Molecular and Cellular Probes (1997) 11, 211–216

Multiplex PCR for avian pathogenic mycoplasmas

Han Wang, A. A. Fadl and M. I. Khan∗

Department of Pathobiology, College of Agriculture and Natural Resources, Universityof Connecticut, 61 North Eagleville Road, Storrs, CT 06269-3089, USA

(Received 2 January 1997, Accepted 25 March 1997)

Mycoplasma infections are of great concern in avian medicine, because they cause economiclosses in commercial poultry production. A multiplex polymerase chain reaction (PCR) wasoptimized to simultaneously detect four pathogenic species of avian mycoplasmas. Four sets ofoligonucleotide primers specific for Mycoplasma gallisepticum (MG), M. synoviae (MS), M.meleagridis (MM) and M. iowae (MI) were used in the test. By using agarose gel electrophoresesfor detection of the PCR-amplified DNA products, the sensitivity of detection was between 1 pgfor MG, 1 pg for MS, 100 fg for MM and 100 pg for MI after 35 cycles of PCR. Similar sensitivityof these primers was achieved with broth cultures of these four organisms.

1997 Academic Press Limited

KEYWORDS: avian, mycoplasma, multiplex PCR, primers, DNA.

INTRODUCTION

Four species of mycoplasmas are known to cause interspecies cross-reactions and non-specific re-actions,10 while isolation of mycoplasma is difficulteconomic losses in commercial poultry production.1–4

Mycoplasma gallisepticum (MG) causes chronic res- and time consuming. Molecular methods such asDNA probes11–13 and polymerase chain reactionpiratory disease (CRD) in chickens and infectious

sinusitis in turkeys,3 M. synoviae (MS) most frequently (PCR)13–21 have been recently developed in an attemptto improve diagnosis of avian mycoplasma infections.occurs as subclinical upper respiratory infection and

synovitis in chickens and turkeys,2 M. iowae (MI) Simultaneous detection of bacterial22–25 and viral26

pathogens and defective genes27 have been describedcauses a decrease in hatchability and high embryomortality in turkeys,1,5 and M. meleagridis (MM) using multiplex PCR amplification techniques. The

present study describes the development and op-causes primary lesions in airsacs in the progeny,which leads to lower hatchability and skeletal ab- timization of a multiplex PCR approach to detect and

differentiate four avian pathogenic mycoplasmas innormalities in young turkeys.4 On the other hand,multiple infections of the avian pathogenic myco- a single PCR reaction mixture.plasma are not uncommon in chicken and turkeyflocks.3–8

MATERIALS AND METHODSRapid identification of any of these avian myco-

plasma organisms is of great importance to the poultry Mycoplasma strains and culture conditionsindustry. Both serologic and isolation procedures havebeen used for the diagnosis of avian mycoplasmas.9 Table 1 shows a list of mycoplasma species, strains

and their original sources. These strains were grownHowever, serological tests are often hampered by

∗ Author to whom all correspondence should be addressed.

0890–8508/97/030211+06 $25.00/0/ll970108 1997 Academic Press Limited

H. Wang et al.212

Table 1. Mycoplasma species and strains utilized

Species Strains Source and history

M. gallisepticum F2F10 Univ. of California—Vaccine strain96-3179 Univ. of Auburn, Alabama—Field isolateK810 PDRCa, Georgia—Vaccine strainA5969 Univ. of Massachusetts—Reference strainY9 SEPRLb, USDA, Georgia—Variant strainY10 SEPRL, USDA, Georgia—Variant strainS6 Univ. of California—Type strainK-1501-S PDRC, Georgia—Field isolateK-1486-S1 PDRC, Georgia—Field isolateK-2221(383T) PDRC, Georgia—Field isolate93-49 Univ. of Connecticut—Experimental

infection93-55 Univ. of Connecticut—Experimental

infectionM. synoviae K3146 PDRC, Georgia—Field isolate

K3505 PDRC, Georgia—Field isolateK3181 PDRC, Georgia—Field isolateAlabama PDRC, Georgia—Field isolateWVU1853 Univ. of California—Reference strainK-1415 PDRC, Georgia—Field isolateFMT Univ. of Auburn, Alabama—Field isolate96-8228 Univ. of Auburn, Alabama—Field isolate

