Molecular and Cellular Probes (1991) 5, 1 0 3-109

Detection of Mycoplasma hyopneumoniae DNA by the polymerase chain reaction Ry8 Harasawa,'* Kaoru Koshimizu,' Osamu Takeda,2 Takashi Uemori, 2 Kiyozo Asada 2 and IkunoShin Kato2 'Animal Center for Biomedical Research, Faculty of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo 113 and 2 Biotechnology Research Laboratories, Takara Shuzo Co ., Otsu-shi, Shiga 520-21, Japan (Received 11 July 1990, Accepted 5 September 1990)

DNA amplification by the polymerase chain reaction (PCR) was examined to detect DNA of Mycoplasma hyopneumoniae, an etiological agent of porcine pneumonia . A pair of synthetic primers was selected that specify the amplification of a 520-basepair DNA fragment in a reiterative sequence of M . hyopneumoniae genome . The PCR product was detected by direct gel electrophoresis or by blot hybridization to a synthetic oligonucleotide probe . The specificity of PCR for M . hyopneumoniae was confirmed by lack of cross-reactivity to DNA from other porcine mycoplasmas .

KEYWORDS : PCR, Mycoplasma hyopneumoniae, DNA detection .

INTRODUCTION Mycoplasma hyopneumoniae (synonym M. suipneumoniae), an etiological agent of mycoplasmal pneumonia in swine (MPS), is the most important pathogen in swine diseases because it causes the growth retardation and the reduction of feeding efficiency . No vaccine has been available against this disease . Therefore, to eradicate and control the disease, a reliable method for diagnosis is required . Conventional diagnosis of MPS has been based on cultivation of the organisms from clinical specimens and/or on serological procedures .' A DNA probe method was recently developed to detect M. hyopneumoniae, 2 but this requires purification of the DNA from the clinical specimens . Besides, the adaptation of the DNA probe method in veterinary laboratories may be hampered because of difficulties in handling radioisotopes . Non-radioactive DNA probes, on the other hand, were found to be less sensitive than radioactive probes.' A novel in vitro DNA amplification through the polymerase chain reaction (PCR) was recently

devised, and this provides great advantages for diagnostic procedures .' The PCR technique has been applied to detect a variety of viruses and bacteria .', " We examined the use of PCR to detect M . hyopneumoniae. This paper describes the suitability of two synthetic oligonucleotide primers for PCR and a synthetic oligonucleotide probe for specific detection of M . hyopneumoniae .

MATERIALS AND METHODS Bacterial strains used and culture conditions Mycoplasma hyopneumoniae strains VPP11 (same progenitor as ATCC25617) and J (same progenitor as ATCC25934), M. hyorhinis BTS-7 (same progenitor as ATCC17981), M . flocculare Ms42 (same progenitor as ATCC27399), and M . hyosynoviae S16 (same progenitor as ATCC25591) were obtained from Dr T . Yagi-

* Author to whom correspondence should be addressed . 0890-8508/91/020103 + 07 $03 .00/0

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hashi of the Nippon Institute of Biological Sciences, Tokyo . Each of the mycoplasma strains was cultured at 37 ° C in broth as described elsewhere .7-1' Escherichia coli strains JM83 and JM109 were grown at 37°C in L broth ."

amplification was achieved with 25 cycles of denaturation at 94 ° C for 30 s, renaturation at 55 ° C for 2 min, and elongation at 72 ° C for 2 min . A 5-µl amount of each amplified product was resolved on 1 . 0% agarose gel electrophoresis and analysed by blot hybridization .

DNA manipulations RESULTS Extraction of chromosomal DNA from the mycoplasma strains and construction of genomic DNA libraries in the plasmid vector pUC18 have been previously described ." Total genomic DNA of M . hyopneumoniae VPP11 was digested with the restriction endonuclease Eco RI . The resulted DNA fragments were inserted into the unique Eco RI site of pUC18 . The recombinant DNAs were subjected to transformation of E . coli JM83 by the method of Cohen et al." Plasmid DNA was isolated by the method of Birnboim & Doly . 14 Probe labelling and Southern blot hybridization were performed by using an enhanced chemiluminescence (ECU kit (Amersham Japan, Tokyo) . The DNA fragment to be sequenced was subcloned in E . coli JM109 with pUC118 and serial deletion derivatives were constructed by use of the kilo-sequence deletion kit (Takara Shuzo Co ., Kyoto) . The size of the deletion of each clone was checked by PCR amplification as described below and appropriate clones were selected to produce a single-stranded DNA for sequencing according to Vieira & Messing ."

Hybridization of a cloned DNA to mycoplasmal chromosomes Among the genomic DNA libraries, we chose a chimeric plasmid, pMhP38, which hybridized only to M . hyopneumoniae DNA but did not hybridize to M . flocculare or M . hyorhinis DNA . The probe fragment derived from the pMhP38 plasmid hybridized to eight bands of the Eco RI-digested M . hyopneumoniae DNA in Southern blot hybridization under moderately stringent conditions, indicating that the probe sequence is reiterative in the M . hyopneumoniae genome (Fig . 1) . This reiterative sequence was assigned within the shorter Pst I-Eco RI fragment on the physical map of a 5 . 4-kb M . hyopneumoniae DNA insert in the pMhP38 plasmid (Fig. 2) . The Psi I-Eco RI fragment of pMhP83 gave the same hybridization patterns with chromosomal DNAs from M . hyopneumoniae strains J and VPP11 (Fig . 3) .

