Attempts were made to develop DNA

probes for M. tuberculosis. Random library of M.

tuberculosis was constructed in plasmid pGEM -4.

Selection of recombinant clones was made by hybridisation

with 32P labelled M. tuberculosis probe.

Ten recombinant clones were selected on the basis

of strong signals from the random library.

These 10 clones named pTRC1-10 were subjected to

tests for specificity and sensitivity. On this basis,

pTRC4 was chosen and this is also, useful in restriction

fragment length polymorphism (RFLP)

studies

Narayanan, P R

Narayanan, Sujatha

Prabhakar, R

Ramanujam, S

Sahadevan, R

NIRT Institutional Repository

Ind. J. Tub., 1992, 40, 99DEVELOPMENT OF DNA PROBES FOR M. TUBERCULOSISSujatha Narayanan, R. Sahadevan, S. Ramunujam, R. Prabhakar and P.R. Narayanan  Summary. Attempts were made to develop DNA  probes for M. tuberculosis. Random library of M.tuberculosis was constructed in plasmid pGEM -4.Selection of recombinant clones was made by hy-bridisation with 32P labelled M. tuberculosis probe.Ten recombinant clones were selected on the ba-sis of strong signals from the random library.These 10 clones named pTRC1-10 were subjected totests for specificity and sensitivity. On this basis,pTRC 4 was chosen and this is also, useful in re-striction fragment length polymorphism (RFLP)studies. IntroductionTuberculosis has been reported with increas-ing frequency in patients with acquired immuno-deficiency and other immunocompromised pa-tients’. Diagnosis of tuberculosis is time consum-ing and depends on isolation of the pathogen,which takes up to eight weeks. Direct microscopylacks sensitivity. The bactec system, even thoughit has reduced the time needed for detection, isstill growth dependent2.In recent times attention has been focussed ondeveloping sensitive and specific tests fordetecting M. tuberculosis. Recombinant DNAtechnology, a powerful tool, has opened up thepossibility of developing specific DNA probeswhich can help in the detection of M. tuberculosisand also to differentiate between the differentstrains of mycobacteria. The technique is there-fore of potential value for basic and epidemiol-ogic studies of tuberculosis. There have beenmany reports of specific DNA probes for diagno-sis of M. tuberculosis using the Polymerase ChainReaction (PCR), latest technique in the recombi-nant DNA field3,4,5,6. Even though the sensitivity ofthe tests using PCR is 100% the specificity is only62.5% compared to culture results7. Other reportstoo have not shown better specificity because inthis kind of approach proper primers have to beused. As of today, there is no single immuno-di-agnostic test or tests based on molecular ap-proaches which could be recommended as a con-firmatory test for tuberculosis Here we report theisolation of a DNA probe from a M. tuberculosislibrary made in plasmid pGEM. This probe hasbeen tested for specificity as a whole probe sincethis probe has also been found to be useful in re-striction fragment length polymorphism (RFLP)studies with various strains of mycobacteria. Theinteresting feature of this probe is that it gives adifferent RFLP pattern with drug resistantstrains.Material and MethodsMycobactetial strains : Reference and clinical iso-lates of M. tuberculosis, reference strains of atypi-cal mycobacteria and non-mycobacterial specieswere from the Bacteriology Department of theCentre.Preparation of mycobacterial DNA : Mycobacteriaw re cultured in Sauton-tween medium. D-glycine was added to a concentration of 1mg perml when the cells were in log phase to enhancelysis. After a drop in density, the cells were har-vested by centrifugation and lysed with sodiumdodecyl sulphate (SDS). DNA was purified by aphenol-chloroform-isoamyl alcohol procedure.Digestion with restriction endonucleases : Reactionmixtures containing 1 to 2 ug of DNA and 4 to 10units of enzyme were incubated at 37°C over-night.Cloning into pGEM-4 : DNA from M. tuberculosisH37Rv was digested with Rsa-1 and ligated with aImmunology Department, Tuberculosis Research Centre, Madras- 600031*Paper presented at the 46th National Conference on Tuberculosis and Chest Diseases, New Delhi : 22-24November, 1991.Correspondence : Director, TB Research Centre, Chetput, Madras- 600031.SUJATHA NARAYANAN ET ALFig. 