M. meleagridis RY 39 Univ. of California—Reference strainE-2 Univ. of California—Field isolatevic-44 Univ. of California—Field isolatevic-60 Univ. of California—Field isolate

M. iowae 695 Univ. of California—Reference strainNL-12 Univ. of California—Reference strainK-1805 Univ. of California—Reference strain96-624-8 Univ. of Connecticut—Field isolate96-633-13 Univ. of Connecticut—Field isolate96-793-4 Univ. of Connecticut—Field isolate

a PDRC: Poultry Diagnostic and Research Laboratory.b SEPRL: Southeastern Poultry Research Laboratory.

in Frey’s medium.28 Mycoplasma synoviae isolates The pellet was resuspended in 20 ll of TE buffer. Thepurity and concentration were determined spec-were cultured in Frey’s medium supplemented with

nicotinamide adenine dinucleotide (NAD). All or- trophotometrically by the 260/280 nm ratios and the260 nm readings,29 respectively. DNA was stored atganisms were incubated at 37°C.−20°C until use.

DNA isolation

Primers selectionTotal DNA was extracted and purified using methodsdescribed by Sambrook et al.29 Briefly, 500 ll ofmycoplasma culture were treated with sodium do- Four sets of primers that specifically amplify MM,

MG, MI and MS were selected and their sequencesdecyl sulfate at a final concentration of 1% to lysethe cells. The mixture was then treated with proteinase are listed in Table 2. All four sets of oligonucleotide

primers were synthesized on a model 380B DNAK (20 lg ml−1) and was incubated at 37°C for 1 h.The chromosomal DNA was then extracted twice synthesizer (Applied Biosystem Inc., Foster City, CA)

with the assistance of the University of Connecticutwith an equal volume of phenol-chloroform-isoamylalcohol (25:24:1). The DNA was precipitated with Biotechnology Center. The primers were desalted

through a Sephadex G-25 column (Pharmacia, Inc.,1/10th volume of 3 sodium acetate and 2 volumes ofabsolute ethanol, and incubated overnight at−20°C. Piscataway, NJ). The concentration of the primers was

determined by spectrophotometry,29 and the primersThe precipitated DNA was pelleted by centrifugationat 14 000 g for 10 min and washed by 70% ethanol. were divided into 25-ll volumes and stored at−20°C.

Mycoplasma multiplex PCR 213

Table 2. Sequences of oligonucleotides used

MG 1a 5′-GGATCCCATCTCGACCAGGAGAAAA-3′MG 2a 5′-CTTTCAATCAGTGAGTAACTGATGA-3′MS 1b 5′-GAAGCAAATAGTGATATCA-3′MS 2b 5′-GTCGTCTCGAAGTTAACAA-3′MM 1c 5′-GGATCCTAATATTAATTTAAACAAATTAATGA-3′MM 2c 5′-GAATTCTTCTTTATTATTCAAAAGTAAAGTAC-3′MI 1d 5′-GAATTCTGAATCTTCATTTCTTAAA-3′MI 2d 5′-CAGATTCTTTAATAACTTATGTATC-3′

a 16, b 14, c 21, d 19.

Optimization multiplex-PCR reaction mycoplasma species in the same reaction, artificialmixtures of DNA ranging from 100 ng to 10 fg DNA

Amplification reaction was carried out in 100 ll vol- with various combinations of all four avian myco-umes, each PCR mixture containing 500 m KCl, plasmas were used as template DNA.100 m Tris-HCl (pH 8·3), 2·5 m MgCl2, 200 l

