RPA-Assisted Cas12a System for Detecting Pathogenic Xanthomonas oryzae, a Causative Agent for Bacterial Leaf Blight Disease in Rice
Received date: 2021-09-06
Accepted date: 2021-11-08
Online published: 2022-04-07
Xanthomonas oryzae pv. oryzae (Xoo) is a widespread pathogen causing bacterial leaf blight (BLB) disease, devastating rice productivity in many cultivated areas of Thailand. A specific and simple method for Xoo detection is required to improve surveillance of disease transmission and outbreak. This study developed a recombinase polymerase amplification (RPA) assay assisted with CRISPR-cas12a assay (RAC) for Xoo detection from bacterial cell suspension of infected rice samples without DNA extraction. The efficiency of the RAC system for Xoo detection using either Xoo80 or Xoo4009 locus was optimized to amplify and determine the sensitivity and specificity using a Xoo DNA template from bacterial cell suspension of infected rice samples without DNA extraction. The RAC system using the Xoo4009 locus gave a higher specificity than Xoo80 locus, because only Xoo species was amplified positive RPA product with fluorescence signal by cas12a digestion, which indicated no cross reactivity. Optimal RAC using the Xoo4009 locus enabled diagnosis of Xoo presence from both plant extracted samples of Xoo artificially inoculated rice leaves within 3 d post-inoculation without symptomatic BLB appearance, and Xoo naturally infected rice. Findings exhibited that RAC using the Xoo4009 locus offered sensitivity, specificity and simplicity for Xoo detection, with low intensities of Xoo-DNA (1 × 103 copies/µL) and Xoo-cell (2.5 × 103 cfu/mL). This developed RAC system showed significantly potential for Xoo detection at point-of-care application for early signs of BLB disease outbreak in rice fields.
Kittisak Buddhachat, Nattaporn Sripairoj, Onchira Ritbamrung, Phithak Inthima, Kumrop Ratanasut, Thanita Boonsrangsom, Tepsuda Rungrat, Pongsanat Pongcharoen, Kawee Sujipuli . RPA-Assisted Cas12a System for Detecting Pathogenic Xanthomonas oryzae, a Causative Agent for Bacterial Leaf Blight Disease in Rice[J]. Rice Science, 2022 , 29(4) : 340 -352 . DOI: 10.1016/j.rsci.2021.11.005
| [1] | Adli M. 2018. The CRISPR tool kit for genome editing and beyond. Nat Commun, 9(1): 1911. |
| [2] | Ai J W, Zhou X, Xu T, Yang M L, Chen Y Y, He G Q, Pan N, Cai Y W, Li Y J, Wang X R, Su H, Wang T, Zeng W Q, Zhang W H. 2019. CRISPR-based rapid and ultra-sensitive diagnostic test for Mycobacterium tuberculosis. Emerg Microbes Infect, 8(1): 1361-1369. |
| [3] | Ali Z, Aman R, Mahas A, Rao G S, Tehseen M, Marsic T, Salunke R, Subudhi A K, Hala S M, Hamdan S M, Pain A, Alofi F S, Alsomali A, Hashem A M, Khogeer A, Almontashiri N A M, Abedalthagafi M, Hassan N, Mahfouz M M. 2020. iSCAN: An RT-LAMP-coupled CRISPR-Cas12 module for rapid, sensitive detection of SARS-CoV-2. Virus Res, 288: 198129. |
| [4] | Aman R, Mahas A, Marsic T, Hassan N, Mahfouz M M. 2020. Efficient, rapid, and sensitive detection of plant RNA viruses with one-pot RT-RPA-CRISPR/Cas12a assay. Front Microbiol, 11: 610872. |
| [5] | Ansari T H, Haque S S, Uddin A. 2020. Pest management decision guide: Green and yellow list: Management of bacterial leaf blight (BLB) of rice. Plantwise knowledge Bank, CAB International. [2 October, 2021]. https://www.plantwise.org/KnowledgeBank/pmdg/20167800391. |
| [6] | Bai J, Lin H S, Li H J, Zhou Y, Liu J S, Zhong G R, Wu L T, Jiang W F, Du H L, Yang J Y, Xie Q M, Huang L Z. 2019. Cas12a-based on-site and rapid nucleic acid detection of African swine fever. Front Microbiol, 10: 2830. |
| [7] | Barra G B, Santa Rita T H, de Almeida Vasques J, Chianca C F, Nery L F A, Santana Soares Costa S. 2015. EDTA-mediated inhibition of DNases protects circulating cell-free DNA from ex vivo degradation in blood samples. Clin Biochem, 48(15): 976-981. |
| [8] | Barrangou R, Horvath P. 2017. A decade of discovery: CRISPR functions and applications. Nat Microbiol, 2: 17092. |
