Species-specific biotinylated DNA probe for the detection of Mycoplasma gallisepticum

Molecular

and

Cellular Probes (1988) 2, 2 3 7-244

Species-specific biotinylated DNA probe for the detection of Mycoplasma gallisepticum

Steven J . Geary,* Richard Intres and Michael G . Gabridge Bionique Laboratories, Inc ., Bloomingdale Road, Saranac Lake, NY 12983, USA (Received 28 April 1988, Accepted 9 May 1988)

A 5 . 5 kilobase DNA fragment from an Eco RI digest of the Mycoplasma gallisepticum genome was specific for the detection of M . gallisepticum . This 5 . 5 kb fragment was cloned into bacteriophage X gtll followed by subcloning into the plasmid vector pCEM-3Z . The incorporation of a biotin label was accomplished by utilizing biotin-1 1dUTP in a nick translation reaction . This probe, designated pMg6, reacts specifically with M. gallisepticum when tested against various mycoplasma DNAs in Southern blot hybridization analysis . Spot-blot hybridization data indicate the pMg6 is capable of detecting 800 pg of M. gallisepticum DNA.

initially

KEYWORDS : biotinylated, DNA probe,

Mycoplasma gallisepticum .

INTRODUCTION Mycoplasma gallisepticum causes respiratory diseases in chickens and turkeys . It is

a major cause of reduced egg production, reduced hatchability and downgrading of carcasses .' Mycoplasma gallisepticum is transmitted through the egg or infected airborne droplets .' Depending on the system of management, the infection may spread rapidly through a flock . Current means of diagnosis depend on serological assays to detect serum antibodies or on the direct isolation and identification of the organism . The isolation of M . gallisepticum requires specialized media and techniques, and may require as long as a month to complete . Analysis of the sera involve methods for evaluating prior exposure . For definitive results, a specific M . gallisepticum antigen to differentiate that pathogen from other closely related mycoplasmas such as M . synoviae is required. Specific M . gallisepticum antigen is commercially available . However, some users have reported cross-reactions, and some recently isolated strains of M . gallisepticum are not detected in the current antigenic assays . This pMg6 biotinylated DNA probe offers a means of discriminating M . gallisepticum from other mycoplasmas as well as the potential for detecting the newly reported, difficult to detect M . gallisepticum strains . *Corresponding author, to whom inquiries should be sent .

0890-8508/88/030237+08 $03 .00/0

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MATERIALS AND METHODS

Organisms and culture conditions Mycoplasma gallisepticum PG31 (ATCC) was cultured in Frey's medium .' Mycoplasma synoviae WVU (ATCC)- .was cultured in Frey's medium supplemented with nicotinamide adenine dinucleotide (NAD) . Mycoplasma pneumoniae PI 1428 (ATCC) was cultured in G199H . 4 Mycoplasma bovis PG45 (ATCC) and Acholeplasma laidlawii PG 8 (ATCC) were cultured in fortified commercial medium . 5 Mycoplasma hyopneumoniae VPP11 (ATCC) was cultured in Friis medium .6 Escherichia coli

Y1090 (Promega) and JM109 (Promega) were grown in Luria broth . Incubation for all of the organisms was done at 37°C.

DNA isolation and quantitation DNAs were extracted by the procedure of Marmur .7 The purities and concentrations were determined spectrophotometrically by the 260/280 nm ratios and the 260 nm readings,' respectively . DNA, from recombinant k gt11 clones were extracted from plate lysates utilizing Lambdasorb (Promega) according to the manufacturer's protocol . DNA fragments were cut from low melting point agarose, and the gel was melted at 65 ° C . It was then extracted twice with phenol, once with phenol/ chloroform and once with chloroform . The DNA was precipitated with two volumes of 95% ethanol .

Restriction enzyme digestion Eco RI, Hind III, Pvu I and Pvu 11 restriction digests were each performed at 37 ° C for 1 h in the buffers recommended by the manufacturer (Bethesda Research Laboratories) .

