Am J Clin Exp Med 2014_10.11648.j.ajcem.20140202.12

8/10/2019 Am J Clin Exp Med 2014_10.11648.j.ajcem.20140202.12

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American Journal of Clinical and Experimental Medicine2014; 2(2): 14-21Published online March 20, 2014 (http://www.sciencepublishinroup.co!/"/a"ce!)doi: 10.11#4$/".a"ce!.20140202.12

The Reproducibility of the multiplex RAPD-PCR assay in

genotyping of Mycobacterium tuberculosis isolates fromula!esi" #ndonesia

Mochammad Hatta1, *

, AndiRofian Sultan1, Ressy Dwiyanti

1, 3, Muhammad Sabir

1, 3,

Andini Febrianty1, 3

, Ahmad Adhyka1, 3

, ur !ndah "urnamasari#, Muhammad Re$a "rima%una

#,

&uhri Sanin%3, 'usriani Man%aren%i

3, Munawir Muhammad

3, ataniel (andiro%an%

),

'adi 'asir), Masyhudi Amir

)

1Molecular %iolo& and '!!unolo& aborator& or 'nectious *iseases, +acult& o Medicine, asanuddin niersit&, Maassar,

'ndonesia2*epart!ent o Microbiolo&, +acult& o Medicine, aluoleo niersit&, endari, 'ndonesia

*epart!ent o Microbiolo&, +acult& o Medicine, adulao niersit&, Palu, 'ndonesia4*epart!ent Microbiolo&, +acult& o Medicine, Mulawar!an niersit&, 3a!arinda, 'ndonesia

mail address+hattara!indosat.net.id (M. atta), ar5sultan2002&ahoo.co! (6. 7. 3ultan), ress&5chan&ahoo.co.id (7. *wi&anti),

destada!ba&ahoo.co! (M. 3abir), andini5ebriant&$8&ahoo.co.id (6. +ebriant&), dh&ao!ail.co! (6. 6dh&a),

nurindahpurna!asari!ail.co! (9. '. Purna!asari), repri!a!ail.co! (M. 7. Pri!auna), "uhri.sanin&ahoo.co! (. 3anin),

&usrianiaris&ahoo.co! (entral 3ulawesiin

eastern reion o 'ndonesia belon to roup M-> ($0.8D, $0.0D and #2.# D respectiel&) with result reproducibilit& as

hih as 100D. >onclusion:Molecular t&pin with !ultiple= 76P*-P>7 is a powerul approach to show the enetic

heteroeneit& o M> isolates. he discri!ination power and reproducibilit& o this !ultiple loci-based 76P* was hiher

than conentional ewer loci-tareted 76P*.

eywords+ 76P*-P>7, ?enot&pin,Mycobacterium Tuberculosis>o!ple=, 3ulawesi 'ndonesia

1- !ntroduction

uberculosis is still one o the leadin causes o death b&

inectious diseases with 1. !illiondeaths per &ear and $.#

!illion new cases annuall&(). Meanwhile, !orethan 2

!illion people are inected with latent tuberculosis

inection (!"#). *espitecontinuous eort in the preention,

accination, !onitorin and treat!ent o tuberculosis, thediseasere!ains a !a"or health proble! in !an& countries


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6!erican ournal o >linical and B=peri!ental Medicine 2014; 2(2): 14-21 1C

($"%), particularl& in deelopin countriesincludin

'ndonesia (&). Ene o the !ain proble!s is insuicient

nowlede about the enetic diersit& o circulatin

Mycobacterium tuberculosis isolates.

%& appl&in !olecular t&pin !ethods as an ad"unct to

the classical epide!ioloical approach, !olecular

epide!iolo& is an i!portant wa& to stud& tuberculosis

trans!ission d&na!ics and to learn !ore about the

epide!iolo& o tuberculosis. he !ethods o !olecular

epide!iolo&, especiall& '3#110 7estricted +ra!ent

enth Pol&!orphis! (7+P) o M. tuberculosis, were

irst introduced in outbrea inestiations and its

application was then raduall& e=panded to a population-

based stud& in apan. '3#110 7+P is obiousl& a powerul

tool or strain dierentiation o Mycobacterium

tuberculosis but its labor-intensieness li!its the

achieable throuhput and !aes it less useul or lon-

ter! prospectie studies. 7ecentl&, a *96 a!pliication-

based !ethod, has appeared as a substitute or or ad"unct tothe '3#110 7+P, i.e., ariable nu!ber o tande! repeats

