PDF
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.
The online version is available at https://link.springer.com/.
Corresponding editor: Tao Xu
The online version contains supplementary material available at https://doi.org/10.1007/s11676-025-01922-y.
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
Keywords
Ginkgo biloba
/
RPA-LFD
/
Biosensor platform
/
Rapid diagnosis
Cite this article
Download citation ▾
Jiahui Zang, Yufang Guo, Fangfang Fu, Fengqi Wang, Tingting Dai, Fuliang Cao.
Detection and field visualization of male and female Ginkgo biloba using RPA-LFD, enhanced by EZ-D and PNADCK technology.
Journal of Forestry Research, 2025, 36(1): 121 DOI:10.1007/s11676-025-01922-y
| [1] |
Al-QurainyF, Al-AmeriAA, KhanS, NadeemM, GaafarAZ, TarroumM. SCAR marker for gender identification in date palm (Phoenix dactylifera L.) at the seedling stage. Int J Genom, 2018, 20183035406.
|
| [2] |
AyfanAKS, MacDonaldJ, HarrisPNA, HeneyC, PatersonDL, TrembizkiE, WangCYT, WhileyDM, ZowawiHM, IrwinAD. 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] |
BaiJH, BaldwinE, LiaoHL, ZhaoW, KostenyukI, BurnsJ, IreyM. Extraction of DNA from orange juice, and detection of bacterium candidatus liberibacter asiaticus by real-time PCR. J Agric Food Chem, 2013, 61(39): 9339-9346.
|
| [4] |
BerthomieuP, MeyerC. Direct amplification of plant genomic DNA from leaf and root pieces using PCR. Plant Mol Biol, 1991, 17(3): 555-557.
|
| [5] |
BianZ, LiuWB, JinJH, HaoYL, JiangLS, XieYH, ZhangHX. 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, 1712. e0278869
|
| [6] |
BranchDW, VreelandEC, McClainJL, MurtonJK, JamesCD, AchyuthanKE. Rapid nucleic acid extraction and purification using a miniature ultrasonic technique. Micromachines, 2017, 87228.
|
| [7] |
ByrnesS, FanA, TruebJ, JareczekF, MazzochetteM, SharonA, Sauer-BudgeAF, KlapperichCM. A portable, pressure driven, room temperature nucleic acid extraction and storage system for point of care molecular diagnostics. Anal Methods, 2013, 5(13): 3177-3184.
|
| [8] |
ChenX, DengX, ZhangW, ZhangY. 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] |
DaherRK, StewartG, BoissinotM, BergeronMG. 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] |
DaiTT, GuoYF, WenTY, YuSN, TaoY, LiuZ. Establishment of a rapid detection technique based on RPA-LFD and RPA-CRISPR/Cas12a on Phytophthora pini. Microorganisms, 2025, 134863.
|
| [11] |
DiJJ, YeW, WuQX, FengK, CaoD, LiQ, ChenY. 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(4): 59-67 . in Chinese
|
| [12] |
DuSH, SangYL, LiuXJ, XingSY, LiJH, TangHX, SunLM. Transcriptome profile analysis from different sex types of Ginkgo biloba L. Front Plant Sci, 2016, 7. 871
|
| [13] |
EmausMN, VaronaM, EitzmannDR, HsiehSA, ZegerVR, AndersonJL. Nucleic acid extraction: fundamentals of sample preparation methodologies, current advancements, and future endeavors. Trac Trends Anal Chem, 2020, 130. 115985
|
| [14] |
FockeF, HaaseI, FischerM. Loop-mediated isothermal amplification (LAMP): methods for plant species identification in food. J Agric Food Chem, 2013, 61(12): 2943-2949.
|
| [15] |
FuMQ, ChenGF, ZhangCY, WangYY, SunR, ZhouJ. 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-GutierrezSV, GoodwinSB. Loop-mediated isothermal amplification for detection of plant pathogens in wheat (Triticum aestivum). Front Plant Sci, 2022, 13. 857673
|
| [19] |
GrundE, DarissaO, AdamG. Application of FTA®cards to sample microbial plant pathogens for PCR and RT-PCR. J Phytopathol, 2010, 158(11–12): 750-757.
