Dataset Information

CRISPR-Based Multiplex Detection of Human Papillomaviruses for One-Pot Point-of-Care Diagnostics.

ABSTRACT: The World Health Organization's global initiative toward eliminating high-risk Human Papillomavirus (hrHPV)-related cancers recommends DNA testing over visual inspection in all settings for primary cancer screening and HPV eradication by 2100. However, multiple hrHPV types cause different types of cancers, and there is a pressing need for an easy-to-use, multiplex point-of-care diagnostic platform for detecting different hrHPV types. Recently, CRISPR-Cas systems have been repurposed for point-of-care detection. Here, we established a CRISPR-Cas multiplexed diagnostic assay (CRISPRD) to detect cervical cancer-causing hrHPVs in one reaction (one-pot assay). We harnessed the compatibility of thermostable AapCas12b, TccCas13a, and HheCas13a nucleases with isothermal amplification and successfully detected HPV16 and HPV18, along with an internal control in a single-pot assay with a limit of detection of 10 copies and 100% specificity. This platform offers a rapid and practical solution for the multiplex detection of hrHPVs, which may facilitate large-scale hrHPV point-of-care screening. Furthermore, the CRISPRD platform programmability enables it to be adapted for the multiplex detection of any two nucleic acid biomarkers as well as internal control.

SUBMITTER: Ghouneimy A

PROVIDER: S-EPMC10949237 | biostudies-literature | 2024 Mar

REPOSITORIES: biostudies-literature

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CRISPR-Based Multiplex Detection of Human Papillomaviruses for One-Pot Point-of-Care Diagnostics.

Ghouneimy Ahmed A Ali Zahir Z Aman Rashid R Jiang Wenjun W Aouida Mustapha M Mahfouz Magdy M

ACS synthetic biology 20240213 3

The World Health Organization's global initiative toward eliminating high-risk Human Papillomavirus (hrHPV)-related cancers recommends DNA testing over visual inspection in all settings for primary cancer screening and HPV eradication by 2100. However, multiple hrHPV types cause different types of cancers, and there is a pressing need for an easy-to-use, multiplex point-of-care diagnostic platform for detecting different hrHPV types. Recently, CRISPR-Cas systems have been repurposed for point-of ...[more]

PMID: 38349963

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Project description:The pandemic due to the outbreak of 2019 coronavirus disease (COVID-19) caused by novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has raised significant public health concerns. Rapid, affordable, and accurate diagnostic testing not only paves the way for the effective treatment of diseases, but also plays a crucial role in preventing the spreading of infectious diseases. Herein, a one-pot CRISPR/Cas13a-based visual biosensor was proposed and developed for the rapid and low-cost nucleic acid detection. By combining Cas13a cleavage and Recombinase Polymerase Amplification (RPA) in a one-pot reaction in a disposable tube-in-tube vessel, amplicon contamination could be completely avoided. The RPA reaction is carried out in the inner tube containing two hydrophobic holes at the bottom. After the completion of amplification reaction, the reaction solution enters the outer tube containing pre-stored Cas13a reagent under the action of centrifugation or shaking. Inner and outer tubes are combined to form an independent reaction pot to complete the nucleic acid detection without opening the lid. This newly developed nucleic acid detection method not only meets the need of rapid nucleic acid detection at home without the need for any specialized equipment, but also fulfils the requirement of rapid on-site nucleic acid detection with the aid of small automated instruments. In this study, CRISPR/Cas13a and CRISPR/Cas12a were used to verify the reliability of the developed one-pot nucleic acid detection method. The performance of the system was verified by detecting the DNA virus, i.e., African swine fever virus (ASFV) and the RNA virus, i.e., SARS-Cov-2. The results indicate that the proposed method possesses a limit of detection of 3 copy/μL. The negative and positive test results are consistent with the results of real-time fluorescence quantitative polymerase chain reaction (PCR), but the time required is shorter and the cost is lower. Thus, this study makes this method available in resource-limited areas for the purpose of large-scale screening and in case of epidemic outbreak.

Project description:BackgroundMelioidosis is a neglected but often fatal tropical disease. The disease has broad clinical manifestations, which makes diagnosis challenging and time consuming. To improve diagnosis, we aimed to evaluate the performance of the CRISPR-Cas12a system (CRISPR-BP34) to detect Burkholderia pseudomallei DNA across clinical specimens from patients suspected to have melioidosis.MethodsWe conducted a prospective, observational cohort study of adult patients (aged ≥18 years) with melioidosis at Sunpasitthiprasong Hospital, a tertiary care hospital in Thailand. Participants were eligible for inclusion if they had culture-confirmed B pseudomallei infection from any clinical samples. Data were collected from patient clinical records and follow-up telephone calls. Routine clinical samples (blood, urine, respiratory secretion, pus, and other body fluids) were collected for culture. We documented time taken for diagnosis, and mortality at day 28 of follow-up. We also performed CRISPR-BP34 detection on clinical specimens collected from 330 patients with suspected melioidosis and compared its performance with the current gold-standard culture-based method. Discordant results were validated by three independent qualitative PCR tests. This study is registered with the Thai Clinical Trial Registry, TCTR20190322003.FindingsBetween Oct 1, 2019, and Dec 31, 2022, 876 patients with culture-confirmed melioidosis were admitted or referred to Sunpasitthiprasong Hospital, 433 of whom were alive at diagnosis and were enrolled in this study. Median time from sample collection to diagnosis by culture was 4·0 days (IQR 3·0-5·0) among all patients with known survival status at day 28, which resulted in delayed treatment. 199 (23%) of 876 patients died before diagnosis and 114 (26%) of 433 patients in follow-up were treated, but died within 28 days of admission. To test the CRISPR-BP34 assay, we enrolled and collected clinical samples from 114 patients with melioidosis and 216 patients without melioidosis between May 26 and Dec 31, 2022. Application of CRISPR-BP34 reduced the median sample-to-diagnosis time to 1·1 days (IQR 0·7-1·5) for blood samples, 2·3 h (IQR 2·3-2·4) for urine, and 3·3 h (3·1-3·4) for respiratory secretion, pus, and other body fluids. The overall sensitivity of CRISPR-BP34 was 93·0% (106 of 114 samples [95% CI 86·6-96·9]) compared with 66·7% (76 of 114 samples [57·2-75·2]) for culture. The overall specificity of CRISPR-BP34 was 96·8% (209 of 216 samples [95% CI 93·4-98·7]), compared with 100% (216 of 216 samples [98·3-100·0]) for culture.InterpretationThe sensitivity, specificity, speed, and window of clinical intervention offered by CRISPR-BP34 support its prospective use as a point-of-care diagnostic tool for melioidosis. Future development should be focused on scalability and cost reduction.FundingChiang Mai University Thailand and Wellcome Trust UK.

Dataset Information

CRISPR-Based Multiplex Detection of Human Papillomaviruses for One-Pot Point-of-Care Diagnostics.

Publications

CRISPR-Based Multiplex Detection of Human Papillomaviruses for One-Pot Point-of-Care Diagnostics.

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