(@97) (').Ether t&pes o assa&s such as M@6 allow

the inestiator to li!it the Fuantit& o reFuired enot&pin

to onl& epide!ioloicall& or ph&loeneticall& inor!atie

!arers, dependin on the branch depth(reardin ti!e) to

which a ien data set should to be anal&ed (). 7apid

chanes in Mycobacterium tuberculosis complex (M>)

enot&pin !ethodoloies hae led to onoin debate

about the choice o the best enot&pin strate& (!( )).

+urther!ore, seeral enetic loci within the M>

eno!es are pol&!orphic and can be used or

!oleculareolutionar& studies with pol&!erase chainreaction (P>7) or an isother!al a!pliication

assa&(#).7ando! 6!pliied Pol&!orphic *96 (76P*),

also nown as arbitraril& pri!ed P>7, allows the detection

o pol&!orphis!s without prior nowlede o the

nucleotide seFuence. he pol&!orphis!s !a& sere as

enetic !arers to construct the enetic !aps. his !ethod

utilies short (>10 nucleotides) pri!ers o arbitrar&

nucleotide seFuence that are annealed in the irst ew c&cles

o P>7 at low strinenc&. >urrentl&, 76P* is increasinl&

appliedin epide!ioloic t&pin o a wider rane o

!icrooranis!s includin M> ($( *). 6lthouh the

techniFue is si!ple and rapid, reproducibilit& issues hae

been reported due to its sensitiit& to pri!er ariation,

*96 concentration, *96 te!plate Fualit&, el

electrophoresis, and t&pe o *96 pol&!erase (+(

%).Preious studies hae shown that !ultiple= 76P* with

ten, seen and ie rando! deca!erolionucleotide pri!ers,

respectiel&,in a sinle P>7 was particularl& useul or

strain dierentiation o Mycobacteria isolates because it is

able to increase the nu!ber oinor!atie enetic !arers

in co!parison with onl& one round o a!pliication(*"%).

'n this prospectie enot&pin stud& coerin tuberculosis

trans!ission in the island o 3ulawesi, Bastern 'ndonesia,

we were able to show that Multiple= 76P*-P>7 usin 11

pri!ers is reproducible; this techniFue reealed a hihdiersit& o M> isolates.

#- Methods

2.1. Sample Collection

+our hundred and ninet&-three sputu! sa!ples ro!

suspected tuberculosis patients were obtained ro! seeral

pri!ar& health cares in 3outh 3ulawesi, 3outheast 3ulawesiand >entral 3ulawesi ro! 200C to 2012. Microscop& and

culture assa& were peror!ed accordin to the standard

dianostic !ethods e!plo&ed at the *epart!ent o Medical

Microbiolo&, Molecular %iolo& and '!!unolo&

aborator& in asanuddin niersit&, Maassar, 'ndonesia.

Giehl-9eelsen stainin with so!e !odiications was used

or !icroscopic detection (&). 3putu! sa!ples were

deconta!inated and cultured on owenstein ensen

!ediu!, which is locall& produced (!'( !).

2.2. DNA Preparation

*96 was e=tracted ro! reshl& collected isolatecolonies o M> accordin to the diato!-

uanidiniu!isothioc&anate (?u3>9) !ethod described b&

attaet al.(!!). a loop ull o the bacterial colonies was

!i=ed with A00 ul o l&sis buer (C0 !Mris->l, C.2C M

?u3>9, 20 !M B*6, 0.1D riton H-100), orte=ed

iorousl&, and centriued at 1,000 rp! or C !in. o

obtain the *96, sa!ples were l&sed b& incubation or 1C

!inutes at 1$I> and 20 Jl o diato! suspension was added.

he diato! containin the bound *96 was centriued at

12,000 K or 1C seconds to obtain diato! pellet. he

diato! pellet was then washed with washin buer (C.2C

M ?u3>9 in 0.1 M ris->l, p #.4), rinsed with 80D

ethanol and acetone, and dried b& incubation at C#I> or 10

!inutes. he pellet was !i=ed with #0 ul o 10 !Mris-

>l, p $.0, 1 !M B*6 buer and the *96 was eluted

b& incubation at C#I> or 10 !inutes. 6ter sedi!entation

o the diato! b& centriuation, the supernatant was

collected and stored at -20I> until P>7 was peror!ed.