|
| [20] |
HassanMM, GristLF, PoirierAC, La RagioneRM. JMM profile: loop-mediated isothermal amplification (LAMP): for the rapid detection of nucleic acid targets in resource-limited settings. J Med Microbiol, 2022.
|
| [21] |
HsiehL. Origin and distribution of Ginkgo biloba. For Chron, 1992, 68(5): 612-613.
|
| [22] |
HuSD, YuH, ZhangCQ. Development of recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) as a rapid on-site detection technique for Fusarium oxysporum. Bio Protoc, 2024, 141. e4915
|
| [23] |
HuesoL, MartorellS, Sena-TorralbaA, FerrandoM, FerriM, MaquieiraA, NtoumiF, MoraisS. Recombinase polymerase amplification technology for point-of-care diagnosis of neglected tropical diseases. Int J Infect Dis, 2025, 153. 107831
|
| [24] |
IkedaN, BautistaNS, YamadaT, KamijimaO, IshiiT. Ultra-simple DNA extraction method for marker-assisted selection using microsatellite markers in rice. Plant Mol Biol Rep, 2001, 19(1): 27-32.
|
| [25] |
JacobsBP, BrownerWS. Ginkgo biloba: a living fossil. Am J Med, 2000, 108(4): 341-342.
|
| [26] |
JamesA, MacDonaldJ. Recombinase polymerase amplification: emergence as a critical molecular technology for rapid, low-resource diagnostics. Expert Rev Mol Diagn, 2015, 15(11): 1475-1489.
|
| [27] |
JiangTT, WangYC, JiaoWW, SongYQ, ZhaoQ, WangTY, BiJ, ShenAD. Recombinase polymerase amplification combined with real-time fluorescent probe for Mycoplasma pneumoniae detection. J Clin Med, 2022, 1171780.
|
| [28] |
JinJ, JiangH, YuSQ, ZhouGM. Sex-linked photosynthetic physiologic research and the evolutionary ecological analysis in living fossil plant Ginkgo Biloba L.. Acta Ecol Sin, 2008, 28(3): 1128-1136.
|
| [29] |
KakiharaY, MatsufujiH, ChinoM, TakedaM. Extraction and detection of endogenous soybean DNA from fermented foods. Food Control, 2006, 17(10): 808-813.
|
| [30] |
KarakkatBB, HockemeyerK, FranchettM, OlsonM, MullenbergC, KochPL. 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] |
KimSY, LeeJP, ShinWR, OhIH, AhnJY, KimYH. Cardiac biomarkers and detection methods for myocardial infarction. Mol Cell Toxicol, 2022, 18(4): 443-455.
|
| [32] |
KlimyukVI, CarrollBJ, ThomasCM, JonesJDG. Alkali treatment for rapid preparation of plant material for reliable PCR analysis. Plant J, 1993, 3(3): 493-494.
|
| [33] |
KochE, JaggyH, ChatterjeeSS. 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] |
LaiFY, ChangKC, ChangCS, WangpH. Development of a rapid sex identification method for newborn pigeons using recombinase polymerase amplification and a lateral-flow dipstick on farm. Animals, 2022, 1221. 2969
|
| [35] |
LangridgeU, SchwallM, LangridgeP. Squashes of plant tissue as substrate for PCR. Nucleic Acids Res, 1991, 19246954.
|
| [36] |
LeeJW. Development of SCAR marker for identifying male trees of Ginkgo biloba using multiplex PCR. J Korean For Soc, 2016, 105(4): 422-428.
|
| [37] |
LeeJP, WooJA, ShinWR, ParkYS, KimHK, AhnJY, KimYH. Distinction of male and female trees of Ginkgo biloba using LAMP. Mol Biotechnol, 2023, 65(10): 1693-1703.
