Abstract:
The present invention provides biosensors and methods of use for detecting the presence or absence of mycoplasma contamination through the detection of hydrolytic enzymes that are conserved among  Mycoplasma  species. Such hydrolytic enzymes include, but are not limited to, proteases, reductases and nucleases.

Description:
RELATED APPLICATION 
       [0001]    This application is a continuation of co-pending PCT international patent application PCT/US2008/081483, filed Oct. 28, 2008, the entire contents of which are incorporated by reference for all purposes. 
     
    
     BACKGROUND OF THE INVENTION 
       [0002]    Mycoplasmas are very small microorganisms (Class Mollicutes) without cell walls that can cause infections in humans, animals, and plants. Mycoplasmas are also commonly found contaminating buffer solutions, and tissue culture media used in life science research. The  Mycoplasma  and  Acholeplasma  species,  Acholeplasma laidlawii, M. hyorhinis, M. orale, M. salivarium, M. arginini , and  M. hominis , account for about 98% of the tissue culture contaminants (McGarrity, G. J., &amp; Carson, D. A., Adenosine phosphorylase-mediated nucleoside toxicity. Application towards the detection of mycoplasmal infection in mammalian cell cultures. Exp Cell Res. 1982 May; 139(1):199-205). As used herein, “mycoplasma” or “mycoplasmas” refers generally to members of the Class Mollicutes, including  Mycoplasma  and  Acholeplasma  species. 
         [0003]    There is a clear unmet need for the real time detection of mycoplasmas for infection control monitoring in hospitals and for quality control of buffers and tissue culture media used in clinical laboratory testing and life science research. 
       SUMMARY OF THE INVENTION 
       [0004]    The present invention provides biosensors and methods of use for detecting the presence or absence of mycoplasma contamination through the detection of hydrolytic enzymes that are conserved among  Mycoplasma  species. Such hydrolytic enzymes include, but are not limited to, proteases, reductases and nucleases 
         [0005]    In preferred embodiments, the present invention provides a biosensor for detecting the presence or absence of  Mycoplasma  contamination comprising a support and a detectably labeled substrate for an enzyme produced and/or secreted by a mycoplasma, wherein the substrate is attached to the support. Typically, the enzyme is a  Mycoplasma -specific hydrolytic enzyme selected from the group consisting of proteases, reductases and nucleases. In certain preferred embodiments, the enzyme is a  Mycoplasma -specific protease selected from the group consisting of the gene product of pepA1 (MCAP — 0157), pepA2 (MCAP — 0195), pepA (leucyl aminopeptidase, such as MHP7448 — 0464), MCAP — 0267 (metalloendopeptidase), pepP (Xaa-Pro endopeptidase, such as MCAP — 0341 or MHP7448 — 0649), MCAP — 0509, mapP (methionine amino peptidase, MCAP — 0675 or MHP7448 — 0173), mixtures thereof and homologous enzymes with at least 40% sequence identity. When the enzyme is a mycoplasma-specific protease, preferred substrates include leucine-(7-methoxycoumarin-4-yl)acetyl (leu-MCA), arginine-(7-methoxycoumarin-4-yl)acetyl (arg-MCA), methionine-(7-methoxycoumarin-4-yl)acetyl (met-MCA), an acetoxymethyl ester or maleimide derivative of blue dye number 1 coupled to a peptide substrate of the mycoplasma-specific protease. 
         [0006]    In other preferred embodiments, the enzyme is a  Mycoplasma -specific reductase selected from the group consisting of the gene product of nrdE (such as MCAP — 0101), MCAP — 0427, trxB (thioredoxin reductase, such as MCAP — 0779 or MHP7448 — 0098), MCAP — 0858 and mixtures thereof. When enzyme is a mycoplasma-specific reductase, suitable substrates include reactive black 5,5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB), BODIPY®FL L-cystine, 2′,7′-difluoro-4′-(2-(5-((dimethyl amino phenyl)azo)pyridyl)dithiopropionyl aminomethyl)fluorescein (DFDMAP-fluorescein), or an azo dye that is sensitive to decolorization by microbial reductases. 
         [0007]    In yet other preferred embodiments, the enzyme is a mycoplasma-specific nuclease selected from the group consisting of the 5′-3′ exonuclease encoded by MCAP — 0047 or MHP7448 — 0581, the gene product of nfo (such as MCAP — 0060 or MHP7448 — 0062), vacB (such as MCAP — 0097 or MHP7448 — 0037), uvrC (such as MCAP — 0252 or MHP7448 — 0066), mc (ribonuclease III, such as MCAP — 0492 or MHP7448 — 0398), MCAP — 0768, uvrB (such as MCAP — 0773 or MHP7448 — 0648), uvrA (such as MCAP — 0774 or MHP7448 — 0091) and mixtures thereof. When the enzyme is a mycoplasma-specific nuclease, a preferred substrate is an acetoxymethyl ester or maleimide derivative of blue dye number 1 coupled to an aminoallyl-dNTP labeled nucleic acid substrate of the mycoplasma-specific nuclease. Typically the substrate is a reagent container, a culture medium container or a cell culture container. 
         [0008]    In other aspects, the present invention provides a method of detecting mycoplasma contamination of a cell culture comprising the steps of providing a cell-permeable detectable label coupled to a cell-impermeant carrier in the culture medium wherein cleavage of the detectable label by a mycoplasma-specific enzyme is followed by uptake of the detectable label into cells; and detecting labeled cells, thereby detecting mycoplasma contamination of the cell culture. In certain embodiments, the mycoplasma-specific enzyme is a protease and the detectable label is an acetoxymethyl ester of derivative of blue dye number 1 coupled to a peptide substrate of the mycoplasma-specific protease. Preferred proteases can be selected from the group consisting of the gene product of pepA1 (MCAP — 0157), pepA2 (MCAP — 0195), pepA (leucyl aminopeptidase, such as MHP7448 — 0464), MCAP — 0267 (metalloendopeptidase), pepP (Xaa-Pro endopeptidase, such as MCAP — 0341 or MHP7448 — 0649), MCAP — 0509, mapP (methionine amino peptidase, MCAP — 0675 or MHP7448 — 0173), and mixtures thereof. In other preferred embodiments, the mycoplasma-specific enzyme is a nuclease and the detectable label is an acetoxymethyl ester of derivative of blue dye number 1 coupled to a nucleic acid substrate of the mycoplasma-specific nuclease Preferred nucleases can be selected from the group consisting of the 5′-3′ exonuclease encoded by MCAP — 0047 or MHP7448 — 0581, the gene product of nfo (such as MCAP — 0060 or MHP7448 — 0062), vacB (such as MCAP — 0097 or MHP7448 — 0037), uvrC (such as MCAP — 0252 or MHP7448 — 0066), mc (ribonuclease III, such as MCAP — 0492 or MHP7448 — 0398), MCAP — 0768, uvrB (such as MCAP — 0773 or MHP7448 — 0648), uvrA (such as MCAP — 0774 or MHP7448 — 0091) and mixtures thereof. 