| [9] | Buddhachat K, Ritbamrung O, Sripairoj N, Inthima P, Ratanasut K, Boonsrangsom T, Sujipuli K. 2021. One-step colorimetric LAMP (cLAMP) assay for visual detection of Xanthomonas oryzae pv. oryzae in rice. Crop Prot, 150: 105809. |
| [10] | Carpenter S C D, Mishra P, Ghoshal C, Dash P K, Wang L, Midha S, Laha G S, Lore J S, Kositratana W, Singh N K, Singh K, Patil P B, Oliva R, Patarapuwadol S, Bogdanove A J, Rai R. 2018. A strain of an emerging Indian Xanthomonas oryzae pv. oryzae pathotype defeats the rice bacterial blight resistance gene xa13 without inducing a clade III SWEET gene and is nearly identical to a recent Thai isolate. Front Microbiol, 9: 2703. |
| [11] | Chaijarasphong T, Thammachai T, Itsathitphaisarn O, Sritunyalucksana K, Suebsing R. 2019. Potential application of CRISPR-Cas12a fluorescence assay coupled with rapid nucleic acid amplification for detection of white spot syndrome virus in shrimp. Aquaculture, 512: 734340. |
| [12] | Chen J S, Ma E B, Harrington L B, da Costa M, Tian X R, Palefsky J M, Doudna J A. 2018. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science, 360: 436-439. |
| [13] | Chen P, Zhou J, Wan Y B, Liu H, Li Y Z, Liu Z X, Wang H J, Lei J, Zhao K, Zhang Y L, Wang Y, Zhang X H, Yin L. 2020. A Cas12a ortholog with stringent PAM recognition followed by low off-target editing rates for genome editing. Genome Biol, 21(1): 78. |
| [14] | Creutzburg S C A, Swartjes T, van der Oost J. 2020. Medium- throughput in vitro detection of DNA cleavage by CRISPR- Cas12a. Methods, 172: 27-31. |
| [15] | Cui Z, Ojaghian M R, Tao Z, Kakar K U, Zeng J, Zhao W, Duan Y, Vera Cruz C M, Li B, Zhu B, Xie G. 2016. Multiplex PCR assay for simultaneous detection of six major bacterial pathogens of rice. J Appl Microbiol, 120(5): 1357-1367. |
| [16] | Daher R K, Stewart G, Boissinot M, Bergeron M G. 2016. Recombinase polymerase amplification for diagnostic applications. Clin Chem, 62(7): 947-958. |
| [17] | Ding X, Yin K, Li Z Y, Lalla R V, Ballesteros E, Sfeir M M, Liu C C. 2020. Ultrasensitive and visual detection of SARS-CoV-2 using all-in-one dual CRISPR-Cas12a assay. Nat Commun, 11(1): 4711. |
| [18] | Garita-Cambronero J, Palacio-Bielsa A, López M M, Cubero J. 2017. Pan-genomic analysis permits differentiation of virulent and non-virulent strains of Xanthomonas arboricola that cohabit Prunus spp. and elucidate bacterial virulence factors. Front Microbiol, 8: 573. |
| [19] | Gootenberg J S, Abudayyeh O O, Lee J W, Essletzbichler P, Dy A J, Joung J, Verdine V, Donghia N, Daringer N M, Freije C A, Myhrvold C, Bhattacharyya R P, Livny J, Regev A, Koonin E V, Hung D T, Sabeti P C, Collins J J, Zhang F. 2017. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science, 356: 438-442. |
| [20] | Kanitchinda S, Srisala J, Suebsing R, Prachumwat A, Chaijarasphong T. 2020. CRISPR-Cas fluorescent cleavage assay coupled with recombinase polymerase amplification for sensitive and specific detection of Enterocytozoon hepatopenaei. Biotechnol Rep, 27: e00485. |
| [21] | Lang J M, Hamilton J P, Diaz M G Q, van Sluys M A, Burgos M R G, Vera Cruz C M, Buell C R, Tisserat N A, Leach J E. 2010. Genomics-based diagnostic marker development for Xanthomonas oryzae pv. oryzae and X. oryzae pv. oryzicola. Plant Dis, 94(3): 311-319. |
| [22] | Lang J M, Langlois P, Nguyen M H R, Triplett L R, Purdie L, Holton T A, Djikeng A, Vera Cruz C M, Verdier V, Leach J E. 2014. Sensitive detection of Xanthomonas oryzae Pathovars oryzae and oryzicola by loop-mediated isothermal amplification. Appl Environ Microbiol, 80(15): 4519-4530. |
| [23] | Lau H Y, Botella J R. 2017. Advanced DNA-based point-of-care diagnostic methods for plant diseases detection. Front Plant Sci, 8: 2016. |
| [24] | le Thanh T, Thumanu K, Wongkaew S, Boonkerd N, Teaumroong N, Phansak P, Buensanteai N. 2017. Salicylic acid-induced accumulation of biochemical components associated with resistance against Xanthomonas oryzae pv. oryzae in rice. J Plant Interact, 12(1): 108-120. |
