Co esponding au ho : Ligia Ion
Copy igh © 2025 Au ho (s) e ain he copy igh o his a icle. This a icle is published unde he e ms o he C ea i e Commons A ibu ion Liscense 4.0.
Resea ch on genes iden i ica ion o an isep ic ac ion in some basil geno ypes
Mădălin Vasile Chiuoa u, Ad ian Chi a, Daniel Nagy, Gab iela Bu na iu and Ligia Ion *
Uni e si y o Ag onomic Sciences and Ve e ina y Medicine o Bucha es , acul y o Ho icul u e, 59 Mă ăș i Bl d, Dis ic
1, Bucha es , Romania.
Wo ld Jou nal o Ad anced Resea ch and Re iews, 2025, 27(02), 1266-1274
Publica ion his o y: Recei ed on 06 July 2025; e ised on 12 Augus 2025; accep ed on 15 Augus 2025
A icle DOI: h ps://doi.o g/10.30574/wja .2025.27.2.2959
Abs ac
The s udies unde aken by he p esen wo k e e o 21 domes ic and o eign geno ypes, in which a sc eening was done
wi h 8 molecula ma ke s, o he in es iga ion o he genome in e ms o highligh ing he gene ha con ols he
an isep ic ac ion and he esis ance o plan s o bio ic ac o s.
The collec ion o basil geno ypes is no e y la ge in Romania. The e a e only 150 au och honous basil geno ypes. The
genomic DNA isola ed om he basil geno ypes, “A oma de Buzau”, “BZ 1”, “Ho Igal 2”, “Gea”, “LBRS 2”, “LBVS 1”, e c.
was used o implemen o RADP molecula ma ke s o highligh he gene ha con ols an isep ic ac ion. In o de o
de e mine, he DNA samples ex ac ed om young lea es by he me hods o Lodhi e al. 1994, modi ied by Pop e al.,
2003 and Doyle & Doyle, 1990, we e quan i ied/measu ed, ampli ied, and he ampli ica ion p oduc s (RAPDs) we e
elec opho e ic ally sepa a ed in aga ose gel. The esea ch aimed o highligh he ObGAPDH gene in basil geno ypes
aken in he s udy using 8 speci ically designed molecula ma ke s.
Keywo ds: Ocimum Basilico; Geno ypes; Pa hogen; Molecula Ma ke s; Samples
1. In oduc ion
Ocimum is a genus o a oma ic plan s and pe ennial and annual sh ubs belonging o he Liliaceae amily, comp ising
nea ly 200 gene a and 3,200 species. The numbe o species in he Ocimum genus is unce ain due o se e al axonomic
di icul ies. The e o e, i could ha e 30–160 species. Ocimum basilicum L., o swee basil, is an impo an species known
o i s medicinal p ope ies and essen ial oil [15], [16]. I s lowe has bila e al symme y, wi h i e pe als and i e sepals;
he s em is e ec , b anched, solid, and hai y; he seeds a e o al-shaped and black; he lea is simple and opposi e, wi h
epide mal glands con aining a oma ic oil [12]. In ecen yea s, Ocimum basilicum has been ex ensi ely s udied o i s
a ious ac i i ies [4] [3].
In silico molecula docking is a apidly de eloping ield o unde s anding and p edic ing possible modes o in e ac ion
be ween a ligand and a a ge biomolecule [8, 9, 5, 11, 1], while molecula dynamics simula ions ha e p esen ed a
plausible way o es ima e he dynamic and ene ge ic s abili y o a p o ein-ligand complex [1, 7, 17, 6, 13, 16, 10].
The e o e, a docking analysis o he main cons i uen s o Ocimum basilica, linalool and es agole, wi h alpha-amylase
and lipase oge he wi h hei inhibi o s was planned.
