Optimization of r andomly amplified polymor phic DNA-polymer ase
chain r eaction for molecular typing of
Salmonella enterica serovar
Typhi
Otimização da reação de amplificação aleatória do DNA polimórfico – reação em cadeia
da polimerase para tipagem molecular de
Sa lm o ne lla e nte rica
sorovar
Typhi
Bianca Ramalho Quintaes1 ,2
, Nilma Cintr a Leal3
, Eliane Mo ur a Falavina Reis1
and Er nesto Ho fer1
ABSTRACT
Op ti m i za ti o n o f th e RAPD re a c ti o n f o r c h a ra c te ri zi n g Salmo ne lla e nte r ic a se ro va rTyp h i stra i n s wa s stu d i e d i n o rd e r to
e n su re the re pro du c i b i li ty a n d the di sc ri m i n a to ry po we r o f thi s te c hn i q u e . Ei ght Salmo ne lla se ro va rTyphi stra i n s i so la te d
f ro m va ri o u s re gi o n s i n Bra zi l we re e x a m i n e d f o r th e f ra gm e n t p a tte rn s p ro d u c e d u si n g d i f f e re n t c o n c e n tra ti o n s o f DNA
te m p la te , p ri m e r, MgCl2 a n d Taq DNA p o lym e ra se . Usi n g two d i f f e re n t lo w stri n ge n c y th e rm a l c yc le p ro f i le s, th e RAPD
f i n ge rp ri n ts o b ta i n e d we re c o m p a re d . A se t o f si x te e n p ri m e rs wa s e va lu a te d f o r th e i r a b i li ty to p ro d u c e a h i gh n u m b e r o f distin c t fra gm e n ts. We fo u n d tha t va ria tio n s a sso c ia te d to a ll o f the te ste d pa ra m e te rs m o difie d the fin ge rprin tin g pa tte rn s.
Fo r th e stra i n s o f Salmo ne lla e nte r ic a se ro va r Typ h i u se d i n th i s e x p e ri m e n t, we h a ve d e f i n e d a se t o f c o n d i ti o n s f o r
RAPD-PCR re a c ti o n , wh i c h re su lt i n a si m p le , f a st a n d re p ro d u c i b le typ i n g m e th o d .
Ke y-wor ds: Opti m i za ti o n . RAPD – PCR. Salmo nella enter ic a se ro va rTyphi . Bra zi l.
RESUMO
A o ti m i za ç ã o d a re a ç ã o d e RAPD p a ra a c a ra c te ri za ç ã o d e c e p a s d e Salmo ne lla e nte r ic a so ro va rTyp h i f o i e stu d a d a c o m o
o b je ti vo d e a sse gu ra r a re p ro d u ti b i li d a d e e o p o d e r d i sc ri m i n a tó ri o d e sta té c n i c a . Oi to c e p a s d e Salmo ne lla so ro va rTyphi
i so la da s de a lgu m a s re gi õ e s do Bra si l f o ra m u sa da s pa ra e xa m i n a r o s pa drõ e s de f ra gm e n ta ç ã o pro du zi do s q u a n do f o ra m
e m p re ga d a s c o n c e n tra ç õ e s d i f e re n te s d o DNA m o ld e , d o i n i c i a d o r, d o MgCl2 e d a e n zi m a Taq DNA p o li m e ra se . Co m a
u ti li za ç ã o de do i s di f e re n te s pe rf i s de c i c lo s te rm a i s de b a i xa e stri n gê n c i a , f o ra m c o m pa ra do s o s pa drõ e s de b a n de a m e n to o b tido s. Um c o n ju n to de de ze sse is in ic ia do re s fo i a va lia do q u a n to à c a pa c ida de de pro du zir e le va do n ú m e ro de fra gm e n to s di sti n to s. Ob se rvo u - se q u e va ri a ç õ e s a sso c i a da s a to do s o s pa râ m e tro s te sta do s m o di f i c a ra m o s pa drõ e s de b a n de a m e n to .
