Boosting Anticancer Activity Ntawm Aspergillus Favus "endophyte Ntawm Jojoba" Taxol Via Conjugation Nrog Kub Nanoparticles Mediated Los ntawm -Irradiation

Taxol Alternative Suav Tshuaj Ntsuab Cistanche

Ntsiab lus:Aspergillus favus · Jojoba · Endophytic fungi · Kub nanoparticles · Taxol · -Irradiation · Khoom noj khoom haus optimization

Taw qhia

Taxol yog ib qho ntawm cov lag luam tshaj plaws broad-spectrumtshuaj anticancer[1]. Cov haujlwm ntawm Taxol elaborates los ntawm nws qhov tshwj xeeb tshwj xeeb rau kev khi nrog cov cellular tubulin -subunits heterodimer, txhawb nqa tubulin polymerization, yog li cuam tshuam qhov kev faib tawm ntawm cov qog hlwb [2]. Taxol tau nthuav tawm cov haujlwm muaj zog tiv thaiv lub mis, ntsws, lub taub hau, thiab caj dab, mob qog noj ntshav hauv tsev menyuam, thiab cov ntaub ntawv siab heev ntawm Kaposi's sarcoma [3]. Taxol yog thawj zaug tsim los ntawm cov tawv ntoo ntawm yew ntoo Taxus brevifolia "tsev neeg Taxaceae" [4, 5]; Txawm li cas los xij, qhov qis dua ntawm Taxol uas yog<0.001%, i.e., to produce 1 g Taxol, it requires~10 kg of plant bark that collected from 3 to 5 trees [6], is the main challenge. In addition, the vulnerability of this plant to unpredicted fluctuations with the environmental conditions strongly influences the Taxol yield, heterogeneity, and reproducibility [7–9]. Exploring the Taxol producing potency of the endophytic fungi inhabiting medicinal plants raises the hope for overcoming the low yield by the above-mentioned method [10, 11], due to their fast growth, cost-effectiveness, independence on climatic changes, and feasibility for genetic manipulation [12, 13]. Subsequently, a plethora of endophytic fungi with metabolic potency to produce Taxol has been reported as reviewed [1, 14–25]. However, the anticipation of these fungi for industrial production of Taxol has been challenged by the attenuation and loss of Taxol productivity by the fungal storage and multiple subculturing [21, 22, 26–28]. Thus, searching for a novel fungal isolate with affordable metabolic stability and sustainability for Taxol production is the challenge. Medicinal plants of well-known ethnopharmacological relevance and traditional pharmaceutical applications could be the repertoire of novel fungal isolates with unique features of metabolic stability for Taxol biosynthesis. Among the most common medicinal plants, jojoba "Simmondsia chinensis" is a monogenetic dioecious grey-green shrub belonging to the Simmondsiaceae family. Jojoba seeds contain up to 65% of light golden and odorless high-viscosity oily metabolites [29]. Jojoba oil has been frequently used for the relief of headaches, throat inflammation, and wound treatment [30, 31]. As well as Jojoba oil has been used as an anti-inflammatory and antimicrobial agent [30, 31]. The leaves of jojoba are rich with antioxidant flavonoid compounds that are traditionally used for treating of various disorders such as mob hawb pob,mob, thiabmob qog noj ntshav[32]. Yog li, lub hom phiaj tseem ceeb ntawm txoj haujlwm no yog txhawm rau tshawb nrhiav cov kab mob fungal tshiab cais tawm los ntawm jojoba cog nrog qhov tshwj xeeb metabolic ruaj khov rau Taxol ntau lawm, txhawm rau tshuaj xyuas cov kev sib txawv kom ua kom lawv cov txiaj ntsig Taxol zoo tshaj plaws, ntxiv rau, txhawm rau txhim kho cov kev ua haujlwm tiv thaiv kab mob ntawm cov tshuaj tua kab mob. ntawm conjugation nrog kub nanoparticles, kho los ntawm gamma irradiation.

