: 10.1136/jcp.52.7.498.
BACKGROUND
The development of a reproducible, sensitive, and standardised human papillomavirus (HPV) polymerase chain reaction (PCR) test is required to implement HPV testing in cervical cancer screening programmes and for triaging women with mild to moderate dysplasia.
AIMS
To determine the intermethod agreement between different GP5+/6+ and MY09/11 PCR based protocols for the detection and typing of high risk (HR) HPV DNA in cervical smears and to assess the intramethod reproducibility of the GP5+/6+ PCR enzyme immunoassay (EIA) for HR-HPV detection.
METHODS
For the intermethod comparison, crude aliquots of 20 well characterised cervical smears comprising five HPV negative samples, and six and nine samples containing single and multiple HPV infections, respectively, were coded and sent from reference laboratory (A) to three other laboratories. One of these (laboratory B) used the GP5+/6+ PCR-EIA and was provided with standard protocols. Another laboratory (C) used GP5+/6+ PCR combined with sequence analysis and type specific PCR, whereas two laboratories (D and E) used MY09/11 PCR followed by restriction fragment length polymorphism (RFLP) analysis for the detection and typing of HR-HPV. The intramethod agreement of GP5+/6+ PCR-EIA was analysed in a subsequent study with four other laboratories (F to I) on crude aliquots of 50 well characterised cervical smears, consisting of 32 HR-HPV positive and 18 HPV negative samples. Standardised protocols, primers, and probes were also provided by the reference laboratory for HR-HPV detection.
RESULTS
In the intermethod comparison, pairwise agreement of the different laboratories with reference laboratory A for the detection of HR-HPV varied between 75% and 100% (kappa values: 0.5 to 1). Typing data revealed a broader range in pairwise agreement rates between 32% and 100%. The highest agreement was found between laboratories A and B using standardised protocols and validated reagents. In the intramethod evaluation, pairwise comparison of the laboratories F to I with reference laboratory A revealed excellent agreement rates from 92% to 100% (kappa values: 0.88 to 1.0) with an overall sensitivity of 97.5% (195/200) and specificity of 99.5% (199/200).
CONCLUSIONS
The detection of HR-HPV as a group is highly reproducible with GP5+/6+ PCR-EIA provided that standardised protocols and validated reagents are used.
背景
在宫颈癌筛查项目中实施人乳头瘤病毒（HPV）检测以及对轻度至中度发育异常的女性进行分流，需要开发一种可重复、灵敏且标准化的HPV聚合酶链反应（PCR）检测方法。
目的
确定基于不同GP5+/6+和MY09/11 PCR的方案在检测宫颈涂片高危（HR）HPV DNA及其分型方面的方法间一致性，并评估GP5+/6+ PCR酶免疫测定（EIA）在检测HR-HPV方面的方法内重复性。
方法
为进行方法间比较，将20份特征明确的宫颈涂片的粗提物等分试样进行编码，这些试样包括5份HPV阴性样本，以及分别含有单一和多重HPV感染的6份和9份样本，从参考实验室（A）发送至其他三个实验室。其中一个实验室（B）使用GP5+/6+ PCR-EIA，并提供了标准方案。另一个实验室（C）使用GP5+/6+ PCR结合序列分析和型特异性PCR，而两个实验室（D和E）使用MY09/11 PCR，随后进行限制性片段长度多态性（RFLP）分析以检测和分型HR-HPV。在随后的一项研究中，与另外四个实验室（F至I）对50份特征明确的宫颈涂片粗提物（包括32份HR-HPV阳性和18份HPV阴性样本）进行了GP5+/6+ PCR-EIA的方法内一致性分析。参考实验室也提供了用于检测HR-HPV的标准化方案、引物和探针。
结果
在方法间比较中，不同实验室与参考实验室A在检测HR-HPV方面的两两一致性在75%至100%之间（kappa值：0.5至1）。分型数据显示两两一致率范围更广，在32%至100%之间。使用标准化方案和经过验证的试剂的实验室A和B之间一致性最高。在方法内评估中，实验室F至I与参考实验室A的两两比较显示一致性率极佳，为92%至100%（kappa值：0.88至1.0），总体灵敏度为97.5%（195/200），特异性为99.5%（199/200）。
结论
如果使用标准化方案和经过验证的试剂，通过GP5+/6+ PCR-EIA对HR-HPV作为一个整体进行检测具有高度可重复性。
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