{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,9]],"date-time":"2026-10-09T07:27:28Z","timestamp":1791530848723,"version":"4.3.4"},"reference-count":34,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2025,12,18]],"date-time":"2025-12-18T00:00:00Z","timestamp":1766016000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100012166","name":"National Key R&D Program of China","doi-asserted-by":"crossref","award":["2024YFC2310203"],"award-info":[{"award-number":["2024YFC2310203"]}],"id":[{"id":"10.13039\/501100012166","id-type":"DOI","asserted-by":"crossref"}]},{"name":"Shenzhen Science and Technology Program","award":["JCYJ20240813113233044"],"award-info":[{"award-number":["JCYJ20240813113233044"]}]},{"DOI":"10.13039\/501100021171","name":"Guangdong Basic and Applied Basic Research Foundation","doi-asserted-by":"crossref","award":["2022A1515220114"],"award-info":[{"award-number":["2022A1515220114"]}],"id":[{"id":"10.13039\/501100021171","id-type":"DOI","asserted-by":"crossref"}]},{"name":"Key Laboratory of Environmental Pollution Monitoring and Disease Control, Ministry of Education, Guizhou Medical University","award":["442"],"award-info":[{"award-number":["442"]}]},{"name":"Common University Innovation Team Project of Guangdong","award":["2021KCXTD041"],"award-info":[{"award-number":["2021KCXTD041"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biosensors"],"abstract":"<jats:p>Mycoplasma hominis (MH) is a prevalent opportunistic pathogen that is strongly associated with a wide range of urogenital tract infections and severe adverse pregnancy outcomes in clinical settings. Current MH detection methods, including microbial culture and qPCR, are time-consuming and rely on complex equipment, making them unsuitable for scenarios requiring rapid or simplified testing. In this study, we developed a visual readout biosensing platform by synergistically integrating recombinase polymerase amplification (RPA), CRISPR\/Cas12a-mediated target nucleic acid recognition, and lateral flow biosensors for the rapid, sensitive, and specific identification of MH. The assay specifically targets the MH-specific 16S rRNA gene, achieving a limit of detection as low as 2 copies\/reaction of recombinant plasmid containing the target gene with a total assay time of 60 min. Critical reaction parameters, including Cas12a-crRNA molar ratio, volume of RPA amplicon input, and Cas12a cleavage time, were systematically optimized to maximize the biosensor\u2019s response efficiency and detection reliability. The platform exhibited exceptional specificity, with no cross-reactivity observed against common co-occurring urogenital pathogens, and effectively minimized aerosol contamination risks via a rigorous decontamination workflow. Furthermore, this work represents the first documented implementation of a contamination-control protocol for an MH-specific CRISPR-LFA assay. Notably, testing results from 18 clinical samples demonstrated the high specificity of this assay, highlighting its promising potential for clinical application.<\/jats:p>","DOI":"10.3390\/bios15120821","type":"journal-article","created":{"date-parts":[[2025,12,18]],"date-time":"2025-12-18T13:45:07Z","timestamp":1766065507000},"page":"821","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Rapid Visual Detection of Mycoplasma Hominis Using an RPA-CRISPR\/Cas12a Assay"],"prefix":"10.3390","volume":"15","author":[{"given":"Jie","family":"Chen","sequence":"first","affiliation":[{"name":"College of Health Science and Environmental Engineering, Shenzhen Technology University, 3002 Lantian Road, Pingshan District, Shenzhen 518118, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shutao","family":"Liu","sequence":"additional","affiliation":[{"name":"Department of Radiology and Nuclear Medicine, Xuanwu Hospital, Capital Medical University, Beijing 100053, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sunyi","family":"Chen","sequence":"additional","affiliation":[{"name":"Department of Dermatology, Huashan