(each) dATP, dCTP, dGTP and dTTP, 1 ll of eachprimer (334 ng ll−1), 2·5 U of AmpliTaq DNA poly- RESULTSmerase, and different concentrations of mycoplasmaDNA in 10 ll volumes were then added to the mixture. We have optimized a mycoplasma specific-multiplexVarious controls were included such as template DNA PCR amplification technique which identifies fourwithout any primers, primers without any template pathogenic avian mycoplasmas such as MM, MG, MIDNAs, and with and without Taq DNA polymerase. and MS in a single PCR reaction of 35 cycles. TheThe reaction mixture was overlaid with 50 ll of min- Mycoplasma-multiplex PCR products consisted oferal oil. All DNA amplifications were performed in a 850 bp (MM), 732 bp (MG), 299 bp (MI) and 207 bpDNA thermal cycler (Model 480 Perkin-Elmer Cetus (MS), and were visualized by gel electrophoresis (Fig.Corporation, Norwalk, CT). Following preliminary 1). Results of using mycoplasma broth cultures fromtrials with different annealing temperatures and times four pathogenic avian mycoplasma in various com-and with various concentrations of Mycoplasma bination mixtures showed amplified multiplex DNADNA, the thermal cycler was programmed for op- bands specific to each mycoplasma present (Fig. 2).timum conditions. Initially, the reaction mixture was The faint bands in lanes 6, 8 and 10 appear todenatured at 94°C for 5 min. Then the PCR was run be primer dimers; however, the specific bands forfor 35 cycles at a melting temperature of 94°C for mycoplasmas were clearly amplified (Fig. 2). Various1 min, an annealing temperature of 50°C for 1 min, annealing and extension temperatures as well as dif-and an extension temperature of 72°C for 2 min. The ferent time periods were carried out with the multiplesample was then heated at 72°C for 10 min for the sets of mycoplasma primers to obtain the best andfinal extension reaction. optimal annealing and extension temperatures and

time period required for each thermal cycle. All thenegative controls were negative. The multiplex PCR

Detection of amplified DNAsassay developed and evaluated in this study wasfound to be a specific assay for MG, MS, MM and

Gel electrophoresis was used to detect amplified DNAMI. The multiplex PCR was able to detect DNA from

products. A volume of 12 ll of amplified PCR productsMG at levels as low as 1 pg, MS as low as 1 pg, MM

was subjected to electrophoresis at 80 V in horizontalas low as 100 fg and MI as low as 100 pg (Table 3).

gels containing 1·5% agarose (Ultrapure; BethesdaNo spurious PCR amplification reactions between

Research Laboratories, Bethesda, MD) with Tris-bor-MG, MS, MM and MI were noticed using various

ate buffer (45 m Tris-borate, 1 m EDTA). The gelamounts of DNA mixtures.

was stained with ethidium bromide (0·5 lg ml−1),exposed to u.v. light to visualize the amplified prod-ucts, and photographed.

DISCUSSION AND CONCLUSIONS

Recent advances in the diagnosis of avian myco-Multi-species PCR sensitivity and specificityplasmas have led to the development of commercialMG and MS DNA-based PCR kits.30 These kits, whichTo determine the ability of the multi-species PCR

technique to detect more than one of the target are needed to differentiate MG and MS, have resulted

H. Wang et al.214

1 2 3 4 5 6 7 8 9

850 bp732 bp

299 bp

207 bp

Fig. 1. Agarose gel electrophoresis of multiplex PCR amplified products from the purified DNAs of known avianmycoplasmas. Lane 1=123 bp marker. Lane 2=MM (RY39), MG (S6), MI (695), MS (WVU1853). Lane 3=MM (RY39),MI (695), MS (WVU1853). Lane 4=MG (S6), MI (695), MS (WVU1853). Lane 5=MM (RY39). Lane 6=MG (S6). Lane7=MI (695). Lane 8=MS (WVU1853). Lane 9=Negative control (PCR buffer).

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

850732

207299

Fig. 2. Agarose gel electrophoresis of multiplex PCR amplified products from the avian mycoplasma cultures. Lane 1=123 bp marker. Lane 2=MM (RY39). Lane 3=MG (S6). Lane 4=MI (695). Lane 5=MS (WVU1853). Lane 6=MM (vic-44), MG (F2F10). Lane 7=MM (vic-60), MI (NL-12). Lane 8=MM (vic-44), MS (K3146). Lane 9=MG (A5969), MI (NL-12). Lane 10=MG (Y9), MS (K3181). Lane 11=MI (vic-44), MS (3505). Lane 12=Negative control (PCR buffer).

in doubling the cost of each clinical sample tested. alternative to currently described PCR methods fordetecting one or more species of mycoplasma in aThe multi-species PCR described by Fan et al.31 and

Garcia et al.32 is capable of testing for more than one sample. Further studies are in progress to test thisprocedure on clinical samples as well as ex-mycoplasma in a sample but the need for RFLP

or species-specific DNA probes to identify the PCR perimentally infected birds.products makes the procedure somewhat cum-bersome. Overall, the data presented above indicatethat the use of the multiplex PCR assay for the iden- ACKNOWLEDGEMENTStification of pathogenic avian mycoplasmas is reliableand specific. The multiplex PCR, being specific, sens- This work was supported in part by funds provided by the

