Rapid and sensitive detection of Mycobacterium leprae using a nested primer gene amplification assay

0095-1137/90/091913-05$02.00/0

CopyrightC 1990,American Societyfor Microbiology

Rapid and Sensitive Detection of

Mycobacterium

leprae

Using

a

Nested-Primer

Gene

Amplification Assay

BONNIE B. PLIKAYTIS,' ROBERT H.

GELBER,23

AND THOMAS M.

SHINNICK1*

Hansen DiseaseLaboratory, Division of Bacterial Diseases, Center for Infectious Diseases, Centers for

Disease Control, Atlanta, Georgia303331; Kuzell Institute for Arthritis and Infectious Diseases, San Francisco, California

941152;

andGillis W. Long Hansen

Disease Center, Carville, Louisiana

7072J3

Received 16 April 1990/Accepted 1June 1990

By using a set of four nestedoligonucleotide primers, a two-step polymerase chain reaction assay for the

detection and identification of Mycobacterium leprae that does not require the use of radioactively labeled hybridization probes was developed. The nested-primerprocedureamplified a347-base-pair product from M.

lepraegenomic DNA. Noamplification products were producedfromDNAs of 19 otherMycobacterium species, 19non-Mycobacterium species, mousecells,or humancells. Minor amplificationproducts were observed with three additional Mycobacterium species, i.e., "M. lufu," M. simiae, and M. smegmatis. These products were easily distinguished from the M. leprae product by size and restriction enzyme cleavage patterns. The assay could amplify the 347-base-pair product from samples containing as little as 3 fg of M. Ieprae genomic DNA-the amount ofDNA in a single bacillus. The assay also amplified targetsequences incrude lysates ofM.

Iepraebacilli isolatedfrom tissue biopsy specimens from infectedanimalsand humans. The entire assay,from

sample preparationtodata analysis, can be completed in less than 8 h.

The diagnosis of leprosy is often based solely on the

observationof acid-fastbacilli ina lesion displaying

charac-teristic histopathologic features. This is due mainly to the

inability to cultivate the etiologic agent of leprosy,

Myco-bacterium leprae, invitro. Such ahistopathologicdiagnostic

procedure is relatively insensitive and does not give a

definitive identification of the infecting organism as M.

leprae.

Several attempts havebeen made in recent years to

improvethesensitivity and specificity of the

detection

of M.

leprae

with

immunologic,

biochemical, and nucleic acid

probes (forareview, see

reference

2). For example,

Clark-Curtiss and Docherty (3)have described a DNAprobe that can be used in a dot blot hybridization assay to detect as

little as 1 pg of purified DNA-the amount of DNA in

approximately 300 bacilli.

Recently, several investigators have studied the use of polymerase chain reactions (PCR) to detect mycobacteria.

In a PCR-based assay, oligonucleotide primers are used to

direct the

replication

(amplification) of a particular target sequence to adetectablelevel(10). Withrespect todetecting

M.leprae, Woods and Cole (18)havedescribedaPCR-based

assayin whichasingle pair of primersto aportion ofthe M.

leprae groEL gene (also called the 65-kilodalton

antigen

gene)or a

single

pair of

primers

toa

portion

ofthe M.

leprae

repetitive sequence is used.

Similarly,

Hartskeerl etal.

(8)

usedasingle

pair

of

primers

to

amplify

a

portion

ofthe gene

encoding

theM.

leprae

36-kilodalton

antigen

from infected

armadillo tissue. For another

Mycobacterium

species,

M.

tuberculosis, primers

to a

portion

of the

groEL

gene have

also been usedtodetectbacilli inpurecultures and inclinical specimens including sputa,

gastric

aspirates,

and

lymph

node

biopsy

specimens

(1,

7).

WealsohavedevelopedaPCRassaytodetect M.

leprae,

based on

amplification

of

portions

ofthe M.

leprae

groEL

gene. The

key

difference between this assay and those

previously reported is thata setoffour nested

primers

was

*Correspondingauthor.

used. Inthis procedure, primers are selected so thatthere are twooutside primersthatwilldirecttheamplification ofa

portion of thetarget genome and twoinside primers that will direct the amplification of sequences contained within the product defined and produced by the outsideprimers (fora

review, see reference 10). Theoretically, this approach

should increase specificity and sensitivity, since successful amplification requires binding of four different primers and since a smaller number of cycles is used with each pair of primers, so that background or nonspecific amplification should be reduced. Also, thisassaydoesnotrequire theuse

of radioactive

probes.

