Specific detection of Mycobacterium tuberculosis complex strains by polymerase chain reaction

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JOURNALOFCLINICAL MICROBIOLOGY,June1990, p. 1204-1213 0095-1137/90/061204-10$02.00/0

Specific Detection of Mycobacterium tuberculosis Complex Strains

by Polymerase Chain Reaction

PETER W. M. HERMANS,1*ANJA R. J. SCHUITEMA,2 DICK VAN SOOLINGEN,' CEES P. H. J. VERSTYNEN,2 ELISABETH M. BIK,' JELLE E. R. THOLE,2 AREND H. J. KOLK,2AND JAN D. A.VAN EMBDEN' National Institute of Public Health and Environmental Protection, P.O. Box 1, 3720 BA Bilthoven,' and N. H. Swellengrebel Laboratory of Tropical Hygiene, Royal Tropical Institute, 1105AZAmsterdam/ TheNetherlands

Received1December 1989/Accepted 12March 1990

During thescreening ofaMycobacterium tuberculosis lambdagt-11genelibrary with monoclonalantibodies, we detected a recombinant clone, lambda PH7311, which contained a mycobacterial DNA insert that hybridized specifically with DNA ofM.tuberculosiscomplexstrains. Partofthisinsertwassequencedandused for the development ofan M. tuberculosis complex-specific polymerase chain reaction (PCR). Only strains belongingtospeciesof the M. tuberculosis complexgroupcontainedanamplifiablefragment of 158 base pairs (bp). This fragment was absent in all strains tested belonging to 15 other mycobacterial species. After amplification by PCR and dot blot hybridization with a digoxigenin-labeled oligonucleotide, the limit of detection ofpurified genomicM. tuberculosis DNA amountedtoaquantity correspondingto20 bacterial cells. By this techniqueabout i03M. tuberculosisbacteriaweredetectable in sputum.UsingPCR,wewerealso able todetectM. tuberculosis cells in clinical material suchaspleuralfluid, bronchial washings, and biopsies, and theseresultswerecomparable with those obtainedbyclassicalbacterial culture. Of 34M.tuberculosis strains, 5 didnot carry theamplifiable 158-bp fragment, whichoccurs usuallyas asingle copy in the chromosome. Evidence is presented that the 158-bp fragment is locatednear arepeatedsequence inthe chromosome. We presumethatstrainswhich do notcarrythe 158-bp fragment havelostachromosomalsegmentbyagenetic rearrangementinduced by the repetitive DNAelement.

Although a presumptive diagnosis of tuberculosis can be madeonthebasis of patient histories, clinical and radiolog-icalfindings, and the presenceofacid-fastbacilli in patient specimens, the isolation of Mycobacterium tuberculosis, Mycobacterium africanum, or Mycobacterium bovis is re-quired for the definitive diagnosis of tuberculosis. Routine cultures are cumbersome and time-consuming. For that reason,thedevelopmentofamorerapid methodtodiagnose thediseaseis highlydesirable.

Microscopic examination ofsmears of acid-fast bacteria by Ziehl-Neelsen staining is the most rapid method for detection ofmycobacteria, but it is insensitive and nonspe-cific. A major limitation of immunological detection of infections with M. tuberculosis by nonculture methods, such as latex agglutination, radioimmunoassay, and enzyme-linked immunosorbent assay, is their lack of sensitivity and/orspecificity (13, 15, 43). Serological techniquesmaybe useful insomeclinical settings, but this approach is limited, in general, due topoor sensitivity and/or specificity (3, 24, 25). Great progress has been made in reducing the time requiredtodetect growth ofmycobacteria bymeasuring the radioactivity of carbon dioxide, produced as a result of 14C-labeled growth substrate of mycobacteria in a highly selective growth medium(BACTECsystem;Johnston Lab-oratories,Inc., Towson, Md.). However, on average, 10to

12daysarestillneededtodetect mycobacterial growth from clinicalspecimensortocarryoutsusceptibilitytests(12, 23, 32).

Recentadvances in DNAtechniques haveprovidedanew approachtorapiddiagnosis of mycobacterial disease by the useofspecies-specific nucleic acid probes (4, 14, 33, 39). An oligonucleotide probe, based on the 16S rRNA gene

se-quenceofM. tuberculosis, is commercially available

(Gen-* Correspondingauthor.

Probe, San Diego, Calif.). This probe permits the identifica-tion of the species M. tuberculosis, and other similar species-specific probes have been developed for

Mycobac-terium avium andMycobacterium intracellulare (7, 30, 35). The sequenceofpartof the 16S rRNAgeneof Mycobacte-riumlepraewasrecentlypublished (6), permitting the

devel-opmentof similar DNA probes for the diagnosis ofleprosy. Although DNA probes are very specific, the methods still suffer from poorsensitivity, becauseatleast 106organisms are required for a reliable identification. Therefore, these methods are not useful for direct detection ofpathogenic mycobacteria in clinical samples (28).

Therecently developed polymerase chain reaction (PCR) permits the in vitroamplification of DNAsegments(34), and this technique has been shown to increase the level of detection enormously. By extracting DNA from clinical samples, amplifyingapathogen-specific DNAsequence,and detecting the amplified sequencewitha labeled oligonucle-otide probe, it is theoretically possible to detect a single microorganism in clinical samples (17). The PCR has already found wide application, such as the detection of specific DNAsegmentsof humanimmunodeficiencyvirustype1, the causative agent of acquired immunodeficiency syndrome, long before seroconversion (9, 16; W. J. A. Krone, J. Sninsky, and J. Goudsmit, submitted forpublication). The sequenceof the M.leprae36-kilodalton(kDa)genewasused forthe specific recognition of M. leprae by using the PCR (10). Hanceetal. (1,8) used primers basedonthe evolution-arily well-conserved 65-kDa heat shockgenefor the ampli-fication by PCR of a small DNA segment. The amplified product was subsequently identified bythe use of oligonu-cleotideprobes that selectively hybridizedwith DNAfrom M.tuberculosis-M.bovis,M.avium-Mycobacterium

paratu-berculosis, orMycobacterium fortuitum.

