Detection and field visualization of male and female Ginkgo biloba using RPA-LFD, enhanced by EZ-D and PNADCK technology

Abstract

Both genders of the dioecious gymnosperm Ginkgo biloba have distinct practical production and application uses, so quick, accurate identification of males and females is important for early seedling breeding. To develop a fast method to identify the sexes, we used the Easy DNA extraction (EZ-D) method to extract DNA from leaves within 1 min for use with the recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) system and identify the sex. A portable nucleic acid detection card kit (PNADCK) was used for on-site analysis. This method facilitates rapid extraction of nucleic acids from a single and can accurately detect 100 pg/µL of G. biloba female genomic DNA within 20 min at 39 °C. The EZ-DRPA-LFD-PNADCK system enables precise on-site determination of G. biloba leaf sex and is rapid, efficient, sensitive, and convenient, greatly enhancing productivity for G. biloba because seedlings with specific sex characteristics can be selected at an earlier stage, planting strategies can be optimized, and production efficiency improved.

Keywords

Ginkgo biloba / RPA-LFD / Biosensor platform / Rapid diagnosis

References

[1] Al-Qurainy F, Al-Ameri AA, Khan S, Nadeem M, Gaafar AZ, Tarroum M. SCAR marker for gender identification in date palm (Phoenix dactylifera L.) at the seedling stage. Int J Genom, 2018, 2018: 3035406

[2] Ayfan AKS, MacDonald J, Harris PNA, Heney C, Paterson DL, Trembizki E, Wang CYT, Whiley DM, Zowawi HM, Irwin AD. Rapid detection of NDM and VIM carbapenemase encoding genes by recombinase polymerase amplification and lateral flow-based detection. Eur J Clin Microbiol Infect Dis, 2021, 40(11): 2447-2453

[3] Bai JH, Baldwin E, Liao HL, Zhao W, Kostenyuk I, Burns J, Irey M. Extraction of DNA from orange juice, and detection of bacterium candidatus liberibacter asiaticus by real-time PCR. J Agric Food Chem, 2013, 61(399339-9346

[4] Berthomieu P, Meyer C. Direct amplification of plant genomic DNA from leaf and root pieces using PCR. Plant Mol Biol, 1991, 17(3): 555-557

[5] Bian Z, Liu WB, Jin JH, Hao YL, Jiang LS, Xie YH, Zhang HX. Development of a recombinase polymerase amplification assay with lateral flow dipstick (RPA-LFD) for rapid detection of Shigella spp. and enteroinvasive Escherichia coli. PLoS ONE, 2022, 17(12 e0278869

[6] Branch DW, Vreeland EC, McClain JL, Murton JK, James CD, Achyuthan KE. Rapid nucleic acid extraction and purification using a miniature ultrasonic technique. Micromachines, 2017, 8(7): 228

[7] Byrnes S, Fan A, Trueb J, Jareczek F, Mazzochette M, Sharon A, Sauer-Budge AF, Klapperich CM. A portable, pressure driven, room temperature nucleic acid extraction and storage system for point of care molecular diagnostics. Anal Methods, 2013, 5(133177-3184

[8] Chen X, Deng X, Zhang W, Zhang Y. Studies on the karyotype and early sex identification of female and male plants in Ginkgo biloba. J Fruit Sci, 1997, 2: 87-90in Chinese

[9] Daher RK, Stewart G, Boissinot M, Bergeron MG. Isothermal recombinase polymerase amplification assay applied to the detection of group B streptococci in vaginal/anal samples. Clin Chem, 2014, 60(4): 660-666

[10] Dai TT, Guo YF, Wen TY, Yu SN, Tao Y, Liu Z. Establishment of a rapid detection technique based on RPA-LFD and RPA-CRISPR/Cas12a on Phytophthora pini. Microorganisms, 2025, 13(4): 863

[11] Di JJ, Ye W, Wu QX, Feng K, Cao D, Li Q, Chen Y. Comparisons of physiological metabolism of female and male Ginkgo biloba trees during the late flower bud differentiation and flowering. J Nanjing For Univ (Nat Sci Edn), 2022, 46(459-67 in Chinese