Pa ra a s a m o stra s de Salmo nella enter ic a so ro va rTyphi u ti li za da s n e ste e xpe ri m e n to , de f i n i u - se u m c o n ju n to de c o n di ç õ e s
p a ra a re a ç ã o d e RAPD- PCR q u e re su lto u n u m m é to d o d e ti p a ge m si m p le s, rá p i d o e re p ro d u tí ve l.
Palavr as-chave s: Oti m i za ç ã o . RAPD – PCR. Salmo nella enter ic a so ro va rTyphi . Bra si l.
1 . Lab o r ató r io de Zo o no se s B ac te r ianas do De par tame nto de B ac te r io lo gia do Instituto Oswaldo Cr uz da Fundaç ão Oswaldo Cr uz, Rio de Jane ir o , RJ. 2 . De par tame nto de Mic r o b io lo gia Mé dic a da Unive r sidade do Estado do Rio de J ane ir o , RJ . 3 . Lab o r ató r io de Mic r o b io lo gia do Ce ntr o de Pe sq uisas Agge u Magalhãe s, Re c ife , PE. This wo r k was suppo r te d b y Natio nal Co unc il fo r Sc ie ntific and Te c hno lo gic al De ve lo pme nt ( CNPq ) .
Addr e ss to: Dr a. B ianc a Ramalho Quintae s. Lab o r ató r io de Zo o no se s B ac te r ianas/DB /FIOCRUZ. Av. B r asil 4 3 6 5 , 2 1 0 4 5 - 9 0 0 Rio de J ane ir o , RJ , B r asil. Re c e b ido par a pub lic aç ão e m 2 /7 /2 0 0 3
Ac e ito e m 8 /1 2 /2 0 0 3
Fo r the study o f the e pide mio lo gy o f typho id fe ve r, ne w
mo lec ular typing metho ds have been develo ped and impro ved
to c o mple me nt Vi phage typing, the mo st use ful te c hnique to distinguish o ne S. e n te ri c a ser o varTyphi str ain fr o m ano ther.
One of them, RAPD-PCR ( Random Amplified Polymorphic DNA) ,
c an generate simple and repro duc ible fingerprints o f geno mic
DNA in a PCR r e a c tio n b y us in g s in gle pr im e r s c h o s e n
ir r e spe c tive o f the ge no me se que nc e to b e finge r pr inte d2 0 2 2.
Thus, RAPD-PCR requires no prior knowledge of the molec ular
b io lo gy o f the o r ganisms to b e inve stigate d. The amplific atio n o c c ur s at lo w str inge nc y, allo wing the pr ime r s to anne al to
several lo c atio ns o n the two strands o f the DNA. These primers
de te c t po lymo r phisms in the ab se nc e o f spe c ific se que nc e
As desc ribed previously1 4. It has been demonstrated that
RAPD-PCR r e ac tio n has the po te ntial to pr o vide a disc r iminato r y, r epr o duc ib le and easy to inter pr et metho d to type S. e n te ri c a
se r o varTyphi str ains. Ho we ve r, in o r de r to use RAPD-PCR fo r
diffe r e ntiatio n b e twe e n b ac te r ial str ains, the o ptimizatio n o f
the r e ac tio n is impe r ative to e liminate mo st o f the var iatio ns that ar e so me time s o b se r ve d in duplic ate DNA pr o file s2 4. The
standardizatio n o f so me parameters suc h as the MgCl
2 and Ta q
DNA po lym e r a s e e n zym e c o n c e n tr a tio n s , th e a n n e a lin g
te mpe r atur e and the the r mal c yc ling pr o file1 0 2 1 may le ad to a
more robust and reliable reac tion c apable of rec ognizing related
str ains and disc r iminating between unr elated str ains5.
I n the pr e se nt study we de fine the c o nditio ns fo r the
o ptimizatio n o f RAPD-PCR to S. e n te ri c a se r o var Typhi DNA
using 1 0 bp primer and demonstrate the effec ts in the fingerprint patte r n c ause d b y var ying the tar ge t DNA, MgCl
2 and Ta q DNA
po lyme r ase e nzyme c o nc e ntr atio ns and the the r mal c yc ling
pr o file . We also e valuate a to tal DNA e xtr ac tio n me tho do lo gy,
observing its time c onsumption and the stability of the resulting genetic material.