During a screening ofa lambdagt-11 gene library ofM.

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TABLE 1. Bacterial strainsusedin this study

Strain(s) Species Relevantproperties Reference or source

1 Mycobacteriumtuberculosis H37Rv RIVMa

2 Mycobacteriumtuberculosis H37Ra RIVM

3-11 Mycobacteriumtuberculosis Clinicalisolates (Ghana) T.v.d. Werfb

12-34 Mycobacteriumtuberculosis Clinical isolates KITc,RIVM

35-38 Mycobacterium africanum Clinical isolates KIT,RIVM

39-42 Mycobacterium bovis Clinical isolates KIT, RIVM

43-45 Mycobacterium bovis BCG RIVM

46 Mycobacterium microti F.Portaelsd

47-51 Mycobacteriumavium KIT, RIVM

52-54 Mycobacteriumkansasài KIT,RIVM

55 Mycobacterium intracellulare KIT

56 Mycobacterium intracellulare ATCC 15895 ATCCe

57 "Mycobacterium lufu" KIT

58-60 Mycobacterium fortuitum KIT, RIVM

61 Mycobacterium gastri KIT

62 Mycobacterium chelonei KIT

63-65 Mycobacteriumscrofulaceum KIT,RIVM

66, 67 Mycobacteriumsmegmatis ATCC 607 andATCC10143, respectively ATCC

68 Mycobacteriumduvalii KIT

69 Mycobacterium vaccae KIT

70-89 Mycobacterium gordonae RIVM

90 Mycobacterium leprae KIT

91 ADMf KIT

M1183 Escherichiacoli POP2136(XcI857) A.Raibaudg

M1494 Escherichia coli M1183containing pEX2 K. Stanley(38)

a RIVM, National Institute of Public Health and Environmental Protection, Bilthoven,TheNetherlands. b Sophia Hospital, Zwolle,TheNetherlands.

C KIT,N.H. Swellengrebel Laboratory ofTropical Hygiene, Royal Tropical Institute, Amsterdam,TheNetherlands.

dPrince Leopold InstituteofTropicalMedicine,Antwerp,Belgium. eATCC, American Type Culture Collection, Rockville, Md.

fADM,Armadillo-derived mycobacterium. gInstitut Pasteur, Paris,France.

tuberculosis with monoclonal antibody species, aparticular recombinant was obtained that carried an M. tuberculosis DNA insert that hybridized virtually specifically to DNA frommycobacterial species belongingtotheM. tuberculosis complex. Inthisstudywe show thatapartof thisparticular insert carries DNA which occurs in many copies in the chromosome. We usedthe sequencefromthenonrepetitive

partof this insertforamplification byPCRand forrapidand sensitivedetection ofmycobacteria from theM.tuberculosis complexgroupinclinical samples.

MATERIALS AND METHODS

Bacterial strains and plasmids. The bacterial strains are listed inTable 1. The M. tuberculosiscomplex strainswere isolated fromDutchpatientsunlessotherwisestated.Media, reagents, and enzymes were used as described previously (41). All mycobacterial strains were cultured in Tween-albumin medium on a rotary shaker at 37°C (protocol: Division of Research and Laboratories, National Jewish Hospital, Denver, Colo.). NZ medium and NZagar supple-mented withappropriate antibiotics were used forgrowing Escherichia coliK-12cells (18).

Preparation of DNA. Mycobacterial cultures of 400 ml were growntoanoptical densityat600nmof 0.6, D-glycine was added to afinal concentration of 1%, and the cultures werereincubated for24h. Cellswere harvested by centrif-ugationandsuspendedin 5mlof50mMTris hydrochloride-5 mM EDTA, pH 8.0. Lysozyme was added to a final concentrationof1mg/ml,and themixturewasincubated for 90 min at 37°C. Proteinase K and sodium dodecyl sulfate were added to final concentrations of0.1 mg/ml and 1%,

respectively,

and theincubation wascontinuedfor 10minat

65°C.

The mixturewas

phenol

extracted andethanol

precip-itated,

and

mycobacterial

DNAwasdissolvedin 0.5 mlof10 mMTris

hydrochloride-1

mM

EDTA, pH

8.0

(18).

Standard

procedures

wereused for theisolation of chromosomalDNA ofE. coli K-12 strain M1183 and

plasmid

DNA ofE. coli K-12 strain M1427

(18).

Synthetic

oligonucleotides.

On the basis of the

partial

sequence of

pPH7301,

three

oligonucleotides

were

synthe-sized, using

aDNA

synthesizer

(Applied

Biosystems,

Inc., Foster

City,

Calif.).

Oligonucleotides

A

(5'GGTCCTGAC

GGTAATGGGGT),

D

(5'CGCCCATCCACATCCCGCCC),

and E

(5'GGACATCTCTGTTCCATCCA)

correspond

to

base

pairs (bp)

31 to

50,

169 to

188,

and 99 to

118,

respec-tively, of the M. tuberculosis DNA sequence. Residue 1

corresponds

to the first

mycobacterial

base downstream from cro-lacZ' in

pPH7301.

Oligonucleotides

A and D are

complementary to the

plus

and minus strands of the pPH7301 sequence,

respectively,

and

positioned

118

bp

apart. Two

homologous

regions

within the genes

encoding

the65-kDa

antigens

ofM.

leprae

(22)

andM.bovis BCG-M. tuberculosis

(36, 41)

wereselectedand usedfor

synthesizing

the

oligonucleotides

1P and

2P,

asdescribed

by

Hartskeerlet

al.

(10).

