Development of Enzymatic Recombinase Amplification Assays for the Rapid Visual Detection of HPV16/18

Abstract

Human papillomavirus (HPV) types 16 and 18 are the major causes of cervical lesions and are associated with 71% of cervical cancer cases globally. However, public health infrastructures to support cervical cancer screening may be unavailable to women in low-resource areas. Therefore, sensitive, convenient, and cost-efficient diagnostic methods are required for the detection of HPV16/18. Here, we designed two novel methods, real-time ERA and ERA-LFD, based on enzymatic recombinase amplification (ERA) for quick point-of-care identification of the HPV E6/E7 genes. The entire detection process could be completed within 25 min at a constant low temperature (35-43°C), and the results of the combined methods could be present as the amplification curves or the bands presented on dipsticks and directly interpreted with the naked eye. The ERA assays evaluated using standard plasmids carrying the E6/E7 genes and clinical samples exhibited excellent specificity, as no cross-reaction with other common HPV types was observed. The detection limits of our ERA assays were 10⁰ and 10¹ copies/μl for HPV16 and 18 respectively, which were comparable to those of the real-time PCR assay. Assessment of the clinical performance of the ERA assays using 114 cervical tissue samples demonstrated that they are highly consistent with real-time PCR, the gold standard for HPV detection. This study demonstrated that ERA-based assays possess excellent sensitivity, specificity, and repeatability for HPV16 and HPV18 detection with great potential to become robust diagnostic tools in local hospitals and field studies.

Keywords: Enzymatic recombinase amplification; human papillomavirus; lateral flow dipstick; rapid visual detection.

Fig. 1. Schematic diagram of real-time ERA and ERA-LFD assays.

Fig. 2. Screening for the optimal primer combinations for HPV16 and 18.

(A, B) 1%AGE of ERA amplified products with nine different primer combinations of HPV16 and 18 and the final fluorescence intensities of real-time ERA assays using each primer set. Fluorescence curves of real-time ERA assays generated by 9 primer combinations of HPV16 (C) and 18 (D). M: DL 2000 DNA marker; N/NC: negative control; 1-9: F1+R1, F1+R2, F1+R3, F2+R1, F2+R2, F2+R3, F3+R1, F3+R2, F3+R3; ****: p < 0.0001.

Fig. 3. Optimization of ERA reaction conditions for the detection of pHPV-16-E6/7.

(A) Fluorescence curves and final fluorescence intensities of real-time ERA assay performed at a broad range of amplification temperatures (35°–43°C) to analyze the optimal reaction temperature. (B) Fluorescence curves and final fluorescence values of real-time ERA assay performed with different concentrations of primers (300, 360, 420, 480, and 540 nM) to evaluate the optimal concentration of primer sets. ERA-LFD assay was conducted with 10¹ copies/μl (C) and 10⁵ copies/μl (D) of HPV16-E6/E7 plasmids to evaluate the optimal reaction time. NC: negative control; ****: p < 0.0001.

Fig. 4. Comparison of the limit of detection (LOD) of real-time ERA, ERA-LFD, and real-time PCR assays.

Fluorescence curves and final fluorescence intensities of real-time ERA performed with a serial dilution from 10⁷ to 10⁰ copies/ μl of HPV16 (A) and 18 (D) plasmids containing E6/7 genes. The threshold is illustrated as red dashed lines at 996.7 and 2053.8 for HPV16 and 18, respectively. ERA-LFD assay was conducted with a series of diluted HPV16 (B) and 18 plasmids (E) from 10⁷ to 10⁰ copies/μl. Detection of HPV16 (C) and 18 (F) using real-time PCR. 1-8: 10⁷ to 10⁰ copies/μl; N/NC: negative control; *: p < 0. 05; ****: p < 0.0001; ns: no significance.

Fig. 5. Specificity analysis of real-time ERA and ERA-LFD assays for HPV16/18 detection.

Specificity analysis of HPV16 (A) and 18 (C) real-time ERA assays for the detection of HPV16, HPV18, and other 9 types of HPV by observing the fluorescence curves and final fluorescence intensities. Specificity analysis of HPV16 (B) and 18 (D) ERA-LFD assays for the detection of HPV16, HPV18, and other 9 types of HPV by observing the bands on the test lines. ****: p < 0.0001; ns: no significance; NC: negative control; 1-12: pHPV-16-E6/E7, pHPV-18-E6/E7, pHPV-31-E6/E7, HPV-33, HPV-35 HPV-39, pHPV-45-E6/E7 HPV-52, HPV-58, pHPV-66-E6/E7, pHPV-73-E6/E7, negative control, respectively (All the mentioned strains were shown in Table 1).