Journal Article

Establishment and application of a CRISPR-Cas12a-based RPA-LFS and fluorescence for the detection of Trichomonas vaginalis

Liu, Juan, Li, Jianhua, Zhang, Nan, Zhang, Xichen, Li, Xin, Cao, Songgao, Li, Shan, Cao, Lili, Yu, Yanhui, Zhao, Panpan, Sun, Min, Wang, Xiaocen, Gong, Pengtao

2022

Overview

Background Infection with Trichomonas vaginalis can lead to cervicitis, urethritis, pelvic inflammatory disease, prostatitis and perinatal complications and increased risk of HIV transmission. Here, we used an RPA-based CRISPR-Cas12a assay system in combination with a lateral flow strip (LFS) (referred to as RPA-CRISPR-Cas12a) to establish a highly sensitive and field-ready assay and evaluated its ability to detect clinical samples. Methods We developed a one-pot CRISPR-Cas12a combined with RPA-based field detection technology for T. vaginalis , chose actin as the target gene to design crRNA and designed RPA primers based on the crRNA binding site. The specificity of the method was demonstrated by detecting genomes from nine pathogens. To improve the usability and visualize the RPA-CRISPR-Cas12a assay results, both fluorescence detection and LFS readouts were devised. Results The RPA-CRISPR-Cas12a assay platform was completed within 60 min and had a maximum detection limit of 1 copy/µl and no cross-reactivity with Candida albicans , Mycoplasma hominis , Neisseria gonorrhoeae , Escherichia coli , Cryptosporidium parvum , G. duodenalis or Toxoplasma gondii after specificity validation. Thirty human vaginal secretions were tested by RPA-CRISPR-Cas12a assays, and the results were read by a fluorescent reporter and LFS biosensors and then compared to the results from nested PCR detection of these samples. Both RPA-CRISPR-Cas12a assays showed 26.7% (8/30) T. vaginalis -positive samples and a consistency of 100% (8/8). The RPA-CRISPR-Cas12a assays had a higher sensitivity than nested PCR (only seven T. vaginalis -positive samples were detected). Conclusions The T. vaginalis RPA-CRISPR-Cas12a assay platform in this study can be used for large-scale field testing and on-site tests without the need for trained technicians or costly ancillary equipment. Graphical abstract

• Publisher: BioMed Central,BioMed Central Ltd,Springer Nature B.V,BMC
• Subject: Acids / Actin / Agglutination tests / ancillary equipment / Assaying / Binding sites / Biomedical and Life Sciences / Biomedicine / Biosensors / Candida albicans / Cervicitis / Cloning / Complications / CRISPR / CRISPR-Cas12a / cross reaction / Cross-reactivity / Cryptosporidium / Cryptosporidium parvum / Design / Detection / detection limit / Diagnosis / Disease transmission / E coli / Entomology / Escherichia coli / Fluorescence / Gene editing / Genes / Genetic aspects / Genomes / Health risks / HIV / Human immunodeficiency virus / humans / Identification and classification / Infections / Infectious Diseases / Inflammatory diseases / Laboratories / Mycoplasma hominis / Neisseria gonorrhoeae / Nucleotide sequence / On-site testing / On-site tests / Parasitic diseases / Parasitology / Pathogens / Pelvic inflammatory disease / Plasmids / polymerase chain reaction / Primers / Prostatitis / Proteins / risk / RPA / Secretions / Sexually transmitted diseases / Specificity / STD / Technicians / Toxoplasma gondii / Trichomonas vaginalis / Trichomoniasis / Tropical Medicine / Urethritis / Vagina / Veterinary Medicine/Veterinary Science / Virology / Visualization detection