1. The 10 recombinant pGEM clones were purified by Cscl2 ensity gradient and run on 1% agarose gel.Lane 1 pGEM; Lane 2 to Lane 11 pTRC 1 to pTRC 10 and Lane 12 M. wt marker.Hinc II digest of pGEM-4. Escherichia coli HB101 were transformed and plated. The recombi-nant colonies appearing on the Ampicillin plateswere screened with 32P labelled M. tuberculosisH37Rv. The various recombinants which lighted upstrongly were picked up, grown in log phase cul-tures of E. coli HB 101 cells. DNA from recombi-nant pGEM plasmids was isolated by alkaline ly-sis method. To excise the insert DNA, the DNAfrom recombinant plasmids was digested withEcoRI and Hind III which cleave at the oppositeends of poly linker region. The sizes of the excisedfragments were determined by agarose gelelectrophoresis.Labelling DNA probes : Total chromosomal DNAand recombinant DNA were labelled with 32 P dCTP or 32 P d ATP(3000m Ci/mmol) by nicktranslation. Specific activity of the probes wasabout 108 cpm/ug of DNA and approximately 106t  107 cpm was used in each hybridisation.Slo  blot hybridisation : DNA was denatured in0.4 N NaOH at room temperature for 10 minutes,ne tralised with an equal volume of 2M Tris HCl,pH (7.0) and loaded on a slot blotter (minifold IISchliecher & Schuell Inc. Keene N.H.) with Genescreen membrane (DuPont NEN Research prod-uct, Boston, Mass.). The pre-hybridisation solu-tion consisted of 10% dextran sulphate, 1% SDS& 1M sodium chloride and 100ug of denaturedsalmon sperm DNA per ml. Labelled probe DNAwas added to the hybridisation solution, Mem-branes were hybridised at 65°C and washed asfollows. 2X SSC (2X) and 0.5% SDS at roomtemperature for 5’ 2X SSC & 1.0% SDS for 1/2hour twice and 0.2 X SSC & 0.1% SDS for 1/2hour.DEVELOPMENT OF DNA PROBES FOR M. TUBERCULOSIS 101Fig. 2. The 10 recombinant clones from the pGEM library were spotted in the slot blot in order of decreasingconcentration as shown above. The 1st row represents standard M. tuberculosis DNA. The rest of the 10rows represent the10 recombinant clones numbering 1-10. The probe used for hybridisation was 32Plabelled- M. tuberculosis DNA.Southern blot hybridisations : Digests were elec-trophoresed on 0.8% agarose gels containingethidium bromide and photographed. DNA frag-ments were denatured and transferred to Genescreen plus membrane (DuPont, NEN Researchproducts, Boston, Mass.) by using either a modi-fied Southern transfer method as described byTransblot method (Trans vat 80 Hoeffer Scien-tific Instruments, San Francisco).ResultsThe criterion used in screening the pGEM re-combinant colonies was selection of those cloneswhich give strong signals when hybridised with   32P labelled M. tuberculosis. A total of 10 suchclones with strong signals was chosen and furthertested for specificity and sensitivity using slot blothybridisation. The sizes of these 10 recombinantclones are shown in Fig 1.Slot blot hybridisation : The 10 recombinantclones, standard strain of M. tuberculosis or nontuberculosis strains were spotted in the slot blotin order of decreasing concentrations (2.0ug,Auto-radiography : Auto-radiographs were pre-pared by exposing the hybridised blots for varyinglengths of time at 70°C to Kodak x-omat AR filmand cronex lightning screens.102 SUJATHA NARAYANAN ET AL.Fig. 3. The 1st 10 rows represent the 10 pGEM recombinant clones at three different concentrations. The 11th,12th and 13th rows show E.coli, Clostridium