Polymerase chain reaction‐based fluorescent Luminex assay to detect the presence of human papillomavirus types

Abstract

Becuase 40% of human papillomavirus (HPV) infections are mixed infections, the accurate identification of high-risk HPV genotypes in mixed infections is important for defining a woman's risk for progression to cervical cancer. Thus, advanced Luminex-based HPV genotyping has been developed to simultaneously detect the presence of multiple HPV types. Here, we describe the development of a Luminex-based HPV genotyping that combines polymerase chain reaction amplification with hybridization to fluorescencelabeled polystyrene bead microarrays (Luminex suspension array technology). New HPV type-specific oligonucleotide probes and YBT L1/GP6-1 primers were used to detect the HPV types in 132 clinical samples. We simultaneously evaluated the usefulness of this technique on clinical samples. We detected 15 specific HPV types (6, 16, 18, 31, 35, 42, 51, 52, 55, 56, 58, 59, 66, 67 and 68) examined with specificity without known cross-reaction to other HPV types. The detection limit for the different HPV types was above 500 plasmids. (1) individuals may be infected with more than one type at a time.(2) Because most HPV infections have no visible signs or symptoms, (3) the development of detection tools for HPV identification in asymptomatic patients has been very important. (4) Several HPV DNA detection methods have been described during the last decade, each of which allows the detection of a wide spectrum of HPV types, such as DNA amplification-based methods. Due largely to routine screening using Pap tests, the number of deaths attributed to cervical cancer continues to drop nearly 4% annually. However, the Pap smear has some weaknesses, not least of which is its limited sensitivity for detection of cancer precursors.(5) Therefore, the false-negative rate of the Pap smear ranges from 20 to 30% when cytology is used alone. In addition, there is a false-positive rate of 5-15%.(6 -8) To carry out HPV genotyping, a reliable, FDA-approved test to distinguish the specific HPV types will need to be developed and validated. A DNA-based technology, the Hybrid Capture II HPV test, has been used to detect 13 high-risk types of HPV. (9,10) However, this routine test is not recommended for women under the age of 30 years unless they have atypical or equivocal Pap test results. In addition, despite its high sensitivity, false-negative results are known to occur for histologically confirmed cervical intraepithelial neoplasia (CIN)2 or CIN3 cases, with its impossibility to perform genotyping. (11,12) Therefore, the current techniques available for detection of HPV types all have shown limited ability for complete detection; there is no single technique that provides complete detection to date. Therefore, a new and improved HPV assay that is highly sensitive and reproducible is required. Multiplex technology is a new method that is based on fluorescent bead technology, and allows simultaneous detection of nucleic acids against up to 100 different HPV types. (13,14) Recently, this technology has been used for the genotyping of HPV types u...

Development and Evaluation of a Liquid Bead Microarray Assay for Genotyping Genital Human Papillomaviruses

“…Several previous studies reported the feasibility of establishing a Luminex-based HPV genotyping assay targeting 15 to 45 different HPVs (9,13,17,22,26,28). Various primer systems were used for the development of these Luminex assays, including PGMY09-PGMY11 (28), GP5ϩ-GP6ϩ (26), MY09-MY11 (13), type-specific primers (9), and YBT L1-GP6-1 (22).…”

Commercially available assays for multiplex detection of alpha human papillomaviruses

“…Positivity for the relevant types is calculated from the median fluorescent intensity over a defined threshold level, and can provide a semi-quantitative numerical output. Several in-house genotyping protocols based on xMAP technology have been developed in the last 5 years [121][122][123][124][125][126][127]; some of them are considered to be the most sensitive HPV detection methods currently available [123]. In addition, at least two commercial assays based on this technology are available at present.…”

Human Papillomavirus Detection in Head and Neck Squamous Cell Carcinomas and Its Clinical Implications

“…Nevertheless, a limitation of the assay is the reduction of signal that occurs for a plasmid target in low abundance when it is amplified with another target that is 2 or 3 logs higher in abundance. This technology has been tested in cervical samples (Oh et al, 2007;Lowe B et al, 2010).…”