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.

References

[1] DADONAITE B,GILBERTSON B,KNIGHT M L,et al.The structure of the influenza A virus genome[J].Nat Microbiol,2019,4(11):1781-1789.
[2] CHEN L J,LIN X D,TIAN J H,et al.Diversity,evolution and population dynamics of avian influenza viruses circulating in the live poultry markets in China[J].Virology,2017,505:33-41.
[3] 翟新验,刘林青,李婷,等.H5亚型高致病性禽流感病毒流行特点及其防控[J].中国兽医杂志,2020,56(2):5-8.
ZHAI X Y,LIU L Q,LI T,et al.Prevalence characteristics and prevention of highly pathogenic Avian Influenza virus H5 subtype[J].Chinese Journal of Veterinary Medicine,2020,56(2):5-8.(in Chinese)
[4] PEIRIS J S M,DE JONG M D,GUAN Y.Avian influenza virus (H5N1):a threat to human health[J].Clin Microbiol Rev,2007,20(2):243-267.
[5] 刘亚东,王育伟,刘卜玮,等.禽流感检测方法研究进展[J].畜牧兽医科学(电子版),2020,77(17):26-27.
LIU Y D,WANG Y W,LIU B W,et al.Research progress on the detection methods of avian influenza[J].Graziery Veterinary Sciences,2020,77(17):26-27.(in Chinese)
[6] 邝振展,肖斌,孙朝晖,等.基于CRISPR/Cas13a技术检测肿瘤驱动基因TP53 R248W的研究[J].南京医科大学学报:自然科学版,2022,42(8):1119-1124.
KUANG Z Z,XIAO B,SUN Z H,et al.Detection of tumor driver gene TP53 R248W by CRISPR/Cas13a[J].Journal of Nanjing Medical University:Natural Sciences,2022,42(8):1119-1124.(in Chinese)
[7] HABIBZADEH P,MOFATTEH M,SILAWI M,et al.Molecular diagnostic assays for COVID-19:an overview[J].Crit Rev Clin Lab Sci,2021,58(6):385-398.
[8] GOOTENBERG J S,ABUDAYYEH O O,LEE J W,et al.Nucleic acid detection with CRISPR-Cas13a/C2c2[J].Science,2017,356(6336):438-442.
[9] CHEN J S,MA E B,HARRINGTON L B,et al.CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity[J].Science,2018,360(6387):436-439.
[10] CHANDRASEKARAN S S,AGRAWAL S,FANTON A,et al.Rapid detection of SARS-CoV-2 RNA in saliva via Cas13[J].Nat Biomed Eng,2022,6(8):944-956.
[11] EAST-SELETSKY A,O'CONNELL M R,KNIGHT S C,et al.Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection[J].Nature,2016,538(7624):270-273.
[12] WANG S C,LI Y,ZHANG F Y,et al.Reverse transcription recombinase-aided amplification assay for H5 subtype avian influenza virus[J].Virol J,2022,19(1):129.
[13] XU C L,DONG L B,XIN L,et al.Human avian influenza A (H5N1) virus infection in China[J].Sci China Ser C Life Sci,2009,52(5):407-411.
[14] 于佳佳,张旭霞,张雨晴,等.PCR扩增技术联合CRISPR-Cas13a系统对MTB DNA检测方法的初步研究[J].中国防痨杂志,2020,42(12):1280-1288.
YU J J,ZHANG X X,ZHANG Y Q,et al.Preliminary study on detection method of MTB DNA by PCR amplification combined with CRISPR-Cas13a system[J].Chinese Journal of Antituberculosis,2020,42(12):1280-1288.(in Chinese)
[15] 潘文雅.利用LAMP-CRISPR/LbCas12a技术检测非洲猪瘟病毒CD2v基因[D].武汉:华中农业大学,2022.
PAN W Y.Detection of ASFV CD2v gene by LAMP-CRISPR/LbCas12a technology[D].Wuhan:Huazhong Agricultural University,2022.(in Chinese)

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

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