Oligonucleotides

1P

(5'CTCAAGGAGCGCAAG-CACCG)

and2P

(5'TTGAAGGCGATCTGCTT) correspond

to residues 1734to 1754 and 1920 to

1937,

respectively,

of the65-kDa heatshock

protein

geneasdescribed

by

Tholeet

al.

(41)

and are

complementary

to the

plus

and minus

strands,

respectively,

of this gene.

Labeling of DNA probes. The

mycobacterial

DNA

frag-ments of

pPH7301

aswellas the

158-bp

amplified

fragment

ofpPH7301 were labeled with

[fr-32P]dCTP,

using

a

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1206 HERMANS ET AL.

tiprime

DNA-labelingkit(Amersham Internationalplc, Am-ersham, United Kingdom). The oligonucleotide E was la-beled with digoxigenin dUTP (Boehringer GmbH,

Mannheim,

FederalRepublic of Germany)

according

to the instructions of the manufacturer.

Southern blot hybridization. Chromosomal digests and PCR-amplified DNA were electrophoretically separated on 0.8 and 1.5%agarose

gels, respectively, containing

ethidium bromide (500

ng/ml).

After denaturation and transfer to a

GeneScreen Plus membrane (Dupont,NEN Research Prod-ucts,Boston, Mass.) byvacuumblotting (27, 37) (MilliblotV system;

Millipore Corp., Bedford,

Mass.), the DNA was

hybridized

as described

by

Noordhoek et al. (26). 32p_ labeledprobes were hybridized and washed at65°C. Mem-braneswereexposed for variable lengths oftime at -70°Cto X-Omat film (Eastman Kodak Co., Rochester, N.Y.).

Digoxigenin-labeled

probes were hybridized and washed at

37°C according

to the instructions of the manufacturer. Dot blothybridization. The

amplified

DNAwasdiluted five times with10 mM Tris

hydrochloride-1

mMEDTA(pH 8.0), denatured for5minat100°C,andkeptonice.Samples of100

idl

were spotted onto GeneScreen Plus filters, using the Minifoldsystem(Schleicher & Schuell GmbH, Dassel, Fed-eralRepublic of Germany).The blots weretreated with 0.4 MNaOHfor3 min and neutralizedwith1MTris hydrochlo-ride (pH

8.0)

for 3 min. Hybridization and washing were done asdescribed above.

PCR. The PCR with thermoresistant DNA polymerase from Thermus aquaticus (Taq polymerase; The Perkin-Elmer

Corp.,

Norwalk, Conn.) was done as recommended

by

themanufacturer. OneunitofTaqpolymerasewasadded to 100 ,ul of50 mM NaCl-5 mM MgCl2-10mMTris hydro-chloride-0.01%

(wt/vol)

gelatin (pH 9.6)-0.2mMeach of the

deoxynucleotides dGTP,

dATP, dTTP, and dCTP

(Pharma-cia, Uppsala, Sweden)

(pH 9.6)

and,

unless otherwise

indi-cated,

100ngoftarget DNAand150ngof each

primer.

DNA was

amplified

in aPCRprocessor(Biomed GmbH, Theres, FederalRepublic of Germany), using 35 cycles asfollows: 2-min denaturation at 94°C, 2-min

annealing

at 55°C, and 3-min

primer

extensionat

72°C.

After 35 cycles of

amplifi-cationthe mixtures were

analyzed by

agarose gel

electro-phoresis,

Southernblotting, and/ordotblotting.

Clinical samples. Clinical

specimens

from tuberculosis

patients

as well as

patients

who were

suspected

to have tuberculosisweredetermined

by

PCR.

Sputum samples

(no.

052, 400629,

400676, 400699, 400739,and400777)anda

lung

tumor

biopsy

(no. 400674) were taken from

patients

with

pulmonary tuberculosis,

which had been confirmed

by

cul-turing

ofM. tuberculosis.

Samples

of different clinical

ori-gins

from

patients

whowere

suspected

to have

pulmonary

tuberculosis (no. 400695, 400750, and 400781) and from

patients

whowere

suspected

tohave

extrapulmonary

tuber-culosis (no. 400635, 400651, and 400723) were also

exam-ined. Samples fromthesepatientswithsuspected pulmonary andextrapulmonary tuberculosis wereM. tuberculosis cul-ture

negative.

DNA extraction from clinical samples. In orderto isolate DNAfromM. tuberculosisbacteria in clinical samples, 500

,ul

of 1 M NaOHwasadded toanequalvolume of thesample and themixturewaskeptatroom

temperature

for 10 min. A

620-,ul

volumeof1 M

NaH2PO4

was

added,

andthe

samples

were

centrifuged

for 10 minat 12,000 x g. Thepelletswere washed and suspended in 400 ,ul of 50 mM Tris hydrochlo-ride-5mMEDTA, pH8.0. Afterthe addition oflysozymeto 1

mg/ml,

the samples were incubated at 37°C for 90 min. ProteinaseKandsodiumdodecylsulfatewereaddedtofinal

concentrationsof 1 mg/ml and 1%, respectively, and incu-bationwas continued for 30 minat60°C. In ordertopurify and concentrate the DNA, the mixtures were phenol ex-tracted and the DNAwasprecipitatedwith ethanol(18).The DNA wasfinallydissolved in 50 ,u1 ofdistilledwater.

RESULTS

Identification ofaDNAfragmentcharacteristic for species of the M. tuberculosiscomplex. After the screening of an M. tuberculosis gene library (44) of about 106 lambda gt-11 clones,the lambda PH7311 recombinantwasselectedasthe onlyonewhichexpressedaproteinthat reacted with mono-clonal antibody F116-5(Hermans et al., unpublished data). Lambda PH7311 contained a mycobacterial DNA insert of 2.4 kilobase pairs (kb), and it expressed a ,-galactosidase fusionproteinof about 126 kDa(datanotshown).The 2.4-kb EcoRImycobacterialDNAfragmentof lambda PH7311was

subcloned into expression vector pEX2, resulting in the recombinant plasmid pPH7301. As expected, pPH7301 in-ducibly expressed afusion protein ofthe same sizeas that expressed by lambdaPH7311.

