Simultaneous Detection, Genotyping, and Quantification of Human Papillomaviruses by Multicolor Real-Time PCR and Melting Curve Analysis

Abstract

c Long-term infection with high-risk human papillomavirus (HPV) is the leading cause of cervical cancer, while infection with low-risk HPV is the major reason for condylomata acuminata. An accurate, rapid, and convenient assay that is able to simultaneously detect, genotype, and quantify HPV would be of great clinical value yet remains to be achieved. We developed a three-color real-time PCR assay that is able to analyze 30 predominant HPV types in three reactions. The amplification curves indicated the presence of HPV, melting curve analysis identified the HPV genotype, and the quantification cycle value determined the quantity. We applied this assay to 647 cervical swab samples, and the results were compared with those obtained with a commercial genotyping system. The proposed assay had a limit of detection of 5 to 50 copies per reaction and a dynamic range of 5 ؋ 10 1 to 5 ؋ 10 6 copies per reaction. A comparison study showed that the overall sample concordance with the comparison method was 91.6% and the type agreement was greater than 98.7%. The quantification study demonstrated that the loads of HPV type 16 in 30 samples with cervical intraepithelial neoplasia grade III (CIN III) lesions were significantly higher than those in samples with CIN I lesions or CIN II lesions, and the results were concordant with those of the comparison method. The increased information content, high throughput, and low cost would facilitate the use of this real-time PCR-based assay in a variety of clinical settings.

Distribution characteristics of different human papillomavirus genotypes in women in Wuhan, China

“…This method, called HybriMax, can be used to simultaneously identify 21 different HPV genotypes, including 14, 5, and 2 genotypes of HR‐HPVs, LR‐HPVs and unknown‐risk HPVs, respectively. This identification is particularly useful for detecting multiple or coinfections of different HPV genotypes, and HybriMax is a reasonable or even ideal technique for performing HPV genotyping in a clinical setting …”

“…All the DNA samples were amplified using the Real‐Time PCR system ABI 7500 (Applied Biosystems, USA) using a PCR reagent kit (Chaozhou Hybribio Biochemical Co., Ltd.). HPV genotyping was performed with a HybriMax HPV GenoArray Test Kit (HybriBio Ltd., Chaozhou, China) according to the manufacturer’s instructions . Positive and negative controls were run in each PCR analysis process to control for possible contamination and accuracy.…”

“…HPV genotyping was performed with a HybriMax HPV GenoArray Test Kit (HybriBio Ltd., Chaozhou, China) according to the manufacturer's instructions. [21][22][23][24][25][26] Positive and negative controls were run in each PCR analysis process to control for possible contamination and accuracy. This method was used to differentiate 21 genotypes of HPVs,…”

Human papillomavirus prevalence and associated factors in women and men in south China: a population-based study

“…GP5+/6+ was used as the general primer rather than the MY09/MY11 or SPF10 primers, which amplified longer or shorter target L1 fragments, respectively. 12, 13, 14 DNA extraction was performed using the Lad-Aid 824 system (Zeesan Biotech Corporation, Xiamen, China) according to the instructions of the manufacturer. In brief, 200 μL aliquots of the clinical material were digested with 3 M guanidine hydrochloride solution for 300 s. The DNA was eluted in 150 μL of 10 mmol/L Tris-HCl buffer (pH 7.5) at room temperature.…”

Rapid Detection of Isoniazid Resistance in Mycobacterium tuberculosis Isolates by Use of Real-Time-PCR-Based Melting Curve Analysis

“…The MeltPro TB/INH assay is the first officially approved assay dedicated solely to detection of INH resistance. This assay is based on melting curve analysis that uses unique dually labeled, selfquenched probes, which have been proved to be accurate, sensitive, and flexible in the detection of multiple mutations in a single reaction (11,(27)(28)(29). The assay can detect 20 mutant sites that involve 30 mutation types in the inhA promoter region (positions Ϫ17 to Ϫ8), katG position 315 (katG 315), the ahpC promoter, and inhA position 94 (inhA 94) of M. tuberculosis, constituting the largest number of INH resistance mutations.…”