         [0009]    In other aspects, the present invention provides a method of determining the presence or absence of mycoplasma in a sample, comprising the steps of contacting the sample with a detectably labeled substrate for an enzyme produced and/or secreted by a mycoplasma under conditions that result in the modification of the substrate by the enzyme; and detecting the modification or the absence of the modification of the substrate wherein modification of the substrate indicates the presence of mycoplasma in the sample, and wherein the absence of modification of the substrate indicates the absence of mycoplasma in the sample. Preferably, the level of the detectable label is quantitatively related to the presence or amount of mycoplasma in the sample. 
         [0010]    In preferred embodiments, the enzyme is a hydrolytic enzyme selected from a protease, a nuclease or a reductase. In certain embodiments, enzyme is a protease selected from group consisting of the gene product of pepA1 (MCAP — 0157), pepA2 (MCAP — 0195), pepA (leucyl aminopeptidase, such as MHP7448 — 0464), MCAP — 0267 (metalloendopeptidase), pepP (Xaa-Pro endopeptidase, such as MCAP — 0341 or MHP7448 — 0649), MCAP — 0509, mapP (methionine amino peptidase, such as MCAP — 0675 or MHP7448 — 0173), and mixtures thereof. In other preferred embodiments, the enzyme is a reductase selected from the group consisting of the gene product of nrdE (such as MCAP — 0101), MCAP — 0427, trxB (thioredoxin reductase, such as MCAP — 0779 or MHP7448 — 0098), MCAP — 0858 and mixtures thereof. In yet other preferred embodiments, the enzyme is a nuclease selected from the group consisting of the 5′-3′ exonuclease encoded by MCAP — 0047 or MHP7448 — 0581, the gene product of nfo (such as MCAP — 0060 or MHP7448 — 0062), vacB (such as MCAP — 0097 or MHP7448 — 0037), uvrC (such as MCAP — 0252 or MHP7448 — 0066), mc (ribonuclease III, such as MCAP — 0492 or MHP7448 — 0398), MCAP — 0768, uvrB (such as MCAP — 0773 or MHP7448 — 0648), uvrA (such as MCAP — 0774 or MHP7448 — 0091) and mixtures thereof. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0011]    The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention. 
           [0012]      FIG. 1  is a photograph of the decolorization of an azo dye, reactive black 5, with supernatants of cultured bacteria. Each well was incubated with 10 μg of reactive black 5 plus 190 μl of culture supernatant from the following bacterium:  E. coli, E. faecalis, S. aureus, P. aeruginosa, S. pyogenes , and  S. marcescens . Such azo dyes were decolorized by most bacteria after incubation with the dye for about 18 hours. The decolorization is indicative of reductases produced by the bacteria. 
           [0013]      FIG. 2A  is a diagrammatic illustration of an embodiment of a contamination biosensor 200 placed on a container 100 for a reagent or culture medium. 
           [0014]      FIG. 2B  a diagrammatic illustration of an embodiment of a contamination biosensor 210 placed on a container 110 for tissue culture. 
           [0015]      FIG. 3  is a diagrammatic illustration of an embodiment of a mycoplasma contamination detection system for cell culture, showing in  FIG. 3A  a cell 300 in an uncontaminated culture, and in  FIG. 3B , a cell 300 in a contaminated culture containing a dye deposit 360 that is indicative of mycoplasma contamination. 
           [0016]      FIG. 4A  shows RNA samples used in RT-PCR. The lanes are: a) 1 kB DNA Ladder, b) BHK-21 cells infected with  Mycoplasma hyorhinis  as a monolayer, c) the pellet of BHK-21 cells medium infected with  Mycoplasma hyorhinis , and d) the pellet of  Mycoplasma hyorhinis  from mycoplasma enrichment broth (not from tissue culture cells). 
           [0017]      FIG. 4B  is a graphical representation of the expression of several  Mycoplasma hyorhinis  genes under conditions A-J: A) lon, 3T3 cells growing as a monolayer, B) lon, BHK-21 cells growing in DMEM, C) map, 3T3 cells growing as a monolayer, D) map, BHK-21 cells growing in DMEM, E) pepA, 3T3 cells growing as a monolayer, F) pepA, BHK-21 cells growing in DMEM, G) trxB, 3T3 cells growing as a monolayer, H) trxB, BHK-21 cells growing in DMEM, I) vacB, 3T3 cells growing as a monolayer, J) vacB, BHK-21 cells growing in DMEM. 
           [0018]      FIG. 5  shows the results of testing of primers with genomic DNA from  Mycoplasma hyorhinis . The PCR products were run on a 1.5% agarose gel. We performed 35 cycles of hot start PCR with an initial melt of 95° C. for 4 minutes, followed by a melt at 95° C. for 45 seconds, an annealing at 50° C. for 45 seconds, and a final extension step 72° C. for 7 minutes. The lane order includes a 1 kB DNA ladder (a), 5′-3′ exonuclease (b &amp;k), gcp (c &amp; l), lon (d &amp; m), map (e &amp; n), nfo (f &amp; o), nox, (g&amp;p), trxB (h &amp;q), uvrA (i &amp;r), p37 (j&amp;s), all using 0.5 μl of DNA template (B-J) or 1 μl of DNA diluted 1:10. 
           [0019]      FIG. 6  shows the results of testing of further PCR primers with genomic DNA from  Mycoplasma hyorhinis . Lanes include 1 kB DNA ladder (a), gcp (b), hrcA (c), lgt (d), pepA (e), pepP pth (g), mc (h), uvrB (i), uvrC (j), vacB (k), 5′-3′ (1), nox (m), p37 (n). The PCR product for pepF amplified the correct size product as well (not shown). 
           [0020]      FIG. 7  shows the results of testing by RT-PCR of all primers that worked under 50° C. AT. The RNA template used was the BHK-21 DMEM pellet that had been previously treated with DNase. For each 41 of a 1:10 dilution of template. RT, 45° C. for 10 minutes, 95° C. for 15 minutes, amp. cycled 35 times 95° C. 15 s 50° C. 45 s, final extension at 72° C. for 7 minutes. Lanes include 1 kB DNA ladder (a), 5′-3′(b), lgt (c), lon (d), map (e), nfo (f), nox (g), pepA (h), pepF (i), pth (j), rnc (k), trxB (1), uvrA (m), uvrB (n), vacB (O), p37 (p), no primer control. 