The2.4-kbDNAinsert ofpPH7301wasusedtohybridize with genomicDNAfrom various strains ofM. tuberculosis and 10 othermycobacterial species. Morethan 10 different-size bands of

BstEII-digested

chromosomal DNA of M. tuberculosis reacted with the labeled 2.4-kb probe

(Fig.

1). Comparable banding patterns were obtained by Southern blot analysis of

BstEII-digested

chromosomal DNA of six other strains of M. tuberculosis (data not shown). This suggests that the M. tuberculosis chromosome contains repeated sequences which sharehomology with the probe.

Consistently, multiple

bands were also observed when M. tuberculosisDNAwascleaved withoneof12other restric-tion enzymes, AvaI, BstEII, ClaI, EcoRI, FokI, MboII, NruI, PstI, PvuI, PvuII, RsaI, and StuI, and

subsequently

hybridized

with the probe (datanotshown).

Allnon-M.tuberculosis strainstested

belonging

to

species

ofthe M. tuberculosis

complex

group, M.

africanum,

M. bovis,andMycobacterium microti,showedaBstEIIbanding pattern

comparable

to that ofM. tuberculosis

(Fig. 1).

In contrast, no

hybridization

of the 2.4-kb

fragment

wasfound with DNA fromM. avium,M.

intracellulare,

M.

fortuitum,

Mycobacterium

scrofulaceum,

and

Mycobacterium

smeg-matis. DNA from the two other

mycobacterial species

tested,

Mycobacterium gordonae

and

Mycobacterium

kan-sasii,

hybridized

with the 2.4-kb EcoRI

fragment

of M.

tuberculosis

(Fig.

1,lanes 16 and

19).

Again, multiple

bands wereobservedontheSouthern

blots;

however, the

banding

pattern differed

completely

from that of

BstEII-digested

DNAofM. tuberculosis

complex

strains. In

addition,

most bandswereless

intense,

indicating

alesser

degree

of homol-ogy with the 2.4-kbprobe.

The2.4-kb

fragment

didnot

hybridize

with the

previously

described

repetitive

M. tuberculosis sequences present in the recombinant

bacteriophages

M13KE37 and M13KE115

(5) (data

not

shown).

In order to obtain information about the size of the

repetitive

sequence in pPH7301, various

fragments

of the 2.4-kb insert were labeled with 32P and used in Southern blotsof

BstEII-digested

M. tuberculosisDNA.Asshown in Fig. 2, hybridizationwith each of thefive subfragments A, B,

C,

D, and E

(Fig.

3) resulted in the samecharacteristic

banding

pattern. However, certain

fragments

hybridized

much more

strongly

with

fragment

A thanwith

fragment

C andvice versa,

indicating

thatin the left arm ofthe 2.4-kb

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DETECTION OF M. TUBERCULOSIS COMPLEX STRAINS BY PCR

5 J O n19 1l0i. 3)13 4l1 16 17 18 19

r.!_j_*

11-a

wi

---.-,~~~_*

*~~~~~~~~tm *

«Min w-

-m -..

_

- -_

_ - _

_

_.

.d-oe

lm

e

FIG. 1. Southernblotanalysis ofBstEII-digestedchromosomalDNAof variousmycobacterialspecies, usingthe2.4-kbEcoRIfragment of pPH7301as aprobe. Lanes: 1,M.tuberculosis8; 2, M.tuberculosis 10; 3,M.tuberculosis 14; 4,M. tuberculosis 15; 5,M. tuberculosis 25; 6, M. tuberculosis22; 7,M. tuberculosis7; 8,M. tuberculosis3; 9,M. tuberculosis11; 10,M.tuberculosis5; 11,M. microti46; 12,M. bovis 41; 13, M. africanum 38; 14,M. avium 50; 15,M.fortuitum 60; 16,M.gordonae 71; 17,M. intracellulare56; 18, M.smegmatis,66; 19,M.kansasii 53. Numbersatleft indicate sizes of standard DNAfragmentsinkilobasepairs.

insertrepeats are presentwhicharepartially different from those in theright arm of this insert. This suggests that the repeatextendsto alarge portion ofthe 2.4-kbinsert.

M. tuberculosis complex-specific DNA amplification by PCR. Part of the 2.4-kb M. tuberculosis DNA insert was sequenced (Fig. 3). On the basis of this sequence two

oligonucleotides,AandD,weresynthesized,with the aimto use them as primers for amplification by PCR of chromo-somal M.tuberculosisDNAcorrespondingtobp 31 to 188 of the cloned 2.4-kb EcoRI fragment (Fig. 3). As expected, a

fragment of approximately158bpwasvisibleby agarose gel electrophoresis after amplification of either chromosomalM. tuberculosis DNAorpPH7301 DNA (Fig. 4A). In orderto confirm the identity of the amplified 158-bp fragment, this DNA washybridized with adigoxigenin-labeled oligonucle-otide, probe E, correspondingto bp99to 118. Indeed, this probe hybridizedwith theamplified fragmentofpPH7301,as well as with the M. tuberculosis amplified fragment (Fig. 4B). Chromosomal DNAs of the three other species ofthe M. tuberculosis complexweresubjected to PCR and South-ern blot analysis, and all were foundto contain the 158-bp amplifiablefragment(Fig.4). No suchfragmentwasfoundin M. avium,M.kansasii, M. intracellulare, M.fortuitum, M.

scrofulaceum,M. smegmatis, M.

leprae,

andM. gordonae. Asapositivecontrol for thesuccessfulDNAamplificationin vitro, the two primers

Pi

and P2, homologous toconserved regionsin the65-kDa heat shockproteingene(45),werealso added to the reaction mixture. Hartskeerl et al. (10) have shown that theseprimers enable theamplificationofa204-bp

DNA fragmentofmycobacteria and also of other bacterial generaand evenofeucaryotic DNA. This 204-bpfragment wasamplified byusing DNA from allmycobacterial strains tested (Fig. 4). This fragment, however, did not hybridize with probe E, confirming that this probe is specific forthe 158-bpsequence inpPH7301.