U.S. Department of Agriculture’s formula Hatch grant. Weitive and cost effective, seems to be an attractive

Mycoplasma multiplex PCR 215

Table 3. Results of sensitivity of multiplex PCR 11. Dohms, J. E., Hnatow, L. L., Whetzel, P., Morgan, R.& Keeler, C. L., Jr. (1993). Identification of the putativecytadhesin gene of Mycoplasma gallisepticum and itsMultiplex PCRuse as a DNA probe. Avian Diseases 37, 380–8.

12. Khan, M. I., Kirkpatrick, B. C. & Yamamoto, R. (1989).DNA template MG MS MM MIMycoplasma gallisepticum species and strain-specificrecombinant DNA probes. Avian Pathology 18, 135–100 ng + + + +

10 ng + + + + 46.13. Razin, S. (1994). DNA probes and PCR in diagnosis1 ng + + + +

100 pg + + + + of mycoplasma infections. Molecular and CellularProbes 8, 497–511.10 pg + + + −

1 pg + + + − 14. Lauerman, L. H., Hoerr, F. J., Sharpton, A. R., Shah,S. M. & Van Santen, V. L. (1993). Development and100 fg − − + −

10 fg − − − − application of a polymerase chain reaction assay forMycoplasma synoviae. Avian Diseases 37, 829–34.

15. Nascimento, E. R., Yamamoto, R. & Khan, M. I. (1993).Mycoplasma gallisepticum F-vaccine strain-specificpolymerase chain reaction. Avian Diseases 37, 203–thank Dr Tom Yang and Dr Richard Yamamoto for their11.constructive suggestions and critical review of the manu-

16. Nascimento, E. R., Yamamoto, R., Herrick, K. R. &script.Tait, R. C. (1991). Polymerase chain reaction for de-tection of Mycoplasma gallisepticum. Avian Diseases35, 62–9.

REFERENCES 17. Silveira, R. M., Fiorentin, L. & Marques, E. K. (1996).Polymerase chain reaction optimization for Myco-plasma gallisepticum and M. synoviae diagnosis.1. Kleven, S. H. (1991). Mycoplasma iowae. In Diseases

of Poultry, 9th ed. (Calnek, B. W., Barnes, H. J., Avian Diseases 40, 218–22.18. Slavik, M. F., Wang, R. F. & Cao, W. W. (1993).Beard, C. W., Reid, W. M. & Yoder, H. W. Jr., eds),

Pp. 231–233. Ames, Iowa: Iowa State University Press. Development and evaluation of the polymerase chainreaction method for diagnosis of Mycoplasma gal-2. Kleven, S. H., Rowland, G. N. & Olson, N. O. (1991).

Mycoplasma synoviae infection. In Diseases of Poultry, lisepticum infection in chickens. Molecular and Cellu-lar Probes 7, 459–63.9th ed. (Calnek, B. W., Barnes, H. J., Beard, C. W.,

Reid, W. M. & Yoder, H. W. Jr., eds), Pp. 223–231. 19. Zhao, S. & Yamamoto, R. (1993). Amplification ofMycoplasma iowae using polymerase chain reaction.Ames, Iowa: Iowa State University Press.

3. Yoder, H. W., Jr. (1991). Mycoplasma gallisepticum Avian Diseases 37, 212–7.20. Zhao, S. & Yamamoto, R. (1993). Detection of Myco-infection. In Diseases of Poultry, 9th ed. (Calnek, B.

W., Barnes, H. J., Beard, C. W., Reid, W. M. & Yoder, plasma synoviae by polymerase chain reaction. AvianPathology 22, 533–42.H. W. Jr., eds), Pp. 198–212. Ames, Iowa: Iowa State

University Press. 21. Zhao, S. & Yamamoto, R. (1993). Detection of Myco-plasma meleagridis by polymerase chain reaction.4. Yamamoto, R. (1991). Mycoplasma meleagridis in-

fection. In Diseases of Poultry, 9th ed. (Calnek, B. W., Veterinary Microbiology 36, 91–7.22. Bej, A. I., Mahbubani, M. H., Miller, R., DiCesare, J.Barnes, H. J., Beard, C. W., Reid, W. M. & Yoder, H.

