|
|
|
| Rapid identification of Brucella with CRISPR/Cas13a |
| HUANG Ming-yao1, LIANG Wen-li1, WU Wan-ting2,3, LIU Zu2,3, XIE Shu-mei2,3, DENG Ying-ying1, FU Jun-fang1, JIANG Chang-hong1, LONG Jun1, JIANG Ling-xiao1 |
1. Division of Laboratory Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China;
2. Vision Medicals, Co., Ltd, Guangzhou 510000, China;
3. Vision Medicals Center for Medical Research, Shenzhen 518000, China |
|
|
|
|
Abstract This study aimed to establish a method based on CRISPR/Cas13a to identify Brucella. We designed specific primers for RPA amplification targeting the conserved region of the Brucella BCSP31 gene to develop a CRISPR/Cas13a method to identify Brucella. The specificity and sensitivity of the method were evaluated by testing pure strains and positive clinical aerobic blood cultures; the lower limit of this method was evaluated with the gradient dilution method. A rapid identification method for Brucella through CRISPR/Cas13a was successfully established, and its detection limit reached 10 fg/μL. On the basis of detection of 64 Brucella strains, 56 non-Brucella strains, human DNA, and 57 cases of clinical aerobic blood culture positive bacteria, the sensitivity and specificity of this method reached 100%. In conclusion, a Brucella identification method based on CRISPR/Cas13a was established and can be applied for rapid identification of Brucella after a positive clinical aerobic blood culture.
|
|
Received: 27 November 2020
|
|
|
| Fund:Supported by the National Key R&D Program of China (No.2017YFC1200801) |
|
Corresponding Authors:
Jiang Ling-xiao, Email: jiang-lingxiao@163.com
|
|
|
|
[1] Poonawala H, Marrs Conner T, Peaper DR.The brief case: misidentification of Brucella melitensis as Ochrobactrum anthropi by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS)[J]. J Clin Microbiol, 2018, 56(6): e00914-17. DOI: 10.1128/JCM.00914-17
[2] Shallal A, Markowitz N, Tibbetts R.Closing the brief case: cough in an immunocompromised patient[J]. J Clin Microbiol, 2020, 58(11): e00369-20. DOI: 10.1128/JCM.00370-20
[3] Hou T, Zeng W, Yang M, et al.Development and evaluation of a rapid CRISPR-based diagnostic for COVID-19[J]. PLoS Pathog, 2020, 16(8): e1008705. DOI: 10.1371/journal.ppat.1008705
[4] Franco MP, Mulder M, Gilman RH, et al.Human brucellosis[J]. Lancet Infect Dis, 2007, 7(12): 775-786. DOI: 10.1016/s1473-3099(07)70286-4
[5] 刘锋, 金福芝, 江伟, 等. 浅析影响我国人间布鲁菌病防控的原因[J]. 中华地方病学杂志, 2016, 35(5): 386-389. DOI: 10.3760/cma.j.issn.2095-4255.2016.05.018
[6] Zhen Q, Lu Y, Yuan X, et al.Asymptomatic brucellosis infection in humans: implications for diagnosis and prevention[J]. Clin Microbiol Infect, 2013, 19(9): 395-397. DOI: 10.1111/1469-0691.12250
[7] Yagupsky P, Morata P, Colmenero JD.Laboratory diagnosis of human brucellosis[J]. Clin Microbiol Rev, 2019, 33(1): e00073-19. DOI: 10.1128/CMR.00073-19
[8] 罗文博, 付梦姣, 焦俊, 等. 布鲁氏菌IV型分泌系统效应蛋白BPE043的功能和致病机制研究[J]. 中国人兽共患病学报, 2020, 36(8): 618-623. DOI: 10.3969/j.issn.1002-2694.2020.00.097
[9] Asaad AM, Alqahtani JM.Serological and molecular diagnosis of human brucellosis in Najran, Southwestern Saudi Arabia[J]. J Infect Public Health, 2012, 5(2): 189-194. DOI: 10.1016/j.jiph.2012.02.001
[10] Fan SY, Ren HT, Wei YP, et al.Next-generation sequencing of the cerebrospinal fluid in the diagnosis of neurobrucellosis[J]. Int J Infect Dis, 2018, 67:20-24. DOI: 10.1016/j.ijid.2017.11.028
[11] Gootenberg J, Abudayyeh O, Lee J, et al.Nucleic acid detection with CRISPR-Cas13a/C2c2[J]. Science, 2017, 356(6336): 438-442. DOI: 10.1126/science.aam9321
[12] Iwasaki RS, Batey RT.SPRINT: a Cas13a-based platform for detection of small molecules[J]. Nucleic Acids Res, 2020, 48(17): e101. DOI: 10.1093/nar/gkaa673
[13] Ai JW, Zhou X, Xu T, et al.CRISPR-based rapid and ultra-sensitive diagnostic test for Mycobacterium tuberculosis[J]. Emerg Microbes Infect, 2019, 8(1): 1361-1369. DOI: 10.1080/22221751.2019.1664939 |
|
|
|
2021, Vol. 37 
