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KMID : 0358420160590060489
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Korean Journal of Obstetrics and Gynecology
2016 Volume.59 No. 6 p.489 ~ p.497
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Combined SYBR Green real-time polymerase chain reaction and microarray method for the simultaneous determination of human papillomavirus loads and genotypes
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Seo Hyun-Hee
Kim Young-Jun
Jeong Mi-Seon
Hong Sung-Ran
Lee In-Ho
So Kyeong-A
Kim Mi-Kyung
Lee Yoo-Kyung
Lee Ki-Heon
Kim Ju-Ree
Kim Sung-Jae
Kim Tae-Jin
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Abstract
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Objective: The aim of this study was to describe the principle of the Cheil HPV DNA Chip assay and evaluate its accuracy. In order to quantify the human papillomavirus (HPV) load and identify HPV genotypes simultaneously, this assay combined the two methods: SYBR Green quantitative real-time polymerase chain reaction (PCR) and DNA microarray.
Methods: We designed novel consensus primer sets that target the conserved region of the HPV L1 gene for quantifying and detecting a broad range of HPV types by quantitative real-time PCR. Subsequently, using the PCR products, DNA microarray was performed with 36 HPV type-specific probes. To validate this method, direct sequencing and correlation analysis among HPV genotype, viral load, and cytological abnormality was performed by Cohen’s kappa values, two-sided McNemar chi-square test, Kruskal-Wallis test, and odds ratios.
Results: The kappa value of the Cheil HPV DNA Chip was 0.963 (95% confidence interval, 0.919 to 0.98), which was significantly higher than the value of 0.527 (95% confidence interval, 0.447 to 0.59) obtained using a conventional HPV DNA Chip. HPV16 (χ2=62.28, P<0.01), HPV33 (χ2=7.18, P<0.01), and HPV58 (χ2=9.52, P<0.01), which are classified as high-risk HPVs, were detected at significant levels in samples with high-grade lesions. And viral loads tended to be higher in groups with high odds ratios.
Conclusion: The Cheil HPV DNA Chip is an effective diagnostic assay for simultaneously detecting HPV genotypes and loads in cervical samples.
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KEYWORD
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Diagnosis, Genotyping, Human papilloma virus, Microarray, Viral load
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