The

nested-primer

approachmayalso reducethelikelihood offalse-positive resultsdue tospurious contamination of the samples by the end products ofprior positiveamplifications, sincearathersmall number ofcycles is used in thesecond round ofamplification toproducethe

end product. Such contamination is likely tobe the major

source of

contaminating

DNAina clinicallaboratory.

MATERIALSANDMETHODS

Bacterial strains andgrowth. Thestrains used in this

study

andtheirsources arelistedinTable1. TheMycobacterium strainswere maintained in the Centers for Disease Control (CDC) HansenDisease

Laboratory

stock culture collection on Lowenstein-Jensen slants

(BBL

Microbiology Systems,

Cockeysville, Md.)

and transferredtoTB Broth with 0.2%

Tween 80 and

supplemented

with Middlebrook oleic acid

dextrose

complex

enrichment

(Difco

Laboratories,

Detroit,

Mich.).

The Rhodococcus and

Corynebacterium

species

were grown on brain heart infusion agar

(Difco

Laborato-ries). The Klebsiella and Pseudomonas

species

weregrown on Trypticase soy agar

(BBL

Microbiology Systems),

and the Legionella and Bordetella strains were grown on

buff-ered

charcoal-yeast

extractagar

(Carr-Scarborough

Micro-biological Inc.,

Decatur,

Ga.).

The

Streptococcus

strains weregrown on

Trypticase

soy agar with 5%

sheep

blood.

Isolationof DNA.DNAwasisolated from M. tuberculosis,

M.

avium,

Mycobacterium

bovis

BCG,

M.

