Huang C C, Qiu J T, Kashima M L, Kurman R J, Wu T C
Department of Pathology, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21205, USA.
Mod Pathol. 1998 Oct;11(10):971-7.
The integration of human papillomavirus (HPV) DNA is associated with the pathogenesis of HPV-associated malignancies. The ability, however, of standard in situ hybridization (ISH) to detect low-copy integrated HPV DNA is limited. We describe the generation of HPV type-specific biotin-labeled DNA probes and a novel ISH method that uses the catalyzed reporter deposition (CARD) system for the detection of single-copy target HPV DNA in formalin-fixed, paraffin-embedded tissue. Consensus primers flanking the noncoding region of HPVs were used to generate biotin-labeled HPV-6b, -11, -16 and -18 probes by polymerase chain reaction (PCR). The probes were used for ISH with the novel technique of CARD to increase the sensitivity of the assay. Tissue blocks were prepared from CaSki (500-600 copies of HPV-16), SiHa (1-2 copies of HPV-16), and HeLa (10-50 copies of HPV-18) cell lines, as well as from an HPV-negative cell line, C33A, and then tested to demonstrate the sensitivity and specificity of the probes. Surgical specimens were used to show the clinical applicability of this technique. We successfully detected HPV-16 DNA in CaSki and SiHa cells but not in HeLa or C33A cells. HPV-18 DNA was detected in HeLa cells but not in CaSki, SiHa, or C33A cells. Sensitivity was increased when ISH was performed using probes with more biotin incorporation or when more cycles of signal amplification were employed, but significant nonspecific background was observed after more than two cycles of signal amplification. The probes generated in this study detected specific types of HPV in surgical specimens with much higher sensitivity than did conventional ISH. We concluded that our new method was highly sensitive and could be applied to formalin-fixed, paraffin-embedded clinical material for the detection of HPV.
人乳头瘤病毒（HPV）DNA的整合与HPV相关恶性肿瘤的发病机制有关。然而，标准原位杂交（ISH）检测低拷贝整合HPV DNA的能力有限。我们描述了HPV型特异性生物素标记DNA探针的生成以及一种新型ISH方法，该方法使用催化报告沉积（CARD）系统检测福尔马林固定、石蜡包埋组织中的单拷贝目标HPV DNA。使用位于HPV非编码区两侧的共有引物，通过聚合酶链反应（PCR）生成生物素标记的HPV-6b、-11、-16和-18探针。这些探针用于ISH，并采用新型CARD技术以提高检测的灵敏度。从CaSki（500 - 600个HPV-16拷贝）、SiHa（1 - 2个HPV-16拷贝）和HeLa（10 - 50个HPV-18拷贝）细胞系以及HPV阴性细胞系C33A制备组织块，然后进行检测以证明探针的灵敏度和特异性。使用手术标本展示该技术的临床适用性。我们成功在CaSki和SiHa细胞中检测到HPV-16 DNA，但在HeLa或C33A细胞中未检测到。在HeLa细胞中检测到HPV-18 DNA，但在CaSki、SiHa或C33A细胞中未检测到。当使用掺入更多生物素的探针进行ISH或采用更多信号放大循环时，灵敏度会提高，但在超过两个信号放大循环后会观察到明显的非特异性背景。本研究中生成的探针在手术标本中检测特定类型的HPV时，灵敏度比传统ISH高得多。我们得出结论，我们的新方法高度灵敏，可应用于福尔马林固定、石蜡包埋的临床材料以检测HPV。
