Abstract Persistent infection with high-risk human papillomavirus (HR-HPV) is the primary and initiating factor for cervical cancer. With over 200 identified HPV types, including 14 high-risk types that integrate into the host cervical epithelial cell DNA, early determination of HPV infection type is crucial for effective risk stratification and management. Presently, on-site immediate testing during the HPV screening stage, known as Point of Care Testing (POCT), remains immature, severely limiting the scope and scenarios of HPV screening. This study, guided by the genomic sequence patterns of HPV, established a multiplex recombinase polymerase amplification (RPA) technology based on the concept of “universal primers.” This approach achieved the multiple amplification of RPA, coupled with the CRISPR/Cas12a system serving as a medium for signal amplification and conversion. The study successfully constructed a POCT combined detection system, denoted as H-MRC12a (HPV—Multiple RPA—CRISPR/Cas12a), and applied it to high-risk HPV typing detection. The system accomplished the typing detection of six high-risk HPV types (16, 18, 31, 33, 35, and 45) can be completed within 40 min, and the entire process, from sample loading to result interpretation, can be accomplished within 45 min, with a detection depth reaching 1 copy/μL for each high-risk type. Validation of the H-MRC12a detection system’s reproducibility and specificity was further conducted through QPCR on 34 clinical samples. Additionally, this study explored and optimized the multiplex RPA amplification system and CRISPR system at the molecular mechanism level. Furthermore, the primer design strategy developed in this study offers the potential to enhance the throughput of H-MRC12a detection while ensuring sensitivity, providing a novel research avenue for high-throughput detection in Point-of-Care molecular pathogen studies. 

A multiplex RPA-CRISPR/Cas12a-based POCT technique and its application in human papillomavirus (HPV) typing assay

Development of a duplex recombinase-aided amplification assay for direct detection of Mycoplasma pneumoniae and Chlamydia trachomatis in clinical samples.

Nucleic acid amplification enhancers suitable for use in a recombinase-aided amplification (RAA) assay were studied for the first time, and amplification of a long-fragment (509 bp) was initially explored. Using recombinant plasmids and clinical samples, RAA fluorescence and basic methods were used to evaluate the efficacy. The fluorescence method was evaluated by threshold time and fluorescence value, and the basic method was characterized by 2% agarose gel electrophoresis. Taking a previously established RAA assay for HPV18 as an example, we demonstrated that the addition of 0.2 M, 0.4 M, and 0.6 M betaine and 10% pullulan could enhance the RAA. The new RAA assays with betaine and pullulan were named B-RAA and P-RAA, respectively. Using the B-RAA and P-RAA fluorescence methods, the threshold time values could be shortened by 1.72–2.32 minutes and 2.60 minutes, respectively, and the fluorescence values could be enhanced by 8847.25–9094.37 mv and 5250 mv, respectively. Using the basic method, the sensitivity could be increased 10-fold. We successfully amplified a long-fragment of 509 bp using a P-RAA assay with a sensitivity of 102 copies/µL (compared with 103 copies/µL in the RAA assay). Thus, we concluded that betaine and pullulan are effective additives to enhance the sensitivity of RAA assays. 

Enhancement of a recombinase-aided amplification assay using betaine and pullulan

Development and evaluation of a sensitive recombinase aided amplification assay for rapid detection of Vibrio parahaemolyticus

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.

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

Cervical cancer is primarily caused by persistent infection with high-risk human papillomavirus (HPV), and 70% of cases are associated with HPV16 and 18 infections. The objective of this study was to establish rapid, simple, and sensitive internally controlled recombinase-aided amplification (IC-RAA) assays for the detection of HPV16 and 18. The assays were performed at 39 ℃ and were completed within 30 min. A total of 277 clinical samples of exfoliated cervical cells were tested by IC-RAA assays and commercial HPV real-time fluorescent PCR kits using extracted DNA and samples treated with nucleic acid releasing agent. The analytical sensitivity of the IC-RAA assay was found to be 10 copies/μL for the detection of HPV16 and 18 when using recombinant plasmids as targets. The optimal concentration of the internal control (IC) plasmid and 18 was 1000 copies/μL for HPV16 and 100 copies/μL for HPV18. The clinical sensitivity of the IC-RAA assays for HPV16 using extracted DNA and samples treated with nucleic acid releasing agent was 98.73% and 97.47%, respectively, with kappa values of 0.977 (P < 0.01) and 0.955 (P < 0.01), respectively, and 100% The specificity in both cases. For HPV18, the sensitivity and specificity were 100%, and the kappa value was 1 for both samples (P < 0.01). The IC-RAA assay is a promising tool for the detection of HPV16 and HPV18, especially in resource-constrained settings.

Internally controlled recombinase-aided amplification (IC-RAA) assays for the detection of human papillomavirus genotypes 16 and 18 using extracted DNA and samples treated with nucleic acid releasing agent.

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Internally controlled recombinase-aided amplification (IC-RAA) assays for the detection of human papillomavirus genotypes 16 and 18 using extracted DNA and samples treated with nucleic acid releasing agent.