           [0021]      FIG. 8  shows an agarose gel loaded with double stranded DNA (dsDNA, lanes b-i) or double stranded RNA (ribosomal RNA, lanes j-q) treated with  M. hyorhinis  extract from a cell culture infection or supernantants from infected or uninfected cell cultures. The respective lanes contain: a) 1 kb DNA ladder, b) dsDNA exposed to an aliquot of  M. hyorhinis  extract for 30 minutes, c) dsDNA exposed to an aliquot of the supernatant of an infected cell culture for 30 minutes, d) dsDNA exposed to an aliquot of the supernatant of an uninfected cell culture for 30 minutes, e) dsDNA exposed to H 2 O for 30 minutes, f) dsDNA exposed to an aliquot of  M. hyorhinis  extract for 0 minutes, g) dsDNA exposed to an aliquot of the supernatant of an infected cell culture for 0 minutes, h) dsDNA exposed to an aliquot of the supernatant of an uninfected cell culture for 0 minutes, i) dsDNA exposed to H 2 O for 0 minutes, j) ribosomal RNA exposed to an aliquot of  M. hyorhinis  extract for 30 minutes, k) ribosomal RNA exposed to an aliquot of the supernatant of an uninfected cell culture for 30 minutes, 1) ribosomal RNA exposed to an aliquot of the supernatant of an uninfected cell culture for 30 minutes, m) ribosomal RNA exposed to exposed to H 2 O 30 minutes, n) ribosomal RNA exposed to an aliquot of  M. hyorhinis  extract for 0 minutes, o) ribosomal RNA exposed to an aliquot of the supernatant of an infected cell culture for 0 minutes, p) ribosomal RNA exposed to an aliquot of the supernatant of an uninfected cell culture for 0 minutes, and q) ribosomal RNA exposed to an aliquot of H 2 O for 0 minutes. 
           [0022]      FIG. 9  is a graph of the results of testing the thioredoxin reductase activities of  M. hyorhinis, E. coli  and  S. aureus . There was no significant activity from 10 4 -10 6  CFU/ml of  S. aureus  or  E. coli  using the DTNB substrate. 
           [0023]      FIG. 10A  and  FIG. 10B  are schematic diagrams of a substrate-linked enzymatic reporter reagent. In  FIG. 10A , a bead 400 is covalently linked to horseradish peroxidase 420 by a molecule DTSSP 410 that is a substrate for a reductase such as trxB. In  FIG. 10B , a bead 400 is covalently linked to luciferase 440 by a molecule Leu 430 that is a substrate for a protease such as pepA. 
           [0024]      FIG. 11  is a graph showing the effect of 1 mM DTT in enhancing the fluorescence of trxB substrates 2′,7′-difluoro-4′-(2-(5-((dimethylaminophenyl) azo)pyridyl)dithiopropionyl aminomethyl)fluorescein (DFDMAP) and BODIPY®FL L-cystine. 
           [0025]      FIG. 12  is a graph showing the effect of digitonin on the trxB assay. 
           [0026]      FIG. 13  is a graph showing the effect of acid pH levels on the leu-MCA assay. 
           [0027]      FIG. 14  is a graph showing the effect of basic pH levels on the leu-MCA assay. 
           [0028]      FIG. 15  is a graph showing the sensitivity of the leu-MCA assay. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0029]    Many of the genomes of the genus  Mycoplasma  have been sequenced. It is apparent that the microorganism has few biosynthetic genes, and the microorganism can only thrive in very rich growth mediums. Using a genomic approach in which we compared the genomes of 10 different mycoplasma ( M. gallisepticum, M. capricolum, M. genitalium, M. hyoppneumonia, M. mobile, M. mycoides, M. penetrans, M. pneumonia, M. pulmonis , and  M. synoviae ) at 40% sequence identity, we have identified 243 genes that are conserved in all  Mycoplasma  species studied to date.  Mycoplasma  species use an array of hydrolytic enzymes to uptake materials that compensates for having very few internal biosynthetic processes. The identified genes are listed in Table 1, below, using the  M. capricolum  notation. 
         [0030]    The common genes included a variety of enzymes that can be grouped into seven classes: synthetic enzymes, hydrolytic enzymes, chaperones, permeases, kinases, transcription factors, and ribosomal proteins. The presence of acetate kinase has been disclosed as an assay for the presence of  Mycoplasma  (U.S. published patent application No. 2004/0265942). However, this assay is an enzyme cascade assay requiring luciferase and is not amenable to a simple and direct method for measuring contamination in culture and in vivo. 
         [0031]    The hydrolytic enzymes are interesting targets both for diagnosis and the treatment of a mycoplasma infection because they are secreted and likely involved in infection and virulence. The common hydrolytic enzymes of  Mycoplasma  species include: proteases, such as the gene products of MCAP — 0157, MCAP — 0195, MCAP — 0267, MCAP — 0341, MCAP — 0509, MCAP — 0675, nucleases, such as the gene products of MCAP — 0047, MCAP — 0060, MCAP — 0097, MCAP — 0252, MCAP — 0492, MCAP — 0768, MCAP — 0773, MCAP — 0774, and reductases, such as the gene products of MCAP — 0101, MCAP — 0427, MCAP — 0779, and MCAP — 0858. 
         [0032]    Reductase activity can be measured through a Azo dye that gets decolorized by the release of reductases from many bacterial cells. An azo dye such as reactive black 5 or DABCYL (4-((4-(dimethylamino)phenyl)azo)benzoic acid) is completely decolorized by many bacterial cultured supernatant after just 18 hours on incubation. A sensor placed on the bottom of a culture dish, buffer container or even on a swab for measuring the presence of mycoplasma in human fluids can be used to ascertain bacterial contamination or infection. The benefit of a simple azo dye sensor is low cost although it may not specifically detect different bacteria.  FIG. 1  is a photograph of a microtiter plate containing reactive black 5 decolorized by incubation with different pathogenic bacteria. Each well was incubated with 10 μg of reactive black 5 plus 190 μl of filtered culture supernatant from the following bacterium:  E. coli, E. faecalis, S. aureus, P. aeruginosa, S. pyogenes , and  S. marcescens . Such azo dyes are decolorized by most bacteria after incubation with the dye for about 18 hours. The decolorization is indicative of reductases produced by the bacteria.  Mycoplasma  reductases such as the gene products of MCAP — 0101. MCAP — 0427, MCAP — 0779, and MCAP — 0858 can also decolorize such substrates. 
         [0033]    In other embodiments, substrates for reductases are reagents that produce a fluorescent signal. Suitable such reagents include DTNB (5,5′-Dithio-bis-(2-nitrobenzoic acid), also known as Ellman&#39;s reagent. 
         [0000]    
       