MATERIAL AND METHODS
The strains o f S. e n te ric a serovarTyphi used in the present
study are listed in Table 1 . These strains iso lated fro m humans
had b e e n maintaine d o n nutr ie nt agar slo pe s in the c ultur e
c o lle c tio n o f the Natio nal Re fe r e nc e Ce nte r fo r Cho le r a and Ente r ic Dise ase s, De par tme nt o f B ac te r io lo gy, Oswaldo Cr uz
Institute/FIOCRUZ, Rio de Janeiro, B razil. The c ultures for DNA
extrac tion were grown in 5 ml Brain Heart Infusion broth ( Difc o)
fo r 1 8 to 2 4 h at 3 7 ° C.
Total DNA was extrac ted as desc ribed by Sambrook et al1 5,
using 1 ml o f e ac h c ultur e . The DNA was q uantifie d, afte r
elec trophoresis in a 1 % agarose gel, by c omparison with known
amounts of Hind III digested by bac teriophage
λ
( lambda) DNA.Using S. e nte rica serovar Typhi genomic DNA as template in the
optimization steps, we first evaluated the DNA extrac tion method. An amount of 1 0 0 to 2 0 0 ng/ml was produced and this was adequate
to perform the amplific ation reac tions. The genetic material
remained stable for about 4 weeks, when stored at -2 0 ° C.
Preliminary assays with o ne, rando mly c ho sen, S. e n te ric a
sero varTyphi strain ( 5 0 1 – Oswaldo Cruz Institute Co llec tio n)
were c arried out with sixteen 1 0 -mer oligonuc leotides primers,
c o mme r c ially synthe size d, and aime d to te st the ir ab ility to
pr o duc e disc r iminato r y RAPD pr o file s in S. e n te ri c a se r o var Typhi. The pr ime r s we r e synthe size d at the Esc o la Paulista de
Medic ina, São Paulo, B razil ( Table 2 ) .
Ta b le 1 - Stra in s o f Salmonella enteric a se ro va rTyphi, a re a , iso la tio n so urc e a nd ye a r o f iso la tio n.
Strain nº Area Isolation sourc e Year 4 9 2 north ( Ac re) blood 1 9 9 5 4 9 6 north ( Ac re) blood 1 9 9 5 5 0 1 north ( Ac re) blood 1 9 9 5 3 0 mid west ( Distrito Federal) blood 1 9 7 2 6 5 5 mid west ( Distrito Federal) blood 1 9 9 5 6 5 6 mid west ( Distrito Federal) blood 1 9 9 5 T6 8 northeast ( Pernambuc o) blood 1 9 3 7 T5 4 northeast ( Pernambuc o) blood 1 9 3 8
Ta b le 2 - Cha ra c te ristic s o f the prim e rs use d fo r the RAPD- PCR re a c tio n with
