Analytical performance of newly developed multiplex human papillomavirus genotyping assay using Luminex xMAP™ technology (Mebgen™ HPV Kit).
Regional differences in human papillomavirus (HPV) genotypes and the presence of mixed HPV infections may affect adversely the efficacy of the HPV vaccine. Therefore, a simple and high-throughput HPV genotyping system is required. Recently, a novel HPV genotyping kit (the Mebgen™ HPV kit) was developed. This kit uses multiplex PCR and Luminex xMAP™ technology to detect 13 types of high-risk HPVs and an internal control in a 96-well format. In the present study, the analytical performance of the kit was examined using HPV plasmid DNA. All 13 types of HPVs were detected with a minimum detection sensitivity of 250 copies/test, and highly specific signals were observed. HPV 16 plasmid was detected in samples containing mixtures with other HPV-type plasmids in ratios ranging from 1:1 to 1:1000. No cross reactivity was observed with DNA from 27 types of other infectious microbes. A clinical evaluation was carried out using cervical samples from 356 patients with persistent abnormal smears diagnosed at mass public health screenings for cervical cancer. The samples were preserved in Tacas™ medium until analysis. HPV was detected in 162 (45.5%) samples including 110 (67.9%) with single infections and 52 (32.1%) with multiple infections. The type distribution of the 13 high-risk HPVs was as follows: 28.4% HPV 16, 11.7% HPV 18, 6.8% HPV 31, 3.1% HPV 33, 3.7% HPV 35, 9.3% HPV 39, 1.9% HPV 45, 8.6% HPV 51, 37.0% HPV 52, 9.3% HPV 56, 16.7% HPV 58, 3.7% HPV 59, and 1.9% HPV 68. To evaluate sample stability over time, changes in the detection of HPV DNA derived from HeLa and SiHa cells were measured in 3 types of liquid-based cytology media. HPV DNA was detected in Tacas and Thinprep™ samples after storage at 4°C or 30°C for 4 weeks and within 1 week of collection in Surepath™ samples. These results suggest that this newly developed HPV genotyping kit is suitable for use in both clinical applications and large-scale epidemiological studies.
Department of Clinical Laboratory Science, Kanazawa University, Kanazawa, Japan. Electronic address: s-ozaki@med.kanazawa-u.ac.jp.
Satoru
S
Ozaki
G&G Science, Fukushima, Japan.
Kana
K
Kato
G&G Science, Fukushima, Japan.
Yukiko
Y
Abe
Medical & Biological Laboratories, Tokyo, Japan.
Hirotaka
H
Hara
Medical & Biological Laboratories, Tokyo, Japan.
Hiroshi
H
Kubota
Department of Obstetrics and Gynecology, Toho University School of Medicine, Ohashi Medical Center, Japan.
Kaneyuki
K
Kubushiro
Department of Clinical Laboratory Science, Kanazawa University, Kanazawa, Japan.
Ei
E
Kawahara
Department of Obstetrics and Gynecology, Kanazawa University, Graduate School of Medical Science, Kanazawa, Japan.
Masaki
M
Inoue
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D060085:Coinfection / Q000821:virology; D005260:Female; D060005:Genotyping Techniques / Q000379:methods*; D006801:Humans; D008875:Middle Aged; D027383:Papillomaviridae / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D030361:Papillomavirus Infections / Q000821:virology*; D013048:Specimen Handling / Q000379:methods; D013696:Temperature; D013997:Time Factors; D055815:Young Adult
Cervical cancer; HPV; HPV genotyping; Luminex; Multiplex PCR