                 
         
             
             
         
       
     
         [0034]    Two other suitable fluorogenic compounds are BODIPY®FL L-cystine, 
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         [0035]    and 2′,7′-difluoro-4′-(2-(5-((dimethylaminophenyl)azo)pyridyl) dithiopropionyl aminomethyl)fluorescein, 
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         [0036]    Specific peptidase substrates can be used to identify a specific bacterium. Published patent applications disclosing both specific and broad-spectrum targets for detection of pathogens include WO 2005/042770, WO2005/012556 and WO2004/087942, which are incorporated herein by reference. Mycoplasmas secrete a lysine-specific endopeptidase, an aminopeptidase and a carboxypeptidase that make it possible to specifically detect the presence of mycoplasma by using a substrate that is specific for these enzymes. Suitable aminopeptidases and carboxypeptidases have been purified by Watanabe and colleagues (Watanabe, T. (1988), Proteolytic activities of  Mycoplasma salivarium , Adv Dent Res 2(2):297-300; Watanabe, T (1985) Proteolytic activity of mycoplasmas and ureaplasmas isolated freshly from human saliva, Medical Microbiology and Immunology 173(5): 251-255; Watanabe, T. et al., (1984) Aminopeptidase and caseinolytic activities of  Mycoplasma  salivarium Medical Microbiology and Immunology, 172 (4): 257-264). In a preferred embodiment, these purified or partially purified enzymes are used in a high-throughput screen to identify potential novel substrates. 
         [0037]    Mycoplasmas produce both secreted and membrane-bound nucleases that are involved in obtaining nucleotides for DNA synthesis. See Minion, C. J. D. Goguen (1986) Identification and Preliminary Characterization of External Membrane-Bound Nuclease Activities in  Mycoplasma pulmonis , Infection And Immunity, 51(1):352-354; Kannan, T. R.,&amp; Baseman, J. B., (2006) ADP-ribosylating and vacuolating cytotoxin of  Mycoplasma pneumoniae  represents unique virulence determinant among bacterial pathogens. PNAS, 103:6724-6729; Bendjennat, M., et al., (1997) Purification and Characterization of  Mycoplasma penetrans  Ca2+/Mg2+-Dependent Endonuclease, Journal of Bacteriology 179:2210-2220; Minion, C. F., et al., (1993) Membrane-Associated Nuclease Activities in Mycoplasmas. Journal of Bacteriology 175:7842-7847. 
         [0038]    RNA or DNA sequences that are efficiently hydrolyzed by  Mycoplasma  nucleases that labeled with a detectable colorimetric or fluorescent dye can be used to detect the presence of mycoplasma contamination. A dye such as blue dye number 1 is not decolorized by microorganisms and would be a good choice for a colorimetric reporter. The dye is labeled with a reactive aminoallyl-dUTP via a Klenow reaction using techniques known to one skilled in the art to covalently attach the dye to a nucleic acid. See Hasseman, J. J., et al., 2006 Microbial Genomic DNA Aminoallyl Labeling For Microarrays, The Institute For Genomic Research Standard Operating Procedure # M009. The aminoallyl groups on the nucleic acid would then be available for labeling with a reactive fluorescent or chromogenic dye molecule. The dye-labeled nucleic acid can be attached to the surface of a sterile bottle. If the bottle after opening became contaminated with mycoplasmas, the spot of color on the inner surface of the bottle would be released, indicating that the bottle is contaminated.  FIG. 2A  is a diagrammatic illustration of a contamination biosensor 200 placed on a container 100 for a reagent or culture medium.  FIG. 2B  a diagrammatic illustration of a contamination biosensor 210 placed on a container 110 for tissue culture. 
         [0039]    Azo dyes such as reactive black 5 and DABCYL are decolorized by bacteria and can be used as a broad spectrum sensor for microbial contamination. Blue dye number 1, which is not decolorized by bacteria, can be used as a label of nucleic acids or a peptide to give a specific probe for mycoplasmas or other contaminating microorganism. Fluorescent probes or the release of fluorescent micro-spheres can be used to indicate contamination. Contamination can be measured by eye, using a fluorimeter or colorimeter or on a microscope stage. 
         [0040]    In another embodiment, a peptide or nucleic acid can be labeled with an acetoxymethyl ester of a dye, such as blue dye number 1, that upon hydrolytic cleavage would be taken up by cells in culture and thereby turn them blue to indicate the presence of mycoplasmas in the culture medium.  FIG. 3  is a diagrammatic illustration of an embodiment of such a mycoplasmas contamination detection system for cell culture, showing in  FIG. 3A  a cell 300 in an uncontaminated culture, and in  FIG. 3B , a cell 300 in a contaminated culture containing a dye deposit 360 that is indicative of mycoplasmas contamination. An acetoxymethyl ester derivative of blue dye number 1 coupled to a peptide or nucleic acid carrier would be impermeable to tissue culture cells until contamination with mycoplasmas. The proteases or nucleases from  Mycoplasma  spp. would cleave the carrier from the acetoxymethyl ester derivative of blue dye number 1, thereby allowing the acetoxymethyl ester derivative of blue dye number 1 to be taken up by the tissue culture cells. Tissue culture cells that become colored blue indicate that the culture was contaminated with mycoplasmas. The colored cells can be observed with a light microscope. Alternatively a cell permeable fluorescent dye can be used and the fluorescing cells can be detected with a fluorescence microscope. 
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                 TABLE 1 
               