Cloning Eco RI restriction fragments of M . gallisepticum DNA were cloned into bacteriophage X gtll . The Protoclone and Packagene systems (Promega) were utilized . Briefly, 0 E2 .tg of M . gallisepticum DNA fragments were ligated to 0 . 5 µg of X gt11 dephosphorylated arms using 0 . 5 Weiss units of T 4 DNA ligase at 16°C for 2 h . The entire reaction was packaged in vitro and lysogenized to E . coli Y1090 and plated out as an overlay using Luria-Bertani (LB) agar containing 100 µg ml -1 ampicillin, MgCl, Plates were incubated at 42°C until plaques 400 µg ml -1 X-Gal and 0 . 01 M . appeared . The plaques were analysed by filter lifts followed by hybridization to biotinylated M . gallisepticum total genome DNA probe or biotinylated A . laidlawii total genome DNA probe . Those plaques hybridizing with the M. gallisepticum probe and not the A . laidlawii probe were picked and propagated for further analysis . X gt11-Mg6 clone containing a DNA sequence specific to M . gallisepticum



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was subcloned into the plasmid vector pGEM-3Z . 0 . 1 lag of Eco RI-digested pGEM-3Z DNA and 1 .tg of Eco RI-digested X gt11-Mg6 DNA were ligated with 1 Weiss unit of T4 ligase and incubated at 15 ° C for 12 h . Transformation of E. coli JM 109 was performed using a standard calcium chloride procedure .' Transformed cells were -1 ampicillin, 0 . 5 mm IPTG, 40 lag plated onto LB agar plates containing 100 Vg ml ml - ' X-Gal and incubated at 37°C . White colonies were picked and amplified by chloramphenicol treatment . 10 The plasmids were harvested" utilizing pZ523 columns (5 Prime-3 Prime, Inc .) A plasmid restriction enzyme map was made using the multiple endonuclease digestion method ."

Probe construction DNA was labelled with biotin-11-dUTP (Bethesda Research Laboratories) in a .12,13 Biotin-labelled DNA was separated from standard nick translation reaction unincorporated nucleotides by either column chromatography on Sephadex G-50 fine (Pharmacia) equilibrated with 10 mm Tris-HC1 (pH 8 . 0), 50 mm NaCl and 0-1 mm EDTA (pH 8 . 0) or with Select-B columns (5 Prime-3 Prime, Inc .) .

Agarose gel electrophoresis DNAs from various mycoplasmas were cleaved with the restriction enzyme indicated . DNA fragments were separated by electrophoresis in 0 . 8% agarose (BioRad Laboratories) at 6 V cm - ' for 2 h or in 0. 6% low melting point agarose (BRL) at 4 V cm - ' for 4 h . Gels were stained after electrophoresis with 0 . 5 µg ml - ' ethidium bromide in TEA [40 mm Tris (pH 7 . 6), 20 mm sodium acetate, 2 mm EDTAI at room temperature, for 45 min . The DNA was visualized with a u .v . illuminator at a 300 nm wavelength .

Sample deposition, hybridization and detection Samples of DNA were diluted in TE (10 mm Tris base and 1 mm EDTA, pH 8 .0) with 200 .tg ml - ' denatured salmon DNA and applied directly onto NTC paper [pre'soaked for 10 min in 10 X SSC (1 . 5 M NaCl, 0 . 15 M sodium citrate, pH 7 . 0) and dried] . Cleaved DNA fragments were transferred from agarose gels to NTC by the method of Southern ." For spot blots, the OTC was washed with 1 . 5 M NaCI-0 . 5 M NaOH for 5 min, followed by two washes with 1 . 5 M NaCI/0. 05 M Tris-HCI (pH 8. 0) for 5 min . The NTC was air-dried for 30 min, followed by incubation for 2 . 5 h at 80°C . The NTC was then placed in 10 ml of prehybridization buffer (6 X SSC, 10 mm EDTA, 5 X Denhardt's+0 . 5% SDS, 200 jag ml - ' denatured salmon DNA) and incubated at 42 ° C in a water-bath for 2 . 5 h . The buffer was drained out, and 10 ml of hybridization buffer (prehybridization buffer with 43% deionized formamide and the biotinylated probe), prewarmed to 42°C was added and incubated at 42°C for 16 h . The NTC was removed and washed twice with 2 X SSC+0 . 1% SDS for 3 min each, twice with 0 . 2 X SSC+011% SDS for 3 min each, and twice at 60 ° C, with