Hospital, Fudan University, Shanghai 200040, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jingwen","family":"Mai","sequence":"additional","affiliation":[{"name":"KingMed School of Laboratory Medicine, Guangzhou Medical University, Guangzhou 511436, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maiwula","family":"Abudukadi","sequence":"additional","affiliation":[{"name":"KingMed School of Laboratory Medicine, Guangzhou Medical University, Guangzhou 511436, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yao","family":"Chen","sequence":"additional","affiliation":[{"name":"KingMed School of Laboratory Medicine, Guangzhou Medical University, Guangzhou 511436, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0425-3921","authenticated-orcid":false,"given":"Jie","family":"Lu","sequence":"additional","affiliation":[{"name":"Department of Radiology and Nuclear Medicine, Xuanwu Hospital, Capital Medical University, Beijing 100053, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guanglei","family":"Li","sequence":"additional","affiliation":[{"name":"College of Pharmacy, Shenzhen Technology University, 3002 Lantian Road, Pingshan District, Shenzhen 518118, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chenchen","family":"Ge","sequence":"additional","affiliation":[{"name":"College of Health Science and Environmental Engineering, Shenzhen Technology University, 3002 Lantian Road, Pingshan District, Shenzhen 518118, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,12,18]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"2179","DOI":"10.1007\/s40121-024-01035-9","article-title":"Mycoplasma hominis as Cause of Extragenital Infection in Patients with Hypogammaglobulinemia: Report of 2 Cases and Literature Review","volume":"13","author":"Russo","year":"2024","journal-title":"Infect. Dis. Ther."},{"key":"ref_2","first-page":"169","article-title":"Implicaciones cl\u00ednicas de las especies del g\u00e9nero Mycoplasma Clinical implications of the genus Mycoplasma","volume":"34","year":"2021","journal-title":"Rev. Esp. Quimioter."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"231","DOI":"10.1097\/QCO.0b013e328360db58","article-title":"Ureaplasma urealyticum, Mycoplasma hominis and adverse pregnancy outcomes","volume":"26","author":"Capoccia","year":"2013","journal-title":"Curr. Opin. Infect. Dis."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Oh, K.Y., Lee, S., Park, J., Park, M.H., Jeong, J.H., Yang, J.B., Lim, C.K., Ha, J.G., and Yang, Y.S. (2024). Vaginal microbiota of pregnant women with Ureaplasma urealyticum and Mycoplasma hominis infections. Front. Cell. Infect. Microbiol., 14.","DOI":"10.3389\/fcimb.2024.1445300"},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Chudzicka-Struga\u0142a, I., Go\u0142\u0119biewska, I., Brudecki, G., Elamin, W., Banaszewska, B., Chudzicka-Adamczak, M., Struga\u0142a, D., and Zwo\u017adziak, B. (2025). Proinflammatory Cytokines in Women with PCOS in Atypical Pathogen Infections. Diagnostics, 15.","DOI":"10.3390\/diagnostics15131669"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"88","DOI":"10.1016\/j.ijmmb.2020.10.020","article-title":"Mycoplasma hominis: An under recognized pathogen","volume":"39","author":"Ahmed","year":"2021","journal-title":"Indian J. Med Microbiol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"749","DOI":"10.1097\/INF.0000000000002306","article-title":"Successful Whole Genome Sequencing-guided Treatment of Mycoplasma hominis Ventriculitis in a Preterm Infant","volume":"38","author":"Huang","year":"2019","journal-title":"Pediatr. Infect. Dis. J."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"2469","DOI":"10.2147\/IDR.S510660","article-title":"Diagnosis of Mycoplasma Hominis Peritonitis in a CAPD Patient with Metagenomic Next-Generation Sequencing: A Case Report","volume":"18","author":"Deng","year":"2025","journal-title":"Infect. Drug Resist."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Huang, S.-M., Tang, Y.-R., Wang, J.-L., Wang, X.-Z., Zhang, Y.