Atotalof91 mycobacterial strains belongingto 19species

weretestedby PCR and dot blothybridization, using oligo-nucleotide probe E (Fig. 5 and Table 2). None of the 45 strains not belonging to species of the M. tuberculosis complex contained the 158-bp fragment. In contrast, all strains of the species M. africanum, M. bovis, and M. microti and 29 of 34 M. tuberculosis strains tested were

positive by PCR analysis. A negative result in the dot blot assay may be due to the fact that the chromosomal DNA doesnotcontainsequencescomplementarytooneofthetwo

amplimers or toprobe E. In these cases, the chromosomal

DNA may still hybridize to the amplified 158-bp fragment. To investigate this possibility, Southern blots were done with BstEII-digested DNAof the five PCR-negative strains and compared with a number ofPCR-positive M. tubercu-losis complex strains. The blots were hybridized with the 158-bp amplified M. tuberculosis fragment. None of the PCR-negative strains hybridized with the labeled 158-bp fragment, indicatingthat thisfragmentwasmissingin these strains. Conversely, in all

PCR-positive

strains a 1.3-kb BstEIIfragment hybridized with the 158-bpprobe

(Fig.

6). The only significant difference between the

PCR-positive

and PCR-negative strains was found in the presence or

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1208 HERMANS ET AL.

1 2 3 4 5

+.e.

.~~~~~~~~..w

_» e

_M me

FIG. 2. Southern blotanalysis ofBstEII-digestedDNAfrom M. tuberculosis 16, hybridized with the following: lane 1, 450-bp

EcoRI-SmaI fragment A; lane 2, 950-bpSmaI-EcoRIfragment B; lane3,1.0-kbSmaI-SmaIfragment C; lane 4,900-bpEcoRI-BssHII fragmentD; lane 5, 1.5-kbBssHII-EcoRI fragment E. Numbers at left indicate sizes ofstandardDNAfragments inkilobasepairs.

EcoPJ Smel BSHI Smai Xnrr EcoRJ

r

r-ro-LacZ

a

c

b d

e

30 60

EcoRI . pRrimer A

GAATTCCGAGTAACTGACGAGCACGGGCGGGGTCCTGACGGTAATGGGGTTGACGGTGAT GluPheArgValThrAspGluHisGlyArgGlyProAspGlyAsnGlyValAspGlyAsp

90 120

probe E

GGAGCCGACATGGACGGCGGGGTCGAGGCCCAAGTGAATGGATGGAACAGAGATGTCCGG GlyAlaAspMETAspGlyGlyValGluAlaGlnValAsnGlyTrpAsnArgAspValArg

150 180

primer D GATGGCGATCGGGCCGATGCCACCGACCGCGGCGAAGCCGACCGGAATGGGCGGGATGTG AspGlyAspArgAlaAspAlaThrAspArgGlyGluAlaAspArgAsnGlyArgAspVal

210 240

GATGGGCGGCAGCACGGTAATCGGGCCGATCCCGCCGCTGACGTCGGCGCCCACCGCGGG

AspGlyArgGlnHisGlyAsnArgAlaAspProAlaAlaAspValGlyAlaHisArgGly FIG. 3. Physicalmapof pPH7301 andDNAsequenceofpartof the mycobacterial insert in pPH7301. The predicted amino acid sequence and the synthetic oligomers are also depicted. a to e, FragmentsA toE asdefined in the legendtoFig. 2.

absenceof the 1.3-kb band. The two species not belonging to the M. tuberculosis complex group, M. gordonae and M. kansasii, which hybridized with the 2.4-kb insert of pPH7301, did nothybridize with the 158-bpprobe.

Sensitivity of the PCR. The sensitivity of the PCR test to detect M.tuberculosisDNAwasdeterminedby PCR ampli-fication of10-foldserialdilutions of pPH7301 and ofpurified chromosomalM. tuberculosisDNA,usingprimersAand D. AmplifiedDNA wasdenatured andprobed with oligonucle-otide E by dot blot and Southern blot hybridization. One femtogram of pPH7301 DNA was clearly detectable after PCR, corresponding to about 100 plasmid DNA molecules (Fig. 7). The

detection

limitofchromosomalM. tuberculosis

DNA was 100fg, a quantitytheoretically corresponding to about 20 bacteria. To investigate the sensitivity of the PCR testwith wholebacteria, 10-fold dilutions of M. tuberculosis cellsweremade ineitherbuffersolution or in sputum from a healthy person. DNAextractions weredone asdescribed in Materials and Methods. After M. tuberculosis complex-specificPCR, samples were analyzed by DNAhybridization with oligonucleotide E. The detection limit amounted to about 200 bacteriafor cells suspended in buffer and 1,000 bacteria for cells suspended in sputum (Fig. 7 and 8).

Detectionof M.tuberculosisinclinical material. In order to investigate the use of the M.

tuberculosis

complex-specific PCR test in clinical samples, DNA was extracted from sputumspecimens, urine specimens, tumor biopsies, pleural fluids, and bronchial washings from patients withdifferent clinical symptoms. All clinical samples which were Ziehl-Neelsenpositive and M. tuberculosis culture positive were alsopositivein thePCRtest(Table 3). We also investigated sputa of a tuberculosis patient during isoniazid-rifampin-pyrazinamide treatment. Theculture-positive sputum of this patient was also positive in the PCR test, and this testwas

positiveupto16weeksaftertreatment,althoughnobacteria couldbeculturedafter4weeks andnobacteriawerevisible after 14 weeks (Table 3). Thisindicates that the mycobac-teria were killed after the onset of the multiple-drug treat-mentand thatnonviablebacteriawerepresentevenafter 16 weeks of treatment.