2.3. Molecular Fingerprint

P>7 usin the 76P* !ethod was peror!ed with 11

pri!ers accordin to ai et al. (*), 3inh et al. L1A and

6ssad M et al (+). *96 a!pliication reaction was

peror!ed in a total olu!e o 2C Nl. he reaction !i=ture

contained 1 unit o aF *96 pol&!erase 0.12C; M>l ,

d9P 0.C; 10 K buer 2.C; te!plate *96 C Nl; dd2E

12,$8C Nl and each pri!er 0 p!ol (62: CO

?>>?6?>? O; 7$: CO->>>??>>-; 10: CO

6>>>??>6> O; 20: CO 6>>>??>6> O; EP9-01:

CO >>6>??? O ; EP9 02: CO 6>>6????>6 O;

EP9-0C: CO6>?66>?>> O ; EP9 0A: CO

?>>??>? O; EP9 20: CO ???>>>? O; %?-

#C: CO >>?6?>??> O; %?-##: CO >?6>?>?>? O ).

6!pliication was carried out in a ther!al c&cler (&baid

EM9 B, ). +ollowin an initial denaturation (pre-P>7)

step or C !in at A4I>, the c&clin condition consisted o

4C c&cles o the denaturation step or 1 !in at A4I>, theannealin step or 1 !in at #I>, and the e=tension step or


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1# Mocha!!ad atta et al.: he 7eproducibilit& o the Multiple= 76P*-P>7 6ssa& in ?enot&pin oMycobacterium

Tuberculosis'solates ro! 3ulawesi, 'ndonesia

1 !in at 82I>, ollowed b& the inal e=tension (post-P>7)

or C !in at 82I>. Blectrophoresis was peror!ed ater

76P*-P>7 usin 2.C D aarose el stained with ethidiu!

bro!ide and 100 bp !arer. 3ubseFuentl&, the el was

isualied and photoraphed usin a el docu!entation and

anal&sis s&ste!. >luster anal&sis was peror!ed b& the

unweihted-pair roup !ethod with an arith!etic !ean

(P?M6) alorith!, and a rooted tree was enerated

(http://!inisatellites.u-psud.r). 'n this stud&, a enot&pe is

deined as a strain with a distinct 76P*-P>7 pattern. he

distance between two enot&pes is deined as the !ini!u!

nu!ber o chanes in the data !atri= co!posed o

nu!erals 1 and 0 (presence: 1,or absence: 0).

o ealuate the stabilit& o the !ethods, each sa!ple o

eno!ic *96 was a!plied in duplicate in repeated P>7s

at dierent ti!es. o test or reproducibilit&, the *96s

ro! 4$# isolates were e=tracted ro! 2 independent

cultures. 6ll a!plications were done with riorousl&

standardied concentrations o reaents, the sa!e ther!alc&cler and the sa!e c&clin conditions.

2.4. Ethical Approval

he pro"ect receied approal ro! the !edical ethical

reiew board o the *epart!ent o 9ational Bducation o

the asanuddin niersit&. Eral inor!ed consent was

obtained ro! all stud& participants ater e=planation o the

procedure and the purpose o the stud&. Eral inor!ed

consent was applied as the collection o the speci!ens did

not aect the surical procedure to an& e=tend and all

clinical data was !ade anon&!ous beore anal&sis. he

collection o inor!ed consent was witnessed b& a nurse

and or the !edical oicer in chare and was recorded in the

patientOs !edical ile. he erbal consent procedure was

approed b& the !edical ethical reiew board.