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0 . 16 X SSC+0 . 1% SDS for 15 min each . The NTC paper was then rinsed with 2 X SS.C+0 . 1ofoSDS . The hybridized probe was detected utilizing the BRL DNA detection system (Bethesda Research Laboratories) . The NTC was washed for 1 min in buffer 1[0 . 1 M Tris-H CI (pH 7 5), 0-1 M NaCL, 2 MM MgCI 2, 0. 05% (vol/vol) Triton X-1001, followed by incubation for 20 min at 42 ° C in buffer 2 [3% (wt/vol) bovine serum albumin (BSA) in buffer 11 . The remainder of the detection procedure was performed at room temperature . The NTC was incubated for 10 min with gentle mixing in streptavidin (2 lag ml - ' in buffer 1) . It was then washed three times in buffer 1 for 3 min each . Biotinylated polyalkaline phosphatase (1 lag ml - ' in buffer 1) was added and incubated for 10 min with gentle mixing . The NTC was washed twice with buffer 1 for 3 min each, and then washed twice with buffer 3 [0-1 M Tris HCI (PH 9 . 5), 0 . 1 M NaCI, and 50 mm MgC1 2 for 3 min each . Nitro-blue tetrazolium and 5-bromo-4chloro-3-indolyl phosphate were diluted in buffer 3 and incubated with the NTC in the dark until the reaction was complete . The NTC was washed in 20 mm Tris (pH 7-5) and 5 mm EDTA, to terminate the reaction, and baked at 80°C for 2 min .

RESULTS The cloning of Eco RI fragments of M . gallisepticum DNA into bacteriophage ~, gt11 resulted in an efficiency of 3 . 4 X 107 recombinants per Itg of M . gallisepticum DNA . The NTC filter lift which was hybridized with the biotinylated M . gallisepticum total genome DNA probe reacted with all of the clear plaques as expected . This verifies that the plaques did contain M. gallisepticum DNA fragments . The identical NTC filter lift, when hybridized with the biotinylated A . Iaidlawii total genome DNA probe, resulted in approximately 90% of the plaques also reacting . This indicated that those plaques contained DNA fragments that are at least in part identical to both M . gallisepticum and A . Iaidlawii . The plaques that hybridized only to the M. gallisepticum probe were propagated and their insert size determined . ? gt11-Mg6 was found to contain a 5-5 kb Eco RI fragment (Fig . 1) . Fig . 2(a) shows the gel of various restriction digested mycoplasma DNAs . Southern blot hybridization using biotinylated ? gt11-Mg6 5 . 5 kb Eco RI fragment as the probe shows the only species of mycoplasma recognized is M . gallisepticum [Fig. 2(b)] . The quantity of DNA obtained by the extraction of the fragment from the low melting point agarose was insufficient to generate a strong hybridization signal, so the 5-5 kb fragment was subcloned into the plasmid pGEM-3Z . The total pMg6 vector was then biotinylated and utilized as a probe . A total of 750 ng of each Eco RI-digested DNA were loaded per well in an agarose gel [Fig . 3(a)] . After Southern blot transfer, the NTC was hybridized with 1 .tg of pMg6 probe . As seen in Fig . 3(b), a single M. gallisepticum band of 5-5 kb was the only fragment recognized . This hybridization reaction was strong and . specific for M. gallisepticum . Spot blot hybridization, used to determine the limit of sensitivity of the biotinylated pMg6 probe against M . gallisepticum target DNA detected 800 pg . This is equivalent to approximately 8 X 10 5 mycoplasma organisms according to calculations based on a genome size of 500 MDa . Fig . 4 shows a partial restriction map of clone pMg6 containing the 5-5 kb M . gallisepticum fragment . As the map indicates, there are two Pvu I sites, one Pvu II site and four Hind III sites that could be located . Two additional Hind III sites are present, but their



Mycoplasma

gallisepticum

biotinylated DNA probe

Fig. 1 0 .6% low melting point agarose gel stained with ethidium bromide . Lane 1, biotinylated % Hind III DNA fragments . Lanes 2-6 X gt11-Mg6 Eco RI DNA fragments. Arrow indicates the 5 . 5 kb Mycoplasma gallisepticum DNA insert .