S. enteric a se ro va r Typhi.
Pr ime r Sequenc e GC ( % ) Tm ( ° C) 7 8 4 5 ’GCG GAA ATA G 3 ’ 5 0 3 0 7 8 5 5 ’CCG CAG CCA A 3 ’ 7 0 3 4 7 8 6 5 ’GCG ATC CCC A 3 ’ 7 0 3 4 7 8 7 5 ’AAC GCG CAA C 3 ’ 6 0 3 2 7 8 8 5 ’GTG GAT GCG A 3 ’ 6 0 3 2 7 8 9 5 ’AGC CAG TTT C 3 ’ 5 0 3 0 7 9 0 5 ’GTC AAC GAA G 3 ’ 5 0 3 0 7 9 1 5 ’GAG ACT CCC C 3 ’ 5 0 3 0 7 9 2 5 ’GGT ACT CCC C 3 ’ 7 0 3 4 7 9 3 5 ’GAC CGA CCC A 3 ’ 7 0 3 4 7 9 4 5 ’ACT GAA CGC C 3 ’ 6 0 3 2 7 9 5 5 ’GAC ACG CAC A 3 ’ 6 0 3 2 7 9 6 5 ’ACC TCA GCT C 3 ’ 6 0 3 2 7 9 7 5 ’AGC GTC ACT C 3 ’ 6 0 3 2 7 9 8 5 ’TGA CCC GCC G 3 ’ 8 0 3 6 7 9 9 5 ’GGC TTG GCC G 3 ’ 8 0 3 6
We c onduc ted this step under non-standardized c onditions: the r e ac tio n was pr e par e d in a to tal vo lume o f 2 5
µ
l pe r tub e ,c ontaining 2 0 ng DNA of the strain 5 0 1 , 3 U Ta q DNA polymerase
( CENB I OT/RS) , 1 0 m M Tr is HCl, 5 0 m M KCl, 1 . 5 m M MgCl
2,
2 0 0 mM o f eac h dNTP and 2 0 pM/ml o f pr imer.
Co nside r ing the amplific atio n pr o duc ts finge r pr int, we selec ted one primer and first evaluated the effec ts of varying the
Ta q DNA polymerase enzyme c onc entration ( 1 U/2 5 ml, 2 U/2 5
µ
l, 3 U/2 5µ
l, 4 U/2 5µ
l and 5 U/2 5µ
l) using str ain numb e r 5 0 1 .Fu r th e r m o r e , th e a s s a ys we r e c a r r i e d o u t a t di ffe r e n t c o nc e ntr atio ns o f MgCl
2 ( 2 mM, 3 mM and 4 mM) , DNA ( 2 0 ng
and 4 0 ng) and pr ime r ( 2 0 pM/ml and 4 0 pM/ml) at the r mal
c yc le A, pr o gr amme d fo r 3 0 c yc le s c o mpo se d o f o ne ste p o f
denaturation for 1 min at 9 4 ° C, one annealing step for 1 min at 3 6 ° C fo llo we d b y a final synthe sis fo r 2 min at 7 2 ° C.
To evaluate the influenc e of thermal c yc le profile, six strains
we r e sub m itte d to the r m a l c yc le A a nd the r m a l c yc le B ,
pr o gr amme d fo r five c yc le s, c o mpo se d o f o ne initial ste p o f
de natur atio n fo r 1 min at 9 4 ° C, o ne ste p o f lo w str inge nc y te mpe r atur e o f anne aling fo r 1 min at 3 6 ° C and o ne ste p o f
synthesis o r amplific atio n fo r 2 min at 7 2 ° C. This was fo llo wed
b y 2 5 c yc le s o f high-str inge nc y te mpe r atur e o f anne aling,
c o nsisting o f 1 min at 9 4 ° C, 1 min at 5 0 ° C and 2 min at 7 2 ° C, finishing with an amplific atio n step fo r 7 min at 7 2 ° C.
The r e ac tio n tub e s we r e c o ve r e d with 6 0
µ
l o f ste r ilize dminer al o il and the amplific atio n to o k plac e in a DNA ther mal
c yc le r ( Pe r k in Elm e r 4 8 0 ) . Am plific a tio n pr o duc ts we r e
sub mitted to elec tr o pho r esis in 1 .5 % agar o se gel, Tr is-b o r ate buffer and a c onstant voltage of 1 0 0 V, followed by staining with
ethidium bro mide and visualizatio n in a UV transilluminato r. A
DNA. The
λ
phage DNA cleaved by Hind III restriction enzyme ( Sigma) and the synthetic DNA, ladder 100 ( Pharmacia) were employed as patterns for band molecules weight.RESULTS
Submitting 2 0 ng/
µ
l DNA template to 2 0 pM/µ
l primer, 1 .5 mM MgCl2 , 1 U/2 5
µ
l Ta q DNA polymerase at thermal cycle A, the primer7 8 4 ( sequence 5 ’GCG GAA ATA3 ’; 5 0 % GC content and Tm = 3 0 ° C) was chosen due to its ability to produce a higher number of bands and a better fingerprinting pattern ( Figure 1 ) . Varying the Ta q DNA polymerase concentrations markedly affected the amplification profiles obtained with primer 7 8 4 ( Figure 2 ) . Thus, reactions performed with inc reasing c onc entrations of the enzyme showed the best performance when using a concentration of 5 U/2 5
µ
l. The loss of fragments in the profiles observed using 1 U/2 5µ
l and 2 U/2 5µ
lhighlights the influenc e o f this parameter in the o ptimizatio n o f
RAPD reac tion.