[12] Du SH, Sang YL, Liu XJ, Xing SY, Li JH, Tang HX, Sun LM. Transcriptome profile analysis from different sex types of Ginkgo biloba L. Front Plant Sci, 2016, 7 871

[13] Emaus MN, Varona M, Eitzmann DR, Hsieh SA, Zeger VR, Anderson JL. Nucleic acid extraction: fundamentals of sample preparation methodologies, current advancements, and future endeavors. Trac Trends Anal Chem, 2020, 130 115985

[14] Focke F, Haase I, Fischer M. Loop-mediated isothermal amplification (LAMP): methods for plant species identification in food. J Agric Food Chem, 2013, 61(12): 2943-2949

[15] Fu MQ, Chen GF, Zhang CY, Wang YY, Sun R, Zhou J. Rapid and sensitive detection method for Karlodinium veneficum by recombinase polymerase amplification coupled with lateral flow dipstick. Harmful Algae, 2019, 84: 1-9

[16] Gautam S, Arora S (2025) Chapter 30-chloroform: risk assessment, environmental, and health hazard. In: Chawla M, Singh J, Kaushik RD (eds.), Hazardous Chemicals: Academic Press. pp 439–451. https://doi.org/10.1016/B978-0-323-95235-4.00033-5

[17] Ge XH, Liu XC, Hao ZM, Wei T, Tang HX, Liu ZN, Huo RX (2025) Screening of SCAR molecular markers for male and female early sex determination in Ginkgo biloba. J Anhui Agric Sci Advance online publication. (in Chinese)

[18] Gomez-Gutierrez SV, Goodwin SB. Loop-mediated isothermal amplification for detection of plant pathogens in wheat (Triticum aestivum). Front Plant Sci, 2022, 13 857673

[19] Grund E, Darissa O, Adam G. Application of FTA®cards to sample microbial plant pathogens for PCR and RT-PCR. J Phytopathol, 2010, 158(11–12): 750-757

[20] Hassan MM, Grist LF, Poirier AC, La Ragione RM. JMM profile: loop-mediated isothermal amplification (LAMP): for the rapid detection of nucleic acid targets in resource-limited settings. J Med Microbiol, 2022

[21] Hsieh L. Origin and distribution of Ginkgo biloba. For Chron, 1992, 68(5): 612-613

[22] Hu SD, Yu H, Zhang CQ. Development of recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) as a rapid on-site detection technique for Fusarium oxysporum. Bio Protoc, 2024, 14(1 e4915

[23] Hueso L, Martorell S, Sena-Torralba A, Ferrando M, Ferri M, Maquieira A, Ntoumi F, Morais S. Recombinase polymerase amplification technology for point-of-care diagnosis of neglected tropical diseases. Int J Infect Dis, 2025, 153 107831

[24] Ikeda N, Bautista NS, Yamada T, Kamijima O, Ishii T. Ultra-simple DNA extraction method for marker-assisted selection using microsatellite markers in rice. Plant Mol Biol Rep, 2001, 19(1): 27-32

[25] Jacobs BP, Browner WS. Ginkgo biloba: a living fossil. Am J Med, 2000, 108(4): 341-342

[26] James A, MacDonald J. Recombinase polymerase amplification: emergence as a critical molecular technology for rapid, low-resource diagnostics. Expert Rev Mol Diagn, 2015, 15(11): 1475-1489

[27] Jiang TT, Wang YC, Jiao WW, Song YQ, Zhao Q, Wang TY, Bi J, Shen AD. Recombinase polymerase amplification combined with real-time fluorescent probe for Mycoplasma pneumoniae detection. J Clin Med, 2022, 11(7): 1780

[28] Jin J, Jiang H, Yu SQ, Zhou GM. Sex-linked photosynthetic physiologic research and the evolutionary ecological analysis in living fossil plant Ginkgo Biloba L.. Acta Ecol Sin, 2008, 28(31128-1136