The lipase inhibi o y ac i i y o plan ex ac s was es ima ed acco ding o a epo ed p o ocol [14]. The DPPH ee
adical sca enging po en ial o pola and nonpola ex ac s om Ocimum basilica lea es and lowe s was de e mined
acco ding o he epo ed es [ 2] PCR which is used o apidly make millions o billions o copies o a speci ic DNA
sample, ampli ying i exponen ially in a se ies o empe a u e change cycles, hus allowing scien is s o ake a e y small
sample o DNA and ampli y i o a quan i y la ge enough o be s udied. PCR echnology was pionee ed in 1983 by
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Ame ican biochemis Ka y Mullis a Ce us Co po a ion. The PCR p ocess is now an indispensable p ocedu e used in
medical labo a o y esea ch in a wide a ie y o applica ions, including biomedical and o ensic esea ch. The e a e
se e al PCR me hods, bu all a e based on he mal cycling. These expose he eac an s o epea ed empe a u e changes
o allow di e en eac ions o occu —in pa icula , he mel ing and enzyma ic eplica ion o DNA.
2. Ma e ial and me hods
2.1. Plan ma e ial consis s o 21 geno ypes o basil
The esea ch was conduc ed o e h ee g owing seasons, om 2021 o 2024, and in ol ed measu emen s o he
pheno ypic cha ac e is ics o di e en basil geno ypes, namely g ow h a e a 15 days, plan heigh , a e age bush
diame e , a e age shoo leng h, and a e age numbe o shoo s pe plan . O he cha ac e is ics s udied include he g een
mass weigh o basil o he 16 geno ypes s udied o e he h ee yea s alloca ed o he esea ch. O he esea ch
add essed he a e age alues o he ge mina ion pe iod o he 16 geno ypes s udied o e he h ee yea s alloca ed o
he esea ch.
Table 1 Plan ma e ial basil plan biome ics o he 16 geno ypes s udied
N ca .
Geno ypes s udied
A e age heigh o he plan (cm)
1.
A oma ic Basil om Buzau
75.50
2
Pu ple basil Se a im
77.10
3
Swee basil L1
62.20
4
Limone o basil L2
73.30
5
G eek basil
30.21
6
Basil wi h salad lea es L4
84.40
7
G and Ve Basil L5
60.30
8
Pe sian basil
52.48
9
Spicy Globe Basil L7
37.51
10
A ican basil L8
118.00
11
Holy basil a . K ishna L9
78.10
12
Macedonian basil
60.85
13
Dwa basil Ema ald
29.40
14
Da k Opal ed basil
56.56
15
Siam Queen Basil
51.48
16
Basil cinnamon
110.50
17
BZ 1
60.22
18
Ho igal 2
71.13
19
Gea
81.11
20
LBVS 1
60.32
21
LBVS 2
62.22
(su as/sou ce: o iginal)
A e he DNA isola ion, he amoun and pu i y o he DNA was de e mined o he 21 a ie ies s udied. These
de e mina ions we e made using he Nano D op ND 33 000 de ice, wi h he Eppendo Bio Pho ome e . The amoun o
DNA was exp essed in ng/all and he pu i y was highligh ed by ealizing he a io be ween wo wa eleng hs,
espec i ely 260/280.
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A e he pheno ypic de e mina ions we e made, geno ypic de e mina ions ollowed, s a ing wi h he isola ion o DNA
om he basil geno ypes s udied. The DNA was quan i ied using a nanod op and hen e ealed in a 2% elec opho esis
gel. An ampli ica ion ki om Applied Biosys ems wi h p ime s speci ic o he Ocimum Basilicum species was used o
pe o m PCR.
Following he op imiza ion o ampli ica ion condi ions, a PCR eac ion was pe o med in a empe a u e g adien . Thus,
a DNA sample was ampli ied a eigh di e en empe a u es: 51°C; 51.8°C; 53.1°C; 54.9°C; 57.4°C; 59.3°C; 60.5°C; 61°C
( able 2). The PCR ampli ica ion p oduc s we e mig a ed in 2% aga ose gel and isualized unde UV ansillumina o .
Conside ing he amplicon p o ile, he op imal p ime hyb idiza ion empe a u e was selec ed. The ampli ica ion
eac ion was pe o med as ollows: 20 all o eac ion mix u e was pipe ed in o each 0.2 all ube and 5 all o DNA was
added, espec i ely 5 all o deionized H2O in he case o he nega i e con ol. The samples we e o exed and gen ly
cen i uged, hen placed in he PCR machine. The PCR machine was se acco ding o he p og am in able 1.
Table 2 PCR eac ion scheme o op imize ampli ica ion condi ions
Tempe a u e / Timp
Dena u ise ini ial
95 ºC, 10 min.