Khoom siv thiab cov txheej txheem

Kev cais tawm thiab kev coj noj coj ua ntawm Endophytic Fungi

Ntau qhov chaw ntawm jojoba (Simmondsia chinensis) raws li nplooj, tawv, twigs, thiab buds tau sau los ntawm Kws Qhia Ntawv ntawm Kev Ua Liaj Ua Teb, Cairo University, thiab siv los ua qhov chaw rau cov kab mob endophytic. Cov nroj tsuag tau sau thiab ntxuav hauv qab dej ntws, qhov chaw kom tsis muaj menyuam nrog 70 feem pua ​​​​ethanol rau 1 min thiab tom qab ntawd yaug nrog dej tsis muaj menyuam [28]. Cov nroj tsuag uas tsis muaj kab mob saum npoo tau txiav mus rau hauv me me raws li cov neeg mob tsis muaj menyuam thiab muab tso rau ntawm daim phiaj ntawm qos yaj ywm dextrose agar (PDA) nruab nrab, Czapek's-Dox, thiab malt extract agar nruab nrab [33–36], thiab cov phaj tau incubated ntawm 30 degree rau. 10 hnub. Qhov ua tau zoo ntawm kev ua kom tsis muaj menyuam saum npoo ntawm cov nroj tsuag tau soj ntsuam los ntawm centrifuging cov dej yaug, tom qab ntawd 500 ul sterile dej tau ntxiv rau hauv precipitate thiab plated rau hauv PDA nruab nrab [37]. Cov purified endophytic fungal cais tawm tau inoculated ntawm PDA slants rau 7 hnub thiab khaws cia ntawm 4 degree

Kev tshuaj xyuas, Extraction, thiab Quantification ntawm Taxol los ntawm Endophytic Fungi

Cov fungi endophytic uas nyob hauv jojoba tau raug tshuaj xyuas rau Taxol ntau lawm los ntawm kev loj hlob ntawm qos yaj ywm dextrose broth (PDB) [38]. Ib lub ntsaws ntawm txhua 7 hnub qub kev lom zem gal cais tawm tau inoculated rau hauv 100 ml ntawm PDB/250 ml Erlenmeyer cov dej num, incubated rau 15 hnub ntawm 30 ± 1 degree, nyob rau hauv co tej yam kev mob (120 rpm). Tom qab incubation, cov kab lis kev cai raug lim, thiab cov lim tau hloov kho nrog 0.2 feem pua ​​​​sodium bicarbonate rau precipitate fatty acids. Taxol tau muab rho tawm nrog dichloromethane, thiab cov organic theem tau sau thiab evaporated rau dryness, thiab cov residues tau rov dissolved hauv methanol [17, 39]. Taxol raug cais thiab txheeb xyuas los ntawm TLC siv Merck 1 hli (20 × 20 cm) pre-coated silica gel daim hlau (TLC Silica gel 60 F254, Darmstadt, Lub teb chaws Yelemees), kuaj pom los ntawm UV illumination ntawm 254 nm [39]. Cov chaw tso quav ntawm Taxol tau muab pov tseg-ntawm TLC silica gel daim hlau thiab yaj hauv methanol, vortexed rau 10 min, thiab centrifuged ntawm 1000 rpm rau 5 min. Cov silica precipitated tau raug tshem tawm, thiab cov supernatant raug coj mus rau Taxol quantification thiab purity checking los ntawm HPLC (YOUNG In, Chromass, 9110 plus Quater nary Pump, Kauslim) ntawm C18 rov qab theem kem (Eclipse Plus C18 4.6 × 150 hli, 3.5 μm, Cat. #959,963–902). Cov theem txawb siv yog methanol / acetonitrile / dej (25: 35: 40, v / v / v) ntawm tus nqi fow ntawm 1.0 ml / min rau 20 min [40], thiab Taxol feem raug ntsuas ntawm 227 nm, thiab lawv Cov tshuaj lom neeg tus kheej thiab cov concentrations tau lees paub los ntawm lub sijhawm khaws cia thiab qhov chaw nqus qhov siab tshaj plaws piv rau cov qauv tseeb.