-Y., and Pan, S.-F. (2023). Case Report: Double trouble: A rare case of successfully treated Mycoplasma hominis and Pseudomonas aeruginosa co-infection. Front. Cell. Infect. Microbiol., 13.","DOI":"10.3389\/fcimb.2023.1159891"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"e01175","DOI":"10.1016\/j.idcr.2021.e01175","article-title":"Intracranial Mycoplasma hominis infection following emergent craniectomy","volume":"25","author":"Qamar","year":"2021","journal-title":"IDCases"},{"key":"ref_11","first-page":"e31491","article-title":"A Rare Case of a Prostatic Abscess Secondary to a Mycoplasma hominis Infection","volume":"14","author":"Jagtap","year":"2022","journal-title":"Cureus"},{"key":"ref_12","first-page":"345","article-title":"Simultaneous Detection of Mycoplasma pneumoniae, Mycoplasma hominis and Mycoplasma arthritidis in Synovial Fluid of Patients with Rheumatoid Arthritis by Multiplex PCR","volume":"18","author":"Ataee","year":"2015","journal-title":"Arch. Iran. Med."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"838","DOI":"10.1016\/j.jmoldx.2023.07.004","article-title":"Development of a Rapid and High-Throughput Multiplex Real-Time PCR Assay for Mycoplasma hominis and Ureaplasma Species","volume":"25","author":"Chan","year":"2023","journal-title":"J. Mol. Diagn."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Tan, M., Liao, C., Liang, L., Yi, X., Zhou, Z., and Wei, G. (2022). Recent advances in recombinase polymerase amplification: Principle, advantages, disadvantages and applications. Front. Cell. Infect. Microbiol., 12.","DOI":"10.3389\/fcimb.2022.1019071"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"459","DOI":"10.1146\/annurev-anchem-061622-015112","article-title":"The Present and Future Landscapes of Molecular Diagnostics","volume":"17","author":"Rolando","year":"2024","journal-title":"Annu. Rev. Anal. Chem."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"De Silva, P.I.T., Hiniduma, K., Canete, R., Bhalerao, K.S., Shawky, S.M., Gunathilaka, H., Rouge, J.L., Mosa, I.M., Steffens, D.C., and Manning, K. (2025). Multiplexed CRISPR Assay for Amplification-Free Detection of miRNAs. Biosensors, 15.","DOI":"10.3390\/bios15060346"},{"key":"ref_17","first-page":"1","article-title":"A multiplex RPA-CRISPR\/Cas12a-based POCT technique and its application in human papillomavirus (HPV) typing assay","volume":"29","author":"Liu","year":"2024","journal-title":"Cell. Mol. Biol. Lett."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Wang, H., Zhou, G., Liu, H., Peng, R., Sun, T., Li, S., Chen, M., Wang, Y., Shi, Q., and Xie, X. (2024). Detection of Porcine Circovirus (PCV) Using CRISPR-Cas12a\/13a Coupled with Isothermal Amplification. Viruses, 16.","DOI":"10.3390\/v16101548"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"6853","DOI":"10.1021\/acs.analchem.4c01452","article-title":"CRISPR\/Cas12a-Amplified Aptamer Switch Microplate Assay for Small Molecules","volume":"96","author":"Zhu","year":"2024","journal-title":"Anal. Chem."},{"key":"ref_20","first-page":"673\u2212679","article-title":"Methodological study on eliminating nucleic acid contamination in molecular diagnostic laboratory","volume":"37","author":"Li","year":"2021","journal-title":"Chin. J. Biotechnol."},{"key":"ref_21","unstructured":"Centers for Disease Control and Prevention, Respiratory Viruses Branch, Division of Viral Diseases (2025, April 09). Real-Time RT-PCR Panel for Detection 2019-Novel Coronavirus. Available online: https:\/\/www.who.int\/docs\/default-source\/coronaviruse\/uscdcrt-pcr-panel-for-detection-instructions.pdf?sfvrsn=3aa07934_2."