3- Results

42 76P* enot&pes were tested in 2CC clinical isolates

ro! 3outh 3ulawesi, 1A ro! 3outheast 3ulawesi, AA

ro! >entral 3ulawesi, and a 87 laborator& strain as

control. 7esults showed that the discri!ination power was

increasin b& dierent loci that associated with each other

and the results reproducibilit& was 100D. he reater the

nu!ber o loci that were co!bined, the bier the unter-

?aston inde= was. +or e=a!ple, the unter-?aston inde=

o EP9 0A associated with EP9 02 or 3outh 3ulawesi was

0.A#, b& which 12# strains could be diided into 42 roups

(anal&ed usin the P?M6 alorith! onhttp://!inisatellites.u-psud.r, data not shown).

6 dendora! was enerated or all 4A enot&pes. he

dendrora! o M> isolates showed a hih deree o

pol&!orphis! thereore the enot&pes were rouped in

our !ain clusters, i.e. eno!ic roups M-6,M-%, M->

and M-* (iure 1).he !a"orit& o the isolates ro!

3outh 3ulawesi, 3outheast 3ulawesi and >entral 3ulawesi

belon to roup M->($0.8D, $0.0D and #2.#D,

respectiel&)(iures2-4).hose M> 'solates reealed a

continuu! o related enot&pes.

Figure 1. The dendro,raph of Mycobacterium tuberculosis complex -MTC ,enotypes. RAPD analysis /as performed for #&) MTC sputum culture

isolates from tuberculosis from eastern part of 0ndonesia -1outh( 1outheast and Central 1ula/esiand 2)+R3 isolates from laboratory stoc4. 5ased on the

3ariation obser3ed at four hi,hly 3ariable loci a rooted tree /as constructed by the un/ei,hted"pair ,roup method /ith an arithmetic mean al,orithmusin, 2)+R3strainas reference and depicted to,ether /ith the RAPD pattern and the number of isolates obtained from each ,enotype.


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Figure 2. The dendro,raph of Mycobacterium tuberculosis complex -MTC ,enotypes. RAPD analysis /as performed for !$$ MTC sputum culture

isolates from tuberculosis from 1outh 1ula/esi( 0ndonesia and2)+R3 isolates from laboratory stoc4. 5ased on the 3ariation obser3ed at four hi,hly

3ariable loci a rooted tree /as constructed by the un/ei,hted"pair ,roup method /ith an arithmetic mean al,orithm usin, 2)+R3strainas reference and

depicted to,ether /ith the RAPD pattern and the number of isolates obtained from each ,enotype.

Figure 3. The dendro,raph of Mycobacterium tuberculosis complex -MTC ,enotypes. RAPD analysis /as performed for )& MTC sputum culture

isolates from tuberculosis from 1outheast 1ula/esi( 0ndonesia and2)+R3 isolates from laboratory stoc4. 5ased on the 3ariation obser3ed at four hi,hly

3ariable loci a rooted tree /as constructed by the un/ei,hted"pair ,roup method /ith an arithmetic mean al,orithm usin, 2)+R3strainas reference anddepicted to,ether /ith the RAPD pattern and the number of isolates obtained from each ,enotype.


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1$ Mocha!!ad atta et al.: he 7eproducibilit& o the Multiple= 76P*-P>7 6ssa& in ?enot&pin oMycobacterium


Figure 4. The dendro,raph of Mycobacterium tuberculosis complex -MTC ,enotypes. RAPD analysis /as performed for && MTC sputum culture isolates

from tuberculosis from Central 1ula/esi( 0ndonesia and2)+R3 isolates from laboratory stoc4. 5ased on the 3ariation obser3ed at four hi,hly 3ariable loci

a rooted tree /as constructed by the un/ei,hted"pair ,roup method /ith an arithmetic mean al,orithm usin, 2)+R3 strain as reference and depicted

to,ether /ith the RAPD pattern and the number of isolates obtained from each ,enotype.

able 1 shows that the enot&pe diersit& was hih,

ranin ro! 2#.2D to $.D o the 42 enot&pes that wereidentiied !ore than once, and bein detected at two or all

three areas. he diersit& in 3outh 3ulawesi was

$.Dersus#4.D in >entral 3ulawesi and 2#.2D in

3outheast 3ulawesi (anal&ed usin the P?M6 alorith!

