猪流行性腹泻病毒RAA-CRISPR/Cas13a检测方法的建立与初步应用

doi:10.11843/j.issn.0366-6964.2023.09.037

Abstract

Abstract: The aim of this paper is to establish a rapid, sensitive, and specific detection method for porcine epidemic diarrhea virus (PEDV) by recombinase aided amplification (RAA) combined with the Clustered Regularly Interspaced Short Palindromic Repeats associated protein 13a (CRISPR-Cas13a), called RAA-Cas13a. The specific primer for RAA and CRISPR RNA (crRNA) of the conserved region of the PEDV N gene were designed. The sample nucleic acids were amplified by RAA, whose amplification products were then detected with CRISPR-Cas13a and use RT-qPCR as a control method to evaluate the sensitivity, specificity, and consistency of this method. The results showed that the method could detect as low as 10¹ copies·μL^-1 and had no cross-reactivity with pig-derived pathogens such as porcine circovirus type 1, porcine circovirus type 2, porcine circovirus type 3, porcine reproductive and respiratory syndrome virus, swine fever virus and pseudorabies virus. The positive coincidence rate of 40 clinical samples detected by RAA-Cas13a and RT-qPCR method was 100%, the negative coincidence rate was 84.6%, the total coincidence rate was 95%, and the Kappa value was 0.881. The established RAA-Cas13a detection method has high sensitivity and strong specificity, providing a reliable technical means for the clinical diagnosis and epidemiological monitoring of PEDV.

Key words: porcine epidemic diarrhea virus, CRISPR/Cas13a, recombinase aided amplification, N gene, detection

CLC Number:

S852.659.6

LIU Hua, YIN Dongdong, SHAO Ying, SONG Xiangjun, WANG Zhenyu, PAN Xiaocheng, TU Jian, HE Changsheng, ZHU Liangqiang, QI Kezong. Detection of Porcine Epidemic Diarrhea Virus by Recombinase Aided Amplification Combined with CRISPR/Cas13a[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(9): 3991-3997.

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Detection of Porcine Epidemic Diarrhea Virus by Recombinase Aided Amplification Combined with CRISPR/Cas13a

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