fortuitum,

and

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TABLE 1. Strains

Species_Species Strain Origina

~~~~~~of

Source_DNAb

Mycobacterium africanum Mycobacteriumavium Mycobacteriumbovis Mycobacteriumchelonae Mycobacteriumfortuitum Mycobacteriumgordonae Mycobacterium intracellulare Mycobacterium kansasii Mycobacterium leprae "Mycobacteriumlufu' Mycobacteriummarinum Mycobacteriummicroti Mycobacterium nonchromo-genicum Mycobacterium phlei Mycobacterium scrofulaceum Mycobacteriumsimiae Mycobacterium smegmatis Mycobacterium szulgai Mycobacteriumtuberculosis Mycobacteriumulcerans Mycobacteriumvaccae ICRC bacillus Mycobacterium sp. Aerococcusviridans Bordetella bronchiseptica Candidaalbicans Clostridiumperfringens Corynebacterium pseudo-tuberculosis Corynebacteriumxerosis Enterococcuscasseliflavus Enterococcusmalodoratus Klebsiellapneumoniae Legionellapneumophila Neisseriameningitidis Pediococcuspentosaceus Pseudomonas aeruginosa Rhodococcus equi Rhodococcusrubropestuctus Rhodococcus sputi Staphylococcusaureus Staphylococcus epidermidis Streptococcuspyogenes TMC5122 TMC 724 BCG-Rosenthal TMC 1544 TMC1529 TMC 1324 TMC 1406 TMC 1204 TMC 1218 TMC 1601 TMC 1481 TMC 1548 TMC 1316 607 954 H37Ra TMC 1617 TMC 1526 w 11563 F6286 H317 F8271 F3785 SS937 SS1226 4809-84 P-1 M1027 SS1259 8 57003 60016 55001 136 SS91 1 B 1 A,B 2 A,B 1 B 1 A,B 1 B 1 B 1 B A, B 12 B 1 B 1 B 1 B 1 B 1 B 3 B 4 B 5 B 1 A,B 1 B 1 B 6 B 7 B 8 A 9 B 8 A 8 A 10 B 10 B 8 A 8 A 9 B 9 B 8 A 8 A 9 B 5 B 5 B 5 B 8 A 8 A il B

a 1, D. D. Gwinn, NationalInstitute ofAllergyand InfectiousDiseases, Bethesda, Md.; 2,Sol R.Rosenthal, UniversityofIllinois Research Founda-tion, Chicago; 3, M. J. Colston, MRC, London, England; 4, Norman Morrison, Johns Hopkins University, Baltimore, Md.; 5, Ray Butler, Division

of BacterialDiseases, CDC; 6, C.V.Bapat,IndianCancer Research Center (ICRC), Bombay, India; 7,P.Talwar, National Institute of Immunology, New Delhi, India; 8, Steve O'Connor, Division of Bacterial Diseases, CDC;9,

Jacquelyn Sampson, Division of Bacterial Diseases, CDC; 10, Robert

Weaver, Division of Bacterial Diseases, CDC;11,RichardFacklam,Division

of Bacterial Diseases, CDC; 12, Daniel L. Shunga, Merck Institute for Therapeutic Research, Rahway,N.J.

bA, CsCl-purified DNA; B, crude celllysate prepared as described in

MaterialsandMethods.

CTheprecise taxonomicpositionof "M.lufu" is uncertain, although it is

clearlyaMycobacterium species(4, 11). Preliminary studiessuggestthat it

belongstothe M.aviumcomplex(R. C.Good, personal communication).

armadillo-grownM. leprae andwas purifiedon CsCl

gradi-entsbypublishedprocedures(17).CsCl-purifiedDNAsfrom Aerococcus, Candida, Clostridium, Neisseria,

Enterococ-cus, Pediococcus, and Staphylococcus species were

ob-tainedfrom Steve O'Connor, Divisionof BacterialDiseases, CDC. HeLacell DNA wasisolated andpurified by phenol-chloroform extraction and ethanol precipitation.

TABLE 2. Sequencesofoligonucleotideprimers

Speciesand Residue' Sequence

primer'

M. leprae

L1 1236-1253 GTGGCTCAGATCCGTACC

L2 1813-1792(C) ATGCCACCGGTCGGGTCGCTCG L3 1458-1476 CTACAGGCTGCTCCGGCTC L4 1804-1782(C) GTCGGGTCGCTCGCCGGAGCTGC M. tuberculosis

Ti 1281-1298 GTGGCCCAGATCCGCCAG

T2 1856-1837(C) CATGTCGCCGCCACCGGGAA T3 1503-1521 TTGCAAGCGGCCCCGACCC T4 1846-1827(C) CCACCGGGAACGGAAGCCTT

aTheprimersequenceis found in thegroEL gene sequence of the indicated

species.

bResidue numbersarelistedbyusingthenumberingschemeof Mehraetal. (9)for M. Ieprae and Shinnick (14) for M.tuberculosis. C indicates that the primer sequencecorrespondstothecomplement of the indicated residues. Thesequencesarewrittenin the 5'to3' direction.

Preparation of crude lysates of bacterial suspensions.

Ap-proximately 5 x

10'

cells were harvested from liquid cul-turesby centrifugation, suspended in 500 ,uI of 10 mMTris

hydrochloride (pH

8.0)-i

mM EDTA-10 mM NaCÎ, and transferred to a 1.5-ml screw-top plastic microfuge tube containing 500

itl

of siliconized 0.1-mm-diameterglassbeads and250,ulofchloroform. The mixture was homogenized for 2 min at roomtemperature; a Mickle apparatus (H. Mickle, Gomshell Surrey, England) was used to disrupt the cells.

Two minutes of homogenization was sufficient to break

