Abstract: Nucleic acids in plant tissue lysates can be captured quickly by a cellulose filter paper and prepared for amplification after a quick purification. In this study, a published filter paper strip method was modified by sticking the filter paper on a polyvinyl chloride resin (PVC) sheet. This modified method is named EZ-D, for EASY DNA extraction. Compared with the original cetyl trimethylammonium bromide (CTAB) method, DNA extracted by EZ-D is more efficient in polymerase chain reaction (PCR) amplification due to the more stable performance of the EZ-D stick. The EZ-D method is also faster, easier, and cheaper. PCR analyses showed that DNA extracted from several types of plant tissues by EZ-D was appropriate for specific identification of biological samples. A regular PCR reaction can detect the EZ-D-extracted DNA template at concentration as low as 0.1 ng/μL. Evaluation of the EZ-D showed that DNA extracts could be successfully amplified by PCR reaction for DNA fragments up to 3000 bp in length and up to 80% in GC content. EZ-D was successfully used for DNA extraction from a variety of plant species and plant tissues. Moreover, when EZ-D was combined with the loop-mediated isothermal amplification (LAMP) method, DNA identification of biological samples could be achieved without the need for specialized equipment. As an optimized DNA purification method, EZ-D shows great advantages in application and can be used widely in laboratories where equipment is limited and rapid results are required.

一种用于实验室和现场检测的高效的植物DNA提取方法的评价及其应用

[1]Chen SL, Pang XH, Song JY, et al., 2014. A renaissance in herbal medicine identification: from morphology to DNA. Biotechnol Adv, 32(7):1237-1244.

[2]Cheng W, Xia ZJ, Feng XZ, et al., 2016. A rapid and nondestructive method for soybean DNA extraction and its application. Chin Bull Bot, 51(1):68-73 (in Chinese).

[3]Fan YH, Wang PL, Fan XS, et al., 2016. Research on application status of Chinese herbal decoction pieces based on clinical survey. China J Chin Mater Med, 41(15):2927-2931 (in Chinese).

[4]Goto M, Honda E, Ogura A, et al., 2009. Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy naphthol blue. Short Tech Rep, 46(3):167-172.

[5]Hou SM, Duan JQ, Liang XN, et al., 2005. Optimized CTAB protocol for extracting the total DNA of ramie. Acta Bot Boreali-Occid Sin, 25(11):2193-2197 (in Chinese).

[6]Jiang C, Huang LQ, Yuan Y, et al., 2013. Rapid extraction of DNA from Chinese medicinal materials by alkaline lysis. Chin J Pharm Anal, 33(7):1081-1090 (in Chinese).

[7]Kiddle G, Hardinge P, Buttigieg N, et al., 2012. GMO detection using a bioluminescent real time reporter (BART) of loop mediated isothermal amplification (LAMP) suitable for field use. BMC Biotechnol, 12:15.

[8]Lan QK, Zhao X, Chen R, et al., 2019. Authentication of Fritillaria cirrhosa based on loop-mediate isothermal amplification. Chin J Pharm Anal, 39(3):551-556 (in Chinese).

[9]Li HJ, Zhao YC, Li LH, 2017. The research progress of Chinese medicine superfine powder quality standard. Mod Anim Husbandry, 1(2):30-32 (in Chinese).

[10]Li JJ, Xiong C, Liu Y, et al., 2016. Loop-mediated isothermal amplification (LAMP): emergence as an alternative technology for herbal medicine identification. Front Plant Sci, 7:1956.

[11]Li M, Zhao X, 2012. RAPD analysis of three species of Fritillaria in southern China. J Zhejiang Univ Technol, 40(6):634-638 (in Chinese).

[12]Li M, Huang LM, Zhao X, et al., 2014. Specific PCR identification of Fritillaria thunbergii . Chin Tradit Herb Drugs, 45(12):1754-1757 (in Chinese).

[13]Liesenfeld O, Lehman L, Hunfeld KP, et al., 2014. Molecular diagnosis of sepsis: new aspects and recent developments. Eur J Microbiol Immunol, 4(1):1-25.

[14]Liu H, Wang JB, Li P, et al., 2020. Rapid detection of P‒35S and T-nos in genetically modified organisms by recombinase polymerase amplification combined with a lateral flow strip. Food Control, 107:106775.

[15]Liu XX, Li J, Zhang YB, et al., 2019. Rapid identification of Fritillariae Cirrhosae Bulbus by polymerase chain reaction (PCR) method. Chin J Pharm Anal, 39(10):1844-1851 (in Chinese).

