Establishment and Implementation of the Point-of-Care RT-RAA-CRISPR/Cas13a Diagnostic Test for Foot-And-Mouth Disease Virus Serotype O in Pigs
Foot and mouth disease virus (FMDV) is a highly pathogenic virus that mainly infects cloven hooved animals, such as pigs. The establishment of a rapid, sensitive and accurate point-of-care detection method is critical for the timely identification and elimination of infected pigs for controlling thi...
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2025-05-01
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| author | Ping Meng Bo Ni Chenyu Li Zhou Sha Chunju Liu Weijie Ren Rong Wei Fuxiao Liu Jinming Li Zhiliang Wang |
| author_facet | Ping Meng Bo Ni Chenyu Li Zhou Sha Chunju Liu Weijie Ren Rong Wei Fuxiao Liu Jinming Li Zhiliang Wang |
| author_sort | Ping Meng |
| collection | DOAJ |
| description | Foot and mouth disease virus (FMDV) is a highly pathogenic virus that mainly infects cloven hooved animals, such as pigs. The establishment of a rapid, sensitive and accurate point-of-care detection method is critical for the timely identification and elimination of infected pigs for controlling this disease. In this study, a RT-RAA-CRISPR/Cas13a method was developed for the detection of FMDV serotype O in pigs. Six pairs of RT-RAA primers were designed based on the conserved gene sequence of FMDV serotype O, and the optimal amplification primers and reaction temperatures were screened. The CRISPR-derived RNA (crRNA) was further designed based on the optimal target band sequence and the most efficient crRNA was screened. The results revealed that FMDV-O-F4/R4 was the optimal primer set, and the optimal temperature for the RT-RAA reaction was 37 °C. Moreover, crRNA4 exhibited the strongest detection signal among the six crRNAs. The established RT-RAA-CRISPR/Cas13a method demonstrated high specificity and no cross-reactivity with other common swine pathogens such as Senecavirus A (SVA), porcine reproductive and respiratory virus (PRRSV), porcine epidemic diarrhea virus (PEDV), porcine circovirus type 2 (PCV2), classical swine fever virus (CSFV), and pseudorabies virus (PRV), additionally, it was observed to be highly sensitive, with a detection limit of 19.1 copies/µL. The repeatability of this method was also observed to be good. This method could produce stable fluorescence and exhibited good repeatability when three independent experiments yielded the same results. A validation test using three types of simulated clinical samples (including swab, tissue, and serum samples) revealed a 100% concordance rate. The detection results could be visualized via a fluorescence reader or lateral flow strips (LFSs). Thus, a highly specific and sensitive RT-RAA-CRISPR/Cas13a detection method was developed and is expected to be applied for the rapid detection of FMDV serotype O in situ. |
| format | Article |
| id | doaj-art-6422c9cb72554d66b2d43fb5253b40b2 |
| institution | OA Journals |
| issn | 1999-4915 |
| language | English |
| publishDate | 2025-05-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Viruses |
| spelling | doaj-art-6422c9cb72554d66b2d43fb5253b40b22025-08-20T02:33:59ZengMDPI AGViruses1999-49152025-05-0117572110.3390/v17050721Establishment and Implementation of the Point-of-Care RT-RAA-CRISPR/Cas13a Diagnostic Test for Foot-And-Mouth Disease Virus Serotype O in PigsPing Meng0Bo Ni1Chenyu Li2Zhou Sha3Chunju Liu4Weijie Ren5Rong Wei6Fuxiao Liu7Jinming Li8Zhiliang Wang9China Animal Health and Epidemiology Center, Qingdao 266011, ChinaChina Animal Health and Epidemiology Center, Qingdao 266011, ChinaCollege of Veterinary Medicine, Shanxi Agricultural University, Jinzhong 030031, ChinaChina Animal Health and Epidemiology Center, Qingdao 266011, ChinaChina Animal Health and Epidemiology Center, Qingdao 266011, ChinaChina Animal Health and Epidemiology Center, Qingdao 