Morphological thiab Molecular Identification ntawm Recovered Endophytic Fungi

Cov kab mob endophytic fungal cais tawm tau txheeb xyuas lawv cov hom kab mob raws li lawv cov macro thiab micro-morphological nta los ntawm kev loj hlob ntawm PDA, Czapek's-Dox, thiab malt extract media raws li kev siv cov yuam sij [33–36]. Tus neeg muaj peev xwm tshaj plaws ntawm Taxol-tsim cov kab mob fungal cais tau ntxiv molecularly paub tseeb raws li qhov sib lawv liag ntawm cov khoom siv hauv nruab nrab (ITS) [41, 42]. Fungal genomic DNA (gDNA) tau muab rho tawm los ntawm pulverizing lub mycelia (~0.2 g) hauv cov kua nitrogen, tom qab ntawd dispensing hauv 1 ml CTAB extraction tsis (2% CTAB, 2% PVP40, { {21}}.2 feem pua ​​2-mercaptoethanol, 20 mM EDTA, 1.4 M NaCl hauv 100 mM Tris−HCl, pH 8.0). PCR primer sets yog ITS4 5′-GGAAGTAAAAGTCGT AACAAGG-3′ and ITS5 5′-TCCTCCGCTTTGATATGC-3′. Cov tshuaj tiv thaiv PCR muaj 10 ul ntawm 2 × PCR master sib tov (i-Taq™, Cat. No. 25027), 2 ul ntawm gDNA, 1 ul ntawm txhua primer (10 pmol / ul), thiab ua tiav rau 20 ul nrog sterile distilled dej. PCR yog programed rau thawj denaturation ntawm 94 degree rau 2 min, denaturation ntawm 94 degree rau 30 s, annealing ntawm 55 degree rau 10 s, extension ntawm 72 degree rau 30 s rau 35 cycles, thiab fnal extension ntawm 72 degree rau 2 min. PCR amplicons tau soj ntsuam los ntawm 1.5 feem pua ​​agarose gel hauv 1 × TBE bufer (Ambion Cat# AM9864), siv 1 kb DNA ntaiv (Cat. # PG010-55DI) thiab pom los ntawm cov ntaub ntawv gel. Cov amplicons tau huv si thiab ua raws los ntawm Applied Biosystems Sequencer, HiSQV Bases, Version 6.0 nrog tib cov primers teeb. Cov kev sib tw tau txais tau BLAST tshawb nrhiav tsis yog rov ua dua ntawm NCBI cov ntaub ntawv, raug xa mus rau hauv MEGA 6.0 software, thiab ua raws li Clustal W leeg algorithm [43] thiab tsob ntoo phylogenetic tau tsim nrog cov neeg nyob sib ze-txoj kev ntawm MEGA 6.0 [44].

Chemical Structure ntawm Extracted Taxol

Cov chaw tso quav ntawm Taxol tau muab pov tseg-los ntawm TLC silica gel daim hlau thiab purified, thiab purity thiab concentration tau txiav txim siab los ntawm UV-Vis tsom xam ntawm λ 227 nm (RIGOL, Ultra-3000 Series) piv rau cov qhabnias Taxol. [39]. Cov xov xwm tsis muaj nyob rau hauv tib yam kev mob tau siv los ua qhov tsis zoo hauv paus rau kev tshuaj ntsuam spectrophotometric. FT-IR spectrum ntawm purified Taxol kuaj tau soj ntsuam los ntawm JASCO FT-IR 3600 spectrophotometer. Cov qauv Taxol yog hauv av nrog KBr pellets, nias rau hauv cov discs hauv lub tshuab nqus tsev, thiab qhov nqus tau ntsuas hauv thaj tsam 400 txog 4000 cm-1 [3], piv rau qhov tseeb. Cov qauv tshuaj ntawm rho tawm Taxol tau lees paub los ntawm HNMR spectroscopy (JEOL, ECA-500II, 500 MHz NMR) piv rau cov qhabnias Taxol. Cov qauv raug daws tsis tau hauv CDCl3, cov tshuaj hloov pauv tau muab rau hauv ppm (δ-scale), thiab qhov sib txuas tsis tu ncua yog qhia hauv hertz (Hz).

Cov nyhuv ntawm ntau hom kev tshaj tawm ntawm Taxol Production

Ob lub agar plugs (9 mm) los ntawm 7 hnub qub kab lis kev cai ntawm txhua tus kab mob fungal cais tau inoculated hauv triplicate rau hauv 100 ml nruab nrab / 250 ml Erlenmeyer hwj ntawm qos dextrose (PDB), Czapek's-Dox (CZD), M1D, thiab malt extract (ME ) cov media. Uninoculated tswj los ntawm txhua qhov kev tshaj tawm uas tsis muaj cov kab mob fungal tau siv los ua kev tswj tsis zoo, incubated ntawm 30 degree rau 15 hnub nyob rau hauv tib yam mob. Tom qab incubation, fungal kab lis kev cai raug lim, thiab Taxol tau muab rho tawm thiab txiav txim siab raws li tau hais los saum toj no.

