Korean J Lab Med.  2007 Feb;27(1):62-68. 10.3343/kjlm.2007.27.1.62.

Human Papilloma Virus Genotyping Assay using Restriction Fragment Mass Polymorphism Analysis, and Its Comparison with Sequencing and Hybrid Capture Assays

• Lee EH
• Chung HJ
• Oh HB
• Chi HS
• Jee MS
• Park SN
• Hong SP
• Yoo W
• Kim SO

• KMID: 1121662
• DOI: http://doi.org/10.3343/kjlm.2007.27.1.62

Abstract

BACKGROUND: Infection with human papilloma virus (HPV) is the main cause of cervical cancer, and HPV genotyping is of increasing importance for determining clinical course and management of the disease based on the HPV genotypes. Here, we established a novel matrix-assisted laser desorption/ ionization time of flight mass spectrometry (MALDI-TOF MS) assay, termed restriction fragment mass polymorphism (RFMP) that is suitable for genotyping multiple HPV in an accurate and high-throughput manner. We evaluated the performance of the RFMP assay in HPV genotyping by comparing the results with those of direct or clonal sequencing and hybrid capture (HC) assays. METHODS: The study population consisted of 50 patients with histologically confirmed cervical lesions and a positive test for HPV DNA. HPV genotyping was performed with RFMP, sequencing, and HC assays. The assigned genotypes and risk groups were compared among the methods. RESULTS: Concordance rates in the genotype level between RFMP vs sequencing, sequencing vs HC, and HC vs RFMP were 98% (49/50), 88% (44/50), and 88% (44/50), respectivley. Especially, RFMP and sequencing were 100% concordant when assigned high-risk group was considered identical in 1 case of mixed genotypes identified only in RFMP. The observed discrepancy between HC and the other two methods is due to the assignment of six cases of low, intermediate, or unassigned risk genotypes as high-risk group in HC method. CONCLUSIONS: RFMP, sequencing, and HC assays were highly concordant with each other in HPV genotyping. Compared to sequencing assay, RFMP assay is found to be advantageous in detecting mixed genotype infections. The accuracy and amenability to high-throughput analysis should make the RFMP assay suitable for reliable screening of HPV genotypes in clinical laboratories.

Keyword

Human papilloma virus (HPV) genotyping; Restriction fragment mass polymorphism (RFMP); Sequencing

MeSH Terms

Female
Genotype
Humans
Nucleic Acid Hybridization/methods
Papillomaviridae/classification/genetics/*isolation & purification
Papillomavirus Infections/*diagnosis
Polymorphism, Restriction Fragment Length
Sequence Analysis, DNA/methods
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/*methods
Uterine Cervical Neoplasms/*diagnosis

Figure

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  Fig. 1. RFMP and sequencing results of patient samples showing discordance with hybrid capture assay. HPV genotype 81 (belonging to low risk group) identified in Patient 20 (upper panel) and HPV genotype 62 (belonging to unassigned risk group) detected in Patient 8 (low panel) by both RFMP and sequencing assays show high risk positivity in HC assay. Sequences in genotype-specific motifs were underlined.

Reference

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