Obstet Gynecol Sci.  2016 Nov;59(6):489-497. 10.5468/ogs.2016.59.6.489.

Combined SYBR Green real-time polymerase chain reaction and microarray method for the simultaneous determination of human papillomavirus loads and genotypes

• Seo HH
• Kim YJ
• Jeong MS
• Hong SR
• Lee IH
• So KA
• Kim MK
• Lee YK
• Lee KH
• Kim J
• Kim SJ
• Kim TJ

• KMID: 2378573
• DOI: http://doi.org/10.5468/ogs.2016.59.6.489

Abstract

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.

Keyword

Diagnosis; Genotyping; Human papilloma virus; Microarray; Viral load

MeSH Terms

Consensus
Diagnosis
Genotype*
Humans*
Methods*
Odds Ratio
Oligonucleotide Array Sequence Analysis
Papillomaviridae
Polymerase Chain Reaction
Real-Time Polymerase Chain Reaction*
Viral Load

Figure

• 

  Fig. 1 Validation of quantitative analysis. Comparison of (A) viral load and (B) relative light unit/cutoff value. Circles indicate the average of measured values in each group, and bars indicate the standard deviation. ASC-US, atypical squamous cells of undetermined significance; ASC-H, atypical squamous cells-cannot exclude high-grade squamous intraepithelial lesion; LSIL, low-grade squamous intraepithelial lesion; HSIL, high-grade squamous intraepithelial lesion; RLU, relative light unit; CO, cut-off.

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