             
             
               
                   
               
               
                 Genes characterized by sequences common to  Mycoplasma  spp. (40% identity) 
               
             
          
           
               
                 Gene 
                 Symbol 
                 Common Name 
               
               
                   
               
               
                 MCAP_0001 
                 dnaA 
                 chromosomal replication initiator protein DnaA 
               
               
                 MCAP_0002 
                 dnaN 
                 DNA polymerase III, beta subunit 
               
               
                 MCAP_0004 
                 ksgA 
                 dimethyladenosine transferase 
               
               
                 MCAP_0008 
                 dnaX 
                 DNA polymerase III gamma-tau subunits 
               
               
                 MCAP_0010 
                 tmk 
                 thymidylate kinase 
               
               
                 MCAP_0011 
                   
                 DNA polymerase III, delta prime subunit 
               
               
                 MCAP_0017 
                 ftsH 
                 ATP-dependent metalloprotease FtsH 
               
               
                 MCAP_0022 
                   
                 acyl carrier protein phosphodiesterase, putative 
               
               
                 MCAP_0026 
                 rpsR 
                 30S ribosomal protein S18 
               
               
                 MCAP_0035 
                 metG 
                 methionyl-tRNA synthetase 
               
               
                 MCAP_0038 
                   
                 ABC transporter, permease protein 
               
               
                 MCAP_0039 
                   
                 ABC transporter, permease protein 
               
               
                 MCAP_0040 
                 gyrA 
                 DNA gyrase, A subunit 
               
               
                 MCAP_0041 
                 gyrB 
                 DNA gyrase, B subunit 
               
               
                 MCAP_0045 
                 secA 
                 preprotein translocase, SecA subunit 
               
               
                 MCAP_0047 
                   
                 5-3 exonuclease family protein 
               
               
                 MCAP_0060 
                   
                 endonuclease IV 
               
               
                 MCAP_0065 
                 rplK 
                 50S ribosomal protein L11 
               
               
                 MCAP_0066 
                 rplA 
                 ribosomal protein L1 
               
               
                 MCAP_0067 
                 rplJ 
                 50S ribosomal protein L10 
               
               
                 MCAP_0068 
                 rplL 
                 50S ribosomal protein L7/L12 
               
               
                 MCAP_0070 
                 rpoB 
                 DNA-directed RNA polymerase, beta subunit 
               
               
                 MCAP_0071 
                 rpoC 
                 DNA-directed RNA polymerase beta subunit 
               
               
                 MCAP_0074 
                   
                 ribose 5-phosphate isomerase B, putative 
               
               
                 MCAP_0075 
                 glyA 
                 serine hydroxymethyltransferase 
               
               
                 MCAP_0076 
                 upp 
                 uracil phosphoribosyltransferase 
               
               
                 MCAP_0078 
                 atpB 
                 ATP synthase F0, subunit A 
               
               
                 MCAP_0079 
                 atpE 
                 ATP synthase F0, subunit c 
               
               
                 MCAP_0082 
                 atpA1 
                 ATP synthase F1, alpha subunit 
               
               
                 MCAP_0083 
                 atpG 
                 ATP synthase F1, gamma subunit 
               
               
                 MCAP_0084 
                 atpD1 
                 ATP synthase F1, beta subunit 
               
               
                 MCAP_0094 
                 ptsG 
                 PTS system, glucose-specific IIABC component 
               
               
                 MCAP_0096 
                 smpB 
                 SsrA-binding protein 
               
               
                 MCAP_0097 
                   
                 Rnase R (VacB) and RNase II family 3-5 exoribonucleases 
               
               
                 MCAP_0101 
                 nrdE 
                 ribonucleoside-diphosphate reductase 2, alpha subunit 
               
               
                 MCAP_0104 
                 prs 
                 phosphoribosylpyrophosphate synthase 
               
               
                 MCAP_0105 
                 pth 
                 peptidyl-tRNA hydrolase 
               
               
                 MCAP_0107 
                 dnaC 
                 replicative DNA helicase 
               
               
                 MCAP_0110 
                 cysS 
                 cysteinyl-tRNA synthetase 
               
               
                 MCAP_0111 
                   
                 RNA methyltransferase, TrmH family, group 3 
               
               
                 MCAP_0114 
                 nusG 
                 transcription antitermination protein NusG 
               
               
                 MCAP_0119 
                   
                 oligopeptide ABC transporter, ATP-binding protein 
               
               
                 MCAP_0120 
                   
                 oligopeptide ABC transporter, ATP-binding protein 
               
               
                 MCAP_0124 
                   
                 hydrolase, TatD family 
               
               
                 MCAP_0130 
                 gltX 
                 glutamyl-tRNA synthetase 
               
               
                 MCAP_0136 
                 fba 
                 fructose-1,6-bisphosphate aldolase, class II 
               
               
                 MCAP_0140 
                 rpmE 
                 ribosomal protein L31 
               
               
                 MCAP_0142 
                   
                 DHH phosphoesterase family protein, putative 
               
               
                 MCAP_0143 
                 tdk 
                 thymidine kinase 
               
               
                 MCAP_0144 
                 prfA 
                 peptide chain release factor 1 
               