Fig . 2 . (a) 0 .8% agarose gel stained with ethidium bromide . Lane 1, biotinylated ?, Hind III fragments ; lane 2, empty; lane 3, Mycoplasma gallisepticum Eco RI DNA fragments ; lane 4, M . synoviae Eco RI DNA fragments; lane 5, Acholeplasma laidlawü Eco RI DNA fragments; lane 6, M. pneumoniae Eco RI DNA fragments ; lane 7, M . .hyopneumoniae-undigested DNA ; lane 8, A . laidlawii-undigested DNA . (b) Southern blot of gel A hybridized with biotinylated X gt11-Mg6 5 . 5 kb DNA probe.

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(a) Fig. 3. (a) 0 .8% agarose gel stained with ethidium bromide . Lane 1, biotinylated . Hind III fragments; lane 2, Mycoplasma gallisepticum Eco RI DNA fragments; lane 3, M . synoviae Eco RI DNA fragments; lane 4, Acholeplasma laidlawii Eco RI DNA fragments ; lane 5, M. pneumoniae Eco RI DNA fragments; lane 6, M . bovis Eco RI DNA fragments . (b) Southern blot of gel A hybridized with biotinylated pMG6 DNA probe .

exact locations could not be determined from this data . Also shown are the locations of transcription initiation sites for phage RNA polymerases SP6 and T7 . These enzymes can be used to generate RNA for use as single-stranded probes or as substrates for in vitro translation reactions . In vitro translation will allow us to characterize the protein(s) coded for by this specific M . gallisepticum DNA fragment.

DISCUSSION AND CONCLUSIONS DNA probes have been developed for the detection of many organisms including mycoplasmas . Taylor et al ." constructed two 32 P-labelled M . hyorhinis DNA probes, 3 . 6 and 9.4 kb in size . Each of these probes reacted specifically with M . hyorhinis and no other mycoplasmas tested . Chan & Ross 16 constructed a radiolabelled 9 kb DNA probe for the detection of M. hyopneumoniae . This probe reacted with both M. hyopneumoniae and M. flocculare . However, it was still useful for the detection of

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4 5 6 7, 8 kb I 2 3 0 Partial restriction digest map of pMg6 locating Eco RI, Pvu I, Pvu II and four of the Hind III sites .

Mycoplasma gallisepticum biotinylated DNA probe

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mycoplasmas in clinical specimens from pigs . A recent article by Santha et al ." reported on the construction of an 800 by M . gallisepticum DNA probe. This 32Plabelled probe was reported to detect 0 . 5 ng of M. gallisepticum DNA and to be specific for M. gallisepticum . This sensitivity is equivalent to approximately 5 x 10 5 mycoplasmas . These probes are all useful for the detection of the mycoplasma for which they were constructed . However, the use of radioisotopes to label the probes limits the scope of use for diagnosis . Our goal is to develop a DNA probe which is specific and sensitive as well as non-hazardous in use . Our laboratory constructed a biotin-labelled M . gallisepticum total genome DNA probe to determine the sensitivity and applicability of the biotin label ." This probe was capable of detecting 75 ng of M . gallisepticum DNA in a spot blot and did not show any background problems often reported for the biotin label,--It reacted strongly with homologous samples but it also reacted weakly with other mycoplasmas . This was not unexpected, since total genome probes contain common genes, such as the highly conserved rRNA genes . The data presented here demonstrate the specificity of our new 5 . 5 kb M . gallisepticum DNA probe . Southern blot hybridization analysis indicated that it hybridizes solely with M . gallisepticum and not with M . pneumoniae, which possesses genes common to M . gallisepticum in addition to the rRNA genes, 19 or with M. synoviae, another avian pathogen . The current level of sensitivity with this probe is 800 pg of target DNA . Efforts are underway to improve the degree of sensitivity to make this a useful tool for evaluation of field samples .

