Development and validation of a multiplex reverse transcript real-time PCR for E6/E7 mRNA detection of high-risk human papillomavirus

Pan, Cai-Xia; Zhou, Jing; Lyu, Jiangfeng; Ren, Xuyi

doi:10.1099/jmm.0.000824

Cited by 3 publications

(2 citation statements)

References 24 publications

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“…Hence, the latter is a potential marker for the identification of women at risk of developing cervical carcinoma. Our review support the argument that the HPV E6/E7 mRNA assay could overcome the shortcoming of low specificity of DNA assays for clinical detection of high-grade cervical lesions [35,[68][69][70][71][72][73][74][75].…”

Section: Discussionsupporting

confidence: 75%

HPV E6/E7 mRNA test for the detection of high grade cervical intraepithelial neoplasia (CIN2+): a systematic review

Derbie

Mekonnen

Woldeamanuel

et al. 2020

Infect Agents Cancer

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Background: Genital infection with certain types of Human papillomavirus (HPV) is a major cause of cervical cancer globally. For early detection of premalignant dysplasia, evidences are coming out on the usefulness of HPV E6/E7 mRNA test as a potential tool compared with cytology and HPV DNA testing. Taking into account shortage of compiled data on this field, the aim of this systematic review was to describe the latest diagnostic performance of HPV E6/E7 mRNA testing to detect high grade cervical lesions (CIN2+) where by histology was taken as a gold standard. Methods: Articles published in English were systematically searched using key words from PubMed/Medline and SCOPUS. In addition, Google Scholar and the Google database were searched manually for grey literature. Two reviewers independently assessed study eligibility, risk of bias and extracted the data. We performed a descriptive presentation of the performance of E6/E7 mRNA test (in terms of sensitivity, specificity, negative and positive predictive values) for the detection of CIN2 +. Results: Out of 231 applicable citations, we have included 29 articles that included a total of 23,576 study participants (age range, 15-84 years) who had different cervical pathologies. Among the participants who had cervical histology, the proportion of CIN2+ was between 10.6 and 90.6%. Using histology as a gold standard, 11 studies evaluated the PreTect HPV Proofer, 7 studies evaluated the APTIMA HPV assay (Gen-Probe) and 6 studies evaluated the Quantivirus® HPV assay. The diagnostic performance of these three most common mRNA testing tools to detect CIN2+ was; 1) PreTect Proofer; median sensitivity 83%, specificity 73%, PPV 70 and NPV 88.9%. 2) APTIMA assay; median sensitivity 91.4%, specificity 46.2%, PPV 34.3% and NPV 96.3%. 3) Quantivirus®: median sensitivity 86.1%, specificity 54.6%, PPV 54.3% and NPV was at 89.3%. Further, the area under the receiver operating characteristics (AU-ROC) curve varied between 63.8 and 90.9%. Conclusions: The reported diagnostic accuracy implies that HPV mRNA based tests possess diagnostic relevance to detect CIN2+ and could potentially be considered in areas where there is no histology facility. Further studies including its cost should be considered.

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Section: Discussionsupporting

confidence: 75%

HPV E6/E7 mRNA test for the detection of high grade cervical intraepithelial neoplasia (CIN2+): a systematic review

Derbie

Mekonnen

Woldeamanuel

et al. 2020

Infect Agents Cancer

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“…HPV screening test is done by molecular diagnostic technology such as PCR, real-time PCR, and NASBA method using HPV DNA or RNA [17][18][19][20]. Especially, real-time PCR is more sensitive and specific molecular diagnostic method to detect live HPV in patients compared to other methods [21][22][23][24]. Real-time PCR for HPV screening test is performed after RNA extraction, complementary DNA (cDNA) synthesis, and amplification of cDNA [25][26][27].…”

Section: Introductionmentioning

confidence: 99%

Evaluation of Commercial Complementary DNA Synthesis Kits for Detecting Human Papillomavirus

Park

Chang

et al. 2019

Korean J Clin Lab Sci.

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Cervical cancer is the fourth most common malignant neoplasm in women worldwide. Most cases of cervical cancer are caused by an infection by the human papillomavirus. Molecular diagnostic methods have emerged to detect the HPV for sensitivity, specificity, and objectivity. In particular, real-time PCR has been introduced to acquire a more sensitive target DNA or RNA. RNA extraction and complementary DNA synthesis are proceeded before performing real-time PCR targeting RNA. To identify an adequate and sensitive cDNA synthesis kit, this study evaluated the two commonly used kits for cDNA synthesis. The results show that the R 2 and efficiency (%) of the two cDNA synthesis kits were similar in the cervical cancer cell lines. On the other hand, the Takara kit compared to Invitrogen kit showed P＜0.001 in the 10 2 and 10 3 SiHa cell count. The Takara kit compared to the Invitrogen kit showed P＜0.001 in the 10 1 and 10 2 HeLa cell count. Furthermore, 8, 4, 2, 1, and 0.5 ml of forty exfoliated cell samples were used to compare the cDNA synthesis kits. The Takara kit compared to the Invitrogen kit showed P＜0.01 in 8, 4, and 1 ml and P＜0.05 in 0.5 mL. The study was performed to identify the most appropriate cDNA synthesis kit and suggests that a cDNA synthesis kit could affect the real-time PCR results.

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Development and validation of a multiplex qPCR assay for detection and relative quantification of HPV16 and HPV18 E6 and E7 oncogenes

Bordigoni

Motte

Tissot‐Dupont

et al. 2021

Sci Rep

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Human papillomaviruses (HPV) play a key role in promoting human anogenital cancers. Current high-risk HPV screening or diagnosis tests involve cytological or molecular techniques mostly based on qualitative HPV DNA detection. Here, we describe the development of a rapid quantitative polymerase chain reaction (qPCR) detection test of HPV16 and HPV18 oncogenes (E6 and E7) normalized on human gene encoding GAPDH. Optimized qPCR parameters were defined, and analytical specificities were validated. The limit of detection was 101 for all genes tested. Assay performances were evaluated on clinical samples (n = 96). Concordance between the Xpert HPV assay and the triplex assay developed here was 93.44% for HPV16 and 73.58% for HPV18. HPV co-infections were detected in 15 samples. The systems developed in the present study can be used in complement to traditional HPV tests for specifically validating the presence of HPV16 and/or HPV18. It can also be used for the follow-up of patients with confirmed infection and at risk of developing lesions, through the quantification of E6 and E7 oncogene expression (mRNA) normalized on the GAPDH expression levels.

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Development and validation of a multiplex reverse transcript real-time PCR for E6/E7 mRNA detection of high-risk human papillomavirus

Abstract: The one-step multiplex RT-PCR for E6/E7 mRNA detection is a simple, fast, universally applicable, sensitive and highly specific method for hrHPV E6/E7 mRNA detection. It is reliable for cervical lesion screening and of potential value in future clinical applications.

Cited by 3 publications

References 24 publications

HPV E6/E7 mRNA test for the detection of high grade cervical intraepithelial neoplasia (CIN2+): a systematic review

HPV E6/E7 mRNA test for the detection of high grade cervical intraepithelial neoplasia (CIN2+): a systematic review

Evaluation of Commercial Complementary DNA Synthesis Kits for Detecting Human Papillomavirus

Development and validation of a multiplex qPCR assay for detection and relative quantification of HPV16 and HPV18 E6 and E7 oncogenes

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