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. 2025 Apr 2;21(1):234.
doi: 10.1186/s12917-025-04711-1.

Rapid and visual detection of transmissible gastroenteritis virus using a CRISPR/Cas12a system combined with loop-mediated isothermal amplification

Haiyang Wang #  1   2 , Zhao Qi #  1   2 , Jiale Wang  1   2 , Zhenjie He  1   2 , Liting Lu  1   2 , Zhe Chen  1   2 , Ying Shao  1   2 , Guijun Wang  1   2 , Zhenyu Wang  1   2 , Jian Tu  1   2 , Xiangjun Song  3   4   5
Affiliations

Rapid and visual detection of transmissible gastroenteritis virus using a CRISPR/Cas12a system combined with loop-mediated isothermal amplification

Haiyang Wang et al. BMC Vet Res. .

Abstract

Background: Transmissible gastroenteritis (TGE) is a highly contagious intestinal disease caused by transmissible gastroenteritis virus (TGEV). The primary techniques for identifying TGEV involve enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and fluorescent quantitative PCR (qPCR). However, these approaches are complex, demanding specialized tools and significant time. Therefore, a precise, swift, and effective differential diagnosis method is crucial for TGEV prevention. In recent years, clustered regularly interspaced short palindromic repeats (CRISPR) and Cas-associated proteins have become popular for their high specificity, unique cleavage activity, and ease of detection. CRISPR-Cas12a, a novel RNA-guided nucleic acid endonuclease, is emerging as a powerful molecular scissor.

Results: In this study, we designed three pairs of crRNA targeting the N gene of TGEV. Following the selection of the most appropriate crRNA, we established the loop-mediated isothermal (LAMP) amplification method with a sensitivity of 102 copies/µL. And based on this, we established the CRISPR-Cas12a fluorescence assay with a sensitivity of 100 copies/µL. Furthermore, we established a CRISPR/Cas12a lateral-flow dipstick assay with a sensitivity of 102 copies/µL. Importantly, none of these methods exhibited cross-reactivity with other related viruses, enabling quicker and more straightforward observation of experimental results.

Conclusions: We have successfully developed a CRISPR-Cas12a fluorescence assay and a CRISPR/Cas12a lateral-flow dipstick assay for clinical TGEV detection. Overall, we created a portable, quick, and sensitive TGEV assay with strong specificity utilizing the CRISPR-Cas12a system.

Keywords: CRISPR-Cas12a; Detection; LAMP; TGEV.

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Conflict of interest statement

Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Results of crRNA screening. (A) Visual inspection under UV light. (B) The values were read by fluorescent quantitative PCR and then analyzed numerically by GraphPad Prism 8
Fig. 2
Fig. 2
LAMP primer screening. (A) Marker: Marker; from TGEV-J1 to TGEV-J3 are LAMP primer set I; from TGEV-J1-NC to TGEV-J3-NC are negative control with ddH2O (B) Marker: Marker; from TGEV-W1 to TGEV-W3 are LAMP primer set II; from TGEV-W1-NC to TGEV-W3-NC are negative control with ddH2O
Fig. 3
Fig. 3
TGEV LAMP sensitivity test results. M: Marker; 106 to 100 indicate tests with varying concentrations of pCI-neo-N recombinant plasmid; NC: negative control with ddH2O
Fig. 4
Fig. 4
Optimization of reaction time and temperature in the CRISPR-Cas12a fluorescence assay. (A) From left to right, the reaction times were 40 min, 35 min, 30 min, 25 min, 20 min, 15 min, and 10 min; NC: negative control of RNase-free ddH2O. (B) From left to right, the reaction temperatures were 43 ℃, 41 ℃, 39 ℃, 37 ℃, 35 ℃, 33 ℃, and 31 ℃; NC: negative control of RNase-free ddH2O
Fig. 5
Fig. 5
Sensitivity of the CRISPR-Cas12a fluorescence assay. (A) Sensitivity assessment using various concentrations of pCI-neo-N. (B) End-point fluorescence intensity to determine sensitivity
Fig. 6
Fig. 6
CRISPR-Cas12a fluorescence assay specificity test results. (A) From 1 to 8 are the experimental groups of TGEV, PCV2, PCV3, PCLV, PRRSV, PPV and PEDV respectively. (B) Determine the endpoint fluorescence intensity for specificity
Fig. 7
Fig. 7
CRISPR-Cas12a lateral-flow dipstick probe concentration optimization results. 500 nM to 30 nM indicate tests with varying concentrations of the probe
Fig. 8
Fig. 8
CRISPR-Cas12a lateral-flow dipstick sensitivity test results. 106 to 100 indicate tests with varying concentrations of pCI-neo-N recombinant plasmid as a template; NC: negative control of RNase-free ddH2O
Fig. 9
Fig. 9
CRISPR-Cas12a lateral-flow dipstick specificity test results. From left to right, the experimental groups were TGEV, PCV2, PCV3, PCLV, PRRSV, PPV and PEDV; NC: negative control of RNase-free ddH2O
Fig. 10
Fig. 10
Test results of clinical samples for the CRISPR-Cas12a fluorescence assay. (A) Results of the CRISPR-Cas12a fluorescence assay. (B) End-point fluorescence. From 1 to 13 are 13 clinical samples. NC: negative control of RNase-free ddH2O
Fig. 11
Fig. 11
Results of clinical sample testing by CRISPR-Cas12a lateral-flow dipstick assay and qPCR. (A) Results of the CRISPR/Cas12a lateral-flow dipstick assay. (B) Results of the q-PCR
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