Nucleotide sequence for in vitro DNA amplification Oligonucleotide Complementary single-stranded oligonucleotides were synthesized in an Applied Biosystem DNA synthesizer (Foster City, California) . The amplimer sequences for PCR were 5'-GTGTATCAAAATTGCCAATC-3' and 5'-TCCCATAACCTTGTGTTCAG-3' (Fig. 4, arrows) . The sequence used for the probe hybridization was 5'-GAATATGCAGAAATCCCCGT AAATCGGTGT-3' (Fig. 4, underline) .

The Pst I-Eco RI fragment was subcloned with pUC118 and sequenced (Fig . 4) . No significant repetition was found within the 1807-bp Pst I-Eco RI fragment . A pair of primer sequences (20 nucleotides as indicated by arrows) for PCR amplification and a probe sequence (30 nucleotides as indicated by underline) for blot hybridization were selected from the 1807-bp Pst I-Eco RI fragment, and then used for synthesis of oligonucleotides . The total G-plus-C content of the primer sequences was 40 mol% .

Polymerase chain reaction (PCR) Evaluation of PCR amplification DNA amplifications were performed with the PerkinElmer Cetus GenAmp kit in a DNA thermal cycler (Perkin-Elmer Cetus, Norwalk, Connecticut) as described previously ." Reaction mixture contained 20 pmol of each primer (20 pmol µI -1 ), 10µl of 10 x reaction buffer, 500 µm deoxynucleotides, and water to a volume of 95 µl . After a 5-min incubation at 94 ° C, 0 . 5 U (5 µl) of Taq polymerase was added and

The specificity of the primers was evaluated using DNAs isolated from M . hyopneumoniae, M. flocculare, M . hyorhinis, M . hyosynoviae, and swine muscle cells by direct gel electrophoresis after PCR amplification or by blot hybridization . As shown in Fig. 5, a 520-bp fragment was apparent when the DNA of M . hyopneumoniae was used as a template, whereas no



PCR for Mycoplasma hyopneUmoniae

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Fig. 1 . Southern blot hybridization of pMhP38 insert probe to the Eco RI- or Hin dIll-digested chromosomal DNA from M. hyopneumoniae VPP11, M. hyorhinis BTS-7, and M. focculare Ms42. Lane a, Eco RI-digested M . hyopneumonia VPP11 DNA ; lane b, Eco RI-digested M. hyorhinis BTS-7 DNA; lane c, Eco RI-digested M . focculare Ms42 DNA ; lane d, Hin dill-digested M . hyopneumoniae VPP11 DNA; lane e, Hin dill-digested M . hyorhinis BTS-7 DNA ; lane f, Hin dIll-digested M. Nocculare Ms42 DNA .

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Fig. 3 . Southern blot hybridization between the 1 . 8 kb Pst I--Eco RI fragment of pMhP38 and the Eco RI- or Hin dill-digested chromosomal DNA from M . hyopneumoniae strains VPP11 and J . Lane a, Eco RI-digested M . hyopneumoniae VPP11 DNA; lane b, Hin dill-digested M . hyopneumoniae VPP11 DNA ; lane c, Eco RI-digested M . hyopneumoniae J DNA ; lane d, Hin dill-digested M . hyopneumoniae J DNA .

other mycoplasma species tested or with swine geno-

amplification was noted with DNAs derived from

grows slowly and weeks can be necessary . Therefore we have examined PCR amplification for detection of

mic DNA . The sequence specificity of this DNA

the organisms . The PCR is a powerful technique for

amplification was confirmed by blot hybridization

amplifying specific DNA sequences from eukaryotes

using an oligonucleotide probe . Although in this

and prokaryotes . Over the past few years PCR has

study 5 ng of M . hyopneumoniae DNA was detected

become the method of choice for the diagnosis of a

by direct gel analysis, a 5-µl aliquot of the dilute

wide range of infections .'

culture containing 103 cfu ml - ' organisms was suffi-

The requirement for in vitro amplification by PCR is

cient as a template for a positive result when it was

that the sequence at both extremities of the DNA

directly subjected to the PCR (data not shown) .

fragment be known and to design primers bracketing the DNA fragment . We have used a repeated sequence in M . hyopneumoniae genome as a target

DISCUSSION

DNA to be amplified by PCR . The repeated sequence

Mycoplasma pneumonia in swine is a disease of

we cloned is present in eight copies in the genome of both strains VPP11 and J . The nature of this repeated

considerable veterinary importance . The causative agent of MPS has been shown to be M . hyopneumoniae . Diagnosis of this disease depends mainly on

repeated DNA sequence which is absent in strain

serological tests such as ELISA . 17 Culture method is

VPP11 . 1920 Repeated sequences have been shown to

not always adequate because the etiological agent

be present in various bacterial species including

DNA sequence is unknown at present . It has been reported that M . hyopneumoniae strain J contains a



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PCR for Mycoplasma hyopneumonlae

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