Nucleic acid detection is vital for agricultural applications including trait detection during breeding, pest surveillance, and pathogen identification. Here, we use a modified version of the CRISPR-based nucleic acid detection platform SHERLOCK to quantify levels of a glyphosate resistance gene in a mixture of soybeans and to detect multiple plant genes in a single reaction. SHERLOCK is rapid (∼15 min), quantitative, and portable, and can process crude soybean extracts as input material for minimal nucleic acid sample preparation. This field-ready SHERLOCK platform with color-based lateral flow readout can be applied for detection and quantitation of genes in a range of agricultural applications.
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References
1.
ZhangDB, GuoJC. The development and standardization of testing methods for genetically modified organisms and their derived products. J Integr Plant Biol, 2011; 53:539–551. DOI: 10.1111/j.1744-7909.2011.01060.x.
LauHY, WuH, WeeEJ, et al.Specific and sensitive isothermal electrochemical biosensor for plant pathogen DNA detection with colloidal gold nanoparticles as probes. Sci Rep, 2017; 7:38896. DOI: 10.1038/srep38896.
4.
KhaterM, de la Escosura-MunizA, MerkociA.Biosensors for plant pathogen detection. Biosens Bioelectron2017; 93:72–86. DOI: 10.1016/j.bios.2016.09.091.
5.
PiepenburgO, WilliamsCH, StempleDL, et al.DNA detection using recombination proteins. PLoS Biol, 2006; 4:e204. DOI: 10.1371/journal.pbio.0040204.
6.
WardLI, HarperSJ. Loop-mediated isothermal amplification for the detection of plant pathogens. Methods Mol Biol, 2012; 862:161–170. DOI: 10.1007/978-1-61779-609-8_13.
7.
SchraderC, SchielkeA, EllerbroekL, et al.PCR inhibitors - occurrence, properties and removal. J Appl Microbiol, 2012; 113:1014–1026. DOI: 10.1111/j.1365-2672.2012.05384.x.
8.
WangY, WangY, ZhangL, et al.Multiplex, rapid, and sensitive isothermal detection of nucleic-acid sequence by endonuclease restriction-mediated real-time multiple cross displacement amplification. Front Microbiol, 2016; 7:753. DOI: 10.3389/fmicb.2016.00753.
9.
WangY, WangY, MaAJ, et al.Rapid and sensitive isothermal detection of nucleic-acid sequence by multiple cross displacement amplification. Sci Rep, 2015; 5:11902. DOI: 10.1038/srep11902.
10.
KiddleG, HardingeP, ButtigiegN, et al.GMO detection using a bioluminescent real time reporter (BART) of loop mediated isothermal amplification (LAMP) suitable for field use. BMC Biotechnol, 2012; 12:15. DOI: 10.1186/1472-6750-12-15.
11.
GootenbergJS, AbudayyehOO, LeeJW, et al.Nucleic acid detection with CRISPR-Cas13a/C2c2. Science, 2017; 356:438–442. DOI: 10.1126/science.aam9321.
12.
AbudayyehOO, GootenbergJS, KonermannS, et al.C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science, 2016; 353:aaf5573. DOI: 10.1126/science.aaf5573.
13.
East-SeletskyA, O'ConnellMR, KnightSC, et al.Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection. Nature, 2016; 538:270–273. DOI: 10.1038/nature19802.
14.
ShmakovS, AbudayyehOO, MakarovaKS, et al.Discovery and functional characterization of diverse class 2 CRISPR-Cas systems. Mol Cell, 2015; 60:385–397. DOI: 10.1016/j.molcel.2015.10.008.
15.
SmargonAA, CoxDB, PyzochaNK, et al.Cas13b is a type VI-B CRISPR-associated RNA-guided RNase differentially regulated by accessory proteins Csx27 and Csx28. Mol Cell, 2017; 65:618–630.e7. DOI: 10.1016/j.molcel.2016.12.023.