W. Jr., eds), Pp. 212–223. Ames, Iowa: Iowa State I., Haff, L. & Atlas, R. M. (1990). Multiplex PCRamplification and immobilized capture probes for de-University Press.

5. Kempf, I., Guittet, M., LeGross, F. X., Toquin, D. & tection of bacterial pathogens and indicators in water.Molecular and Cellular Probes 4, 353–65.Bennejean, G. (1989). Mycoplasma iowae: field and

laboratory studies to evaluate egg transmission in tur- 23. Kulski, J. K., Khinsoe, C., Pryce, T. & Christiansen, K.(1995). Use of multiplex PCR to detect and identifykeys. Avian Pathology 18, 229–305.

6. Bradbury, J. M. & McClenaghan, M. (1982). Detection Mycobacterium avium and M. intercellulare in bloodculture fluids of AIDS patients. Journal of Clinicalof mixed mycoplasma species. Journal of Clinical

Microbiology 16, 314–8. Microbiology 33, 668–74.24. Lawrence, L. M. & Gilmour, A. (1994). Incidence of7. Rott, M., Pfutzner, H., Gigas, H. & Rott, G. (1989).

Diagnostic experiences in the routine restrained in- Listeria spp. and Listeria monocytogenes in a poultryprocessing environment and in poultry products andspection of turkey stock for mycoplasma infections.

Archives of Experimental Veterinary Medicine 43, their confirmation by multiplex PCR. Applied andEnvironmental Microbiology 60, 4600–4.743–6.

8. Yoder, H. W., Jr. & Hofstad, M. S. (1964). Charac- 25. Way, J. S., Josephson, K. L., Pillai, S. D., Abbaszadegan,M., Gerba, C. P. & Pepper, I. L. (1993). Specificterization of avian mycoplasma. Avian Diseases 8,

481–512. detection of Salmonella spp. by multiplex polymerasechain reaction. Applied and Environmental Micro-9. United States Department of Agriculture. National

poultry improvement plan and auxiliary provisions. biology 59, 1473–9.26. Karlsen, F., Kalantari, M., Jenkins, A. et al. (1996). UseUSDA, APHIS. VS 91–40. August 1989.

10. Sahu, S. P. & Olson, N. O. (1981). Characterization of of multiplex PCR primer sets for optimal detection ofhuman papillomavirus. Journal of Clinical Mi-an isolate of Mycoplasma WVU 907 which possesses

common antigens to Mycoplasma gallisepticum. Avian crobiology 34, 2095–100.27. Chamberlain, J. S., Gibbs, R. A., Ranier, J. E., Nguyen,Diseases 25, 943–53.

H. Wang et al.216

P. N. & Caskey, C. T. (1981). Deletion screening of gallisepticum and Mycoplasma synoviae: a field re-port. Proceedings of the 42nd Western Poultry Diseasethe duchenne muscular dystrophy locus via multiplex

DNA amplification. Nucleic Acid Research 16, 11141– Conference, Sacramento, CA, pp. 80.31. Fan, H. H., Kleven, S. H., Jackwood, M. W., Johnson,56.

28. Frey, M. L., Hanson, R. P. & Anderson, D. P. (1968). K. E., Peterson, B. & Levisohn, S. (1995). Speciesidentification of avian Mycoplasmas by polymeraseA medium for the isolation of avian mycoplasmas.

American Journal of Veterinary Research 29, 2163–71. chain reaction and restriction fragment length poly-morphism analysis. Avian Diseases 39, 398–407.29. Sambrook, J., Fritsch, E. T. & Maniatis, T. (1989).

Molecular cloning: a laboratory manual. Pp. 1.25, 32. Garcia, M., Jackwood, M. W., Levisohn, S. & Kleven,S. H. (1995). Detection of Mycoplasma gallisepticum,1.85, 6.3, 9.47, A.1, E.3, E.5. Cold Spring Harbor, NY:

Cold Spring Harbor Laboratory Press. M. synoviae, and M. iowae by multi-species poly-merase chain reaction and restriction fragment length30. Campbell, G., Van-Dam, B. & Tyrell, P. I. (1993).

Commercial DNA probe test kits for Mycoplasma polymorphism. Avian Diseases 39, 606–16.