Multiplex recombinase polymerase amplification for detection of high-risk and low-risk HPV types as a potential topical application in one tube Scientific Reports

Cervical cancer is a major public health problem in developed and developing countries. According to the World Health Organization (WHO), there were 604,000 new cases and 342,000 deaths from cervical cancer worldwide in 2020, with 90% of these cases occurring in low- and middle-income countries.^1.2. The reasons were mainly found to be less access to cervical cancer vaccination as a preventive measure and to regular cervical cancer screening programs (and thus women with a positive screening could be adequately treated before developing cancer) compared to high-income countries². In Thailand and other Southeast Asian countries, cervical cancer is the most common cancer in women. The Institute of Oncology/International Agency for Research on Cancer reported that ~9,158 women were diagnosed with cervical cancer and 4,705 women died each year, and an estimated 3.4% of the Thai female population carried high-risk HPV types 16 or 18 (HPV).³. Additionally, the cost of cervical cancer radiation therapy and treatment of side effects such as gastrointestinal or genitourinary side effects is up to ~$63,103 per patient. The economic burden for radiation therapy alone in Thailand in 2018 was ~US$131 million and for diagnosing these patients with advanced cervical cancer ~US$129 million⁴. Consequently, an effective, simple, easily accessible and inexpensive screening method for early prevention is important in order to minimize the number of cases and the economic burden of patients with advanced cervical cancer.

More than 95% of cervical cancer is caused by HPV. HPV is also associated with more than 60-90% of rectal, vaginal, oropharyngeal, and penile cancers^5,6,7. HPV has more than 150 types and is divided into high-risk (HR) and low-risk (LR) for the development of cervical cancer. Common HPV HR types include 16, 18, 31, 33, 34, 35, 39, 45, 51, 52, 58, 59, 68, and 82, with types 16 and 18 generally accounting for 70% of cervical cancers. Common HPV LR types include 6, 11, 32, 40, 42, 43, 44, 54, 61, 70, 72, 81, 84, and 87^2,5,8,9.

Cytological examination with the Papanicolaou test (Pap smear) is traditionally simple and established. However, this procedure is inconvenient, cannot be performed in settings limited by local resources and physicians, and its sensitivity (i.e., detection limit) and specificity for detecting precancerous or cancerous changes in cells are relatively low compared to the nucleic acid amplification test^10,11. For example, Spence et al.¹² reported a Pap smear false-negative rate of 35.5% on average, with sensitivities ranging from 30 to 87%¹¹. In addition, Pap smear may require additional HPV specific nucleic acid test such as Cobas 4800 HPV test (Roche Diagnostics, New Jersey, USA) and REBA HPV-ID (Molecules and Diagnostics, Wonju, Republic of Korea) to accompany and identify HR types . Therefore, HPV-specific tests have been proposed as an alternative screening tool, and some have even suggested that these tests could allow screening intervals to be extended to ≥ 5 years.^11,13,14,15. The Cobas 4800 HPV test (price ~$8.5, reaction time ~5h) uses polymerase chain reaction (PCR) amplification of target DNA and nucleic acid hybridization to detect HPV HR types 16, 18 and pool 31, 33, 35, 39 , 45, 51, 52, 56, 58, 59, 66 and 68¹⁵. REBA HPV-ID (price ~$25.2, reaction time ~6 h) is a PCR-based reverse blot hybridization assay for 19 HPV HR probes (types 16, 18, 26, 31, 33, 34, 35, 39, 45 , 51, 52, 53, 56, 58, 59, 66, 68, 69 and 73) and 13 LR probes (types 6, 11, 32, 40, 42, 43, 44, 54, 70, 72, 81, 84 and 87) for HR and LR genotyping¹⁶. Although the Cobas and REBA assays are commercially available and widely used in clinical laboratories in vitro, they require an expensive thermal cycler for PCR amplification of HPV DNA and a special instrument for product-probe detection.^15,16.

Recently, an isothermal amplification assay such as recombinase polymerase amplification has emerged to address this issue. Amplification by recombinase polymerase utilizes 3 enzymes, recombinase, single-stranded DNA-binding protein, and strand displacement polymerase, allowing isothermal amplification of a specific primer-binding target at 37-42°C. This allows the reaction to be simply performed using a heat block or water bath, or even holding a tube in the hands, supporting low-cost, rapid, point-of-care molecular testing.¹⁷. For HPV detection, Ma et al.¹⁸ developed RPA for HPV HR types 16 and 18, and Gong et al.¹⁹ developed RPA for 13 HPV HR types based on the L1 gene. Therefore, we further developed a multiplex recombinase polymerase amplification (mRPA) assay with an attempt to cover broad HR and LR HPV types, as well as normal and precancerous stages of the HPV cycle in humans, as a potential local one-tube application for broad and precancerous cervical cancer screening. We determined the limit of detection, verified the correct specificity for positive controls including some types of HR and LR and negative controls containing urogenital bacteria, other viruses, human and mouse, and evaluated the performance of the assay (e.g., sensitivity, specificity, and accuracy) on real 130 clinical cervical samples. swabs compared to Cobas and REBA results. In the HPV developmental cycle, the late gene L1 encodes the major viral capsid protein, while the early genes E6 and E7 encode oncoproteins, and their detection indicates an increased risk of developing cancer (e.g., higher expression of E6/E7 than L1 mRNA was found in cancer patients). E6 degrades human growth suppressor protein p53 and E7 degrades human retinoblastoma tumor suppressor protein pRb, thus E6 and E7 induce cancer development by evading human growth suppressors, resisting cell death, maintaining proliferative signaling, enabling replicative immortality, angiogenesis induction and activation invasion and metastasis²⁰. Consequently, this study designed multiplex degenerate primers based on HPV L1 and E6/E7 genes that cover up to 20 HR and 14 LR types, and the E6/E7 amplification product could indicate increased cancer risk.