>90%of thebacilli, as determined bymicroscopy. The glass beads were allowed to settleout, and the aqueous superna-tant was transferred to a fresh tube and boiled for 10 min. Twenty micrograms of RNase (Sigma Chemical Co., St. Louis, Mo.) was added to each sample, and the samples

were incubated at 37°C for 30 min. Portions of the crude lysates were electrophoresed on 0.75% agarose gels

contain-ing 0.75 ,ug of ethidium bromide per ml to estimate DNA concentration.

Processingof tissuesamples.Tissuehomogenatesof mouse

footpad tissue, armadillolungtissue, and fivebiopsy speci-mensfrom threelepromatous leprosy patientswereprepared as previously described (12, 13).

Briefly,

the tissue was

minced and thenhomogenized in a Mickle apparatus for 1 min in the presence of3-mm-diameterglass beadsin 2 ml of

Hanksbalanced saltsolution. Bacteriawereharvestedfrom

the tissue homogenates bycentrifugation, and crude bacte-rial lysates were prepared as described above, except that chloroform was notpresentduringthehomogenization step and the sampleswerenottreatedwith RNase.

Oligonucleotide primers. Primers corresponding to

por-tions ofthe sequenceof theM. leprae(Li through L4)and

M. tuberculosis groEL (Tl through T4) genes were synthe-sized andprovidedby Brian Holloway, CDC. The primers

correspondedtosequencespresent in theM.lepraegene but notin the M.tuberculosisgene orvice versa. Thesequences

andlocations oftheseprimers are listed in Table 2. Primers

L1 and L2 shouldamplify a 578-base-pair (bp)piece of the M. leprae groEL gene, and L3 and L4 should amplify a

347-bp piece

of this 578-bp product. Primers Tl and T2

shouldamplifya576-bppieceof the M. tuberculosisgroEL gene thatcontains sites for primersT3and T4, which should

amplifya344-bppiece.

PCR. Thetemplate DNA in10,uIofH20 was added to 90

,uIofreactionmixcontaining each deoxynucleoside

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1 9 .r 4 5 g

578bp-%-

347bp-_-A B

FIG. 1. Amplification of M. Ieprae DNA by using primers Li through L4. (A) One nanogram of M.lepraeCsCl-purifiedgenomic DNA wasamplified with primersLiand L2 (lane 1) orprimers L3 andL4for25cycles(lane 2) or with primersLM and L2 for 25cycles followed by L3 and L4 for 15 cycles (lane 3). Size markers are a pBR322 Hinfl digest (lane 4). (B) Samples were amplified with primersLM and L2 for 25 cycles and L3 andL4for 15 cycles. Target DNAwasused asfollows: Lane 1, 1pg; lane 2, 0.4 pg; lane 3, 0.2 pg;lane 4, 0.1 pg; lane 5, none. Lanes 6 and 7, The amplification products after 30 cycles in round 1 and 30 cycles in round 2 starting with 0.1 pg of DNA (lane 6) or 0.003 pg of DNA (lane 7).

phate at 200 ,uM, each primer at 1.0 puM, 2.5 U of Taq

polymerase, 10 mM Tris hydrochloride (pH 8.3), 50 mM

KCl,

1.5mMMgCl2,and0.01%gelatin asrecommendedby

theGeneAmp Kit manufacturer (Perkin Elmer Cetus,

Nor-walk, Conn.). The amplifications were carried out in a

programmablethermal controller (MJ Research, Watertown,

Mass.) inatwo-stepcycleof75 sat94°C followedby 3 min at 68°C. The samples were usually amplified through 25

cycles

byusing the outside pair of primers

(Li

and L2); 10%

of theamplified mixturewasthentransferredto a fresh tube

containing

reaction mixture with the inside primers (L3 and

L4). The secondamplificationwas usually allowed to

pro-ceedthrough 15cycles.Ten microliters ofthe reaction mix waselectrophoresedon1.5%agarose gels, and the reaction

productswerevisualizedbyethidiumbromidefluorescence.

Restriction enzyme digestion. Nucleic acids were

recov-ered from the reaction mixture by chloroform extraction followed by ethanol

precipitation.

Each sample was

sus-pended in20pl of10 mM

Tris-i

mMEDTA(pH 8.0), and 5

,ul of the suspension was digested with PstI (International Biotechnologies, Inc., New Haven, Conn.) or RsaI (Be-thesda Research Laboratories, Gaithersburg, Md.), as de-scribed by the

supplier.

The digested DNA was

electro-phoresed on

1.5%

agarose gels and visualizedby ethidium bromide fluorescence.

RESULTS

Amplificationof M.

leprae

DNAusingprimers

Lt

through

L4.

CsCl-purified

M.

leprae

genomic

DNA

(1 ng)

was

amplified with each

pair

of

primers

for 25

cycles.

As

ex-pected,primers

Li

and L2produceda

578-bp fragment

(Fig.