[29] Kakihara Y, Matsufuji H, Chino M, Takeda M. Extraction and detection of endogenous soybean DNA from fermented foods. Food Control, 2006, 17(10): 808-813

[30] Karakkat BB, Hockemeyer K, Franchett M, Olson M, Mullenberg C, Koch PL. Data for designing two isothermal amplification assays for the detection of root-infecting fungi on cool-season turfgrasses. Data Brief, 2018, 20: 471-479

[31] Kim SY, Lee JP, Shin WR, Oh IH, Ahn JY, Kim YH. Cardiac biomarkers and detection methods for myocardial infarction. Mol Cell Toxicol, 2022, 18(4): 443-455

[32] Klimyuk VI, Carroll BJ, Thomas CM, Jones JDG. Alkali treatment for rapid preparation of plant material for reliable PCR analysis. Plant J, 1993, 3(3): 493-494

[33] Koch E, Jaggy H, Chatterjee SS. Evidence for immunotoxic effects of crude Ginkgo biloba L. leaf extracts using the popliteal lymph node assay in the mouse. Int J Immunopharmacol, 2000, 22(3): 229-236

[34] Lai FY, Chang KC, Chang CS, Wang pH. Development of a rapid sex identification method for newborn pigeons using recombinase polymerase amplification and a lateral-flow dipstick on farm. Animals, 2022, 12(21 2969

[35] Langridge U, Schwall M, Langridge P. Squashes of plant tissue as substrate for PCR. Nucleic Acids Res, 1991, 19(24): 6954

[36] Lee JW. Development of SCAR marker for identifying male trees of Ginkgo biloba using multiplex PCR. J Korean For Soc, 2016, 105(4422-428

[37] Lee JP, Woo JA, Shin WR, Park YS, Kim HK, Ahn JY, Kim YH. Distinction of male and female trees of Ginkgo biloba using LAMP. Mol Biotechnol, 2023, 65(10): 1693-1703

[38] Lei R, Li Y, Li LM, Wang JY, Cui ZH, Ju R, Jiang L, Liao XL, Wu PS, Wang XY. A crispr/Cas12a-based portable platform for rapid detection of Leptosphaeria maculans in Brassica crops. Front Plant Sci, 2022, 13 976510

[39] Li R, Mock R, Huang Q, Abad J, Hartung J, Kinard G. A reliable and inexpensive method of nucleic acid extraction for the PCR-based detection of diverse plant pathogens. J Virol Methods, 2008, 154(1–2): 48-55

[40] Li ZT, Yi YX, Luo XM, Xiong N, Liu Y, Li SQ, Sun RL, Wang YQ, Hu BC, Chen W, Zhang YC, Wang J, Huang BF, Lin Y, Yang JS, Cai WS, Wang XF, Cheng J, Chen ZQ, Sun KJ, Pan WM, Zhan ZF, Chen LY, Ye F. Development and clinical application of a rapid IgM-IgG combined antibody test for SARS-CoV-2 infection diagnosis. J Med Virol, 2020, 92(9): 1518-1524

[41] Liao QG, Du R, Gou JB, Guo LJ, Shen H, Liu HL, Nguyen JK, Ming R, Yin TM, Huang SW, Yan JB. The genomic architecture of the sex-determining region and sex-related metabolic variation in Ginkgobiloba. Plant J, 2020, 104(5): 1399-1409

[42] Liao C, Pan LL, Tan MY, Zhou ZH, Long SP, Yi XL, Li XB, Wei GJ, Liang LN. A dual RPA-LFD assay for the simultaneous detection of Salmonella typhimurium and Salmonella enteritidis. Front Bioeng Biotechnol, 2024, 12: 1379939

[43] Lin H, Zhao S, Liu YH, Shao L, Ye YY, Jiang NZ, Yang K. Rapid visual detection of Plasmodium using recombinase-aided amplification with lateral flow dipstick assay. Front Cell Infect Microbiol, 2022, 12 922146

[44] Ling LX, Liang LY, Wang HF, Lin XL, Li CH. Real-time monitoring on the Chinese giant salamander using RPA-LFD. Int J Mol Sci, 2024, 25(9 4946

