Extract-Free One-Pot Ambient RPA-CRISPR Detection of Plasmodium in Whole Blood

Extract-Free One-Pot Ambient RPA-CRISPR Detection of Plasmodium in Whole Blood | medRxiv Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search Extract-Free One-Pot Ambient RPA-CRISPR Detection of Plasmodium in Whole Blood Jipei Liao , Yun Su , View ORCID Profile Feng Jiang doi: https://doi.org/10.64898/2026.03.17.26348654 Jipei Liao 1 Department of Pathology, University of Maryland School of Medicine , Baltimore, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yun Su 2 Department of Medicine, Nanjing University of Chinese Medicine , Nanjing, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Feng Jiang 1 Department of Pathology, University of Maryland School of Medicine , Baltimore, USA 3 BIOTARGET DX LLC , Baltimore, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Feng Jiang For correspondence: fjiang{at}som.umarylnd.edu fengjiang{at}biotargetdxllc.onmicrosoft.com Abstract Full Text Info/History Metrics Data/Code Preview PDF ABSTRACT Background Malaria caused by Plasmodium is a major global health burden. Rapid and accurate malaria detection is essential for effective disease control. However, most molecular diagnostics require DNA extraction, thermal cycling, and specialized infrastructure, limiting use in resource-limited settings. This study aimed to develop an extraction-free, one-pot CRISPR-based platform for rapid Plasmodium detection directly from whole blood under ambient conditions. Methods Crude blood lysates were directly applied to a one-pot recombinase polymerase amplification (RPA)-CRISPR reaction coupled with lateral flow readout. Analytical sensitivity and specificity were evaluated using purified DNA and whole-blood lysates. Diagnostic performance was assessed using a clinical cohort comprising 116 malaria cases and 109 negative controls. Results The extract-free RPA-CRISPR assay achieved an analytical limit of detection of 100 copies/µL from crude blood lysates, corresponding approximately to 12-20 parasites/µL. No cross-reactivity was observed across Plasmodium species. In clinical evaluation, the assay demonstrated strong concordance with reference PCR, achieving 93.1% sensitivity and 100% specificity. Sensitivity varied with parasite burden, reaching 97.8% for high-density infections, 95.1% for moderate density, and 82.8% for low-density infections. The whole workflow was completed within 40 minutes at room temperature without specialized instrumentation. Conclusions An extract-free, one-pot ambient-temperature RPA-CRISPR platform allows rapid detection of Plasmodium directly from whole blood with high specificity and sensitivity across parasite burdens, supporting its potential for decentralized malaria diagnostics. Larger multicenter field studies are needed to validate performance under real-world conditions. Competing Interest Statement The authors have declared no competing interest. Funding Statement No Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The study protocol (IRB 231876) was reviewed and approved by Jiangsu Province Hospital of Chinese Medicine. All procedures were conducted under institutional review board-approved protocol, following the ethical standards. Written informed consent was obtained from all participants prior to sample collection and study enrollment. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Data Availability All data produced in the present study are available upon reasonable request to the authors Copyright The copyright holder for this preprint is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license . 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Share Extract-Free One-Pot Ambient RPA-CRISPR Detection of Plasmodium in Whole Blood Jipei Liao , Yun Su , Feng Jiang medRxiv 2026.03.17.26348654; doi: https://doi.org/10.64898/2026.03.17.26348654 Share This Article: Copy Citation Tools Extract-Free One-Pot Ambient RPA-CRISPR Detection of Plasmodium in Whole Blood Jipei Liao , Yun Su , Feng Jiang medRxiv 2026.03.17.26348654; doi: https://doi.org/10.64898/2026.03.17.26348654 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Infectious Diseases (except HIV/AIDS) Subject Areas All Articles Addiction Medicine (638) Allergy and Immunology (918) Anesthesia (351) Cardiovascular Medicine (5007) Dentistry and Oral Medicine (496) Dermatology (428) Emergency Medicine (676) Endocrinology (including Diabetes Mellitus and Metabolic Disease) (1693) Epidemiology (16267) Forensic Medicine (36) Gastroenterology (1241) Genetic and Genomic Medicine (7280) Geriatric Medicine (751) Health Economics (1093) Health Informatics (5291) Health Policy (1467) Health Systems and Quality Improvement (1832) Hematology (599) HIV/AIDS (1382) Infectious Diseases (except HIV/AIDS) (16492) Intensive Care and Critical Care Medicine (1203) Medical Education (700) Medical Ethics (156) Nephrology (752) Neurology (7598) Nursing (378) Nutrition (1130) Obstetrics and Gynecology (1296) Occupational and Environmental Health (1040) Oncology (3746) Ophthalmology (1093) Orthopedics (409) Otolaryngology (465) Pain Medicine (501) Palliative Medicine (143) Pathology (721) Pediatrics (1858) Pharmacology and Therapeutics (756) Primary Care Research (786) Psychiatry and Clinical Psychology (6133) Public and Global Health (9994) Radiology and Imaging (2532) Rehabilitation Medicine and Physical Therapy (1520) Respiratory Medicine (1270) Rheumatology (661) Sexual and Reproductive Health (804) Sports Medicine (597) Surgery (803) Toxicology (109) Transplantation (319) Urology (308)

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