Abstract.A real-time polymerase chain reaction (PCR) assay using hybridization probes on a LightCycler platform was developed for detection of Mycoplasma bovis from individual bovine mastitis milk and pneumonic lung tissues. The detection limit was 550 colony forming units (cfu)/ml of milk and 650 cfu/25 mg of lung tissue. A panel of bovine Mycoplasma and of other bovine-origin bacteria were tested; only M. bovis strains were positive, with a melting peak of 66.6ЊC. Mycoplasma agalactiae PG2 was also positive and could be distinguished because it had a melting peak of 63.1ЊC. In validation testing of clinical samples, the relative sensitivity and specificity were 100% and 99.3% for individual milks and 96.6% and 100% for the lung tissue. Using M. bovis real-time PCR, the M. bovis culture-positive milk samples were estimated to contain between 5 ϫ 10 4 and 7.7 ϫ 10 8 cfu/ml and the M. bovis culture-positive lungs between 1 ϫ 10 3 and 1 ϫ 10 9 cfu/25 mg. Isolation, confirmed with the real-time PCR and colony fluorescent antibody test, showed that at the herd level, the proportion of samples positive for M. bovis isolation in mastitis milk samples submitted to the Mastitis Laboratory, Animal Health Laboratory, University of Guelph, Ontario, Canada, was 2.4% (5/201). We conclude that this probe-based real-time PCR assay is a sensitive, specific, and rapid method to identify M. bovis infection in bovine milk and pneumonic lungs.

Minichromosome maintenance (MCM) proteins are part of the replication licensing factor (RLF-M), which limits the initiation of DNA replication to once per cell cycle. We have previously reported that higher order complexes of mammalian pol II and general pol II transcription factors, referred to as pol II holoenzyme, also contain MCM proteins. In the present study we have analyzed in detail the interaction between MCM2 and pol II holoenzyme. N-and C-terminal deletions were introduced into epitope-tagged MCM2 and the truncated proteins were transiently expressed in 293 cells. Affinity chromatography was used to purify RNA pol II holoenzyme and histone binding MCM complexes. We found that amino acids 168-230 of MCM2 are required for its binding to pol II holoenzyme in vivo. We also showed that bacterially expressed amino acids 169-212 of MCM2 associate with pol II and several general transcription factors in vitro. Point mutations within the 169-212 domain of MCM2 disrupted its interaction with pol II holoenzyme both in vitro and in vivo. This region is distinct from the previously characterized histone H3 binding domain of MCM2.Keywords: MCM2; RNA polymerase II holoenzyme; histone.Large protein complexes, which contain RNA polymerase II as well as the general pol II transcription factors (GTFs) TFII A, B, D, E, F, and H [1] and other proteins have been isolated from yeast, mammalian and amphibian cells [2][3][4][5][6][7][8]. They are referred to as pol II ÔholoenzymeÕ. Some of the components of pol II holoenzyme make direct or indirect contacts with the C-terminal domain (CTD) of the largest subunit of pol II. Antibodies against the CTD disrupt the yeast holoenzyme into core pol II and a mediator subcomplex, which contains the SRB and MED proteins [7][8][9]. A similar treatment of pol II holoenzyme from HeLa cells also disrupts its interaction with several of the GTFs [6]. In higher eukaryotes the CTD mediates the interaction with complexes that contain homologues of the yeast SRB and MED proteins such as SMCC [10] or NAT [11]. It is believed that pol II holoenzyme is a functionally significant complex, which is responsible for transactivator-stimulated transcription in vivo. It has been shown that the srb4 and srb6 genes are essential for expression of most mRNAs in budding yeast [12]. Other holoenzyme components such as SRB 2, 5, 7-11, SWI/SNF proteins, SIN4, RGR1, MED2, MED9/CSE2, MED10/NUT2, MED11, GAL11, PGD1 and ROX3 [7,9,[13][14][15][16] are not essential for transcription of most genes but do contribute to the response to transactivators and repressors (reviewed in [17,18]). In addition to its role in the response to transcriptional regulators, pol II holoenzyme may be involved in integrating transcription with RNA processing, DNA repair and replication. In support of this idea, the DNA repair factors DNA pol e, XPC, XPF, XPG, Ku, RAD51 [3], BRCA1 [19]; RNA helicase A [20]; the replication factors RP-A, RP-C [3] and MCM proteins [6]; and the cleavage/polyadenylation factors CPSF and CstF [21] have been id...

A Mycoplasma iowae real-time polymerase chain reaction (PCR) assay using primers and probes targeting the 16S rRNA gene was developed and field-validated in this study. The assay specifically identified M. iowae with a detection limit of 80 colony-forming units (cfu) per turkey cloacal swab sample (3.2 cfu per PCR reaction). It was validated by testing 154 field turkey cloacal swab samples in parallel with culture isolation. The diagnostic sensitivity of the PCR was 97.6%, and the specificity was 95.5%. The real-time PCR developed in this study is a rapid, sensitive, and cost-effective alternative to culture isolation for detecting M. iowae from cloacal swab samples.

A PCR assay was validated for the detection of Mycoplasma hyopneumoniae in porcine lung tissue. The detection limit of the assay was 0.18 colony-forming units/g of lung sample spiked with M. hyopneumoniae. In field validation, 426 pigs from 220 cases were examined for M. hyopneumoniae infection by M. hyopneumoniae PCR and a fluorescent antibody (FA) test. In total, 103 pig lungs (24.2%) were positive in the PCR test, and 69 pig lungs (16.2%) were positive in the FA test, among which, 62 pigs were positive for both PCR and FA test. Most of the PCR-positive but FA test-negative cases had lesions compatible with M. hyopneumoniae infection. With Bayesian modeling, the diagnostic sensitivity and specificity of the PCR were determined to be 97.3% and 93.0%, respectively.

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