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Development and evaluation of a CRISPR-Cas13a system-based diagnostic for hepatitis E virus

  • Manyu Li EMAIL logo , Qiyu He , Tingting Li , Wenjun Wan and Haiwei Zhou EMAIL logo
Published/Copyright: December 29, 2023
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Clinical Chemistry and Laboratory Medicine (CCLM)
From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 62 Issue 6

Abstract

Objectives

Hepatitis E virus (HEV) is the leading cause of acute viral hepatitis worldwide. HEV RNA detection is the gold standard for HEV infection diagnosis and PCR methods are commonly used but are usually time-consuming and expensive, resulting in low detection efficiency and coverage, especially in low-income areas. Here, we developed a simpler and more accessible HEV RNA detection method based on CRISPR-Cas13a system.

Methods

A total of 265 samples of different types and sources, including 89 positive samples and 176 negative samples, were enrolled for evaluations. The sensitivity and specificity of the Cas13a-crRNA detection system were evaluated. The World Health Organization reference panel for HEV genotypes was used to evaluate the capability for detecting different HEV genotypes. The validity of the assay was compared with RT-qPCR.

Results

The 95 % limits of detection (LOD) of Cas13a-crRNA-based fluorescence assay and strip assay were 12.5 and 200 IU/mL, respectively. They did not show cross-reactivity with samples positive for hepatitis A virus, hepatitis B virus, hepatitis C virus, coxsackievirus A16, rotavirus, enterovirus 71, norovirus or enteropathic Escherichia coli. Different HEV genotypes (HEV1–4) can be detected by the assay. Compared to RT-qPCR, the positive predictive agreements of Cas13a-crRNA-based fluorescence and strip assay were 98.9 % (95 % CI: 93.9–99.8 %) and 91.0 % (95 % CI: 83.3–95.4 %), respectively. The negative predictive agreements were both 100 % (95 % CI: 97.8–100 %).

Conclusions

In conclusion, we established a rapid and convenient HEV RNA detection method with good sensitivity and specificity based on CRISPR-Cas13a system, providing a new option for HEV infection diagnosis.

Keywords: hepatitis E virus; CRISPR-Cas13a; nucleic acid detection; recombinase polymerase amplification; diagnosis
Corresponding authors: Manyu Li, PhD, Assistant Research Fellow and Haiwei Zhou, Professor, Division I of In Vitro Diagnostics for Infectious Diseases, Institute for In Vitro Diagnostics Control, National Institutes for Food and Drug Control, 2 Tiantanxili Rd, Dongcheng District, Beijing 100050, P.R. China, Phone: +86 10 67095614, +86 10 67095485, E-mail: (M. Li), (H. Zhou)
Manyu Li and Qiyu He contributed equally to this work and share first authorship.

  1. Research ethics: Research involving human subjects complied with all relevant national regulations, institutional policies, and is in accordance with the tenets of the Helsinki Declaration (as revised in 2013). This study was approved by the Ethics Committee of Zhengzhou University Fifth Affiliated University (KY2021031).

  2. Informed consent: Informed consent was obtained by Zhengzhou University Fifth Affiliated University from all individuals included in this study.

  3. Author contributions: ML: conceptualization, methodology, formal analysis, data curation, investigations, visualization, funding, writing – original draft preparation; QH: conceptualization, methodology, formal analysis, data curation, investigations, writing – reviewing and editing; TL: investigations, data curation; WW: formal analysis, writing – reviewing and editing; HZ: conceptualization, methodology, supervision, writing – reviewing and editing. All authors have accepted responsibility for the entire content of this manuscript and approved its submission.

  4. Competing interests: The authors state no conflict of interest.

  5. Research funding: This study was supported by National Natural Science Foundation of China, grant number 82202504; the China Postdoctoral Innovation Talent Support Program, grant number 88032Y0008; the National Institutes for Food and Drug Control, grant number 2022C4; the China Postdoctoral Science Foundation, grant number 88014Y0236.

  6. Data availability: The raw data can be obtained on request from the corresponding author.

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Supplementary Material

This article contains supplementary material (https://doi.org/10.1515/cclm-2023-1007).