1A, lane 1), and primers L3 and L4

generated

a

347-bp

fragment (Fig. 1A,lane2).A

347-bp

bandwasalso

produced

when 1 ng ofM.

leprae

DNA was

amplified by

the nested

primer

procedure (Fig. 1A,

lane

3).

To confirm that the

347-bp

product

did

correspond

tothe

expected

portion

ofthe M.

leprae

groEL gene, the

amplified product

was

digested

with either PstI or RsaI. PstI

digestion

yielded

254- and

347bp _ I

-FIG. 2. Specificity of M. leprae primers in the presence of extraneousDNA.Each template wasamplified through 25 cycles in round 1 and 15 cycles in round 2, by using the indicated primers. Lane1, 1ngof M. tuberculosisgenomic DNA, primersTithrough T4; lane 2, 25 ng of M. tuberculosis genomic DNA, primers

Li

through L4; lane 3, 1

pug

of M.tuberculosisgenomic DNA plus 10 pg ofM.lepraegenomic DNA, primers

Li

through L4;lane 4, 1 ngof M. leprae genomic DNA, primers Li through L4; lane 5, 1 p.g of HeLacell DNA, primersLithrough L4; lane6, 1,ugof HeLa cell DNAplus 0.03 pg of M.lepraegenomic DNA(101HeLa genomes to 1 M. leprae genome), primers

Li

through L4.

93-bp fragments, andRsaIdigestion yielded 193-and 154-bp fragments,aspredicted fromthe sequenceofthe M.

leprae

gene (datanotshown).

Sensitivity of the

nested-primer

assay. Serial twofold dilu-tions ofM.

leprae

genomicDNA(1ng to 3fg)wereamplified for 25 cycles in the first round and 15 cycles in the second round. The 347-bp product was observed starting with as

little as0.2 pg ofpurified genomic DNA (Fig. 1B, lane 3).

Given that the M.

leprae

genome has a size of2.2 x

109

daltons or aweight of -0.0036 pg (5), 0.2 pg corresponds with the amount of DNA in -60 cells. By increasing the

numberof cycles to 30 inboththefirst and secondrounds,

positive

signals

could begenerated withaslittle as 0.003 pg

of

genomic

DNA

(Fig.

1B, lane 7).Ofcourse, notallsamples

containing 0.003 pg ofDNAproduced apositive result. In

fact, the distribution of positive and negative results in

samples

containing

0.003pg ofDNA was consistentwith a

Poissondistribution fora singletarget

(i.e., single

genome) in these

samples (data

notshown).

Thus,

itappearsthat the

procedure

iscapable ofdetectingasingletarget sequencein

a sample.

Specificity

of thenested-primer assay. Asaninitial testof

specificity,

25 ng of

CsCl-purified

M. tuberculosis

genomic

DNA or 1 ,ugof

purified

DNAfromHeLacellswas

amplified

by

using primers L1 through

L4.No

amplified

products

were

observed with M. tuberculosis DNA

(Fig.

2,

lane

2)

or human DNA

(Fig.

2, lane

5);

the

expected products

were

amplified

from1ngofM.

leprae

DNA

(Fig.

2,

lane

4) (the

M.

tuberculosisDNA used here was

amplifiable,

since

primers

Ti

throughT4

[Table 2]

were able to

amplify

the

expected

344-bp band from1 ngofM.tuberculosis

DNA; Fig. 2,

lane

1).

In addition,

primers L1

through

L4 could

specifically

amplify

M.

leprae

target

sequences

in mixtures

containing

these DNAs. That

is,

primers

Li

through

L4 were able to

amplify

theM.

leprae

sequences

from

samples

containing

M.

leprae

genomic

DNA andeitherM.

tuberculosis

orhuman

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123 4 i 2 ;3 4 45 `u

13

*2ff .s1 i

{

FIG. 3. Cross-reactivities of the M. leprae-nested primers. (A) Amplification products obtained by using primers Li and L2 (25 cycles) and L3 and L4 (15 cycles) and crude cell lysates from "M. lufu" (lane1),M. smegmatis (lane 2),M. simiae(lane 3),and

M. Ieprae(lane 4). (B) Amplification products by using primersLi

and L2 (25 cycles) and L3 and L4 (15 cycles) and mixtures containing 0.2pgofacontrol plasmid, pRL47, andportions (_105 cells) of celllysatesof M. avium(lane 1), M. bovis BCG(lane2), M.fortuitum (lane 3), M. gordonae (lane 4), M. microti (lane5), Bordetella bronchiseptica (lane 6), orRhodococcus equi (lane 7).

Theplasmid contains thebindingsites for primersLi through L4

anda70-bpinternal deletion which allowsitsamplification product (277 bp)tobe distinguishedfrom that of M. Ieprae genomicDNA

(347 bp) (lane 8).

DNAatratiosashighas105foreigngenomesto1 M. leprae genome(Fig. 2, lanes 3 and 6). Thus,notonlydo Li through L4 seem tobe specific for M. leprae, butlarge amountsof extraneousDNA do notappearto interfere with the ampli-fication of theM. Ieprae target sequences.