Fi gu re 1 - RAPD- PCR f i n ge rp ri n ti n g o f S. e nte r ic a se ro va r Typ h i stra i n 5 0 1 DNA u si n g si x te e n a rb i tra ry p ri m e rs f o r se le c ti o n . a ) La n e s: 1, n e ga ti ve c o n tro l; 2, pha ge l di ge ste d wi th Hind III; 3 to 10, p ri m e rs 7 8 4 to 7 9 1 ; 1 1 , la d d e r 1 0 0 DNA. b ) La n e s: 1 , p h a ge λ
d i ge ste d w i th Hind III ; 2 to 9 , p ri m e rs 7 9 2 to 7 9 9 ; 1 0 , la d d e r 1 0 0 ; 1 1 , n e ga ti ve c o n tro l .
thermal c yc le A. Higher MgCl
2 c onc entrations ( 3 .0 and 4 .0 mM)
had signific ant e ffe c ts upo n the RAPD pr o file s pr o duc e d, sho wing fewer and indistinguishable bands.
Whe n we inc r e ase d the DNA te mplate c o nc e ntr atio n fr o m
2 0 to 4 0 ng/
µ
l and the primer concentration from 2 0 to 4 0 pM/µ
l, o nly a few o r no amplific atio n fr agments wer e o b ser ved.The analysis o f two different thermal c yc le pro files applied to 6 str ains o f S. e n te ri c a se r o var Typhi sho we d that 3 0 lo w
str inge nc y c yc le s, name d c yc le A was suffic ie nt to pr o mo te pr im e r /te m plate inte r ac tio ns and ge ne r ate disc r im inato r y
a m plific a tio n fr a gm e n ts . Ho we ve r, a la c k o f b a n ds a n d so metimes no amplific atio n was o bserved when applying c yc le B ( Figur es 3 a and 3 b) .
DISCUSSION
Various molec ular biologic al tec hniques suc h as ribotyping1
and pulse d- fie ld ge l e le c tr o pho r e sis ( PFGE)1 1 1 2 1 8 1 9 have
rec ently been applied to Sa lm o n e lla e n te ric a serovar Typhi for
epidemiologic al purposes. Nevertheless, there is still no rapid, r e liab le and suffic ie ntly disc r iminative me tho d fo r lab o r ato r y inve stigatio n o f the e pide mio lo gy o f typho id fe ve r8. Thus, the
use of random amplific ation of polymorphic DNA fingerprinting te c hnique ( RAPD) , a mo dific atio n o f the po lyme r ase c hain
Fi g u r e 2 - RAPD p a t t e r n s va r i a t i o n s a s s o c i a t e d t o Ta q DNA p o lym e ra se e n zym e c o n c e n tra ti o n o b ta i n e d f ro m S. e nte r ic a se ro va r Typ h i stra i n 5 0 1 DNA u si n g p ri m e r 7 8 4 . La n e s: 1 , la d d e r 1 0 0 DNA; 2 , 1 U/ 2 5 µ l; 3 , 2 U/ 2 5 µ l; 4 , 3 U/ 2 5 µ l; 5 , 4 U/ 2 5 µ l; 6 , 5 U/ 2 5 µ l.