               
                 MCAP_0145 
                   
                 modification methylase, HemK family 
               
               
                 MCAP_0151 
                 rpsL 
                 30S ribosomal protein S12 
               
               
                 MCAP_0152 
                 rpsG 
                 30S ribosomal protein S7 
               
               
                 MCAP_0153 
                 fusA 
                 translation elongation factor G 
               
               
                 MCAP_0154 
                 tuf 
                 translation elongation factor Tu 
               
               
                 MCAP_0157 
                 pepA1 
                 cytosol aminopeptidase 
               
               
                 MCAP_0159 
                 alaS 
                 alanyl-tRNA synthetase 
               
               
                 MCAP_0163 
                   
                 oligopeptide ABC transporter, ATP-binding protein 
               
               
                 MCAP_0195 
                 pepA2 
                 cytosol aminopeptidase 
               
               
                 MCAP_0200 
                   
                 spermidine/putrescine ABC transporter, permease protein 
               
               
                   
                   
                 and spermidine/putrescine-binding protein 
               
               
                 MCAP_0201 
                   
                 spermidine/putrescine ABC transporter, permease protein 
               
               
                 MCAP_0203 
                 rplT 
                 50S ribosomal protein L20 
               
               
                 MCAP_0205 
                 infC 
                 translation initiation factor IF-3 
               
               
                 MCAP_0208 
                 gmk 
                 guanylate kinase 
               
               
                 MCAP_0213 
                 eno 
                 enolase4.2.1.11 
               
               
                 MCAP_0216 
                 hpt1 
                 hypoxanthine phosphoribosyltransferase 
               
               
                 MCAP_0220 
                 pfkA 
                 Phosphofructokinase 
               
               
                 MCAP_0221 
                 pyk 
                 pyruvate kinase 
               
               
                 MCAP_0222 
                 thrS 
                 threonyl-tRNA synthetase 
               
               
                 MCAP_0223 
                   
                 NADH oxidase 
               
               
                 MCAP_0224 
                   
                 lipoate-protein ligase 
               
               
                 MCAP_0225 
                 pdhA 
                 pyruvate dehydrogenase complex, El component, 
               
               
                   
                   
                 alpha subunit 
               
               
                 MCAP_0226 
                 pdhB 
                 pyruvate dehydrogenase complex, E1 component, 
               
               
                   
                   
                 beta subunit 
               
               
                 MCAP_0228 
                 pdhD 
                 dihydrolipoamide dehydrogenase 
               
               
                 MCAP_0229 
                 pta 
                 phosphate acetyltransferase 
               
               
                 MCAP_0230 
                 ackA 
                 acetate kinase 
               
               
                 MCAP_0233 
                 ptsI 
                 phosphoenolpyruvate-protein phosphotransferase 
               
               
                 MCAP_0234 
                 crr 
                 PTS system, glucose-specific IIA component 
               
               
                 MCAP_0237 
                 rpsD 
                 30S ribosomal protein S4 
               
               
                 MCAP_0245 
                   
                 GTP-binding conserved hypothetical protein 
               
               
                 MCAP_0251 
                 greA 
                 transcription elongation factor GreA 
               
               
                 MCAP_0252 
                 uvrC 
                 excinuclease ABC, C subunit 
               
               
                 MCAP_0255 
                   
                 conserved hypothetical protein 
               
               
                 MCAP_0258 
                 valS 
                 valyl-tRNA synthetase 
               
               
                 MCAP_0260 
                 rpe 
                 ribulose-phosphate 3-epimerase 
               
               
                 MCAP_0261 
                 rsgA 
                 ribosome small subunit-dependent GTPase A 
               
               
                 MCAP_0267 
                   
                 metalloendopeptidase, putative 
               
               
                 MCAP_0318 
                 proS 
                 prolyl-tRNA synthetase 
               
               
                 MCAP_0321 
                 lepA 
                 GTP-binding protein LepA 
               
               
                 MCAP_0323 
                 aspS 
                 aspartyl-tRNA synthetase 
               
               
                 MCAP_0324 
                 hisS 
                 His-tRNA synthetase 6.1.1.21 
               
               
                 MCAP_0330 
                 rpsO 
                 30S ribosomal protein S15 
               
               
                 MCAP_0333 
                 infB 
                 translation initiation factor IF-2 
               
               
                 MCAP_0336 
                   
                 transcription elongation protein nusA, putative 
               
               
                 MCAP_0339 
                 polC 
                 DNA polymerase III, alpha subunit, Gram-positive type 
               
               
                 MCAP_0340 
                 cdsA 
                 phosphatidate cytidylyltransferase 
               
               
                 MCAP_0341 
                   
                 Xaa-Pro peptidase 
               
               
                 MCAP_0342 
                 trpS 
                 tryptophanyl-tRNA synthetase 
               
               
                 MCAP_0358 
                 atpA2 
                 ATP synthase F1, alpha subunit 
               
               
                 MCAP_0359 
                 atpD2 
                 ATP synthase F1, beta subunit 
               
               
                 MCAP_0364 
                   
                 RNA methyltransferase, TrmH family 
               
               
                 MCAP_0365 
                   
                 hydrolase of the HAD superfamily, putative 
               
               
                 MCAP_0367 
                 hrcA 
                 heat-inducible transcription repressor HrcA 
               
               
                 MCAP_0369 
                 dnaK 
                 Chaperone protein dnaK (Heat shock protein 70) 
               
               
                   
                   
                 (Heat shock 70 kDaprotein) (HSP70) 
               
               
                 MCAP_0371 
                 rpsB 
                 30S ribosomal protein S2 
               
               
                 MCAP_0372 
                 tsf 
                 translation elongation factor Ts 
               
               
                 MCAP_0374 
                 pyrH 
                 uridylate kinase 
               
               
                 MCAP_0375 
                 frr 
                 ribosome recycling factor 
               
               
                 MCAP_0376 
                 argS 
                 arginyl-tRNA synthetase 
               
               
                 MCAP_0383 
                 pheS 
                 phenylalanyl-tRNA synthetase, alpha subunit 
               
               
                 MCAP_0384 
                 pheT 
                 phenylalanyl-tRNA synthetase, beta subunit 
               
               
                 MCAP_0388 
                 mraW 
                 S-adenosyl-methyltransferase MraW 2.1.1.- 479149 
               
