Establishment of a One–Pot RAA–CRISPR/Cas13a Assay-Based TGEV S Gene Detection
Porcine transmissible gastroenteritis virus (TGEV) is a highly contagious pathogen causing severe diarrhea in pigs, particularly piglets, leading to significant economic losses. Distinguishing TGEV from the genetically similar porcine respiratory coronavirus (PRCV) remains challenging due to their h...
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| Format: | Article |
| Language: | English |
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MDPI AG
2025-05-01
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| Series: | Veterinary Sciences |
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| Online Access: | https://www.mdpi.com/2306-7381/12/5/464 |
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| author | Lindan Lv Hao Mu Shaomei Li Jieqi Gao Mingni Liu Shuizhu Niu Guoyang Xu Lizhi Fu Zhenhui Song Liu Yang |
| author_facet | Lindan Lv Hao Mu Shaomei Li Jieqi Gao Mingni Liu Shuizhu Niu Guoyang Xu Lizhi Fu Zhenhui Song Liu Yang |
| author_sort | Lindan Lv |
| collection | DOAJ |
| description | Porcine transmissible gastroenteritis virus (TGEV) is a highly contagious pathogen causing severe diarrhea in pigs, particularly piglets, leading to significant economic losses. Distinguishing TGEV from the genetically similar porcine respiratory coronavirus (PRCV) remains challenging due to their high genomic homology. In this study, we developed a one–pot assay combining recombinase-aided amplification (RAA) and CRISPR/Cas13a technology, targeting the TGEV S gene. This method was optimized for sensitivity and specificity, with orthogonal tests determining the optimal reagent concentrations. The assay achieved a detection limit of 4.13 copies/µL within 40 min at 37 °C, demonstrating no cross-reactivity with other porcine viruses. Clinical validation on 140 samples showed 100% concordance with RT–qPCR and RT–PCR results. Since the established method is completed in a single reaction tube, it eliminates the need for step-by-step operations, simplifying the process and reducing the risk of cross–contamination and false positives in subsequent tests. Overall, this assay shows promising potential for TGEV detection. |
| format | Article |
| id | doaj-art-5d516d27127f4bdd8b294ff6ddf3199c |
| institution | OA Journals |
| issn | 2306-7381 |
| language | English |
| publishDate | 2025-05-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Veterinary Sciences |
| spelling | doaj-art-5d516d27127f4bdd8b294ff6ddf3199c2025-08-20T02:33:51ZengMDPI AGVeterinary Sciences2306-73812025-05-0112546410.3390/vetsci12050464Establishment of a One–Pot RAA–CRISPR/Cas13a Assay-Based TGEV S Gene DetectionLindan Lv0Hao Mu1Shaomei Li2Jieqi Gao3Mingni Liu4Shuizhu Niu5Guoyang Xu6Lizhi Fu7Zhenhui Song8Liu Yang9National Center of Technology Innovation for Pigs, Chongqing 402460, ChinaNational Center of Technology Innovation for Pigs, Chongqing 402460, ChinaNational Center of Technology Innovation for Pigs, Chongqing 402460, ChinaNational Center of Technology Innovation for Pigs, Chongqing 402460, ChinaNational Center of Technology Innovation for Pigs, Chongqing 402460, ChinaNational Center of Technology Innovation for Pigs, Chongqing 402460, ChinaNational Center of Technology Innovation for Pigs, Chongqing 402460, ChinaNational Center of Technology Innovation for Pigs, Chongqing 402460, ChinaCollege of Veterinary Medicine, Southwest University, Chongqing 400715, ChinaNational Center of Technology Innovation for Pigs, Chongqing 402460, ChinaPorcine transmissible gastroenteritis virus (TGEV) is a highly contagious pathogen causing severe diarrhea in pigs, particularly piglets, leading to significant economic losses. Distinguishing TGEV from the genetically similar porcine respiratory coronavirus (PRCV) remains challenging due to their high genomic homology. In this study, we developed a one–pot assay combining recombinase-aided amplification (RAA) and CRISPR/Cas13a technology, targeting the TGEV S gene. This method was optimized for sensitivity and specificity, with orthogonal tests determining the optimal reagent concentrations. The assay achieved a detection limit of 4.13 copies/µL within 40 min at 37 °C, demonstrating no cross-reactivity with other porcine viruses. Clinical validation on 140 samples showed 100% concordance with RT–qPCR and RT–PCR results. Since the established method is completed in a single reaction tube, it eliminates the need for step-by-step operations, simplifying the process and reducing the risk of cross–contamination and false positives in subsequent tests. Overall, this assay shows promising potential for TGEV detection.https://www.mdpi.com/2306-7381/12/5/464TGEVRAACRISPR/Cas13adetection method |
| spellingShingle | Lindan Lv Hao Mu Shaomei Li Jieqi Gao Mingni Liu Shuizhu Niu Guoyang Xu Lizhi Fu Zhenhui Song Liu Yang Establishment of a One–Pot RAA–CRISPR/Cas13a Assay-Based TGEV S Gene Detection Veterinary Sciences TGEV RAA CRISPR/Cas13a detection method |
| title | Establishment of a One–Pot RAA–CRISPR/Cas13a Assay-Based TGEV S Gene Detection |
| title_full | Establishment of a One–Pot RAA–CRISPR/Cas13a Assay-Based TGEV S Gene Detection |
| title_fullStr | Establishment of a One–Pot RAA–CRISPR/Cas13a Assay-Based TGEV S Gene Detection |
| title_full_unstemmed | Establishment of a One–Pot RAA–CRISPR/Cas13a Assay-Based TGEV S Gene Detection |
| title_short | Establishment of a One–Pot RAA–CRISPR/Cas13a Assay-Based TGEV S Gene Detection |
| title_sort | establishment of a one pot raa crispr cas13a assay based tgev s gene detection |
| topic | TGEV RAA CRISPR/Cas13a detection method |
| url | https://www.mdpi.com/2306-7381/12/5/464 |
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