Received: 2023-09-11
Accepted: 2023-12-14
Published Online: 2023-12-29
Published in Print: 2024-05-27

© 2023 Walter de Gruyter GmbH, Berlin/Boston

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https://doi.org/10.1515/cclm-2023-1007
Keywords for this article
hepatitis E virus; CRISPR-Cas13a; nucleic acid detection; recombinase polymerase amplification; diagnosis

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  1. Frontmatter
  2. Editorial
  3. SARS-CoV-2 is here to stay: do not lower our guard
  4. Reviews
  5. SARS-CoV-2 subgenomic RNA: formation process and rapid molecular diagnostic methods
  6. Prognostic value of anti-SARS-CoV-2 antibodies: a systematic review
  7. Presence of SARS-CoV-2 RNA in COVID-19 survivors with post-COVID symptoms: a systematic review of the literature
  8. Opinion Papers
  9. Harmonizing the post-analytical phase: focus on the laboratory report
  10. Blood-based biomarkers in Alzheimer’s disease – moving towards a new era of diagnostics
  11. A comprehensive review on PFAS including survey results from the EFLM Member Societies
  12. General Clinical Chemistry and Laboratory Medicine
  13. Report from the HarmoSter study: different LC-MS/MS androstenedione, DHEAS and testosterone methods compare well; however, unifying calibration is a double-edged sword
  14. An LC–MS/MS method for serum cystatin C quantification and its comparison with two commercial immunoassays
  15. CX3CL1/Fractalkine as a biomarker for early pregnancy prediction of preterm premature rupture of membranes
  16. Elevated S100B urine levels predict seizures in infants complicated by perinatal asphyxia and undergoing therapeutic hypothermia
  17. The correlation of urea and creatinine concentrations in sweat and saliva with plasma during hemodialysis: an observational cohort study
  18. Tubular phosphate transport: a comparison between different methods of urine sample collection in FGF23-dependent hypophosphatemic syndromes
  19. Reference Values and Biological Variations
  20. Monocyte distribution width (MDW): study of reference values in blood donors
  21. Data mining of reference intervals for serum creatinine: an improvement in glomerular filtration rate estimating equations based on Q-values
  22. Hematology and Coagulation
  23. MALDI-MS in first-line screening of newborns for sickle cell disease: results from a prospective study in comparison to HPLC
  24. Cardiovascular Diseases
  25. To rule-in, or not to falsely rule-out, that is the question: evaluation of hs-cTnT EQA performance in light of the ESC-2020 guideline
  26. Temporal biomarker concentration patterns during the early course of acute coronary syndrome
  27. Diabetes
  28. Proteomic analysis of diabetic retinopathy identifies potential plasma-protein biomarkers for diagnosis and prognosis
  29. Infectious Diseases
  30. Serum biomarkers of inflammation and vascular damage upon SARS-Cov-2 mRNA vaccine in patients with thymic epithelial tumors
  31. A high throughput immuno-affinity mass spectrometry method for detection and quantitation of SARS-CoV-2 nucleoprotein in human saliva and its comparison with RT-PCR, RT-LAMP, and lateral flow rapid antigen test
  32. Evaluation of inflammatory biomarkers and vitamins in hospitalized patients with SARS-CoV-2 infection and post-COVID syndrome
  33. The CoLab score is associated with SARS-CoV-2 viral load during admission in individuals admitted to the intensive care unit: the CoLaIC cohort study
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  35. Letters to the Editor
  36. Crioplast® is a reliable device to ensure pre-analytical stability of adrenocorticotrophin (ACTH)
  37. Falsely decreased Abbott Alinity-c gamma-glutamyl transferase-2 result from paraprotein and heparin interference: case report and subsequent laboratory experiments
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  41. Acute myeloid leukemia with blue-green neutrophilic inclusions have different outcomes: two cases and review of the literature
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  43. The 10+1 Santorini Conference
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  45. 15th National Congress of the Portuguese Society of Clinical Chemistry, Genetics and Laboratory Medicine
  46. ISMD2024 Thirteenth International Symposium on Molecular Diagnostics
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