Extraction-free RT-RPA assay for detection of HPV16, HPV18, and HPV45 mRNA expression
Abstract Messenger RNA (mRNA) from high-risk genotypes of human papillomavirus (hrHPV) is a more specific biomarker for cervical cancer risk than hrHPV DNA due to its ability to differentiate clinically significant infections from transient ones. Detecting mRNA from exfoliated cervical cells, howeve...
Saved in:
| Main Authors: | , , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Nature Portfolio
2025-08-01
|
| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-025-13583-2 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1849226394708475904 |
|---|---|
| author | Emilie Newsham Novak Ariel Ma Kathryn A. Kundrod Megan M. Chang Cannon J. Hansen Jane Richards Montealegre Michael E. Scheurer Philip E. Castle Ming Guo Mila P. Salcedo Kathleen Schmeler Rebecca Richards-Kortum |
| author_facet | Emilie Newsham Novak Ariel Ma Kathryn A. Kundrod Megan M. Chang Cannon J. Hansen Jane Richards Montealegre Michael E. Scheurer Philip E. Castle Ming Guo Mila P. Salcedo Kathleen Schmeler Rebecca Richards-Kortum |
| author_sort | Emilie Newsham Novak |
| collection | DOAJ |
| description | Abstract Messenger RNA (mRNA) from high-risk genotypes of human papillomavirus (hrHPV) is a more specific biomarker for cervical cancer risk than hrHPV DNA due to its ability to differentiate clinically significant infections from transient ones. Detecting mRNA from exfoliated cervical cells, however, is a complex, expensive, and equipment-intensive process, making it infeasible in resource-limited settings. Here we describe a sensitive, specific, and minimally instrumented method to detect HPV16, HPV18, and HPV45 mRNA in exfoliated cervical cells. First, reverse transcription recombinase polymerase amplification (RT-RPA) exo assays were designed to amplify and detect HPV16, HPV18, and HPV45 E7 mRNA ≥100 copies per reaction in real time using a portable fluorimeter, which is on par with the only FDA-approved hrHPV mRNA test. Next, an extraction-free sample preparation method using only an enzymatic reaction was developed to lyse cells and degrade cellular DNA in cultured HPV16, HPV18, and HPV45-positive cells. The RT-RPA assays specifically amplified mRNA from this crude lysate across a clinically relevant range of concentrations. Eleven cervicovaginal samples were tested at the point of care using these methods and showed 100% agreement between RT-RPA and RT-qPCR results, including two samples that were positive for HPV16 mRNA. Additionally, nine negative samples spiked with HPV16, HPV18, and HPV45-positive cells successfully detected each respective HPV type. In total, this prototype assay has potential to make sensitive and specific hrHPV mRNA testing more accessible in resource-limited settings. |
| format | Article |
| id | doaj-art-fc7dfa1c993a41f3a348463f83b9a808 |
| institution | Kabale University |
| issn | 2045-2322 |
| language | English |
| publishDate | 2025-08-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj-art-fc7dfa1c993a41f3a348463f83b9a8082025-08-24T11:23:42ZengNature PortfolioScientific Reports2045-23222025-08-0115111110.1038/s41598-025-13583-2Extraction-free RT-RPA assay for detection of HPV16, HPV18, and HPV45 mRNA expressionEmilie Newsham Novak0Ariel Ma1Kathryn A. Kundrod2Megan M. Chang3Cannon J. Hansen4Jane Richards Montealegre5Michael E. Scheurer6Philip E. Castle7Ming Guo8Mila P. Salcedo9Kathleen Schmeler10Rebecca Richards-Kortum11Department of Bioengineering, Rice UniversityDepartment of Bioengineering, Rice UniversityDepartment of Bioengineering, Rice UniversityDepartment of Bioengineering, Rice UniversityDepartment of Bioengineering, Rice UniversityDepartment of Behavioral Science, The University of Texas MD Anderson Cancer CenterDepartment of Pediatrics Hematology/Oncology, Baylor College of Medicine, Texas Children’s HospitalDivisions of Cancer Prevention and Cancer Epidemiology and Genetics, National Cancer InstituteDepartment of Anatomical Pathology, The University of Texas MD Anderson