The number and the intensity of fragments produc ed varied
in func tio n o f the the r mal c yc ling pr o file , the MgCl2, DNA te m p l a te , p r i m e r a n d Ta q D NA p o l ym e r a s e e n zym e
c o n c e n tr a ti o n s . Ma i n ta i n i n g th e DNA a n d p r i m e r 7 8 4
c o nc e ntr atio ns at 2 0 ng/
µ
l and 2 0 pM/µ
l, r e spe c tive ly, andvar ying the MgCl2 c o nc e ntr atio n, we o b se r ve d that the b e st finge r pr inting patte r n was o b taine d using 2 .0 mM MgCl
r e ac tio n ( PCR) in whic h ar b itr ar y o ligo nuc le o tide s pr ime r s are used to promote DNA synthesis at low stringenc y c onditions
in o r de r to de te r mine ge no mic dive r sity, may b e c o nside r e d a
pro mising alternative typing metho d c apable o f disc riminating
b e twe e n S. e n te ri c a se r o var Typhi str ains o f the same phage type1 4. B esides differentiating isolates of serovar Typhi and other
Sa lm o ne lla isolates,RAPD-PCR proved c apable of disc riminating
b e twe e n S. e n te ri c a ser o var Typhi str ains1 6. RAPD-PCR assays
are simpler, faster, more c onvenient and easier to perform than mo st o the r mo le c ular typing me tho ds. Ho we ve r, o nly whe n
use d unde r we ll de fine d and o ptimize d c o nditio ns RAPD is
c apable o f repro duc ing the amplific atio n o f rando m fragments
o f DNA and ge ne r ating high de gr e e s o f po lymo r phism6 1 7.
The DNA extr ac tio n metho d r epo r ted her e was tec hnic ally e asy to pe r fo r m and no t time c o nsuming. The to tal mate r ial
obtained was maintained stable when stored at -2 0 ° C for about
four weeks, reproduc ing the same profiles without lac k of bands
or c learness. However, whole c ell reac tion produc ts if analyzed afte r 2 4 h o f am plific atio n m ay pr o duc e sm e ar e d pr o file s
pr o b a b ly due to th e pr e s e n c e o f de gr a da tive e n zym e s th a t
a r e s ta b le dur in g th e c yc lin g r e a c tio n a n d a c tive dur in g sto r age6.
The standar dizatio n o f DNA te m plate c o nc e ntr atio n is
important to avoid artifac ts on the band patterns. Using 2 0 ng/
µ
lDNA template, RAPD produc ed well resolved profiles, but many
b ands we r e lo st whe n using 4 0 ng/
µ
l. As r e po r te d b e fo r e , an e xtr ac tio n k it to e xtr ac t DNA fr o m the c e lls wo r k e d we ll withRAPD analysis and altho ugh it did no t quantify the amo unt o f
DNA, the te mplate c o nc e ntr atio n o ve r a wide r ange had no
signific ant effec t on the RAPD profiles produc ed1 6 2 3. The primer
c o nc e ntr atio n influe nc e was also e valuate d. Inc r e asing the
pr ime r c o nc e ntr atio n fr o m 2 0 pM/
µ
l to 4 0 pM/µ
l pr o duc e dsimilar finger pr int pr o files.
We fo und that magnesium io n c o nc entr atio n was a c r itic al
element in determining the performance of amplification reaction and signific antly varied the profiles produc ed. Low magnesium
io n c o nc e ntr atio ns may r e sult in po o r r e ac tio n e ffic ie nc y and
high c o nc e ntr atio ns may r e sult in po o r r e ac tio n spe c ific ity1 3.
B e c ause we we r e wo r k ing with no nspe c ific pr ime r-te mplate inte r ac tio n we e xpe c te d that inc r e asing the c o nc e ntr atio n o f
magnesium ion had the net effec t of dec reasing the stringenc y of
pr ime r b inding. Sur pr isingly, a no to r io us lac k o f b ands was
no tic e d whe n using 3 .0 mM MgCl2 and the b e st pe r fo r manc e o c c ur r e d at a lo we r c o nc e ntr atio n, 2 .0 mM MgCl
2. The same
r e sults we r e o b se r ve d fo r Ye rsi n i a e n te ro c o li ti c a str ains,
whic h have the best performanc e for RAPD reac tion at 2 .0 mM9.