on http://!inisatellites.u-psud.r, data not shown). he

hihest prealent enot&pe was EP9 20 loci; the lowest

was EP9 0.2 loci (able 1). he EP920loci was !ost

prealent in enot&pe M-> and three other er& si!ilar

enot&pes !ain up a distinct branch o the enetic tree,

indicatin that the EP920 loci !a& be captured ro!

another bacteriu! on a rare occasion and subseFuentl&spread b& ertical trans!ission. he spatial cluster o

EP920 positie isolates in >entral 3ulawesi or!ed b& the

two er& si!ilar enot&pes M-6 and the M-% indicates

that in that particular area either o these enot&pes was

introduced and that the other enot&pe eoled ro! its

proen&. +urther!ore, the EP9 0.2 loci was rarel&

prealent in enot&pe M->, in line with indins ro! a

preious stud& (+).

Tale 1.6ourty t/o ,enotypes on loci from #&) MTC isolates in eastern part of 0ndonesia.

umber of

isolate

A# R. /10 /#0 "201 "20# "2#0 "20 "2 04 5627 56277

8 0 0 0 0 0 0 1 1 0 0 1

8 1 0 0 0 0 0 1 1 0 0 1

8 0 1 0 0 0 0 1 1 0 0 1

8 0 0 1 0 0 0 1 1 0 0 1

8 0 0 0 1 0 0 0 1 0 0 1

8 0 0 0 1 0 0 1 1 0 0 1

$ 1 1 1 0 1 1 0 1 0 1 0

8 1 0 1 0 1 0 1 1 1 1 1

8 0 1 1 1 1 0 1 0 0 0 0

8 1 0 1 0 1 0 1 0 1 1 0

8 1 1 1 1 1 0 1 0 0 1 0

8 0 1 1 1 1 1 1 1 1 1 1

8 0 1 1 0 1 1 0 1 0 1 1

8 0 1 1 1 1 1 1 1 1 0 1

11 0 0 1 0 1 0 1 0 1 1 011 0 1 1 1 1 0 1 0 0 1 0


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umber of

isolateA# R. /10 /#0 "201 "20# "2#0 "20 "2 04 5627 56277

11 1 0 1 1 1 1 1 1 1 1 1

11 1 1 1 0 1 0 1 1 1 1 1

11 0 1 0 0 1 0 0 0 1 0 0

11 1 0 1 1 1 1 1 1 1 0 1

12 0 0 1 1 1 0 1 0 0 0 0

11 0 0 1 1 1 0 1 1 1 0 1

11 1 1 1 0 1 0 1 0 1 1 0

11 1 0 1 1 1 0 1 0 0 1 0

11 1 1 0 1 1 1 1 1 1 1 1

11 1 1 0 0 1 1 0 1 0 1 1

11 1 0 0 0 1 0 1 1 1 1 0

14 1 1 0 1 1 1 1 1 1 0 1

14 0 1 0 1 1 0 1 0 0 0 0

14 0 1 0 1 1 0 1 1 1 0 1

1C 1 0 0 0 1 0 1 0 1 1 0

14 1 1 0 1 1 0 1 0 0 1 1

14 1 1 1 0 1 1 1 1 1 1 1

14 1 0 1 1 1 0 1 1 1 1 1

14 0 1 0 1 0 0 1 0 0 0 0

14 1 1 1 0 1 1 1 1 1 0 11$ 0 1 1 0 1 0 1 0 0 0 0

1$ 0 1 1 0 1 0 1 1 1 0 1

21 1 0 1 1 1 0 1 0 1 1 0

21 1 1 1 0 1 0 1 0 0 1 1

2C 1 0 1 0 0 0 0 0 1 0 0

2C 1 0 1 0 1 0 0 0 1 0 0

Percentae C4.$ C4.$ #4. 48.# $1.0 2#.2 $. C8.1 C4.$ C0.0 C8.1

)- Discussion

'n a preious stud& (Maidin, et al. 201), enetic diersit&

o 8 strains ro! the sa!e areas in 3ulawesi were anal&ed

b& nu!erical anal&sis o 76P* P>7 iner printins with

ie olionucleotide pri!ers(18).'t was ound that 76P* is

aster and technicall& less de!andin than !ost other

!olecular t&pin !ethods and urther!ore, no *96

seFuence inor!ation is necessar&. 6lso, !uch s!aller

a!ounts o puriied *96 are reFuired than or !ethods such

as 7+P. 76P* is relatiel& si!ple and useul or

epide!ioloical anal&sis, but standardiation o its P>7

!i=ture and conditions are essential or its reproducibilit&

(1$). +urther!ore, een with this standardiation,

reproducible proiles are diicult to obtain, and it is

necessar& to peror! duplicate anal&sis or the true proile

dierences to be dierentiated ro! e=peri!ental ariation.