ACKNOWLEDGEMENTS The authors thank Martha Gladd for technical assistance and Cynthia Gendron for typing this manuscript. This research was supported in part by research grants from the US Department of Agriculture (#85-SBIR-8-0072) and the New York State Science and Technology Foundation (#SBIR 86-62) .

REFERENCES 1 . Jordan, F . T. W. (1979) . Avian mycoplasmas . In The Mycoplasmas Il. Human and Animal Mycoplàsmas . (Tully, J . G. & Whitcomb, R . F., eds) pp . 1-40 . New York : Academic Press . 2 . Yoder, H . W., Jr (1984) . Mycoplasma gallisepticum infection . In Diseases of Poultry. (Hofstad, M. S ., ed .) 8th edn, pp . 190-202 . Ames, Iowa : Iowa State University Press . 3 . Frey, M . L ., Hanson, R. P. & Anderson, D . R . (1968). A medium for the isolation of avian mycoplasmas . American Journal of Veterinary Research 29, 2163-71 . 4 . Gabridge, M . G ., Singer, S . E . & Esposito, R. A . (1976) . Cultivation of mycoplasmas in a modified tissue culture medium . Applied Environmental Microbiology 31, 986-9 . 5 . Macy, M . L . (1980) . Tests for mycoplasmal contamination of cultured cells as applied at the ATCC . Tissue Culture Association Manual 5, 1151-5. 6 . Friis, N . F . (1975) . Some recommendations concerning primary isolation of Mycoplasma suipneumoniae and Mycoplasma flocculare . A survey. Nordic Veterinary Medicine 27, 337-9. 7 . Marmur, J . A . (1969) . A procedure for the isolation of deoxyribonucleic acid from microorganisms . Journal of Molecular Biology 3, 208-18. 8 . Maniatis, T ., Fritsch, E . F . & Sambrook, J . (1982) . Molecular Cloning : A Laboratory Manual, p . 468. Cold Spring Harbor, New York : Cold Spring Harbor Laboratory . 9 . Mandel, M. & Higa, A . (1970) . Calcium dependent bacteriophage DNA infection . Journal of Molecular Biology 53, 154 .



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10 . M,aniatis, T., Fritsch, E . F . & Sambrook, J . (1982) . Molecular Cloning: A Laboratory Manual, p . 88 . Cold Spring Habor, New York: Cold Spring Harbor Laboratory . 11 . Ausubel, F. M., Brent, R ., Kingston, R . E ., Moore, D . D ., Seidman, J . G ., Smith, J . A . & Struhl, K . (1987) . ,Current'Protocols in . Molecular Biology. 3 .2 .1 . New York : Greene Publishing Associates & WileyInterscience. 12 . Kelly, R . G ., Cozzarelli, N ., Deutscher, M . P., Lehman, I . R . & Kornberg, A . (1970) . Enzymatic synthesis of deoxyribonucleic acid . journal of Biological Chemistry 245, 39. 13 . Rigby, P . W. J ., Dieckmann, M ., Rhodes, C . & Berg, P . (1977) . Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I . journal of Molecular Biology 113, 237-51 . 14 . Southern; E. (1975) . Detection of specific sequences among DNA fragments separated by gel electrophoresis . journal of Molecular Biology 98, 503 . 15 . Taylor, M . A ., Wise, K. S . & McIntosh, M . A . (1985). Selective detection of Mycoplasma hyorhinis using cloned genomic DNA fragments . Infection and Immunity 47, 827-30 . 16 . Chan, H . W. & Ross, R. F . (1984) . Restriction endonuclease analysis of two porcine mycoplasma deoxyribonucleic acids : sequence-specific methylation in the Mycoplasma hyopneumoniae genome . International Journal of Systematic Bacteriology 34, 16-20 . 17. Santha, M ., Burg, K ., Rasko, I . & Stipkovits, L . (1987) .'A. species-specific DNA probe for the detection of Mycoplasma gallisepticum . Infection and Immunity 55, 2857-9 . 18. Geary, S . J. (1987) . Development of a biotinylated probe for the rapid detection of Mycoplasma gallisepticum . Israel journal of Medical Science 23, 747-51 . 19. Razin;, S . (1985) . Molecular, biology and genetics of mycoplasmas (mollicutes). Microbiological Reviews 49, 419-55 .