|
| [38] |
LeiR, LiY, LiLM, WangJY, CuiZH, JuR, JiangL, LiaoXL, WuPS, WangXY. A crispr/Cas12a-based portable platform for rapid detection of Leptosphaeria maculans in Brassica crops. Front Plant Sci, 2022, 13. 976510
|
| [39] |
LiR, MockR, HuangQ, AbadJ, HartungJ, KinardG. 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] |
LiZT, YiYX, LuoXM, XiongN, LiuY, LiSQ, SunRL, WangYQ, HuBC, ChenW, ZhangYC, WangJ, HuangBF, LinY, YangJS, CaiWS, WangXF, ChengJ, ChenZQ, SunKJ, PanWM, ZhanZF, ChenLY, YeF. 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] |
LiaoQG, DuR, GouJB, GuoLJ, ShenH, LiuHL, NguyenJK, MingR, YinTM, HuangSW, YanJB. The genomic architecture of the sex-determining region and sex-related metabolic variation in Ginkgobiloba. Plant J, 2020, 104(5): 1399-1409.
|
| [42] |
LiaoC, PanLL, TanMY, ZhouZH, LongSP, YiXL, LiXB, WeiGJ, LiangLN. A dual RPA-LFD assay for the simultaneous detection of Salmonella typhimurium and Salmonella enteritidis. Front Bioeng Biotechnol, 2024, 121379939.
|
| [43] |
LinH, ZhaoS, LiuYH, ShaoL, YeYY, JiangNZ, YangK. Rapid visual detection of Plasmodium using recombinase-aided amplification with lateral flow dipstick assay. Front Cell Infect Microbiol, 2022, 12. 922146
|
| [44] |
LingLX, LiangLY, WangHF, LinXL, LiCH. Real-time monitoring on the Chinese giant salamander using RPA-LFD. Int J Mol Sci, 2024, 259. 4946
|
| [45] |
LorenzTC. Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies. J Vis Exp, 2012, 63. e3998
|
| [46] |
LuWJ, WangJD, WuQ, SunJS, ChenYP, ZhangL, ZhengCS, GaoWN, LiuY, JiangXY. High-throughput sample-to-answer detection of DNA/RNA in crude samples within functionalized micro-pipette tips. Biosens Bioelectron, 2016, 75: 28-33.
|
| [47] |
MajorRT. 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] |
MeiN, GuoXQ, RenZ, KobayashiD, WadaK, GuoL. 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] |
MingR, BendahmaneA, RennerSS. Sex chromosomes in land plants. Annu Rev Plant Biol, 2011, 62: 485-514.
|
| [50] |
MommaertsK, SanchezI, BetsouF, MathiesonW. Replacing β-mercaptoethanol in RNA extractions. Anal Biochem, 2015, 479: 51-53.
|
| [51] |
NdunguruJ, TaylorNJ, YadavJ, AlyH, LeggJP, AvelingT, ThompsonG, FauquetCM. Application of FTA technology for sampling, recovery and molecular characterization of viral pathogens and virus-derived transgenes from plant tissues. Virol J, 2005, 2145.
|
| [52] |
NetoMF, ButzlerMA, ReedJL, RuiX, FisherMJ, KelsoDM, McFallSM. 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] |
ParkJW. Principles and applications of loop-mediated isothermal amplification to point-of-care tests. Biosensors, 2022, 1210857.
|
| [54] |
ParkI, YangS, KimWJ, NohP, LeeHO, MoonBC. Authentication of herbal medicines Dipsacus asper and Phlomoides umbrosa using DNA barcodes, chloroplast genome, and sequence characterized amplified region (SCAR) marker. Molecules, 2018, 2371748.
|
| [55] |
PaulR, OstermannE, WeiQS. Advances in point-of-care nucleic acid extraction technologies for rapid diagnosis of human and plant diseases. Biosens Bioelectron, 2020, 169. 112592
|
| [56] |
PiepenburgO, WilliamsCH, StempleDL, ArmesNA. DNA detection using recombination proteins. PLoS Biol, 2006, 47. e204
|
| [57] |
PikeAM, FriendCM, BellSP. Distinct RPA functions promote eukaryotic DNA replication initiation and elongation. Nucleic Acids Res, 2023, 51(19): 10506-10518.