Cyclu a
Dena u ise: 95 ºC, 30 sec.
Alime a p ime : 51- 61 ºC, 30 sec.
Ex ende : 72 ºC, 1 min.
N . Cyclu a
35
Ex ensae inal
72 ºC, 15 min.
(su as/sou ce: o iginal)
3. Resul s and discussion
Valida ion o amplicon iden i y is necessa y o e i y ha he ampli ied DNA co esponds o he chosen a ge sequence.
Aga ose gel elec opho esis is a simple me hod o e i ying he size o PCR p oduc s. Howe e , sequencing he
amplicons and compa ing he esul s wi h hose in da abases is he mos e ec i e me hod o e i ying he esul s
ob ained by PCR. The eac ion p oduc s ob ained by con en ional PCR we e sequenced a e pu i ica ion wi h he
Wiza d PCR P eps DNA Pu i ica ion Sys em (P omega). The ma ke s and p ime s used o PCR ampli ica ion we e
labeled wi h Big Dye (ABI PRISM® Big Dye™ Te mina o Cycle Sequencing Ready Reac ion Ki , Applied Biosys ems).
Resul s ega ding he quan i ica ion o genomic DNA a nanod op o he s udied basil geno ypes (2021-2023) a e
summa ized in Table 3.
Table 3 Resul s ega ding he quan i ica ion o genomic DNA a nanod op o he s udied basil geno ypes
N .
Ca .
Geno ype
Genomic and
concen a ion (ng/
all)
Repo abs.
260/280 1.98
Repo abs.
260/230 2,01
Abso p ion D260
24.532
1.
A oma ic Basil om
Buzau
1226.6
1.98
2.01
24.532
2.
Pu ple basil Se a im
2108.2
1.96
2.02
42.164
3.
Swee basil L1
1630.2
1.97
1.98
32.603
4.
Lemon basil L2
4847.3
1.7
1.64
96.945
5.
G eek basil
259.3
1.71
1.12
5.185
6.
Basil wi h salad lea es
L4
429.7
1.71
0.97
8.593
7.
G and Ve Basil L5
870.6
1.71
1.05
17.411
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1269
8.
Pe sian basil
1481.2
1.86
1.43
29.625
9.
Spicy Globe Basil L7
164
1.71
0.98
3.279
10.
A ican basil L8
442.8
1.72
1.21
8.856
11.
Holy basil a . K ishna
L9
479.5
1.67
1.01
9.59
12.
Macedonian basil
1478.7
1.82
1.36
29.575
13.
Dwa basil Ema ald
261.6
1.82
1.38
5.231
14.
Da k Opal ed basil
224.2
1.34
1.71
4.485
15
Siam Queen Basil
794.6
1.82
1.26
15.892
16
Basil cinnamon
222.4
1.25
1.23
12.852
17
BZ 1
462.2
1.52
1.12
4.241
18
Ho igal 2
481.1
1.61
1.22
3.123
19
Gea
391.5
1.45
1.14
4.472
20
LBVS 1
473.4
1.71
1.21
4.732
21
LBVS 2
444.2
1.62
1.11
3.751
(su as/sou ce: o iginal)
Table 3 shows he esul s o genomic DNA quan i ica ion by nanod op o he basil geno ypes s udied. The amoun o
genomic DNA a ied g ea ly om geno ype o geno ype, wi h he highes alue eco ded o he Limone o basil geno ype,
wi h a alue o 4847. The lowes alues we e eco ded o he Swee Basil geno ype wi h alues o 1630.2 ng/μl and he
Pe sian Basil geno ype wi h alues o 1481.2 ng/all. The lowes alues we e eco ded o he Spicy Globe basil geno ypes
wi h alues o 164 ng/all, he Da k Opal ed basil geno ype wi h alues o 224.2 ng/all, ollowed by he G eek basil
geno ype wi h alues o 259.3 ng/all and Basil cinnamon 222.4 ng/all
The inal DNA solu ion is p epa ed acco ding o he speci ic p epa a ion me hod, ei he by esuspending he DNA
sedimen a e i s p ecipi a ion wi h alcohols (e hanol o isop opanol) in he p esence o sal s. The gene ic ma e ial,
DNA, was s o ed (a -20ºC) in TE solu ion, bu i can also be s o ed in s e ile deionized wa e . In he p esen expe imen ,
he sedimen was esuspended in 100 all TE pH 8.0.