perfringenes and human placental DNA. The probe used forhybridisation was32P labelled pooled DNA from E. coli, Clostridium perfringenes and human placenta.0.2ug, 0.02ug). These were hybridised with 32 Plabelled M. tuberculosis. Those clones whichlighted up strongly at the lowest concentrationwere chosen for further evaluation (Fig 2). A du-plicate of the above slot blot was hybridised witha non mycobacterial probe (32 P labelled DNA ofa pool of E. coli, Clostridium pefringenes, andhuman placenta). Those recombinant cloneswhich reacted least with non-mycobacterial DNAwere chosen for further work. (Fig 3) Among theclones chosen according to the above criteria, oneclone- pTRC4 is promising. As a further step, thespecificity was studied using DNA from E.c li,E.coli Kk6, Bacillus subtilis, Proteus mirabilis,DEVELOPMENT OF DNA PROBES FOR M. TUBERCULOSIS 103Fig. 4.- Various non mycobacterial species were spotted in the slot blot at 4 different concentrations (20.0, 2.0, 0.2& 0.02 ug) probe - 32P labelled pTRC4Proteus vulgaris, Clostridium perfringenes S. hemo-lytica and human placenta. The DNA from thesespecies were spotted in concentrations rangingfrom 20, 2.0, 0.2, and 0.02 ug of DNA. pTRC4 didnot cross react with any of the non-mycobacterialDNA used except with human placental DNA, atthe highest concentration used i.e. 20 & 2 ug (Fig.4). The mycobacterial fragment of pTRC4 is 2.2kb in size and when it was further subjected to re-striction digestion with various enzymes, gavesites only for Sal-I.pTRC4 was restriction digested with EcoR I,Hind III and Sal I and run on 1% agarose gelelectrophoresis. The gel was Southern transferredto Gene screen membrane. Three lanes were hy-bridised with 32P labelled M. tuberculosis, 32P la-belled pTRC4 and 32P labelled human placentalDNA respectively. The individual fragments didnot cross react with pTRC4 (Fig. 5). The individ-ual fragments are being sequenced for furtherevaluation of their sensitivity and specificity.The potentiality of this clone in RFLP studiesand specificity was evaluated. The DNA fromvarious strains isolated from patients were re-striction digested with Pst I and Sal I and run on1.0% agarose gel, Southern transferred on toGen  screen membrane and probed with 32 P la-belled pTRC4 (Fig.6). Except BCG & M. tubercu-losis none of the atypical mycobacteria reactedwith the probe. The various atypical mycobacteriaused were M. kansasii, M. avium, M. gordonae, M.phlei, M. xenopi, M. chelonei, M. fortuitum, M.chitae, M. bovis BCG and BCG strain. The inter-esting feature of the probe is that it distinguishesthe drug resistant strain from the other strains ofmycobacteria.SUJATHA NARAYANAN ET ALFig. 5.Fig. 6.pTRC4 clone restriction digested with three agents.( triplicate) and probed with 32P pTRC4, 32P labelledM. tuberculosis and 32P labelled human placental DNA.DNA from clinical isolates of M. tuberculosis and atypical mycobacteria restriction digested with Sal1 andPst1 run on 1% agarose gel, Southern transferred and probed with 32p labelled pTRC4. Lanes l-7 clinicalisolates of M. tuberculosis. 8 M. kansasii 9. M. avium 10. M. phlei 11. M. simiae 12. M. smegmatis 13. M.gondonae 14. M. chelonei 15 M. fortuitum 16. M. chitae 17. M. xenopi 18. M. bovis BCG. 20. Rifampicinresistant clinical isolate.DEVELOPMENT OF DNA PROBES FOR M. TUBERCULOSIS 105DiscussionWe have isolated 10 recombinant clones froma library of M. tuberculosis in pGEM. These wereselected on the basis of their strong hybridisationsignals with32 P labelled M. tuberculosis. Theseclones were evaluated for their specificity andsensitivity by doing a slot blot hybridisation withnon mycobacteria DNA and DNA from M. tuber-culosis at concentrations from 20 ug to 0.002ug.Out of these 10 clones, 4 seem to be promisingwith regard to specificity and sensitivity. Amongthese four clones one, pTRC4, is also a repetitivefragment, helpful in studying the restriction frag-ment length polymorphism (RFLP) with variousstrains of mycobacteria.Several repetitive sequences have been de-scribed in the genome of members of the myco-           bacterium tuberculosis complexes8,9,10,11,12.   