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1417","DOI":"10.3109\/21691401.2015.1031905","article-title":"Gold Nanoparticle-Oligonucleotide Conjugate to Detect the Sequence of Lung Cancer Biomarker","volume":"44","author":"Daraee","year":"2016","journal-title":"Artif. Cells Nanomed. Biotechnol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"870","DOI":"10.1038\/s41587-020-0513-4","article-title":"CRISPR\u2013Cas12-based detection of SARS-CoV-2","volume":"38","author":"Broughton","year":"2020","journal-title":"Nat. Biotechnol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1797","DOI":"10.1021\/acs.bioconjchem.4c00375","article-title":"RPA-CRISPR-Cas-Mediated Dual Lateral Flow Assay for the Point-of-Care Testing of HPV16 and HPV18","volume":"35","author":"Zhang","year":"2024","journal-title":"Bioconjugate Chem."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"50","DOI":"10.1186\/s13568-023-01554-7","article-title":"Point-of-care detection of Neisseria gonorrhoeae based on RPA-CRISPR\/Cas12a","volume":"13","author":"Tu","year":"2023","journal-title":"AMB Express"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"19445","DOI":"10.1021\/acs.analchem.5c01400","article-title":"Development of a CRISPR\/Cas-based detection platform for tracking decreased susceptibility to cephalosporins in Neisseria gonorrhoeae","volume":"97","author":"Zhao","year":"2025","journal-title":"Anal. Chem."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"5597","DOI":"10.1038\/s41467-025-60917-9","article-title":"Cas12a Cis-cleavage mediated lateral flow assay enables multiplex and ultra-specific nucleic acid detection","volume":"16","author":"Lin","year":"2025","journal-title":"Nat. Commun."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"342797","DOI":"10.1016\/j.aca.2024.342797","article-title":"A highly specific and ultrasensitive approach to detect Prymnesium parvum based on RPA-CRISPR-LbaCas12a-LFD system","volume":"1315","author":"Huang","year":"2024","journal-title":"Anal. Chim. Acta"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"350","DOI":"10.1186\/s13071-022-05475-5","article-title":"Establishment and application of a CRISPR-Cas12a-based RPA-LFS and fluorescence for the detection of Trichomonas vaginalis","volume":"15","author":"Li","year":"2022","journal-title":"Parasites Vectors"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"117221","DOI":"10.1016\/j.bios.2025.117221","article-title":"Development of a single-tube RPA\/CRISPR-cas12a detection platform for monkeypox virus","volume":"278","author":"Liu","year":"2025","journal-title":"Biosens. Bioelectron."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"341757","DOI":"10.1016\/j.aca.2023.341757","article-title":"One-tube RPA-CRISPR Cas12a\/Cas13a rapid detection of methicillin-resistant Staphylococcus aureus","volume":"1278","author":"Liu","year":"2023","journal-title":"Anal. Chim. Acta"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"113994","DOI":"10.1016\/j.bios.2022.113994","article-title":"A one-pot CRISPR\/Cas13a-based contamination-free biosensor for low-cost and rapid nucleic acid diagnostics","volume":"202","author":"Hu","year":"2022","journal-title":"Biosens. Bioelectron."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"98","DOI":"10.1016\/j.tibtech.2024.07.007","article-title":"Trends in developing one-pot CRISPR diagnostics strategies","volume":"43","author":"Chen","year":"2025","journal-title":"Trends Biotechnol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"9724","DOI":"10.1021\/acs.analchem.2c01193","article-title":"Photoactivatable CRISPR\/Cas12a Strategy for One-Pot DETECTR Molecular Diagnosis","volume":"94","author":"Chen","year":"2022","journal-title":"Anal. Chem."}],"container-title":["Biosensors"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2079-6374\/15\/12\/821\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,12,25]],"date-time":"2025-12-25T05:10:22Z","timestamp":1766639422000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2079-6374\/15\/12\/821"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,12,18]]},"references-count":34,"journal-issue":{"issue":"12","published-online":{"date-parts":[[2025,12]]}},"alternative-id":["bios15120821"],"URL":"https:\/\/doi.org\/10.3390\/bios15120821","relation":{},"ISSN":["2079-6374"],"issn-type":[{"value":"2079-6374","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,12,18]]}}}