The species specificity of primers Li through L4 was

further assessed in nestedprimer amplifications of41species of bacteria.Inthesestudies, the starting templateswere1 jxg

(four Mycobacterium species)or1ng(nine

non-Mycobacte-rium species) of CsCl-purified DNA (Table 1) orportions of

crude lysates containing DNAfrom

i105

bacteria (22 My-cobacterium and 10non-Mycobacterium species) (Table 1). The crude bacterial lysates were made by homogenization

with0.1-mm-diameter glass beads,asdescribed in Materials

and Methods. None of these amplifications produced the 347-bpM. leprae product. However, assays of three of the

species, "M.lufu," Msmegmatis, andM.simiae, didyield detectable products. The products were 750, 1600 and 500,

and 350bp, respectively (Fig. 3A, lanes 1 through 3). Each could beeasilydistinguished from theM.leprae productby

sizeorrestriction fragmentpatternproducedby RsaIorPstI (data notshown).

Toensurethatthe crude lysates didnotcontaina

compo-nent(s) thatinhibited amplification,0.2pgofacontrol DNA

was added to each of seven of the cell lysates, and the

amplification reactions were then carried out in the usual

manner.The controlDNAwasaplasmid (pRL47) carryinga

690-bp fragment of theM. lepraegroELgene thatcontains

the binding sites for all four primers and a 70-bp internal

deletion (residues 1514 through 1583). This deletion allows the products of the amplification of the control DNA (508 and 277 bp) to be easily distinguished from the amplified productsofM.IepraegenomicDNA (578 and347bp). Inall

seven reactions, amplification of the 277-bp fragment was

accomplished at an expected rate of efficiency (Fig. 3B,

FIG. 4. Amplification of M. leprae from tissue homogenates.

Lanes 1through3 show theamplification products usingprimersLi and L2 for 25cyclesfollowedbyL3 and L4 for 15cyclesandacrude lysate of acid-fast bacilli harvested from an M. leprae-infected

mouse footpad (lane 1), a biopsy specimen from a lepromatous leprosypatient(lane 2),andabiopsyoflungtissue froma tubercu-losis patient (lane 3). Lane 4 contains theamplification productby usingM.tuberculosisprimersTl throughT4 and thelysatefromthe tuberculosis patientbiopsy.

lanes 1through 7). Thus, the crude lysates didnotcontaina

componentthat inhibited theamplificationprocess.

Detection ofM. leprae in tissues. Bacilli were harvested

from tissues ofinfectedmice,and crudelysatesof thebacilli

were prepared as described in Materials and Methods.

Portions of the lysates were then assayed by the

nested-primer procedure. The expected 347-bp amplification prod-uctwas obtained when 10,ulofthe celllysates(Fig. 4, lane

1), as well as dilutions of the lysate, was used. Positive

signalsweregenerated in dilutions containingasfewas1,000

bacilli. By increasing the number of cycles, the detection limitimprovedto20organisms persample. Thus,

amplifica-tion oftargetsequencesin crude lysatesappearstobeabout 20-fold less sensitive thanamplification of purified DNA.

Primers Li through L4 could also amplify M. leprae sequencesthathad beenrecovered from tissuehomogenates

of five biopsy specimens from three lepromatous leprosy patients. Approximately 105 to 106 bacilli were harvested

from eachof the homogenates by centrifugation, and crude bacterial lysates were made as described in Materials and

Methods. Portionsof the crude lysateswere thenamplified

with the nested primers Li through L4. The 347-bp amplifi-cation productwas obtained in samples of each of the five

homogenates. A representative amplification product is shown in Fig. 4, lane 2. As negative controls for this experiment, lung biopsy specimens from three tuberculosis patients previously shown to contain M. tuberculosis by culture and microscopywere amplified by using the nested

primers Li through L4. None of these biopsy samples producedanamplification product (Fig.4,lane 3). However, each of thelung specimenswaspositive forM. tuberculosis when tested with the M. tuberculosis nested primers Ti throughT4 inaPCR-based assay (Fig. 4, lane4).

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DISCUSSION

