Assessing genetic evolution and detecting human papillomavirus by matching two complementary highly sensitive approaches, nested-qPCR and sequencing

Alirezaei, Melika; Movahhed, Tahereh Komeili; Khazani, Mohammad; Mansour, Fahimeh Nemati; Zarean, Mehdi; Hamta, Amir; Fotouhi-Ardakani, Reza

doi:10.1016/j.meegid.2020.104274

Cited by 1 publication

(7 citation statements)

References 35 publications

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“…The melting temperatures of HPV18, HPV16, and HPV6 were 81.67 °C, 81.04 °C, and 81.52 °C, respectively. HPV18, a high risk type being the second most common type found in cervical cancer and, HPV6—a low-risk genotype being the most common genotype in our study population 37 , did not show a unique profile and therefore were not distinguishable from one another. However, five HPVs, 16, 52, 59, 66 and 89, with unique characteristics were distinguished.…”

Section: Discussionmentioning

confidence: 67%

“…The separation of two main carcinogenic genotypes, HPV18 and HPV16, as well as the predominant genotype, HPV 6, in the research region, was a significant hurdle in our investigation 37 . Preliminary studies on temperature melting analysis of these three genotypes with insilico assays showed that bioinformatically, it is impossible to separate HPV18 and HPV6 despite the difference in six nucleotides among these two genotypes in studied gene region with a temperature difference of 0.03 °C.…”

Section: Discussionmentioning

confidence: 97%

“…In population-based cervical screening, human papillomavirus (HPV) types 16, 18, 31, 33, 45, and 52 are associated with 85% of HPV-associated cervical cancers 44 , and we anticipated that we would be able to clone more genotypes as the diagnosis standard, the discrepancy among the results obtained by the microarray method and the sequencing prevented us from achieving this goal. Genotypes cloned in this study represent common low-risk and high-risk HPVs in the Middle East 37 , 45 . In this regard, the study of Lee et al showed eight HPV genotypes 16, 18, 39, 45, 52, 56, 58, and 68 with a prevalence of over 75% in Asia, Europe, and the United States.…”

Section: Discussionmentioning

confidence: 99%

“…The study protocol was reviewed and approved by Ethics Committee of Qom University of Medical Sciences (IR.MUQ.REC.1396.40), Iran, and all experiments were performed in accordance with relevant guidelines and regulations. A total of 57 HPV positive samples were randomly selected from 486 Pap smears analyzed in a previous study 37 . Informed consent was obtained from all women who were referred for conventional Pap tests to the medical centers of Qom Province.…”

Section: Methodsmentioning

confidence: 99%

“…Informed consent was obtained from all women who were referred for conventional Pap tests to the medical centers of Qom Province. For optimization of the qPCR-HRM performance, known HPVs DNA were obtained from the previous study or microarray method in clinical laboratory 37 (Fig. 9 ).…”

Section: Methodsmentioning

confidence: 99%

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Discrimination of human papillomavirus genotypes using innovative technique nested-high resolution melting

Alirezaei

Mosawi²,

Afgar

et al. 2022

Sci Rep

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The prompt detection of human papillomavirus and discrimination of its genotypes by combining conventional methods in new molecular laboratories is essential to achieve the global call of eliminating cervical cancer. After predicting the melting temperature of an approximately 221 bp region of the L1 gene from different HPV genotypes by bioinformatics software, an innovative technique based on the nested- high resolution melting was designed with three approaches and using conventional PCR, qPCR, and diagnostic standards. HPV-positive samples identified by microarray along with diagnostic standards were evaluated by qPCR-HRM and discordant results were subjected to sequencing and analyzed in silico using reference types. In addition to screening for human papillomavirus, nested-qPCR-HRM is one of the modified HRM techniques which can discriminate some genotypes, including 6, 16, 18, 52, 59, 68 and 89. Despite the differences in diagnostic capabilities among HRM, microarray and sequencing, a number of similarities between HRM, and sequencing were diagnostically identified as the gold standard method. However, the bioinformatics analysis and melting temperature studies of the selected region in different HPV genotypes showed that it could be predicted. With numerous HPV genotypes and significant genetic diversity among them, determining the virus genotype is important. Therefore, our goal in this design was to use the specific molecular techniques with several specific primers to increase sensitivity and specificity for discriminating a wide range of HPV genotypes. This approach led to new findings to evaluate the ability of different approaches and procedures in accordance with bioinformatics.

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Section: Discussionmentioning

confidence: 67%

Section: Discussionmentioning

confidence: 97%