Table 4 Resul s o he uni ac o ial analysis o he ac o ega ding he quan i ica ion o genomic DNA a nanod op, o
he geno ypes s udied
SOURCE OF
THE VARIANT
VARIANT
GRADE o
Libe y
CORRECTED
DISPERSION
FISHER
FACTOR
CRITICAL FACTOR
(SPA)
(L)
(S2)
(F calcula e)
10 %
5 %
1 %
0.1 %
BETWEEN
GROUPS
(SYSTEMATICS)
5232179.07
14
373727.0763
0.5877
1.6620
1.9200
2.5200
3.3600
WITHIN
GROUPS
(RESIDUAL)
28618013.96
45
635955.8657
TOTAL
33850193.02
59
DIFERENCE
LIMIT (DL)
1894.688
3
2271.3704
3033.7568
3969.8231
(su as/sou ce: o iginal)
Thus, Table 4. p esen s he uni a ia e analysis be ween he ac o s o a ia ion ega ding he quan i ica ion o genomic
DNA a nanod op o he s udied basil geno ypes, which shows us a limi di e ence o 5% a 2271.3704, 1% a
3033.7568, and a 0.1% o 3969.8231, meaning he in luence o ac o s on he esul ing cha ac e is ics.
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(su as/sou ce: o iginal)
Figu e 1 Resul s o he uni ac o ial analysis o he ac o ega ding he quan i ica ion o genomic DNA a nanod op,
o he geno ypes s udied
A e DNA ex ac ion, he samples we e quan i ied spec opho ome ically using he Eppendo Bio Pho ome e in he
i s pa o he expe imen and he Nanod op NA 33 000 in he second pa o he expe imen . The spec opho ome ic
me hod is based on he ac ha mos biological subs ances ha e a cha ac e is ic abso p ion a e in he ul a iole
adia ion ange. Thus, he abso p ion a e o 260 nm co esponds o nucleic acids, ha o 280 nm o p o eins, and 230
nm o a ious con aminan s. The op ical densi y was measu ed a he abso p ion a es A 260 nm and A 280 nm,
calcula ing he a io be ween he wo abso p ions. (Figu e1.)
The uni a ia e ANOVA analysis o he amoun o genomic DNA ex ac ed om di e en Ocimum basilicum geno ypes
e ealed la ge a ia ions be ween geno ypes. The highes alue was eco ded in he Basil Limone o geno ype (4847.3
ng/all), ollowed by Basil iole Se a im (2108.2 ng/all), Swee basil (1630.2 ng/all), and Basil pe son (1481.2 ng/all). In
con as , he lowes alues we e ound in Spicy Globe basil (164 ng/all), Da k Opal ed basil (224.2 ng/all), and G eek basil
(259.3 ng/all).
Howe e , only he di e ence be ween he Limone o basil geno ype and he o he geno ypes exceeded he Limi
Di e ence (LD) a p < 0.05 o 2271.3704 ng/all, indica ing a s a is ically signi ican di e ence only in his case. The e o e,
Basil Limone o s ands ou as ha ing a signi ican ly supe io gene ic capaci y in e ms o genomic DNA accumula ion o
s abili y, which may indica e a highe po en ial o bio echnological o gene ic applica ions.
Rega ding he quan i ica ion o genomic DNA, he esul s we e qui e une en, wi h alues anging om 4847.3 ng/µl o
he "Basil Limone o" a ie y o 164 ng/µl o he "Basil Spicy Globe" a ie y.
Table 5 P ime s es ed a Og-USAMVB Ho in es
N Ca
P ime s
Sequence (5’-3’)
1.
Og A07
GAG ACG GGT A
2.
ObGA08
ATG ACG TAG G
3.
Og A09
CGG TAA CGC C
4.
ObGA10
CTG ATC GCA C
5.
Og A11
GAA TGC CCG T
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6.
ObGA12
CTG CAC CCA C
7.
Og A13
ATG TGA CCG T
8.
ObGB04
CGA CAG GTG A
(su as/sou ce: o iginal)
Howe e , gi en ha small amoun s o DNA in he o de o 10–20 ng/µl a e su icien o SSR ma ke analysis, i is
impo an o know he ini ial alues in o de o de e mine he necessa y dilu ions.