ThegroELgene waschosenasthetarget for amplification because its nucleotide sequence had been determined for both M.

leprae

and M. tuberculosis (9, 14). Therefore,

sequences that were differentbetween the two species and thattherefore

might

bespecific foreach could be identified.

Also,

since this geneishighlyconserved(15) andappears to

be required for viability in bacteria (6), there is strong selective pressure to maintain the amino acid sequence of

theGroEL

protein.

Inaddition, itappearsthat theM.leprae

genome is

exceedingly

stable. Clark-Curtiss and Walsh (5)

estimate thatthere is

only

0.25% sequencevariationamong thefour M.

leprae

isolates examined. Thissuggeststhatthe sequence of the groEL gene should be quite stable, and

false-negative

results due to mutation shouldbe rare.

A

nested-primer amplification

approach was used to

in-crease

specificity

and

sensitivity,

toavoidthe use of

radio-active

probes,

andto shorten thetimerequired toobtaina result.

Sensitivity

and

specificity

are increased because (i)

successful

amplification

requires the binding of four primers;

(ii)

freshreagents are addedafter 25 cycles; and (iii)

back-ground

bands are

reduced,

since each pair of primers is

responsible

for

only

asmall numberof

amplification

cycles.

In

addition, by using

nestedprimers, a sensitivity of

detec-tion

comparable

tothatobtained byusing

32P-labeled

hybrid-ization

probes

to detect the amplification products can be

obtained.

Thus,

the use of

radioactivity

(and concerns

re-garding

the shelf-life ofaradioactive reagent)aswellasthe

need to transferthe

products

to nitrocellulose andperform

hybridization

reactions and autoradiography is avoided.

This,

inturn, reducesto some extentthe amountof

experi-mental

manipulation required

and theoverall time for

anal-ysis.

The entire

nested-primer

assay,frompreparation of the crude

lysate

to

analysis

of the

gel,

canbe finished in lessthan 8 h, incontrast tothe24to48 h needed forthePCRassays

involving

radioactive

probes (1, 7).

The

specificity

of the assay was assessed by using 22

Mycobacterium species

and19

non-Mycobacterium species.

These

particular species

werechosen for

study

becausethey

are

phylogenetically closely

relatedtoM.

leprae

ortheyare

species

that

might

be found inclinical

samples

suchassputa, nasal

secretions,

orskin

biopsies.

None ofthese

organisms

produced

the 578- or

347-bp

M.

leprae product; however,

"M.

lufu"

produced

amoderateamountofa

750-bp product,

and M. simiaeand M.

smegmatis

produced marginally

vis-ible amounts of a

350-bp product

and

1,600-

and

500-bp

products, respectively.

The cross-reactivities with "M.

lufu"

and M. simiaeare

curious,

since "M.

lufu"

has been

usedas amodelsystemfor

antileprosy drug

studies

(11)

and M. simiaehasbeen

reported

toshare

antigenic

determinants with M.

leprae

and to

provide immunologic

protection

against

M.

leprae

infection

(16). Although

the

origin

of these

bands is

uncertain, they

should not be confused with the

expected

amplification products,

since

they

are

easily

distin-guished by

their sizes and

by fragments produced by

the

restriction enzyme PstI orRsaI.

Overall,

the results indicate that

nested-primer

PCR can

be usedto

specifically

amplify

sequences

ofM.

leprae.

The assay can detect as few as 20

organisms

in crude

lysates

without the need to

purify

the DNA or use radioactive

probes,

when a total of60

cycles

of

amplification

is used.

This is much more sensitivethan clinical tests

currently

in

use.

Moreover,

thetestidentifiesthe

organism

in the

sample

as M.

leprae. Thus,

the results

reported

here are

quite

encouraging for the potential useof PCRtechnologyin the rapid detection and definitive identification ofsmallnumbers of M. lepraein clinical specimens.

ACKNOWLEDGMENTS

We thank Ray Butler, Jacquelyn Sampson, Robert Weaver,and Richard Facklam forproviding cultures; Steve O'Connor for pro-viding purified DNA; Vijay Varma for propro-viding clinicalspecimens; Rosalind Van Landingham and LauraWalker for providingmouse tissue homogenates; and Sathish Mundayoor for providingpRL47 DNA.

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