[45] Lorenz TC. Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies. J Vis Exp, 2012, 63 e3998

[46] Lu WJ, Wang JD, Wu Q, Sun JS, Chen YP, Zhang L, Zheng CS, Gao WN, Liu Y, Jiang XY. High-throughput sample-to-answer detection of DNA/RNA in crude samples within functionalized micro-pipette tips. Biosens Bioelectron, 2016, 75: 28-33

[47] Major RT. The Ginkgo, the most ancient living tree. The resistance of Ginkgo biloba L. to pests accounts in part for the longevity of this species. Science, 1967, 157(3794): 1270-1273

[48] Mei N, Guo XQ, Ren Z, Kobayashi D, Wada K, Guo L. Review of Ginkgo biloba-induced toxicity, from experimental studies to human case reports. J Environ Sci Health C Environ Carcinog Ecotoxicol Rev, 2017, 35(1): 1-28

[49] Ming R, Bendahmane A, Renner SS. Sex chromosomes in land plants. Annu Rev Plant Biol, 2011, 62: 485-514

[50] Mommaerts K, Sanchez I, Betsou F, Mathieson W. Replacing β-mercaptoethanol in RNA extractions. Anal Biochem, 2015, 479: 51-53

[51] Ndunguru J, Taylor NJ, Yadav J, Aly H, Legg JP, Aveling T, Thompson G, Fauquet CM. Application of FTA technology for sampling, recovery and molecular characterization of viral pathogens and virus-derived transgenes from plant tissues. Virol J, 2005, 2(1): 45

[52] Neto MF, Butzler MA, Reed JL, Rui X, Fisher MJ, Kelso DM, McFall SM. Immiscible phase filter extraction and equivalent amplification of genotypes 1–6 of hepatitis C RNA: the building blocks for point-of-care diagnosis. J Virol Methods, 2017, 248: 107-115

[53] Park JW. Principles and applications of loop-mediated isothermal amplification to point-of-care tests. Biosensors, 2022, 12(10857

[54] Park I, Yang S, Kim WJ, Noh P, Lee HO, Moon BC. Authentication of herbal medicines Dipsacus asper and Phlomoides umbrosa using DNA barcodes, chloroplast genome, and sequence characterized amplified region (SCAR) marker. Molecules, 2018, 23(71748

[55] Paul R, Ostermann E, Wei QS. Advances in point-of-care nucleic acid extraction technologies for rapid diagnosis of human and plant diseases. Biosens Bioelectron, 2020, 169 112592

[56] Piepenburg O, Williams CH, Stemple DL, Armes NA. DNA detection using recombination proteins. PLoS Biol, 2006, 4(7 e204

[57] Pike AM, Friend CM, Bell SP. Distinct RPA functions promote eukaryotic DNA replication initiation and elongation. Nucleic Acids Res, 2023, 51(1910506-10518

[58] Porebski S, Bailey LG, Baum BR. Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components. Plant Mol Biol Rep, 1997, 15(1): 8-15

[59] Razavi Z, Soltani M, Pazoki-Toroudi H, Chen P. Crispr-microfluidics nexus: advancing biomedical applications for understanding and detection. Sens Actuat A Phys, 2024, 376 115625

[60] Shimali CS, Kumar P (2024) Chapter 12-Current challenges and future prospects of next-generation microfluidics. In: Reza Khondakar Kamil, Kaushik Ajeet Kumar (eds), Next-Generation Smart Biosensing: Academic Press. pp 307–315 https://doi.org/10.1016/B978-0-323-98805-6.00009-9

[61] Shin SK, Lee Y, Kwon H, Rhee JS, Kim JK. Validation of direct boiling method for simple and efficient genomic DNA extraction and PCR-based macroalgal species determination. J Phycol, 2021, 57(4): 1368-1372

[62] Siegel CS, Stevenson FO, Zimmer EA. Evaluation and comparison of FTA card and CTAB DNA extraction methods for non-agricultural taxa. Appl Plant Sci, 2017, 5(2 apps.1600109