266011, ChinaChina Animal Health and Epidemiology Center, Qingdao 266011, ChinaCollege of Veterinary Medicine, Qingdao Agricultural University, Qingdao 266109, ChinaChina Animal Health and Epidemiology Center, Qingdao 266011, ChinaChina Animal Health and Epidemiology Center, Qingdao 266011, ChinaFoot and mouth disease virus (FMDV) is a highly pathogenic virus that mainly infects cloven hooved animals, such as pigs. The establishment of a rapid, sensitive and accurate point-of-care detection method is critical for the timely identification and elimination of infected pigs for controlling this disease. In this study, a RT-RAA-CRISPR/Cas13a method was developed for the detection of FMDV serotype O in pigs. Six pairs of RT-RAA primers were designed based on the conserved gene sequence of FMDV serotype O, and the optimal amplification primers and reaction temperatures were screened. The CRISPR-derived RNA (crRNA) was further designed based on the optimal target band sequence and the most efficient crRNA was screened. The results revealed that FMDV-O-F4/R4 was the optimal primer set, and the optimal temperature for the RT-RAA reaction was 37 °C. Moreover, crRNA4 exhibited the strongest detection signal among the six crRNAs. The established RT-RAA-CRISPR/Cas13a method demonstrated high specificity and no cross-reactivity with other common swine pathogens such as Senecavirus A (SVA), porcine reproductive and respiratory virus (PRRSV), porcine epidemic diarrhea virus (PEDV), porcine circovirus type 2 (PCV2), classical swine fever virus (CSFV), and pseudorabies virus (PRV), additionally, it was observed to be highly sensitive, with a detection limit of 19.1 copies/µL. The repeatability of this method was also observed to be good. This method could produce stable fluorescence and exhibited good repeatability when three independent experiments yielded the same results. A validation test using three types of simulated clinical samples (including swab, tissue, and serum samples) revealed a 100% concordance rate. The detection results could be visualized via a fluorescence reader or lateral flow strips (LFSs). Thus, a highly specific and sensitive RT-RAA-CRISPR/Cas13a detection method was developed and is expected to be applied for the rapid detection of FMDV serotype O in situ.https://www.mdpi.com/1999-4915/17/5/721point-of-care diagnostic testRT-RAACRISPR/Cas13aFMDV serotype O |
| spellingShingle | Ping Meng Bo Ni Chenyu Li Zhou Sha Chunju Liu Weijie Ren Rong Wei Fuxiao Liu Jinming Li Zhiliang Wang Establishment and Implementation of the Point-of-Care RT-RAA-CRISPR/Cas13a Diagnostic Test for Foot-And-Mouth Disease Virus Serotype O in Pigs Viruses point-of-care diagnostic test RT-RAA CRISPR/Cas13a FMDV serotype O |
| title | Establishment and Implementation of the Point-of-Care RT-RAA-CRISPR/Cas13a Diagnostic Test for Foot-And-Mouth Disease Virus Serotype O in Pigs |
| title_full | Establishment and Implementation of the Point-of-Care RT-RAA-CRISPR/Cas13a Diagnostic Test for Foot-And-Mouth Disease Virus Serotype O in Pigs |
| title_fullStr | Establishment and Implementation of the Point-of-Care RT-RAA-CRISPR/Cas13a Diagnostic Test for Foot-And-Mouth Disease Virus Serotype O in Pigs |
| title_full_unstemmed | Establishment and Implementation of the Point-of-Care RT-RAA-CRISPR/Cas13a Diagnostic Test for Foot-And-Mouth Disease Virus Serotype O in Pigs |
| title_short | Establishment and Implementation of the Point-of-Care RT-RAA-CRISPR/Cas13a Diagnostic Test for Foot-And-Mouth Disease Virus Serotype O in Pigs |
| title_sort | establishment and implementation of the point of care rt raa crispr cas13a diagnostic test for foot and mouth disease virus serotype o in pigs |
| topic | point-of-care diagnostic test RT-RAA CRISPR/Cas13a FMDV serotype O |
| url | https://www.mdpi.com/1999-4915/17/5/721 |
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