DISCUSSION

The 2.4-kb mycobacterial DNA fragment of pPH7301 hybridized specifically with genomic DNA of all species belonging to the M. tuberculosis complex and hybridized weaklywithtwospecies,M. gordonae and M. kansasii, not belonging to this complex. No hybridization was detected with genomic DNA of M. avium, M. intracellulare, M. fortuitum,M. scrofulaceum, andM. smegmatis. Because of thespecificityofpPH7301, this clonewasusedtodevelopan M. tuberculosis complex-specific PCR. Partofthe 5'-prox-imal M. tuberculosis DNA insert of pPH7301 was se-quenced, and this sequence was used to develop a PCR-based test to detectM. tuberculosisinbacterial cultures. Of 46strainsbelongingtothe M. tuberculosis

complex,

includ-ingM.

tuberculosis,

M.bovis,M. bovisBCG,M.africanum, and M. microti,41werepositivein thePCR test. Noneof45 non-M.

tuberculosis

complex strains of14 different species

werereactive in the PCR test,indicatingthe greatspecificity of thetest.

Wedeterminedthesensitivity of detectionofM. tubercu-losis by PCR in sputum, which was about 1,000 bacteria. This sensitivity compares favorably with the sensitivity of examination ofsmearsby Ziehl-Neelsen staining. The find-ing that sputum samples froma treated patient becameM.

tuberculosis

Ziehl-Neelsennegativebutremainedpositivein the PCR indicates the degree of sensitivity of this test. J. CLIN. MICROBIOL.

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A 1.33.

9. )8

J

. 8

)3. b

-) 3

iX

B

i 4 5 b 7 8 9 1:... 12a_ 1sI45 16 1. 1 3 a. ' - bQ

Il i 5 l'

15n bp

FIG. 4. Agarose gel electrophoresis (A) and Southern blot analysis (B) of amplified DNAs of different mycobacterial strains. The oligonucleotide amplimer pairsA-Dand1P-2Pwereused in thePCR.Lanes: 1, pPH7301; 2, M.tuberculosis 1; 3, M. tuberculosis 16;4, M. tuberculosis 15; 5, M.tuberculosis8; 6, M. bovis 41; 7, M. bovisBCG45; 8, M. africanum 38;9, M. microti 46; 10, M. avium50; 11, M. kansasii53; 12,M.intracellulare56; 13, M.fortuitum60; 14, M.scrofulaceum64; 15, M. smegmatis 66; 16, M.gordonae71; 17, M.leprae

90.Oligonucleotide Ewasusedas aprobe forhybridization. Numbersatleft indicate sizes of standard DNA fragments in kilobasepairs.

Because the sensitivity of the PCR test was much greater

when purified chromosomal DNA was used, we conclude that our procedure to extract DNA from mycobacteria in clinical material isnot yet optimal. Nevertheless, the PCR

testperformed quite well when clinical samples of various origins were analyzed for the presence of mycobacterial amplifiable DNA.

1 2 3 4 5 6 7 8 9 10 11 12

B cD

D

-

H-FIG. 5. Dot blot analysis of PCR-amplified DNAs of several mycobacterial strains (Table 1) and hybridized with oligonucleotide

E. The various wells contained DNA from the following strains: A-1,distilledwater;A2, 12; A3, 1; A4, 3;A5, 4; A6, 13; A7, 14; A8, 15; A9, 16; A10,5; Ail, 17; A12, 6; Bi, 18; B2, 7; B3, 8; B4, 9; B5, 10; B6,11; B7, 32; B8, 19; B9, 20; B10, 21; B11, 22; B12, 23; Cl, 24; C2,25;C3, 26; C4, 27; C5, 28; C6, 29; C7, 30; C8, 31; C9, 33; C10, 34; Cil, 2; C12, 35; Dl, 36; D2, 37; D3, 38; D4, 39; D5, 40; D6, 41; D7, 42; D8, 43; D9, 44; D10, 45; Dli, 46; D12, 48; El, 49; E2, 53; E3, 54; E4, 56;E5,57;E6,59; E7, 60; E8, 61; E9, 62; E10, 64; E1, 66; E12, 68; Fl, 69; F2, 71; F3, 90; F4, pPH7301.

We think that the PCR test described here will be more sensitive than the PCR test describedbyHance etal. (1,8), because in the latter case the oligonucleotides used for priming the PCR amplify DNA ofthe evolutionarily

well-TABLE 2. Detection ofthe158-bpamplifiable fragment amongMycobacterium species

No.of No. ofstrains

Species strains tested carryingthe

158-bpfragment

M.tuberculosis 34 29

M.africanum 4 4

M.bovis 4 4

M. bovis BCG 3 3

M.microti 1 1

M. avium 5 0

M. kansasii 3 0

M. intracellulare 2 0

"M. lufu" 1 o

M.fortuitum 3 0

M.gastri 1 0

M. chelonei 1 o

M. scrofulaceum 3 0

M. smegmatis 2 0

M. duvalii 1 o

M. vaccae 1 o

M.gordonae 20 0

M. Ieprae 1 o

ADMb 1 0

aSpecific amplification ofthe158-bpfragmentbyamplimersAand Dwas

determinedbyusing probeEin the dotblothybridizationassay. Asapositive

control,amplimers1Pand 2Pwerealso addedtothePCR mixture. The204-bp

fragmentof the 65-kDaproteingene was visibly amplified, asdetectedby

agarosegelelectrophoresis.

bADM, Armadillo-derivedmycobacterium. ic

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1210 HERMANS ET AL.