               
                 MCAP_0393 
                 ileS 
                 isoleucyl-tRNA synthetase 
               
               
                 MCAP_0395 
                   
                 ribosomal large subunit pseudouridine synthase, RluA family 
               
               
                 MCAP_0410 
                   
                 conserved hypothetical protein, TIGR00096 
               
               
                 MCAP_0412 
                 rplU 
                 50 ribosomal protein L21 
               
               
                 MCAP_0414 
                 rpmA 
                 50S ribosomal protein L27 
               
               
                 MCAP_0423 
                   
                 ImpB/MucB/SamB family protein 
               
               
                 MCAP_0427 
                   
                 pyridine nucleotide-disulphide oxidoreductase 
               
               
                 MCAP_0439 
                 ldh 
                 L-lactate/malate dehydrogenase 
               
               
                 MCAP_0445 
                   
                 triacylglycerol lipase 
               
               
                 MCAP_0446 
                   
                 triacylglycerol lipase, putative 
               
               
                 MCAP_0449 
                   
                 lipoate-protein ligase 
               
               
                 MCAP_0454 
                 gtsA 
                 glycerol ABC transporter, ATP-binding protein 
               
               
                 MCAP_0456 
                 parC 
                 DNA topoisomerase IV, A subunit 
               
               
                 MCAP_0457 
                 parE 
                 DNA topoisomerase IV, B subunit 
               
               
                 MCAP_0462 
                   
                 RNA methyltransferase, TrmH family 
               
               
                 MCAP_0465 
                 pgi 
                 glucose-6-phosphate isomerase 
               
               
                 MCAP_0469 
                   
                 aminotransferase, class V 
               
               
                 MCAP_0472 
                   
                 HIT family protein 
               
               
                 MCAP_0474 
                 ung 
                 uracil-DNA glycosylase 
               
               
                 MCAP_0476 
                 gid 
                 glucose inhibited division protein 
               
               
                 MCAP_0478 
                 metK 
                 S-adenosylmethionine synthetase 
               
               
                 MCAP_0479 
                   
                 conserved hypothetical protein TIGR00282 
               
               
                 MCAP_0481 
                 ftsY 
                 signal recognition particle-docking protein FtsY 
               
               
                 MCAP_0488 
                 rpmB 
                 50S ribosomal protein L28 
               
               
                 MCAP_0492 
                   
                 ribonuclease III 
               
               
                 MCAP_0495 
                   
                 structural maintenance of chromosomes 
               
               
                   
                   
                 (SMC) superfamily protein 
               
               
                 MCAP_0497 
                 apt 
                 adenine phosphoribosyltransferase 
               
               
                 MCAP_0503 
                 rpoD 
                 RNA polymerase sigma factor RpoD 
               
               
                 MCAP_0504 
                 dnaG 
                 DNA primase 
               
               
                 MCAP_0505 
                 glyS 
                 glycyl-tRNA synthetase 
               
               
                 MCAP_0507 
                 era 
                 GTP-binding protein Era 
               
               
                 MCAP_0509 
                   
                 Peptidase C39 family protein 
               
               
                 MCAP_0510 
                   
                 DJ-1 family protein 
               
               
                 MCAP_0516 
                 lon 
                 ATP-dependent protease La 
               
               
                 MCAP_0517 
                 tig 
                 trigger factor 
               
               
                 MCAP_0519 
                 efp 
                 translation elongation factor P 
               
               
                 MCAP_0521 
                   
                 conserved hypothetical protein 
               
               
                 MCAP_0523 
                 trmU 
                 tRNA (5-methylaminomethyl-2-thiouridylate)- 
               
               
                   
                   
                 methyltransferase 
               
               
                 MCAP_0529 
                   
                 nicotinate (nicotinamide) nucleotide adenylyltransferase/ 
               
               
                   
                   
                 conserved hypothetical domain 
               
               
                 MCAP_0532 
                   
                 Spo0B-associated GTP-binding protein, putative 
               
               
                 MCAP_0544 
                 rplS 
                 50S ribosomal protein L19 
               
               
                 MCAP_0545 
                 trmD 
                 tRNA(guanine-N1)-methyltransferase 
               
               
                 MCAP_0547 
                 rpsP 
                 30S ribosomal protein S16 
               
               
                 MCAP_0549 
                 ffh 
                 signal recognition particle protein 
               
               
                 MCAP_0551 
                 recA 
                 recA protein 
               
               
                 MCAP_0577 
                 engA 
                 GTP-binding protein engA 
               
               
                 MCAP_0578 
                 cmk 
                 cytidylate kinase 
               
               
                 MCAP_0581 
                 ppa 
                 inorganic pyrophosphatase 
               
               
                 MCAP_0589 
                   
                 ribulose-phosphate 3-epimerase, putative 
               
               
                 MCAP_0601 
                 scpB 
                 chromosomal segregation and condensation protein B 
               
               
                 MCAP_0606 
                   
                 hydrolase, alpha/beta fold family 
               
               
                 MCAP_0609 
                   
                 Uncharacterised membrane protein, UPF0154 family 
               
               
                 MCAP_0610 
                 tkt 
                 transketolase 
               
               
                 MCAP_0613 
                   
                 glucose-inhibited division protein, putative 
               
               
                 MCAP_0616 
                   
                 fructose/tagatose bisphosphate aldolase, class II 
               
               
                 MCAP_0623 
                   
                 metallo-beta-lactamase superfamily protein 
               
               
                 MCAP_0631 
                 pgk 
                 phosphoglycerate kinase 
               
               
                 MCAP_0632 
                 gap 
                 glyceraldehyde-3-phosphate dehydrogenase 
               
               
                 MCAP_0635 
                 mutM 
                 formamidopyrimidine-DNA glycosylase 
               
               
                 MCAP_0636 
                 polA 
                 DNA polymerase I 
               
               
                 MCAP_0637 
                 dnaE 
                 DNA-directed DNA polymerase III (polc) 
               
