CRISPR/Cas12a Technology Combined With RPA for Rapid and Portable SFTSV Detection

Abstract

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a new tick-borne pathogen that can cause severe hemorrhagic fever. Fever with thrombocytopenia syndrome caused by SFTSV is a new infectious disease that has posed a great threat to public health. Therefore, a fast, sensitive, low-cost, and field-deployable detection method for diagnosing SFTSV is essential for virus surveillance and control. In this study, we developed a rapid, highly sensitive, instrument-flexible SFTSV detection method that utilizes recombinase polymerase amplification and the CRISPR/Cas12a system. We found that three copies of the L gene from the SFTSV genome per reaction were enough to ensure stable detection within 40 min. The assay clearly showed no cross-reactivity with other RNA viruses. Additionally, our method demonstrated 100% agreement with Q-PCR detection results for SFTSV in 46 clinical samples. We simplified the requirements for on-site detection instruments by combining the CRISPR/Cas12a tool and immunochromatographic strips to create a system that can reliably detect one copy/μl sample of the L gene, which showed extremely high sensitivity and specificity for detecting the virus. Taken together, these findings indicate that the new SFTSV detection method is a powerful and effective tool for on-site detection, which can contribute to diagnosing SFTSV quickly and sensitively.

Dual-gene detection in a single-tube system based on CRISPR-Cas12a/Cas13a for severe fever thrombocytopenia syndrome virus

“…Unlike the conventional nucleic acid detection methods, our CRISPR/Cas-based methods for SFTSV involve a substantially shorter operation time and avoid the need for thermal circulation apparatus. The whole detection process was easily conducted at 37°C within 1 h. Until now, the Cas12a-based detection method for SFTSV has been reported and similar work was carried out in this study ( Huang et al, 2022 ). Moreover, we first proposed the detection method based on CRISPR/Cas13a for SFTSV, and the lowest detection limit was 0.75 cp/μl.…”

NLR, A Convenient Early-Warning Biomarker of Fatal Outcome in Patients With Severe Fever With Thrombocytopenia Syndrome

“…The results showed that SFTSV viral load and NLR had higher AUC, sensitivity, and specificity, which indicated that SFTSV viral load and NLR were better than the other three indicators in predicting the incidence of fatal outcomes. The importance of the detection of SFTSV viral load had been repeatedly mentioned in previous studies (Huang et al, 2022 ). It is worth mentioning that only 143 patients among the enrolled 228 patients with SFTS had accepted SFTS viral load test in this study.…”

Accuracy of reverse‐transcription polymerase chain reaction and loop‐mediated isothermal amplification in diagnosing severe fever with thrombocytopenia syndrome: A systematic review and meta‐analysis

“…After reading the full text of the articles, 103 were discarded, including 48 that did not use clinical patient samples or lacked data, 35 that were not on nucleic acid‐related diagnostic techniques, 14 that lacked reference standards, and 6 that were not on or related to SFTS. A final total of 16 articles were included, seven on RT‐PCR 19 , 20 , 24 , 25 , 26 , 27 , 28 and seven on RT‐LAMP, 16 , 21 , 29 , 30 , 31 , 32 , 33 and two that were on both RT‐PCR and RT‐LAMP. 15 , 34 A flowchart of the research process is shown in Figure 1 .…”