16.
GootenbergJS, AbudayyehO, KellnerM, et al.Multiplexed and portable nucleic acid detection platform with Cas13, Cpf1, and Csm6. Science, 2018; 360:439–444. DOI: 10.1126/science.aaq0179.
17.
AbudayyehOO, GootenbergJS, EssletzbichlerP, et al.RNA targeting with CRISPR-Cas13. Nature, 2017; 550:280–284. DOI: 10.1038/nature24049.
18.
CoxDBT, GootenbergJS, AbudayyehOO, et al.RNA editing with CRISPR-Cas13. Science, 2017; 358:1019–1027. DOI: 10.1126/science.aaq0180.
19.
WangR, ZhangF, WangL, et al.Instant, visual, and instrument-free method for on-site screening of GTS 40-3-2 soybean based on body-heat triggered recombinase polymerase amplification. Anal Chem, 2017; 89:4413–4418. DOI: 10.1021/acs.analchem.7b00964.
20.
YeJ, CoulourisG, ZaretskayaI, et al.Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics, 2012; 13:134. DOI: 10.1186/1471-2105-13-134.
21.
WhithamSA, QiM, InnesRW, et al.Molecular soybean–pathogen interactions. Annu Rev Phytopathol, 2016; 54:443–468. DOI: 10.1146/annurev-phyto-080615-100156.
22.
BotelhoSR, MartinsTP, DuarteMF, et al.Development of methodologies for virus detection in soybean and wheat seeds. MethodsX, 2016; 3:62–68. DOI: 10.1016/j.mex.2016.01.005.
23.
WangXM, TengD, GuanQF, et al.Detection of Roundup Ready soybean by loop-mediated isothermal amplification combined with a lateral-flow dipstick. Food Control, 2013; 29:213–220. DOI: 10.1016/j.foodcont.2012.06.007.
24.
WuHH, ZhangY, ZhuCQ, et al.Presence of CP4-EPSPS component in Roundup Ready soybean-derived food products. Int J Mol Sci, 2012; 13:1919–1932. DOI: 10.3390/ijms13021919.
25.
GuanXY, GuoJC, ShenP, et al.Visual and rapid detection of two genetically modified soybean events using loop-mediated isothermal amplification method. Food Anal Method, 2010; 3:313–320. DOI: 10.1007/s12161-010-9132-x.
26.
KazlauskieneM, KostiukG, VenclovasC, et al.A cyclic oligonucleotide signaling pathway in type III CRISPR-Cas systems. Science, 2017; 357:605–609. DOI: 10.1126/science.aao0100.
27.
NiewoehnerO, Garcia-DovalC, RostolJT, et al.Type III CRISPR-Cas systems produce cyclic oligoadenylate second messengers. Nature, 2017; 548:543–548. DOI: 10.1038/nature23467.
28.
ChanduD, PaulS, ParkerM, et al.Development of a rapid point-of-use DNA test for the screening of Genuity® Roundup Ready 2 Yield® soybean in seed samples. Biomed Res Int, 2016; 2016:3145921. DOI: 10.1155/2016/3145921.
29.
WangX, JiangD, YangD. Fast-tracking determination of homozygous transgenic lines and transgene stacking using a reliable quantitative real-time PCR assay. Appl Biochem Biotechnol, 2015; 175:996–1006. DOI: 10.1007/s12010-014-1322-3.
30.
ClemmonsBA, VoyBH, MyerPR. Altering the gut microbiome of cattle: considerations of host–microbiome interactions for persistent microbiome manipulation. Microb Ecol, 2019; 77:523–536. DOI: 10.1007/s00248-018-1234-9.
31.
MathewAG, CissellR, LiamthongS. Antibiotic resistance in bacteria associated with food animals: a United States perspective of livestock production. Foodborne Pathog Dis, 2007; 4:115–133. DOI: 10.1089/fpd.2006.0066.
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