[63] Singh B, Kaur P, Singh RD, Ahuja PS. Biology and chemistry of Ginkgo biloba. Fitoterapia, 2008, 79(6): 401-418

[64] Šmarda P, Horová L, Knápek O, Dieck H, Dieck M, Ražná K, Hrubík P, Orlóci L, Papp L, Veselá K, Veselý P, Bureš P. Multiple haploids, triploids, and tetraploids found in modern-day “living fossil” Ginkgo biloba. Hortic Res, 2018, 5: 55

[65] Tang RH, Yang H, Gong Y, You ML, Liu Z, Choi JR, Wen T, Qu ZG, Mei QB, Xu F. A fully disposable and integrated paper-based device for nucleic acid extraction, amplification and detection. Lab Chip, 2017, 17(7): 1270-1279

[66] Vashisth P, Smith CL, Amarasekara DL, Dasanyake GS, Singh G, Chism CM, Hamadani CM, Shaikh T, Grovich N, Gamboa B, Fitzkee NC, Hammer NI, Tanner EEL. Choline carboxylic acid ionic liquid-stabilized anisotropic gold nanoparticles for photothermal therapy. ACS Appl Nano Mater, 2024, 7(2326332-26343

[67] Wang HY, Zhang YQ. The main active constituents and detoxification process of Ginkgo biloba seeds and their potential use in functional health foods. J Food Compos Anal, 2019, 83 103247

[68] Wang H, Qi M, Cutler AJ. A simple method of preparing plant samples for PCR. Nucleic Acids Res, 1993, 21(17): 4153-4154

[69] Wang Q, Shen XX, Qiu T, Wu W, Li L, Wang ZA, Shou HX. Evaluation and application of an efficient plant DNA extraction protocol for laboratory and field testing. J Zhejiang Univ Sci B, 2021, 22(2): 99-111

[70] Wang YF, Zheng X, Li DF, Tian JY, Wu H, Zhang YZ. Comparison of membrane fouling induced by protein, polysaccharide and humic acid under sodium and calcium ionic conditions. Desalination, 2023, 548 116236

[71] Wen YY, Yi TM, Wang YG, Qin ZX. Difference of isoenzyme and adenosine between male and female Ginkgo biloba L. J Shanxi Agric Sci, 2008, 36(06): 84-85in Chinese

[72] Xia WL, Chen K, Liu WS, Yin Y, Yao Q, Ban Y, Pu YW, Zhan XM, Bian HC, Yu SP, Han KP, Yang L, Wang HL, Fan ZJ. Rapid and visual detection of Mycoplasma synoviae by recombinase-aided amplification assay combined with a lateral flow dipstick. Poult Sci, 2022, 101(7 101860

[73] Yang XH, Zhang X, Wang Y, Shen H, Jiang G, Dong JQ, Zhao PP, Gao S. A real-time recombinase polymerase amplification method for rapid detection of Vibrio vulnificus in seafood. Front Microbiol, 2020, 11 586981

[74] Zeng FW, Chen XJ, Zhong WH, Chen T, Sa JQ, Wang GQ, Zhang SQ, Peng SM. Recombinase-aid amplification combined with lateral flow detection assay for sex identification of the great white pelican (Pelecanus onocrotalus). Sci Rep, 2024, 14(1): 21332

[75] Zhang Y, Zhu SN, Jin XF, Hong YH, Chen JM. Identification of male and female Ginkgo plants by 45S rDNA-FISH. Acta Hortic Sin, 2007, 34(61520-1524 in Chinese

[76] Zhou TT, Yang XM, Fu FF, Wang GB, Cao FL. Selection of suitable reference genes based on transcriptomic data in Ginkgo biloba under different experimental conditions. Forests, 2020, 11(111217

[77] Zou YP, Mason MG, Wang YL, Wee E, Turni C, Blackall PJ, Trau M, Botella JR. Nucleic acid purification from plants, animals and microbes in under 30 seconds. PLoS Biol, 2017, 15(11 e2003916

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