Fromthe reported size and map of the clones, oursseems to be different.The data presented indicate that pTRC4 haspotential as a diagnostic probe for identificationof M. tuberculosis. The pTRC4 clone is being sub-cloned. Nucleotide sequencing is in progress tofacilitate PCR amplification of target DNA andthereby increase the sensitivity of the probe. Thesubclones also would be used in RFLP studieswith mycobacterial strains.The present study also indicates that pTRC4 isrepeated around ten times in the genome of vari-ous strains of M. tuberculosis. The repetitive frag-ment did not show cross hybridisation with DNAfrom atypical mycobacterial strains used exceptM. bovis BCG which showed similar pattern as M.tuberculosis. The interesting feature of this cloneis that it has given different banding pattern with   drug resistant isolates of M. tuberculosis. Sincethere is RFLP between the normal and drug re-sistant strains which has been detected with thisprobe, further investigations could be carried outto find out whether this difference has arisen dueto mutation. Mycobacterium has been a challengebecause many facts about its virulence, pathogen-icity and other properties have been a mystery inspite of the long years of work on it.References1. Centres for Disease Control . A strategy planfor the elimination of tuberculosis in the United2.3.4.5.6.7.8.9.10.11.12.States. Morbid mortal weekly Rep. 1989, 38 ,269.Hoeffner S.E. Improved detection of myco-bacterium avium complex with Bactec Radio-metric system diagn. Microbiol Infect. Dis.;1988, 10, 1.Rubina. J. Patel, Jochen W.U. Fries, Willy. F.Piessens and Dyann. F. wirth. Sequence analysesand amplification of PCR of a cloned DNAfragment for identification of M. tuberculosis.Journal of Clinical Microbiology Mar. 1989,p513-518.D. Dewit. L. Steyn, S. Shoemaker and M. Sogin.Direct detection of M. tuberculosis n clinicalspecimens by DNA amplification. J. Clin Micro-biology Nov.; 1990. 2431.Peter. W.M. Hermans, Anja. R.J. Schuitema,Dick Van Soolingan Cees, P.H.J. Verstynen,Elizabeth. M. Bik, Jelle E.R. Thole, Arend.H.J.K.olk and Jan. D.A. Van Embden. J. ClinMicrobiology June. 1990; 1204-1213.Sjobring, Ulf, Mecklenburg, Michael, Anderson,Bengard Ase, and Miorner, Hakan. Polymerasechain reaction for detection of M. tuberculosis J.Clin. Microbiology Oct.; 1990, 2200-2204.Chia, C. Pao., T.S. Benedict, Yen Jinn Bang,You Juehn-Shin Maa, Ellen. H. Fiss and Chau-Hsiung C hang.. Detection and identification ofM. tuberculosis by DNA amplification. Journalof Clinical Microbiology Sept. 1990, 1877-1880.Eisenack. K, Crawford J.T. and Bates. J.H. Re-petitive DNA sequences as probes for M. tuber-culosis. J. Clin. Microbiology.; 1988, 26, 2240.Clark Curtis J.E., and Gerald. P.W. Conserva-tion of genomic sequences among isolates ofMycobacterium leprae. J. Bacteriology.; 1989,171, 4844Dick Van Soolingen, Peter. W.M. Hermans,Petra E.W. de Hass, David R. Soll and Jan.D.A. Van Emdden (1991). Evaluation of IS de-pendent DNA polymorphism as a tool in theepidemiology of tuberculosis.Isabel otal, Carlos Martin, Veronique Vincent,Levy Frebault, Dominique Thierry and BrigitteGicquel. J. Clin Microbiology. 1991; 29 : 1252Thierry, D., Cave, M.D., Eisenach, K.D.,Crawford, J.T., Bates, J.H., Gicquel, B. andGuesdon, J.L. IS 6110 and IS like element of M.tuberculosis complex Nucleic acid Research.1990, 18, 188.

Development of DNA probes for M. tuberculosis

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Development of DNA probes for M. tuberculosis

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