基于RT-RAA的禽流感H5亚型核酸CRISPR-Cas13a检测方法的建立

doi:10.11843/j.issn.0366-6964.2023.09.020

Abstract

Abstract: A new rapid, highly sensitive and highly specific nucleic acid detection method for avian influenza virus(AIV) H5 subtype was established by using reverse transcription-recombinase aided amplification(RT-RAA) and clustered regularly interspaced short palindromic repeats(CRISPR). By analyzing the HA gene sequences of 342 avian influenza virus H5 subtype genes, the conserved regions were selected to design highly specific CRISPR RNA(crRNA) sequences and RT-RAA primers. The contents of main components in the detection system were optimized, including LwaCas13a protein, crRNA, TaqMan probe, T7 RNA Polymerase and NTP Buffer Mix.Thus, the RT-RAA based detection method of AIV H5 subtype nucleic acid CRISPR-Cas13a was established.The sensitivity and specificity of the method were evaluated by taking 10⁵~1 copies·μL^-1 of standard plasmid containing the target gene and other subtypes of avian influenza viruses and other avian disease viruses. The results showed that the RT-RAA primer and crRNA designed in this paper were specific and sensitive. The main components in the optimal reaction system were LwaCas13a protein (60 μg·mL^-1) 2.0 μL, crRNA (100 μmol·L^-1) 3.2 μL, TaqMan probe (50 μmol·L^-1) 1.28 μL, T7 RNA Polymerase 0.25 μL and NTP Buffer Mix 2.0 μL, respectively. The detection sensitivity of this method was up to 1 copy·μL^-1, and there was no cross reaction with AIV H3, H6, H7, H9, H10 subtypes, neither with NDV, IBV, IBDV or DTMUV. The method established in this study and fluorescence quantitative RT-PCR were used to detect 56 clinical samples, and the coincidence rate of the two methods was 98.2%. In conclusion, a rapid detection method for AIV H5 subtype was established by using RT-RAA isothermal amplification technology combined with CRISPR-Cas13a gene editing technology in this study. The results were obtained within 1 h and 20 min. It provides a new technique for the rapid clinical detection of AIV H5 subtype with high sensitivity and high specificity, and has a good application prospect.

Key words: avian influenza virus H5 subtype, CRISPR, isothermal amplification, nucleic acid testing

CLC Number:

S852.65

YANG Zhiyi, WANG Xinkai, SHI Yuting, FU Siyuan, ZHANG Yuxin, CAO Chenfu, JIA Weixin. Establishment of Nucleic Acid Detection Methods for Avian Influenza H5 Subtype Based on CRISPR-Cas13a and RT-RAA[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(9): 3803-3811.

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Establishment of Nucleic Acid Detection Methods for Avian Influenza H5 Subtype Based on CRISPR-Cas13a and RT-RAA

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