Fo molecula ma ke analysis, he eac ion mix u e was p epa ed unde a lamina low hood unde s e ile condi ions.
A con ol sample con aining all componen s o he eac ion mix u e excep DNA was also aken in o accoun . In o de
o ampli y he samples ob ained in he i s pa o he expe imen , eigh Deca nucleo ide p ime s we e used, each
p ime ha ing 10 nucleo ides (Table 5.) They we e chosen om among hose ha p esen ed polymo phic bands. These
p ime s a i e a he labo a o ies in lyophilized o m, each wi h a speci ic ecipe o dilu ion when used.
3.1. Resul s ob ained in he case o ampli ica ion o DNA ex ac ed using he Doyle and Doyle me hod
O he p ime s s udied, i e p ime s (ObGA07, ObGA08, ObGA11, ObGA13, ObGA18, and ObGB04) ampli ied bands in
only one a ie y, and wo o he p ime s, ObGA09 and ObGA10, did no ampli y any agmen s in any o he a ie ies
(Figu e 1.6), and he ampli ied bands we e unclea , making i impossible o assess hem accu a ely.
(su as/sou ce: o iginal)
Figu e 2 Resul s conce ning he bands ob ained om h ee a ie ies o he es p ime s Og
The e could be an explana ion ela ed o he DNA samples, which did no ha e he necessa y quan i y o pu i y o
p oduce polymo phism wi h he p ime s es ed, e en hough hey a e uni e sal and should ha e p oduced esul s. The
polyme ase used was Apli e Polyme ase (S o el) and no P omega, and he ampli ica ion powe may ha e been lowe .
(Figu e 2.)
3.2. Analysis and p ocessing o esul s ob ained by RAPD
The esul s e e o a o al o 8 samples, o which 2 e ealed polymo phism be ween geno ypes, while he o he 6
p esen ed monomo phic bands, wi h a o al o 75 bands gene a ed, o which 66 we e polymo phic (Table 6).
Table 6 RAPD da a ob ained om image analysis (2023)
N .
Ca .
P ime
To al numbe o
lanes
Lanes/p ime
No. o polymo phic
bands
Bands/p ime
No. o monomo phic
bands
Bands/p ime
%
polymo phism
1.
Og A07
12
11
1
99.6
2.
Og A08
9
8
1
88.8
3.
Og A09
7
6
1
85.7
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4.
Og A10
7
5
2
71.4
5.
Og A11
11
10
1
92.8
6.
Og A12
10
9
1
93.3
7.
Og A13
8
8
0
100
8.
Og B04
11
10
1
90.9
(su as/sou ce: o iginal)
In he labo a o y, band de ec ion was pe o med using he Faliscan p og am, by compa ing hem wi h a s anda d DNA,
wi h a size be ween 100 and 4000 bp. All agmen s esul ing om ampli ica ion wi h polymo phic p ime s we e
be ween 200 and 2000 bp in leng h, wi h mos be ween 300 and 1200 bp (in he i s ampli ica ion case).
3.3. In e p e a ion o esul s
In e p e a ion o he esul s ob ained by he RAPD me hod in ol es he genomic DNA isola ion p o ocol, he PCR
eac ion, he ampli ica ion p og am, and image acquisi ion using he Alpha Image 2200 p og am. The smalle he
gene ic dis ance alues in he able (bina y ma ix), he mo e closely ela ed he geno ypes a e. These p ima y da a
we e en e ed in o he RAP Dis ance 1.04 p og am, which calcula ed he gene ic dis ances based on he Jacca d
coe icien (Table 7.). The o mula used o calcula e he Jacca d coe icien (Jij) is as ollows:
Jiji = Cin/ (in + Nj – Cin)
whe e: Cin - ep esen s he numbe o iden ical bands p esen ed in he wo geno ypes; nj - ep esen s he o al numbe
o bands iden i ied in geno ype I and j, espec i ely.
Table 7 Resul s ega ding he gene ic dis ance o basil geno ypes
N ca .
Geno ypes
Gene dis ance (come)
Ma ke Og A07
Gene dis ance (come)
Ma ke Og B04
1.
