Establishment Of Rapid Detection Method For Porcine Infectious Gastroenteritis Virus Based On CRISPR-Cas12a Technology

Transmissible gastroenteritis of pigs(TGE)is an acute,highly contagious intestinal infection caused by Transmissible Gastroenteritis virus of swine(TGEV).The high rate of infection and mortality of TGEV has caused significant losses to the development of the pig industry.A sensitive,rapid and accurate differential diagnosis method can achieve a better prevention of early TGEV transmission.In recent years,the CRISP system and related proteins have gained the attention of many researchers because of their high specificity,sensitivity and ease of field detection.In this study,we established the CRISPR-Cas12 a fluorescence method and CRISPR-Cas12 a test strip method based on LAMP amplification for clinical detection of TGEV,respectively,which are more portable and rapid compared with the traditional molecular diagnostic techniques for TGEV,and have important implications for the differential diagnosis and clinical prevention of TGEV.In this study,primer design and screening of the N gene of TGEV-pCI-neo recombinant plasmid kept in laboratory,reaction conditions,optimization of reaction system,specificity test and sensitivity test were performed to establish LAM-TGEV assay,CRISPR-Cas12 a fluorescence method and CRISPR-Cas12 a test strip method,respectively.The PCR technique and q PCR technique were also used to do parallel tests for comparative analysis.The specific experimental methods and results are as follows:1、Establishment and optimization of TGEV-LAMP assayTwo sets of six primer pairs were designed to screen the amplification effect of TGEV conserved gene N gene as the target gene of LAMP assay.The optimal reaction time was50 min,the optimal reaction temperature was 63 ℃,and the optimal internal and external primer concentration ratio was 2:1,TGEV has no cross-reactivity with PEDV,PCV2,PCV3,PCLV,PPV and PPRS and has significant specificity.2.Establishment and optimization of TGEV-CRISPR-Cas12 a fluorescence assayThe conserved gene N gene of TGEV was selected as the target gene for detection,and three pairs of crRNA primers and one fluorescent probe were designed respectively.The recombinant plasmid TGEV-pCI-neo was used as the template,and the pair of primers with better effect was screened.Then the reaction system was optimized with LAMP amplification product as the detection template respectively,and the optimal reaction time of 25 min and temperature of 39 ℃ were screened;the minimum detection limit of sensitivity test was 1 copies/μL;the results of specificity test showed no cross-reactivity and significant specificity with PEDV,PCV2,PCV3,PCLV,PPV and PPRS.3、Establishment and optimization of TGEV-CRISPR-Cas12 a test strip methodA new technique for presenting results based on CRISPR-Cas12a-LAMP amplification combined with flowmetric chromatography strip technology.Using N gene as the target and LAMP amplification product as the template,the optimum probe concentration of 25 n M/μL,optimum reaction time of X min and optimum reaction temperature of X℃ were selected,The specificity test showed no cross-reactivity and significant specificity with PEDV,PCV2,PCV3,PCLV,PPV and PPRS.In this study,we optimized and established the LAMP assay for TGEV and based on this,we successfully established the CRISPR-Cas12a-LAMP fluorescence assay and test strip assay in combination with CRISPR system.The assay is portable,rapid and sensitive,and provides a new technical means for the detection of TGEV at the grassroots level.