[16]Lu LH, Han Q, Li L, et al., 2014. Establishment of an efficient transformation protocol for soybean using glyphosate as selective agent and the development of glyphosate-tolerant transgenic soybean lines. Sci Sin Vitae, 44(4):406-415.

[17]Moeller JR, Moehn NR, Waller DM, et al., 2014. Paramagnetic cellulose DNA isolation improves DNA yield and quality among diverse plant taxa. Appl Plant Sci, 2(10):1400048.

[18]Murray MG, Thompson WF, 1980. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res, 8(19):4321-4326.

[19]Notomi T, Okayama H, Masubuchi H, et al., 2000. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res, 28(12):e63.

[20]Paul R, Saville AC, Hansel JC, et al., 2019. Extraction of plant DNA by microneedle patch for rapid detection of plant diseases. ACS Nano, 13(6):6540-6549.

[21]Peterson DG, Boehm KS, Stack SM, 1997. Isolation of milligram quantities of nuclear DNA from tomato (Lycopersicon esculentum), a plant containing high levels of polyphenolic compounds. Plant Mol Biol Rep, 15(2):148-153.

[22]Ren LZ, Zhang CB, Zhao HK, et al., 2012. An improved method to rapid and high-quality of genomic DNA extraction from soybean. Chin Agric Sci Bull, 28(9):38-41 (in Chinese).

[23]Tomita N, Mori Y, Kanda H, et al., 2008. Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nat Protoc, 3(5):877-882.

[24]Tu JM, Zhang GA, Datta K, et al., 2000. Field performance of transgenic elite commercial hybrid rice expressing Bacillus thuringiensis δ-endotoxin. Nat Biotechnol, 18(10):1101-1104.

[25]Varma A, Padh H, Shrivastava N, 2007. Plant genomic DNA isolation: an art or a science. Biotechnol J, 2(3):386-392.

[26]Wang HM, Li YX, Weng HL, et al., 2012. Extracted Larix gmelinii DNA with improved DNA extraction kits. J Anhui Agric Sci, 40(35):17029-17030, 17041 (in Chinese).

[27]Wang SD, Yokosho Y, Guo RZ, et al., 2019. The soybean sugar transporter GmSWEET15 mediates sucrose export from endosperm to early embryo. Plant Physiol, 180(4):2133-2141.

[28]Wang SD, Li L, Ying YH, et al., 2020. A transcription factor OsbHLH156 regulates Strategy II iron acquisition through localising IRO2 to the nucleus in rice. New Phytol, 225(3):1247-1260.

[29]Wang SS, Wang F, Zhang H, et al., 2020. Rapid extraction of DNA from Chinese herbal medicines by filter paper method. Mod Chin Med, 22(2):213-218 (in Chinese).

[30]Wu YY, Dai DY, Cai CM, 2015. A modified UREA protocol for soybean DNA extraction. Soybean Sci, 34(1):112-115 (in Chinese).

[31]Yang L, Wu WR, Fu F, et al., 2019. Exploration and application of a new method for rapid extraction of DNA from Chinese medicinal materials. Chin Tradit Herbal Drugs, 50(2):502-509 (in Chinese).

[32]Yu ZX, Wei CJ, Li YM, et al., 2016. Study on DNA extraction method of genetically modified soybean. Guangdong Chem Ind, 43(18):23-24 (in Chinese).

[33]Zhang JJ, Xu H, Zhao SJ, 2017. Rapid identification of medicinal herbs through plant Direct-PCR. Acta Pharm Sin, 52(11):1763-1769 (in Chinese).

[34]Zhang LF, Li SG, Han SY, et al., 2013. Establishment of larch transgenic breeding system and its industrial application. Biotechnol Bus, (3):7-11 (in Chinese).

[35]Zhang XZ, Lowe SB, Gooding JJ, 2014. Brief review of monitoring methods for loop-mediated isothermal amplification (LAMP). Biosens Bioelectron, 61:491-499.

[36]Zhao ZP, Yan Y, Zheng MM, et al., 2019. Genetic evaluation of Gentiana rigescens based on the chloroplast psbA-trnH, trnL-trnF sequence polymorphism. Lishizhen Med Mater Med Res, 30(5):1203-1206 (in Chinese).

[37]Zou YP, Mason MG, Wang YL, et al., 2017. Nucleic acid purification from plants, animals and microbes in under 30 seconds. PLoS Biol, 15(11):e2003916.