A oma de Buzău basil
1/04
1/42
2.
BZ 1
1/04
1/42
3.
Ho Igal 2
1/04
1/42
4.
Gea
1/04
1/42
5.
LBRS 2
1/12
1/62
6.
LBVS 1
1/12
1/62
7.
Se a im basil
1/12
1/62
8.
Swee basil
1/04
1/42
9.
Basil Limone o
1/12
1/62
10.
G eek basil L3
1/12
1/62
11.
Basil F unze de sala L4
1/21
1/69
12.
Basil G and Ve L5
1/21
1/69
13.
Pe sin basil
1/21
1/69
14.
Basil Spicy Globe L7
1/21
1/69
15.
A ican basil
1/21
1/69
16.
Basil K ishna L9
1/21
1/69
17.
Basil Macedon
1/21
1/69
18.
Basil Pi i Ema ald
1/21
1/69
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19.
Da k Opal basil
1/21
1/69
20.
Basil Siam Queen
1/21
1/69
21.
Basil Cinnamon
1/21
1/69
(su as/sou ce: o iginal)
Table 7. shows he esul s o he gene dis ance calcula ion o he wo ma ke s ha showed he highes pe cen age o
polymo phism, ObGA07 and Og B04. Bo h molecula ma ke s es ablish he p esence o he ObGAPDH gene, bu he
signi icance o i s exp ession co e s mo e dis an cha ac e s o o eign geno ypes.
(su as/sou ce: o iginal)
Figu e 3 Resul s conce ning he bands ob ained om h ee a ie ies o he es p ime s Og
Among he s udied p ime s, i e ampli ied bands in only one a ie y Ho igal 2, (ObGA07, ObGA08, ObGA11, ObGA13,
ObGA18 and ObGB04), and 2 o he p ime s: ObGA09 and ObGA10 did no ampli y any agmen in any o he a ie ies
o basil ( igu e 3.), and he ampli ied bands had poo cla i y, no being able o make a ai assessmen o hem. The e
could be an explana ion ela ed o he DNA samples no ha ing he quan i y o pu i y equi ed o gi e polymo phism
wi h he p ime s es ed, al hough hey a e uni e sal and should ha e gi en esul s, he polyme ase used was Apli e
Polyme ase (S o el) and no P omega, and he ampli ica ion powe may ha e been lowe .
4. Conclusion
In he labo a o y, he bands we e de ec ed using he Faliscan p og am, by compa ing hem wi h a s anda d DNA sample,
wi h a size be ween 100 and 4000 bp. All he agmen s esul ing om he ampli ica ion wi h he polymo phic p ime s
we e be ween 200 and 2000 bp in leng h, mos o hem be ween 300 and 1200 bp (in he i s case o ampli ica ion).
The lowe he alues o he gene ic dis ances in he able (bina y ma ix), he mo e closely ela ed he geno ypes a e.
These p ima y da a we e en e ed in o he RAPD dis ance 1.04 p og am which calcula ed he gene ic dis ances based on
he Jacca d coe icien . R esul s ega ding he calcula ion o he gene ic dis ance o he wo ma ke s ha showed he
highes pe cen age o polymo phism ObGA07 and Og B04. Bo h molecula ma ke s es ablish he p esence o he
ObGAPDH gene, bu he signi icance o i s exp ession co e s mo e dis an cha ac e s o o eign geno ypes. The choice
o p ime s p o ed qui e di icul , ou o 8 p ime s used in o al, only 2 ga e dis inc polymo phic bands, among hem:
ObGA07 and ObGB04.
I u ned ou ha he RAPD echnique is a simple and as me hod o e ealing gene ic polymo phism in basil geno ypes
acco ding o he Doyle Doyle p o ocol o he CTAB me hod.
The RAPD echnique is ex emely sensi i e o wo king condi ions (appa a us, eagen s, solu ion concen a ions, hei
pH, e c.). Op imizing he RAPD p o ocol can be an ex emely di icul p oblem, changing he eac ion componen s o he
p og am used can ha e unp edic able consequences.
Compliance wi h e hical s anda ds
Disclosu e o con lic o in e es
No con lic o in e es o be disclosed.
Wo ld Jou nal o Ad anced Resea ch and Re iews, 2025, 27(02), 1266-1274
1274
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