|
| [58] |
PorebskiS, BaileyLG, BaumBR. 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] |
RazaviZ, SoltaniM, Pazoki-ToroudiH, ChenP. 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] |
ShinSK, LeeY, KwonH, RheeJS, KimJK. 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] |
SiegelCS, StevensonFO, ZimmerEA. Evaluation and comparison of FTA card and CTAB DNA extraction methods for non-agricultural taxa. Appl Plant Sci, 2017, 52. apps.1600109
|
| [63] |
SinghB, KaurP, SinghRD, AhujaPS. Biology and chemistry of Ginkgo biloba. Fitoterapia, 2008, 79(6): 401-418.
|
| [64] |
ŠmardaP, HorováL, KnápekO, DieckH, DieckM, RažnáK, HrubíkP, OrlóciL, PappL, VeseláK, VeselýP, BurešP. Multiple haploids, triploids, and tetraploids found in modern-day “living fossil” Ginkgo biloba. Hortic Res, 2018, 555.
|
| [65] |
TangRH, YangH, GongY, YouML, LiuZ, ChoiJR, WenT, QuZG, MeiQB, XuF. A fully disposable and integrated paper-based device for nucleic acid extraction, amplification and detection. Lab Chip, 2017, 17(7): 1270-1279.
|
| [66] |
VashisthP, SmithCL, AmarasekaraDL, DasanyakeGS, SinghG, ChismCM, HamadaniCM, ShaikhT, GrovichN, GamboaB, FitzkeeNC, HammerNI, TannerEEL. Choline carboxylic acid ionic liquid-stabilized anisotropic gold nanoparticles for photothermal therapy. ACS Appl Nano Mater, 2024, 7(23): 26332-26343.
|
| [67] |
WangHY, ZhangYQ. 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] |
WangH, QiM, CutlerAJ. A simple method of preparing plant samples for PCR. Nucleic Acids Res, 1993, 21(17): 4153-4154.
|
| [69] |
WangQ, ShenXX, QiuT, WuW, LiL, WangZA, ShouHX. 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] |
WangYF, ZhengX, LiDF, TianJY, WuH, ZhangYZ. Comparison of membrane fouling induced by protein, polysaccharide and humic acid under sodium and calcium ionic conditions. Desalination, 2023, 548. 116236
|
| [71] |
WenYY, YiTM, WangYG, QinZX. Difference of isoenzyme and adenosine between male and female Ginkgo biloba L. J Shanxi Agric Sci, 2008, 36(06): 84-85in Chinese
|
| [72] |
XiaWL, ChenK, LiuWS, YinY, YaoQ, BanY, PuYW, ZhanXM, BianHC, YuSP, HanKP, YangL, WangHL, FanZJ. Rapid and visual detection of Mycoplasma synoviae by recombinase-aided amplification assay combined with a lateral flow dipstick. Poult Sci, 2022, 1017. 101860
|
| [73] |
YangXH, ZhangX, WangY, ShenH, JiangG, DongJQ, ZhaoPP, GaoS. A real-time recombinase polymerase amplification method for rapid detection of Vibrio vulnificus in seafood. Front Microbiol, 2020, 11. 586981
|
| [74] |
ZengFW, ChenXJ, ZhongWH, ChenT, SaJQ, WangGQ, ZhangSQ, PengSM. Recombinase-aid amplification combined with lateral flow detection assay for sex identification of the great white pelican (Pelecanus onocrotalus). Sci Rep, 2024, 14121332.
|
| [75] |
ZhangY, ZhuSN, JinXF, HongYH, ChenJM. Identification of male and female Ginkgo plants by 45S rDNA-FISH. Acta Hortic Sin, 2007, 34(6): 1520-1524. in Chinese
|
| [76] |
ZhouTT, YangXM, FuFF, WangGB, CaoFL. Selection of suitable reference genes based on transcriptomic data in Ginkgo biloba under different experimental conditions. Forests, 2020, 11111217.
|
| [77] |
ZouYP, MasonMG, WangYL, WeeE, TurniC, BlackallPJ, TrauM, BotellaJR. Nucleic acid purification from plants, animals and microbes in under 30 seconds. PLoS Biol, 2017, 1511. e2003916
|
RIGHTS & PERMISSIONS
Northeast Forestry University