'n this stud&, 76P* was !odiied and applied as a!olecular tool or enot&pin 4Aclinical isolates o M>

ro! ende!ic area in 3ulawesi 'ndonesia. he resolution o

this Multiple= 76P*-P>7 was assessed on the basis o

traditional epide!ioloical tracin inor!ation and 76P*

data. 'n contrast to indins ro! preious studies,

Multiple= 76P*-P>7 assa& was also able to show hih

reproducible results, which was one o the !ain issues

surroundin conentional 76P*-P>7.

his stud& ies eidence that supports the use o 76P*

anal&sis in deter!inin ariabilit& within a population. his

!ethod o inerprintin is aluable in that it is relatiel&

eas& to obtain aluable data. 't allows or a !oreintrospectie interpretation o diersit& within a population.

his stud& can also sere as a reerence point or uture

e=a!inations o enetic ariation within populations o

M>. +urther!ore, it can be a !odel or other studies

inestiatin enetic diersit& within a M> strain. 'n this

stud&, the use o pri!er EP9 20 reealed the speciic *96

ra!ent that was !ost prealent o M> isolates. >lusteranal&sis o distinct enot&pes !a& also be used to loo or

!a"or sources o trans!ission o M>. oweer, the lare

diersit& in enot&pes ound in the inestiated reions in

3ulawesi shows that the nu!ber o tuberculosis

trans!ission can be lare. raelin and increased

!iration sti!ulated b& oern!ent-sponsored

trans!iration prora!s !a& hae pro!oted the spread o

particular enot&pes. he stabilit& o the 76P* loci is not

nown and so!e closel& related enot&pes !a& hae arisen

independentl& at the areas. Possibl&, uniFue enot&pes,

unrelated to those ound in the present stud&, with a !ore

li!ited eoraphic distribution !a& be encountered in!ore re!ote areas.

- 8onclusion

Molecular t&pin o bacterial isolates based on

pol&!orphis!s in eno!ic *96 proides a powerul

approach or distinuishin M> and !a& proide

aluable insiht into the i!portance o dierent hosts and

eoraphic reions in the !aintenance and trans!ission o

tuberculosis inection. he discri!ination power and

reproducibilit& o !ultiple loci 76P* was !uch hiher

than that o ewer loci.


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20 Mocha!!ad atta et al.: he 7eproducibilit& o the Multiple= 76P*-P>7 6ssa& in ?enot&pin oMycobacterium


Acknowled%ments

Qe are rateul to all participants o this stud& or their

oluntar& cooperation. Qe than the sta ro! the hospitals

and health centers in Maassar, 3outh 3ulawesi proince,

endari, 3outheast 3ulawesi proince and Palu, central

3ulawesi proince or their enthusiasticsupport o the stud&.Qe are indebted to the heads o *istrict ealth

*epart!ents in 3outh, 3outheast and >entral

3ulawesi,*r.6neswenan,*r.3&a!sulrial,*r.'rene,Mr.

6li7otib,Mr.a!id,Mrs.7ini,Mrs.'na,Gr.Btt&,Mrs.Ba,Mrs.

Purwaninsih, Mr. Marus, and Mr. Mus ubaru or

dedicatin their precious ti!e to the superision o clinical

and laborator& e=a!inations.

+inall&, we than Mr. 7o!i s!an, Mr. Marwani, Mr.

3&ari, and Mr. 7och!at o the asanuddin niersit& or

their dedicated contributions to the ield wor actiities and

peror!in the blood cultures and sa!ple preparations or

!olecular testin.

8om9etin% interests

he authors declare that the& hae no co!petin interests.

Authors: contributions

M, 67M3 and 7* conceied and desined the

e=peri!ents.M, 67, 7*, 6+, 66, 9'P, M7P, 9 and M3

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