Two Color Multiplexing and Typing
of Human Papillomavirus Types 16, 18 and 45
on LightCycier
MONICA L. HENRIQUEZ, BRIAN E. CAPLIN*, RANDY P. RASMUSSEN
Introduction
Cervical cancer is the second leading cause of cancer in women worldwide [1,2].
Human Papillomaviruses (HPV) have been identified as the most important viral
group associated with benign and malignant neoplasia in humans [3]. Several
studies have demonstrated that infection with certain HPV types may progress
over a period of years through the various stages of cervical intraepithelial neoplasia (CIN) to invasive squamous carcinoma [4,5,6]. There are more than 80 different HPV types that have been identified [7,8]. Currently, there are more than
20 HPV types that have been found linked to cervical cancer [9]. This group is further subdivided into three categories designated as low, medium and high to differentiate between the relative risk for developing cervical carcinoma from any
particular HPV type infection [10]. From the high risk category HPV 16 ranks
first followed by HPV 18 in being found at a higher frequency in CIN and cervical carcinomas [I,II].As of today, HPV 45 is considered a medium/high risk type
but there is supporting evidence indicating that HPV 45 follows HPV 18 in frequency and that it should be classified as a high risk type [1,12].
Unfortunately, to this date, there is no method of detection or characterization
that identifies HPV infections by type. The Hybrid Capture I Assay (Digene Corp,
Bellsville, MD), the current diagnostic test for the screening of cervical cancer
characterizes HPV infections as low risk or medium/high risk but fails to identify HPV infections by type [13].
Polymerase Chain Reaction (PCR) is a highly sensitive method that has been
shown to be an effective tool and the most sensitive method for the detection of
several HPV types [14,15,16]. However, analysis of PCR products is for the most
part tedious and at many times ambiguous. Conventional methods such as ethidium bromide stained agarose gel electrophoresis may show complicated patterns
(multiple bands or smears) making it difficult to interpret gel data [17]. Therefore, the task of designing an assay that will detect and identify HPV infection by
type is challenging and difficult.
We have developed a method that uses a homogenous multiplex real-time peR
technique that amplifies and detects HPV 16, HPV 18 and HPV 45 all in one reac* Brian E. Caplin (~) (e-mail: brianc@idahotech.com)
Idaho Technology, 390 Wakara Way, Salt Lake City, UT 84108, USA
of Human Papillomavirus Types 16, 18 and 45
on LightCycier
MONICA L. HENRIQUEZ, BRIAN E. CAPLIN*, RANDY P. RASMUSSEN
Introduction
Cervical cancer is the second leading cause of cancer in women worldwide [1,2].
Human Papillomaviruses (HPV) have been identified as the most important viral
group associated with benign and malignant neoplasia in humans [3]. Several
studies have demonstrated that infection with certain HPV types may progress
over a period of years through the various stages of cervical intraepithelial neoplasia (CIN) to invasive squamous carcinoma [4,5,6]. There are more than 80 different HPV types that have been identified [7,8]. Currently, there are more than
20 HPV types that have been found linked to cervical cancer [9]. This group is further subdivided into three categories designated as low, medium and high to differentiate between the relative risk for developing cervical carcinoma from any
particular HPV type infection [10]. From the high risk category HPV 16 ranks
first followed by HPV 18 in being found at a higher frequency in CIN and cervical carcinomas [I,II].As of today, HPV 45 is considered a medium/high risk type
but there is supporting evidence indicating that HPV 45 follows HPV 18 in frequency and that it should be classified as a high risk type [1,12].
Unfortunately, to this date, there is no method of detection or characterization
that identifies HPV infections by type. The Hybrid Capture I Assay (Digene Corp,
Bellsville, MD), the current diagnostic test for the screening of cervical cancer
characterizes HPV infections as low risk or medium/high risk but fails to identify HPV infections by type [13].
Polymerase Chain Reaction (PCR) is a highly sensitive method that has been
shown to be an effective tool and the most sensitive method for the detection of
several HPV types [14,15,16]. However, analysis of PCR products is for the most
part tedious and at many times ambiguous. Conventional methods such as ethidium bromide stained agarose gel electrophoresis may show complicated patterns
(multiple bands or smears) making it difficult to interpret gel data [17]. Therefore, the task of designing an assay that will detect and identify HPV infection by
type is challenging and difficult.
We have developed a method that uses a homogenous multiplex real-time peR
technique that amplifies and detects HPV 16, HPV 18 and HPV 45 all in one reac* Brian E. Caplin (~) (e-mail: brianc@idahotech.com)
Idaho Technology, 390 Wakara Way, Salt Lake City, UT 84108, USA