| [25] | Li S Y, Cheng Q X, Wang J M, Li X Y, Zhang Z L, Gao S, Cao R B, Zhao G P, Wang J. 2018a. CRISPR-Cas12a-assisted nucleic acid detection. Cell Discov, 4: 20. |
| [26] | Li S Y, Cheng Q X, Liu J K, Nie X Q, Zhao G P, Wang J. 2018b. CRISPR-Cas12a has both cis- and trans-cleavage activities on single-stranded DNA. Cell Res, 28(4): 491-493. |
| [27] | Liu H, Wang J B, Zeng H J, Liu X F, Jiang W, Wang Y, Ouyang W B, Tang X M. 2021. RPA-Cas12a-FS: A frontline nucleic acid rapid detection system for food safety based on CRISPR- Cas12a combined with recombinase polymerase amplification. Food Chem, 334: 127608. |
| [28] | Liu P P, Luk K, Shin M, Idrizi F, Kwok S, Roscoe B, Mintzer E, Suresh S, Morrison K, Frazão J B, Bolukbasi M F, Ponnienselvan K, Luban J, Zhu L J, Lawson N D, Wolfe S A. 2019. Enhanced Cas12a editing in mammalian cells and zebrafish. Nucleic Acids Res, 47(8): 4169-4180. |
| [29] | Lu W, Pan L Q, Zhao H J, Jia Y L, Wang Y L, Yu X P, Wang X Y. 2014. Molecular detection of Xanthomonas oryzae pv. oryzae, Xanthomonas oryzae pv. oryzicola, and Burkholderia glumae in infected rice seeds and leaves. Crop J, 2(6): 398-406. |
| [30] | Mahas A, Hassan N, Aman R, Marsic T, Wang Q C, Ali Z, Mahfouz M M. 2021. LAMP-coupled CRISPR-Cas12a module for rapid and sensitive detection of plant DNA viruses. Viruses, 13(3): 466. |
| [31] | Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T. 2000. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res, 28: E63. |
| [32] | Piepenburg O, Williams C H, Stemple D L, Armes N A. 2006. DNA detection using recombination proteins. PLoS Biol, 4(7): e204. |
| [33] | Rudresha G V, Urs A P, Manjuprasanna V N, Suvilesh K N, Sharanappa P, Vishwanath B S. 2019. Plant DNases are potent therapeutic agents against Echis carinatus venom-induced tissue necrosis in mice. J Cell Biochem, 120(5): 8319-8332. |
| [34] | Sullivan T J, Dhar A K, Cruz-Flores R, Bodnar A G. 2019. Rapid, CRISPR-based, field-deployable detection of white spot syndrome virus in shrimp. Sci Rep, 9(1): 19702. |
| [35] | Wang X J, Ji P P, Fan H Y, Dang L, Wan W W, Liu S Y, Li Y H, Yu W X, Li X Y, Ma X D, Ma X, Zhao Q, Huang X X, Liao M. 2020. CRISPR/Cas12a technology combined with immune- chromatographic strips for portable detection of African swine fever virus. Commun Biol, 3(1): 62. |
| [36] | Xiao G H, Zhang S, Liang Z H, Li G Q, Fang M T, Liu Y Y, Zhang J J, Ou M, He X, Zhang T Y, Zeng C C, Liu L, Zhang G L. 2020. Identification of Mycobacterium abscessus species and subspecies using the Cas12a/sgRNA-based nucleic acid detection platform. Eur J Clin Microbiol Infect Dis, 39(3): 551-558. |
| [37] | Xu H P, Zhang X L, Cai Z X, Dong X Q, Chen G, Li Z L, Qiu L M, He L, Liang B, Liu X L, Liu J F. 2020. An isothermal method for sensitive detection of Mycobacterium tuberculosis complex using clustered regularly interspaced short palindromic repeats/ Cas12a cis and trans cleavage. J Mol Diagn, 22(8): 1020-1029. |
| [38] | Xu L Q, Dai Q Q, Shi Z Y, Liu X T, Gao L, Wang Z Z, Zhu X Y, Li Z. 2020. Accurate MRSA identification through dual- functional aptamer and CRISPR-Cas12a assisted rolling circle amplification. J Microbiol Methods, 173: 105917. |
| [39] | Yasmin S, Hafeez F Y, Mirza M S, Rasul M, Arshad H M I, Zubair M, Iqbal M. 2017. Biocontrol of bacterial leaf blight of rice and profiling of secondary metabolites produced by rhizospheric Pseudomonas aeruginosa BRp3. Front Microbiol, 8: 1895. |
| [40] | Yuan M, Ke Y G, Huang R Y, Ma L, Yang Z Y, Chu Z H, Xiao J H, Li X H, Wang S P. 2016. A host basal transcription factor is a key component for infection of rice by TALE-carrying bacteria. eLife, 5: e19605. |
| [41] | Zetsche B, Gootenberg J S, Abudayyeh O O, Slaymaker I M, Makarova K S, Essletzbichler P, Volz S E, Joung J, van der Oost J, Regev A, Koonin E V, Zhang F. 2015. Cpf1 is a single RNA- guided endonuclease of a class 2 CRISPR-Cas system. Cell, 163(3): 759-771. |
| [42] | Zhang Y M, Zhang Y, Xie K. 2020. Evaluation of CRISPR/ Cas12a-based DNA detection for fast pathogen diagnosis and GMO test in rice. Mol Breed, 40(1): 1-12. |
/
| 〈 |
|
〉 |