Study on the Detection of Human Papillomavirus Types 16 and 18 by Multiplex Nested Polymerase Chain Reaction Based on E6 , E7 , and E1 Gene

Kim, Jae Weon; Lee, Hyo Pyo

Abstract

Epidemiological and experimental studies have established that specific human papillo-mavirus(HPV) types are strongly associated with cervical cancer. In understanding the role of HPV in the development of cervical cancer, more accurate and efficient detection method of various HPV types is warranted. Currently polymerase chain reaction(PCR)-based methods are the most commonly used one. Recently developed PCR systems, mainly, amplify a region of the L1 or E1 genes of HPVs which may not be integrated into host chromosome and more likely to produce false negative results. In this paper we describe the design and use of a simplified PCR system with high sensitivity in which all primers are multiplexed in the same PCR mixture. The full genomic sequence of HPV types were obtained from the HPV Sequence Database(http://hpv-web.lanl.gov). Matrix, homology and alignment analyses were carried out by using computer programs such as CLUSTAL V, DNASIS(TM) and Amplify. To determine the detection sensitivity of our PCR system, we have performed experiments using cloned HPV DNAs of type 16 and 18 serially diluted(10-fold dilutions from 10 picogram to 0.01 femtogram per assay) and in a cocktail. By alignment and comparing the sequences we located regions of outer and inner primer sequences based on the E6, E7 and E1 open reading frames which is usually integrated into host chromosome. Amplimer size were determined in order to be below than 400 base pair. The sensitivity of our PCR system ranged from the 1 copy per cell for HPV 16 and 10 copies per cell for HPV 18. This sensitivity was maintained when cloned HPV 16 and 18 DNAs were applied both individually and in a cocktail with variable mixing ratio. Our PCR system shows high sensitivity and specificity with minimal competitive inhibition between primers. This multiplex nested PCR system may be efficiently used for simultaneous detection and typing of HPV 16 and 18 in paraffin-embedded sections or rapid prepared samples.