i', .1 1-- 1 i l

--1

.. 1 1

.14 _-.4 ₁-_l' i .q 1'-il

i

-, 1 . -.1 ii-) L

_ u_

FIG. 6. Southern blot ofBstEII-digested DNAof mycobacterial strains, probed with the 158-bp PCR-amplified fragment ofpPH7301. Lanes:1, M. tuberculosis8; 2, M. tuberculosis 10; 3, M.tuberculosis 14; 4, M.tuberculosis15; 5, M.tuberculosis 25; 6, M. tuberculosis 22; 7,M.tuberculosis7; 8, M.tuberculosis3; 9,M. tuberculosis 11; 10,M.tuberculosis5; 11,M.microti46; 12, M. bovis 41; 13,M.africanum 38; 14,M.avium50; 15, M.fortuitum 60; 16,M.gordonae 71; 17,M.intracellulare 56; 18,M.smegmatis, 66; 29,M.kansasii 53. Numbers atleft indicate sizesof standard DNAfragments in kilobasepairs.

conserved sequence ofthe65-k] of any mycobacterial species a

(45). Thus, other non-M. tubers clinical samples might compete tuberculosisDNAduring the PC

A

1 i

FIG. 7. Sensitivityof DNA detect PCRamplification of highly purified obtainedby the short DNA extraction (CandD). Tenfold serial dilutions(1 to A10) and 10-fold serial dilution purifiedM. tuberculosis DNA(Bi ti

andsamplesweresubjectedtodot bl

was isolated from 10-fold serial dilt

(2.106to2.10-3)in 100 mM Tris hyc 8.0)(CltoC10) and from 10-foldser

cells(1.107to1.10-2) insputumfront Samples wereamplified by PCRanc

withprobe E. M. tuberculosis 12w2

Daheat shockprotein gene Of the 34M.tuberculosisstrainstested,5werenegative in Lnd other bacterial species the PCR test, and these 5 strains all lacked a 1.3-kb BstEII -ulosis cells also present in DNAfragment which was present in the 29 other strains and for amplification with M. which hybridized with the M. tuberculosis DNA insert of

,R. pPH7301. Although this fragment seems to be unique for

strains within the M. tuberculosis complex, monoclonal antibody F116-5,which recognizes the 126-kDa fusion pro-teinexpressed bypPH7301, isreactive witha24-kDaantigen present in allM. tuberculosisstrains,including the five PCR test-negative strains that lack the 1.3-kb BstEII fragment (datanotshown). Anexplanationfor thesefindings mightbe that monoclonal antibody F116-5 recognizes an epitope of the fusion protein expressed by pPH7301, created by the fusion of

P-galactosidase

and a mycobacterial polypeptide which is unrelated to the 24-kDa mycobacterial antigen. Similarly, Thole et al. (42) described the selection of a lambda gt-11 recombinant reactive with a monoclonal anti-ction bydot blot analysis after..

DNA(Aand B) andof DNA body

recognizing

the M.

leprae

36-kDa

antigen.

Detailed

nprocedurefor clinicalsamples

analysis

of

this

recombinant

showed that the antibody rec-.ngto 0.001fg)ofpPH7301

(Ai

ognized an epitope at thejunction of the cro-lacZ fusion s (10 ng to 0.01 fg) of highly which,by chance, sharedhomology with the naturalepitope

oB10) wereamplified by PCR, of the36-kDa M. leprae antigen (42).

lotanalysiswithprobeE.DNA Themultiple banding patterns observed in Southern

blots

utions of M. tuberculosis cells of

chromosomal

M.

tuberculosis

DNA

probed

with the drochloride-10 mM EDTA(pH 2k

chrosof

M. indicate

DNA

p

the

rial dilutions of M. tuberculosis 2-4-kb insert ofpPH73O1 indicate that thisinsert contains a

aahealthycontrol(DltoD10). repetitive sequence which is present in more than 10 copies

d subjectedto dotblotanalysis perchromosome. The banding patterns observed with sub-as used to extract DNA. fragments of the 2.4-kb insert indicate that the

repetitive

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DETECTION OF M. TUBERCULOSIS COMPLEX STRAINS BY PCR

i 2 3 4 5 6 7 à 9 10

XH 12 13 14 15 1b 17 15 19 L0

l 2 3 4

``

5 e 7

:;"X

8 k 1O

e; ..rA a, `, .,X 3E

.'i .xt

*_$#injj

.::

.' "

*

uu

.",uu,`;m'A-eA

l 12 3 14 15 16 ;7 18 19 20

FIG. 8. Sensitivityof detection of M. tuberculosis 12 after

am-plification by PCR. Agarose gel electrophoresis (A) and Southern blotanalysis(B)ofDNAisolatedfrom 10-fold serial dilutions ofM.

tuberculosis cells in either buffer (100 mM Tris hydrochloride-10 mM EDTA, pH 8.0) (lanes 1 to 10) or sputum from a healthy individual(lanes11to20). Lanes 1to10and 11to20correspondto

10-fold dilutions beginning with an equivalent of 2.105 bacteria.

DNAwashybridized with probeE.Numbersatleft indicatesizes of standard DNAfragments in kilobase pairs.

DNA isnotonlypresentin thecentral 1-kb SmaI fragment butextends to bothflanking regions.

The repetitive DNA described in this study differs from theM. tuberculosisrepeats identified by Eisenachetal. (5), as the 2.4-kb fragment did not hybridize to this repetitive DNA. Zainuddin and Dale (46) described a repetitive

se-quencethatwasdiscovered inM.tuberculosis because of its homology with a plasmid of M. fortuitum. This 1.3-kb element has recently been sequenced (40; R. A. McAdam, P. W. M. Hermans, D. van Soolingen, Z. F. Zainuddin, D. Catty, J. D. A. vanEmbden, and J. W. Dale, submitted forpublication), anditwasfoundtobeamember of theIS3 family of insertion elements occurring in gram-negative bacteria.Allof these repetitive genetic elementsare specific for species ofthe M. tuberculosis complex, and Southern blots demonstrated restriction fragment-length polymor-phisms which would be consistent with a putative role of these genetic elements in genetic rearrangements (11, 46; P. W. M. Hermans, D. vanSoolingen, J. W. Dale, A.R. J. Schuitema, R. A. McAdam, D. Catty, and J. D. A. van

Embden, submitted forpublication).