Bioprocess Optimization ntawm cov khoom noj khoom haus kom ua tiav cov txiaj ntsig Taxol

Kev ua kom zoo ntawm qhov nruab nrab muaj pes tsawg leeg rau kev ua kom tiav cov txiaj ntsig Taxol los ntawm qhov muaj zog lom zem gal cais tau ua los ntawm cov lus teb saum npoo txheej txheem siv Placket-Burman tsim ua raws li cov qauv hauv nruab nrab [17–20, 45]. Los ntawm RSM cov qauv tsim, qhov zoo thiab qhov tseem ceeb hloov pauv cuam tshuam rau kev tsim cov Taxol los ntawm cov kab mob fungal uas muaj zog tau raug soj ntsuam los ntawm kev siv cov ntaub ntawv txheeb cais los ntawm Design-Expert 7.0 (Stat Ease Inc., Minneapolis, USA). Txhua qhov kev sim tau ua nyob rau hauv peb cov khoom siv roj ntsha thiab cov txiaj ntsig nruab nrab tau txiav txim siab. Tom qab incubation ntawm qhov xav tau, fungal biomass tau pom, thiab Taxol tau muab rho tawm thiab ntsuas los ntawm TLC thiab HPLC raws li tau piav qhia saum toj no.

Plaket-Burman Design

Lub placket-Burman tsim tau nquag siv rau kev ua kom zoo ntawm cov khoom siv xov xwm rau kev loj hlob ntawm cov kab mob fungal thiab tsim cov khoom siv bioactive theem nrab metabolites, ntsuas qhov txawv txav tseem ceeb cuam tshuam rau Taxol ntau lawm [18, 20, 46]. Qhov kev xaiv ntawm qhov tseem ceeb yog raws li kev tshaj tawm uas siv hauv kev tshuaj ntsuam xyuas kom zoo thiab muaj nuj nqis. Kaum ib yam tseem ceeb tau suav nrog; malt extract, peptone, sucrose, soytone, glutamine, nqaij nyug extract, thiab kub, pH, incubation lub sij hawm, thiab co ceev qhov tseem ceeb thiab yam tseem ceeb tau varied nyob rau hauv ob theem, thiab qhov tsawg kawg nkaus thiab siab tshaj plaws theem tau xaiv. Tus Txheej Txheem Tsim-Cov Kws Paub 7.0 tau siv los tsim cov txheej txheem ntawm 12 qhov kev sim. Rau txhua qhov kev sim, Taxol ntau lawm tau txiav txim siab nyob rau hauv peb cov khoom siv roj ntsha, thiab qhov nruab nrab ntawm Taxol tawm los tau txiav txim siab.

Kev txheeb xyuas cov ntaub ntawv rov qab tau ua los ntawm kev siv cov ntaub ntawv txheeb cais. Cov txiaj ntsig ntawm txhua qhov sib txawv tau suav nrog (Biometrika, 2020), siv cov kab zauv hauv qab no:

Qhov twg, E yog cov txiaj ntsig ntawm qhov kev sim sib txawv, M ntxiv, thiab M- yog Taxol concentration ntawm kev sim ntawm qhov ntsuas qhov ntsuas tau nyob ntawm nws qib siab dua thiab qis dua, thiab N yog tus naj npawb ntawm cov kev sim uas tau ua. Cov txiaj ntsig ntawm txhua qhov sib txawv ntawm kev tsim khoom tau txiav txim siab los ntawm kev suav lawv cov E-tus nqi.

Qhov twg Tot siab yog tag nrho cov lus teb nyob rau theem siab, Tot qis yog tag nrho cov lus teb nyob rau theem qis, thiab Tsis yog tus lej ntawm kev sim.