Cancer CenterDepartment of Gynecologic Oncology & Reproductive Medicine, The University of Texas MD Anderson Cancer CenterDepartment of Gynecologic Oncology & Reproductive Medicine, The University of Texas MD Anderson Cancer CenterDepartment of Bioengineering, Rice UniversityAbstract Messenger RNA (mRNA) from high-risk genotypes of human papillomavirus (hrHPV) is a more specific biomarker for cervical cancer risk than hrHPV DNA due to its ability to differentiate clinically significant infections from transient ones. Detecting mRNA from exfoliated cervical cells, however, is a complex, expensive, and equipment-intensive process, making it infeasible in resource-limited settings. Here we describe a sensitive, specific, and minimally instrumented method to detect HPV16, HPV18, and HPV45 mRNA in exfoliated cervical cells. First, reverse transcription recombinase polymerase amplification (RT-RPA) exo assays were designed to amplify and detect HPV16, HPV18, and HPV45 E7 mRNA ≥100 copies per reaction in real time using a portable fluorimeter, which is on par with the only FDA-approved hrHPV mRNA test. Next, an extraction-free sample preparation method using only an enzymatic reaction was developed to lyse cells and degrade cellular DNA in cultured HPV16, HPV18, and HPV45-positive cells. The RT-RPA assays specifically amplified mRNA from this crude lysate across a clinically relevant range of concentrations. Eleven cervicovaginal samples were tested at the point of care using these methods and showed 100% agreement between RT-RPA and RT-qPCR results, including two samples that were positive for HPV16 mRNA. Additionally, nine negative samples spiked with HPV16, HPV18, and HPV45-positive cells successfully detected each respective HPV type. In total, this prototype assay has potential to make sensitive and specific hrHPV mRNA testing more accessible in resource-limited settings.https://doi.org/10.1038/s41598-025-13583-2 |
| spellingShingle | Emilie Newsham Novak Ariel Ma Kathryn A. Kundrod Megan M. Chang Cannon J. Hansen Jane Richards Montealegre Michael E. Scheurer Philip E. Castle Ming Guo Mila P. Salcedo Kathleen Schmeler Rebecca Richards-Kortum Extraction-free RT-RPA assay for detection of HPV16, HPV18, and HPV45 mRNA expression Scientific Reports |
| title | Extraction-free RT-RPA assay for detection of HPV16, HPV18, and HPV45 mRNA expression |
| title_full | Extraction-free RT-RPA assay for detection of HPV16, HPV18, and HPV45 mRNA expression |
| title_fullStr | Extraction-free RT-RPA assay for detection of HPV16, HPV18, and HPV45 mRNA expression |
| title_full_unstemmed | Extraction-free RT-RPA assay for detection of HPV16, HPV18, and HPV45 mRNA expression |
| title_short | Extraction-free RT-RPA assay for detection of HPV16, HPV18, and HPV45 mRNA expression |
| title_sort | extraction free rt rpa assay for detection of hpv16 hpv18 and hpv45 mrna expression |
| url | https://doi.org/10.1038/s41598-025-13583-2 |
| work_keys_str_mv | AT emilienewshamnovak extractionfreertrpaassayfordetectionofhpv16hpv18andhpv45mrnaexpression AT arielma extractionfreertrpaassayfordetectionofhpv16hpv18andhpv45mrnaexpression AT kathrynakundrod extractionfreertrpaassayfordetectionofhpv16hpv18andhpv45mrnaexpression AT meganmchang extractionfreertrpaassayfordetectionofhpv16hpv18andhpv45mrnaexpression AT cannonjhansen extractionfreertrpaassayfordetectionofhpv16hpv18andhpv45mrnaexpression AT janerichardsmontealegre extractionfreertrpaassayfordetectionofhpv16hpv18andhpv45mrnaexpression AT michaelescheurer extractionfreertrpaassayfordetectionofhpv16hpv18andhpv45mrnaexpression AT philipecastle extractionfreertrpaassayfordetectionofhpv16hpv18andhpv45mrnaexpression AT mingguo extractionfreertrpaassayfordetectionofhpv16hpv18andhpv45mrnaexpression AT milapsalcedo extractionfreertrpaassayfordetectionofhpv16hpv18andhpv45mrnaexpression AT kathleenschmeler extractionfreertrpaassayfordetectionofhpv16hpv18andhpv45mrnaexpression AT rebeccarichardskortum extractionfreertrpaassayfordetectionofhpv16hpv18andhpv45mrnaexpression |