Ourobservation that 5 ofthe 34M. tuberculosis isolates lackeda1.3-kb BstEIInonrepetitive chromosomal segment,

whichflanked the repetitiveDNA, stronglysupportstheidea ofa role of suchrepeats ingenetic rearrangements. Other mycobacterium species-specific repetitive DNA has been identified in M. leprae (2) and in M.paratuberculosis (19).

Onlyin thelatterspecies has thepresenceofrepetitiveDNA

TABLE 3. Detection ofM. tuberculosisinclinical material by PCR

Detection of mycobacteriaby: Patient Sourceofclinical

(wk)' specimen Ziehl-Neelsen d

sannb Culture' PCRd

staining

400629 Sputum 3+ + +

400635 Urine ND -

-400651 Urine ND

400674 Tumorbiopsy 1+ + +

400676 Sputum 5+ + +

400695 Pleuralfluid

-400699 Sputum 3+ + +

400723 Urine ND

400739 Sputum 2+ + +

400750 Bronchial wash ND

400777 Sputum 1+ + +

400781 Bronchialwash

-052(0) Sputum 2+ + +

052(4) Sputum 1+ - +

052(6) Sputum 1+ - +

052(14) Sputum - - +

052(16) Sputum - - +

aWeeks afterstartof treatment.

b1+,105bacilliperml;2+,5.105bacilliperml;3+,10'bacilliperml;5+,

107bacilli per ml;-, noacid-fast bacillidetectable in 1-mlclinical sample, ND,Notdone.

CInallculture-positive samplesM. tuberculosiswasidentified.

dClinicalsampleswereanalyzed byPCR, using amplimersAandD. To verifytheamplificationof the158-bpfragment,oligonucleotideE wasusedin the dotblothybridizationassay.

been found to be associated with a particular phenotype: repeat-carrying strains differed from non-repeat-containing

onesin theirrequirementofmycobactin for growth, presum-ably because of insertional gene inactivation (19). Many insertion sequences occur in multiple copies, and a great number of these have been characterized in detail. Except for their ability to transpose and cause other genetic rear-rangements, they usually are not known to be associated with particularphenotypes of the hosts. Recently described repetitive DNA insertion elements have been identified in pathogens such as Bordetella pertussis (20, 21, 29) and Corynebacterium diphtheriae(31).Deletion ofDNAflanking

aninsertion element isonetypeofrearrangementmediated by insertionelements.Therefore, the absence in five strains ofa nonrepetitive 1.3-kb BstEII fragment flanking the M.

tuberculosis repeat stronglysuggests a role of the repeated DNA element in deletion of this adjacent chromosomal DNA.

Since fiveoftheM. tuberculosiscomplexstrains did carry a significant chromosomal deletion, one might speculate whether this deletion is phenotypically detectable. All five strains have been tested for biochemical properties and antimicrobial susceptibilities. However, no special deviant phenotypewasfound. Thefourinitially investigated strains weremissing the 1.3-kbBstEII chromosomal fragment, and all

originated

fromindividuals in West Africa. As the prev-alenceofhumanimmunodeficiency virusis veryhighinthis area, we speculated on the possibility that these strains mightlackcertainfunctionswhichareessentialfor survival of the bacterium in immunocompetent individuals. There-fore,twoof these strainswereinoculated inguineapigs,but no decreased virulencefor these animals wasobserved(D.

vanSoolingen, unpublishedobservations). Thispreliminary resultcertainly does notexclude adecreasedpathogenicity of these strains. The other isolate lacking the 1.3-kb

frag-ment(M.

tuberculosis

25)wasfrom a Dutchpatientwhohad

A

0.

12 9

0.

12

O.

2

-1

B

15c»

ba

158 b-nà

1211

VOL.28, 1990

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1212 HERMANS ET AL.

no clinical symptoms of acquired immunodeficiency syn-drome. No data are available about anti-human immunode-ficiency virus antibodies of this patient.

Inorder not tomiss1.3-kb BstEII fragment-lacking strains of M. tuberculosis, one probably could successfully use

primers derived from sequences solelywithin the repetitive DNA. We are in the progress ofsequencing the complete repeated DNA element,part ofwhich is present in recom-binant plasmid pPH7301. The presence of repeated se-quences in M. gordonae and M. kansasii which hybridize with thatidentified in M. tuberculosis, asmentionedinthis study, will probably not affect the design of M. tuberculosis complex-specific primersfor such aPCR, as the repeats of theformertwospeciesareonlypartiallyhomologous to that of M. tuberculosis.

Whatever the function might be ofrepetitive DNA ele-mentsfound in M.tuberculosis, they may be of great use for therapid detectionand identification of thispathogen whose characterizationbyclassicalmicrobiological techniquesisso

time-consuming because of its extremely slow growth.

ACKNOWLEDGMENTS

We thankE. Veringa (Leiden, The Netherlands), R. van Ketel (Amsterdam, The Netherlands), andL.G. Berwald(Bilthoven, The Netherlands) for providing us with clinical samples, F. Portaels (Antwerp, Belgium) andT. vander Werf(Zwolle, The Netherlands) forprovidinguswithmycobacterial strains,and A.Vilasi forperfect secretarial assistance.

Thisstudywasfinanciallysupported bythe WorldHealth Organ-isation Programme for Vaccine Development and the RoyalDutch

Organisation AgainstTuberculosis.

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