Central Composite Design thiab kev sib cuam tshuam ntawm cov xwm txheej cuam tshuam rau kev tsim cov Taxol

Qhov tseem ceeb tshaj plaws zoo cuam tshuam rau Taxol ntau lawm los ntawm kev xaiv fungal iso lig tau optimized siv lub teb nto hom CCD qauv sim tsim [47]. Los ntawm kev siv CCD, qhov ntau ntawm cov khoom nruab nrab tau ua kom zoo dua qub, thiab lawv cov kev kawm sib cuam tshuam tau siv los tsim tag nrho 20 qhov kev sim rau peb qhov sib txawv. Txhawm rau txiav txim siab qib siab ntawm qhov hloov pauv rau Taxol ntau lawm los ntawm cov muaj zog fungal cais tawm, peb-dimensional (3D) cov lus teb nkhaus nkhaus tau npaj los kawm txog kev sib cuam tshuam ntawm ntau yam thiab txiav txim siab qhov sib txawv ntawm txhua yam cuam tshuam rau Taxol ntau lawm. Cov duab 3D tau ua tiav los ntawm kev tuav peb yam 'qhov tsis tu ncua nyob rau hauv qib zoo tagnrho thiab npaj cov lus teb tau txais los ntawm Taxol yield rau qib sib txawv ntawm ob yam.

Cov nyhuv ntawm Gamma Irradiation ntawm Taxol Yield

Lub zog endophytic isolates uas tsim Taxol tau raug -irradiation nrog 60Cobalt qhov chaw (Gamma cell 4000-A-India) ntawm cov koob tshuaj gamma sib txawv (0.25–3.0 kGy) piv mus rau kev tswj ntawm cov kab lis kev cai uas tsis yog irradiated; ib koob tshuaj 1.2 kGy / h thaum lub sijhawm sim. Cov xov xwm optimized tau inoculated los ntawm irradiated kab lis kev cai nyob rau hauv tus qauv kab lis kev cai tej yam kev mob, piv rau cov non-irradiated spore lub inoculum raws li kev tswj. Cov kab lis kev cai tau tsim nyob rau ntawm 30 ± 2 degree rau 15 hnub ntawm kev sib hloov shaker (120 rpm). Tom qab incubation, cov kab lis kev cai raug lim thiab Taxol raug muab rho tawm, huv si, thiab quantified los ntawm TLC thiab HPLC raws li tau piav saum toj no.

Synthesis thiab Characterization of Gold Nanoparticles (AuNPs); Conjugation nrog Taxol

Anticancer Activity ntawm Taxol

Cov kev ua ntawm cov purified Taxol thiab Taxol-PVP-AuNPs conjugates tiv thaiv daim siab carcinoma (HPG2), thiab mis carcinoma (MCF7) yog txiav txim los ntawm 3- (4,5-dimethylthiazol- 2-yl) -2, 5-diphenyl tetrazolium bromide (MTT) assay [48]. 96- lub phaj zoo tau muab noob nrog 103 lub hlwb rau ib lub qhov dej, thiab incubated hmo ntuj ntawm 37 degree, ces sib txawv ntawm cov tshuaj tau ntxiv, thiab cov phaj tau rov incubated rau 48 teev. MTT reagent (25 ul) tau ntxiv, thiab incubated rau 2 h, thiab cov xim liab ntawm cov tsim formazan complex yog ntsuas ntawm λ570 nm. Tus nqi IC50 tau qhia los ntawm tus nqi ntawm cov tshuaj txo qhov kev loj hlob ntawm 50 feem pua ​​​​ntawm thawj tus lej ntawm cov qog hlwb normalizing rau kev tswj tau zoo.

Antimicrobial Activity ntawm Taxol thiab Taxol-AuNPs Conjugates

Cov tshuaj tiv thaiv kab mob ntawm Taxol thiab Taxol-AuNPs conjugates tau soj ntsuam tawm tsam cov kab mob sib txawv; Bacillus subtilis ATCC 6633 thiab Staphylococcus epidermidis, Pseudomonas aeruginosa, Escherichia coli, thiab Enterobacter agglomerans, ntxiv rau Candida albicans. Cov kab mob kuaj tau raug tshem tawm hauv dej tsis huv peptone kom tau txais cov qauv inoculum ntawm ~0.5 McFarland (1–1.5) × 1{{10}}8 CFU/ml ntawm λ6{{18 }}0 nm. Kev loj hlob inhibition (mm) ntawm microbial pathogens kev loj hlob tau soj ntsuam los ntawm agar disc diffusion txoj kev. Sterile standard antibiotic disks nrog ib txoj kab uas hla ntawm 6.0 mm tau siv raws li kev tswj zoo. Sterile antibiotic discs (6.0 mm) tau ntim nrog 20 ul ntawm methanol thiab amoxicillin-clavulanic acid (AMC) raws li kev tswj tsis zoo thiab zoo. Cov discs tau ntim nrog tib qhov concentration ntawm Taxol, Taxol-PVP-AuNPs, thiab AuNPs (1.0 ug / ml). Peb cov khoom siv roj ntsha tau npaj tseg. Cov phaj tau incubated ntawm 37 degree rau 24 teev, thiab cov cheeb tsam ntawm inhibition raug ntsuas. Amoxicillin clavulanic acid (AMC) thiab nystatin tau siv los normalize cov tshuaj tiv thaiv kab mob ntawm Taxol. Qhov inhibition cheeb tsam ntawm kev loj hlob yog txiav txim los ntawm ib tug vernier caliper (mm).

