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. 2025 Apr 4:13:1451125.
doi: 10.3389/fbioe.2025.1451125. eCollection 2025.

Rapid molecular detection of Senecavirus A based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) and CRISPR/Cas12a

Chenghui Jiang #  1   2 , Huibao Wang #  3 , Rongxia Guo #  1 , Rui Yang  2 , Xiaoming Li  2 , Ping Liu  2 , Jing Wang  2 , Jincai Yang  2 , Yanyan Chang  1   2 , Qiaoying Zeng  1
Affiliations

Rapid molecular detection of Senecavirus A based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) and CRISPR/Cas12a

Chenghui Jiang et al. Front Bioeng Biotechnol. .

Abstract

Introduction: Senecavirus A (SVA), an emerging vesicular pathogen, is responsible for porcine idiopathic vesicular disease (PIVD). This disease is closely associated with porcine vesicular disease and acute neonatal piglet mortality, presenting a substantial threat to the global swine industry. At present, the absence of effective drugs or vaccines for treating the disease makes accurate diagnosis of SVA of paramount importance for the effective prevention and control of the disease.

Methods: In this study, we combined reverse transcription loop-mediated isothermal amplification (RT-LAMP) and Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein12a (CRISPR/Cas12a) using a dual-labelled fluorescence quencher or fluorescent biotin single-stranded DNA reporter molecule to develop two rapid, reliable, and portable visual SVA assays: RT-LAMP-Cas12a-FQ and RT-LAMP-Cas12a-FB.

Results: The two methods exhibited comparable detection limits, with 9.6 copies/μL achieved in 40 and 45 minutes, respectively. They did not cross-react with non-target nucleic acids extracted from other related viruses and showed high specificity for SVA RNA detection. Furthermore, the methods demonstrated satisfactory performance in detecting 69 porcine adventitious samples, with no significant differences from that of quantitative reverse transcription polymerase chain reaction (RT-qPCR).

Discussion: In summary, the RT-LAMP-Cas12a-FQ and RT-LAMP-Cas12a-FB methods developed are promising for early detection and routine surveillance of porcine SVA in resource-poor areas.

Keywords: CRISPR/Cas12a; RT-LAMP; Senecavirus A; nucleic acid detection; visualisation.

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Conflict of interest statement

Authors CJ, RY, XL, PL, JW, JY, and YC were employed by China Agricultural Veterinary Biological Science and Technology Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Schematic of RT-LAMP-Cas12a-FQ and RT-LAMP-Cas12a-FB assays for SVA.
FIGURE 2
FIGURE 2
The screening of highly active crRNA by RT-LAMP-Cas12a-FQ method. (A) Histogram of fluorescence intensity values of the CRISPR/Cas12a reaction for crRNAs screening (Fluorescence intensity values were calculated using ImageJ software). (B) Visual fluorescence results of the CRISPR/Cas12a reaction for crRNAs screening. (C) Analysis of conserved sequences of crRNA1 different SVA strains (only 18 strains are shown here). (D) Schematic diagram of crRNA1 target sequence design.
FIGURE 3
FIGURE 3
Optimisation of reaction conditions for the RT-LAMP-Cas12a-FQ method. (A) Histogram of fluorescence intensity values of the CRISPR/Cas12a reaction at varying concentrations of ssDNA FQ reporter (Fluorescence intensity values were calculated using ImageJ software). (B) Visual fluorescence outcomes of the CRISPR/Cas12a reaction at varying ssDNA FQ reporter concentrations. (C) Histogram of fluorescence intensity values of the CRISPR/Cas12a reaction at varying temperatures. (D) Visual fluorescence results of the CRISPR/Cas12a reaction at varying temperatures. (E) Visual fluorescence outcomes of the CRISPR/Cas12a reaction times. (F) Line graphs of the fluorescence intensity values of the CRISPR/Cas12a reaction times.
FIGURE 4
FIGURE 4
Optimisation of ssDNA FB reporter concentration and CRISPR/Cas12a reaction time for the RT-LAMP-Cas12a-FB method. (A) The findings on changes in lateral flow strips at different concentrations of ssDNA FQ reporters. (B) The findings on changes in lateral flow strips at different times of the CRISPR/Cas12a reaction. “C” indicates control line, “T” indicates test line.
FIGURE 5
FIGURE 5
Evaluation of the sensitivity of RT-LAMP-Cas12a to detect SVA. (A) Histogram of fluorescence intensity values of the sensitivity of the RT-LAMP-Cas12a-FQ method (ST-RNA gradient ranging from 9.6 × 108 to 9.6 × 10−1 copies). (B) Visual fluorescence outcomes of the sensitivity of the RT-LAMP-Cas12a-FQ method (ST-RNA gradient ranging from 9.6 × 108 to 9.6 × 10−1 copies). (C) Sensitivity of the RT-LAMP-Cas12a-FB method (ST-RNA gradient ranging from 9.6 × 106 to 9.6 × 10−1 copies).
FIGURE 6
FIGURE 6
Evaluation of the specificity of RT-LAMP-Cas12a to detect SVA. (A) Histogram of fluorescence intensity values of RT-LAMP-Cas12a-FQ method to SVA detection specificity. (B) Visual fluorescence outcomes of RT-LAMP-Cas12a-FQ method to SVA detection specificity. (C) Specificity of RT-LAMP-Cas12a-FB method to detect SVA.
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