In c ontrast, for S. e n te ric a serovar Enteritidis, no differenc e in the RAPD finge r pr int pr o file s was o b taine d var ying MgCl
2
c o nc entratio n fro m 2 .0 mM to 4 .0 mM7.
R e a c ti o n b u ffe r p H wa s m a i n ta i n e d a t 8 . 0 i n a l l
e xpe r im e nts altho ugh a var iatio n in pH as sm all as 0 . 4 c an
make the differenc e between a disc riminatory array of fragments and no amplific ation6.
B ec ause RAPD variations assoc iated to thermal c yc le profile
( Figur e s 3 a and 3 b ) may o c c ur, this par ame te r sho uld b e
e valuate d whe n standar dizing RAPD r e ac tio n. The Ta q DNA
polymerase preparations c an be c lassified as a major sourc e of variations ( Figure 2 ) . B esides the c onc entration, we also found
var iatio ns whe n c hanging the pr o duc tio n lo t o f Ta q DNA
po lyme r ase o f the same b r and, sho wing the impo r tanc e o f
ac quir ing an appr o pr iate quantity o f this e nzyme to c o mple te the r e se ar c h. As pe r fo r me d pr e vio usly, the use o f diffe r e nt
b r ands o f Ta q DNA po lyme r ase r e ve ale d maj o r var iatio ns
between the patterns o btained1 0.
If RAPD is to b e use ful as a typing me tho d, a suffic ie nt dis c r im in a to r y n um b e r o f a m plifie d fr a gm e n ts m us t b e pr o duc e d, whic h e sse ntially de pe nds o n the pr ime r-te mplate
inte r ac tio n6. Eac h finge r pr int r e fle c ts the suc c e ssful pr ime
r-direc t targeting of a set of sites in the genome2. For this reason,
the c ho ic e o f an appr o pr iate set o f 1 0 -mer o ligo nuc leo tides is impe r ative o r the disc r iminato r y po we r o f RAPD analysis may
dec rease. Thus, eac h primer gave a different fingerprint pattern altho ugh e ac h had the po te ntial o f de te c ting po lymo r phisms
b e twe e n str ains, the r e b y allo wing the diffe r e ntiatio n o f e ve n Fi gu re 3 - RAPD p a tte rn s va ri a ti o n s a sso c i a te d to th e th e rm a l
c losely related strains. From 1 6 arbitrary primers examined for
suitability, primer 7 8 4 was fo und to be spec ially useful fo r optimization steps and generated the best fingerprint patterns. In
addition, due to the arbitrary selec tion of primers, RAPD reac tion
may produce many amplification fragments or no fragments to any
extent ( Figure 1 ) . The use of ERIC ( Enterobac terial Repetitive Intergenic Consensus) primer in the RAPD reaction of S. e nte rica
serovarTyphi resulted in indistinguishable fingerprint patterns
that were unable to disc riminate between strains from different
geographic al origins3 4.
RAPD-PCR finge r pr int str ate gy sho uld b e applic ab le to b ac te r ial typing fo r its r apidne ss, simplic ity, lo w c o st and
po tential to gener ate po lymo r phisms. Our r esults suggest that
ther e is a c o nsider able po ssibility fo r inc r easing the effic ienc y
o f the RAPD-PCR reac tio n if a prec ise standardizatio n pro to c o l is determined. We have proposed a model that c an be used as a
suppo r t fo r typing str ains o f S. e n te ri c a se r o varTyphi.
ACKNOWLEDGEMENTS
To Mrs Deise Paranhos Feitosa (in m e m o ria m) , Mr Junair
Rib e ir o , Mr Evaldo So ar e s da Silva, Mr s Yar a Mar ia Maia
Nak asawa and Mr s Silvana Santo s fo r e xc e lle nt te c hnic al
assistanc e . To Dr Alzir a Mar ia Paiva de Alme ida and Dr Dália do s Pr aze r e s Ro dr igue s fo r the fac ilitie s o ffe r e d fo r c ar r ying
o ut this r e se ar c h.
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