Kev Tshawb Fawb Txog Kev Tshawb Fawb

Morphological thiab Molecular Identification ntawm cov muaj peev xwm Taxol Producers

Morphological nta ntawm cov muaj zog fungal cais tawm ua Taxol tau tshuaj xyuas raws li macroscopical thiab microscopical piav qhia cov yuam sij, raws li tau piav qhia hauv Cov Khoom Siv thiab Cov Txheej Txheem, thiab nthuav tawm nws qhov sib thooj morphological nrog A. favus (Fig. 2). Cov fungal cais tawm tau loj hlob ntawm PDA ntawm 30 degree rau 10 hnub thiab macroscopical thiab microscopical nta qhia nws tus kheej rau xws li lub taub hau conidial, hom ntawm ceg, tus kheej ntawm stigma, thiab conidial ontology, thiab fruiting lub cev tsim, raws li universal. Cov yuam sij morphological [33], thiab tau pom tias zoo ib yam rau Aspergillus favus. Lub zog Taxol-tsim cais A. favus tau txheeb xyuas ntxiv raws li lawv cov kab ke, siv gDNA ua tus qauv. PCR amplicons (~ 550 bp) ntawm A. favus raug daws, ntxuav, thiab ua ntu zus (Fig. 2). Lub ITS ib ntus ntawm A. favus yog qhov tsis muaj kev tshawb nrhiav hauv NCBI database, qhia txog 99 feem pua ​​​​zoo ib yam nrog A. favus, nrog xoom E. qhov tseem ceeb, thiab 95 feem pua ​​​​cov lus nug. Yog li, los ntawm microscopical thiab molecular analyses, lub hom phiaj cais tau lees paub tias yog A. favus thiab tso rau ntawm GenBank nrog tus lej nkag MW485934.1, nrog rau, kev cais tawm tau muab tso rau ntawm Assiut University Mycological Center (AUMC), Egypt nrog rau Tus lej xov tooj AUMC13892. Kev cais tawm tam sim no muaj 99 feem pua ​​​​zoo sib xws nrog A. favus cais MW485934, MT446145, KJ863514, MW522551,

Fig. 1 Ib qho Morphological saib ntawm jojoba tsob nroj. B Phaj kab lis kev cai ntawm lub zog Taxol-tsim endophytic fungi; A. favus Bd1 (13), A. niger Lv1 (21), Penicillium polonium (23), thiab A. oryzae Bd (25) ntawm PDA tom qab 8 hnub o incubation ntawm 30 degree . Cov fungal cais tawm tau loj hlob ntawm PDB, thiab incubated ntawm cov txheej txheem, thiab Taxol tau muab rho tawm thiab tshuaj xyuas los ntawm TLC (C). D HPLC chromatogram ntawm Taxol los ntawm cov muaj zog fungal cais tawm. E Yield of Taxol raws li ntsuas los ntawm HPLC. F, UV-Vis spectral tsom xam ntawm rho tawm Taxol los ntawm fungal cais. G FT-IR tsom xam ntawm rho tawm Taxol piv rau qhov tseeb

Fig. 2 A Macromorphological nta ntawm A. favus ib qho endophyte ntawm jojoba tom qab 3, 5, thiab 8 hnub ntawm kev loj hlob ntawm PDA. Micro-morphological nta, conidial taub hau ntawm A. favus los ntawm 400X magnification. C PCR amplicon ntawm A. favus ITS cheeb tsam ntawm 500 bp, normalizing rau 1 kb ntaiv (Cat.#. SM0312). D Phylogenetic tsom xam ntawm ITS A. favus los ntawm qhov siab tshaj plaws txoj kev [44]

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