               
                 MCAP_0639 
                 tyrS 
                 tyrosyl-tRNA synthetase 
               
               
                 MCAP_0659 
                 leuS 
                 leucyl-tRNA synthetase 
               
               
                 MCAP_0662 
                 rpsI 
                 30S ribosomal protein S9 
               
               
                 MCAP_0663 
                 rplM 
                 50S ribosomal protein L13 
               
               
                 MCAP_0666 
                   
                 cobalt ABC transporter, permease protein 
               
               
                 MCAP_0667 
                   
                 cobalt ABC transporter, ATP-binding protein, putative 
               
               
                 MCAP_0668 
                   
                 cobalt ABC transporter, ATP-binding protein, putative 
               
               
                 MCAP_0669 
                 rplQ 
                 50S ribosomal protein L17 
               
               
                 MCAP_0670 
                 rpoA 
                 DNA-directed RNA polymerase, alpha chain 
               
               
                 MCAP_0671 
                 rpsK 
                 30S ribosomal protein S11 
               
               
                 MCAP_0672 
                 rpsM 
                 30S ribosomal protein S13 
               
               
                 MCAP_0675 
                 map 
                 methionine aminopeptidase, type I 
               
               
                 MCAP_0676 
                 adk 
                 adenylate kinase 
               
               
                 MCAP_0677 
                 secY 
                 preprotein translocase, SecY subunit 
               
               
                 MCAP_0678 
                 rplO 
                 50S ribosomal protein L15 
               
               
                 MCAP_0679 
                 rpsE 
                 30S ribosomal protein S5 
               
               
                 MCAP_0680 
                 rplR 
                 50S ribosomal protein L18 
               
               
                 MCAP_0681 
                 rplF 
                 50S ribosomal protein L6 
               
               
                 MCAP_0682 
                 rpsH 
                 ribosomal protein S8 
               
               
                 MCAP_0684 
                 rplE 
                 ribosomal protein L5 
               
               
                 MCAP_0686 
                 rplN 
                 ribosomal protein L14 
               
               
                 MCAP_0687 
                 rpsQ 
                 ribosomal protein S17 
               
               
                 MCAP_0689 
                 rplP 
                 ribosomal protein L16 
               
               
                 MCAP_0690 
                 rpsC 
                 ribosomal protein S3 
               
               
                 MCAP_0691 
                 rplV 
                 ribosomal protein L22 
               
               
                 MCAP_0692 
                 rpsS 
                 30S ribosomal protein S19 
               
               
                 MCAP_0693 
                 rplB 
                 50S ribosomal protein L2 
               
               
                 MCAP_0694 
                 rplW 
                 50S ribosomal protein L23 
               
               
                 MCAP_0695 
                 rplD 
                 50S ribosomal protein L4 
               
               
                 MCAP_0696 
                 rplC 
                 50 ribosomal protein L3 
               
               
                 MCAP_0697 
                 rpsJ 
                 30S ribosomal protein S10 
               
               
                 MCAP_0707 
                   
                 potassium uptake protein, TrkH family, putative 
               
               
                 MCAP_0708 
                   
                 potassium uptake protein, TrkA family, putative 
               
               
                 MCAP_0709 
                 gatB 
                 glutamyl-tRNA(Gln) amidotransferase, B subunit 
               
               
                 MCAP_0710 
                 gatA 
                 glutamyl-tRNA(Gln) amidotransferase, A subunit 
               
               
                 MCAP_0712 
                 ligA 
                 DNA ligase, NAD-dependent 
               
               
                 MCAP_0714 
                   
                 ribosomal large subunit pseudouridine synthase, RluA family 
               
               
                 MCAP_0716 
                 ptsH 
                 phosphocarrier protein hpr 
               
               
                 MCAP_0750 
                 tpiA 
                 triosephosphate isomerase 
               
               
                 MCAP_0751 
                   
                 HAD-superfamily hydrolase subfamily IIB, protein 
               
               
                 MCAP_0752 
                 gpmI 
                 2,3-bisphosphoglycerate-independent 
               
               
                   
                   
                 phosphoglycerate mutase 
               
               
                 MCAP_0755 
                 deoC 
                 deoxyribose-phosphate aldolase 
               
               
                 MCAP_0757 
                 deoA 
                 pyrimidine-nucleoside phosphorylase 
               
               
                 MCAP_0761 
                 trmB 
                 tRNA (guanine-N(7)-)-methyltransferase 
               
               
                 MCAP_0765 
                 hpt2 
                 hypoxanthine phosphoribosyltransferase 
               
               
                 MCAP_0768 
                   
                 deoxyribonuclease, TatD family 
               
               
                 MCAP_0773 
                 uvrB 
                 excinuclease ABC, B subunit 
               
               
                 MCAP_0774 
                 uvrA 
                 excinuclease ABC, A subunit 
               
               
                 MCAP_0778 
                 lgt1 
                 prolipoprotein diacylglyceryl transferase 
               
               
                 MCAP_0779 
                 trxB 
                 thioredoxin reductase 
               
               
                 MCAP_0780 
                 lgt2 
                 prolipoprotein diacylglyceryl transferase 
               
               
                 MCAP_0781 
                   
                 conserved hypothetical protein 
               
               
                 MCAP_0792 
                 topA 
                 DNA topoisomerase I 
               
               
                 MCAP_0805 
                 engD 
                 GTP-dependent nucleic acid-binding protein engD 
               
               
                 MCAP_0807 
                 gidB 
                 methyltransferase gidB 
               
               
                 MCAP_0808 
                 pgsA 
                 CDP-diacylglycerol--glycerol-3-phosphate 3- 
               
               
                   
                   
                 phosphatidyltransferase 
               
               
                 MCAP_0814 
                   
                 transporter protein, putative 
               
               
                 MCAP_0818 
                 rpsT 
                 30S ribosomal protein S20 
               
               
                 MCAP_0819 
                 trmE 
                 tRNA modification GTPase TrmE 
               
               
                 MCAP_0821 
                   
                 glycoprotease family protein 
               
               
                 MCAP_0824 
                 asnS 
                 asparaginyl-tRNA synthetase 
               
               
                 MCAP_0834 
                   
                 hydrolase, haloacid dehalogenase-like family, putative 
               
               
                 MCAP_0836 
                 lysS 
                 lysyl-tRNA synthetase 
               
               
                 MCAP_0839 
                 serS 
                 seryl-tRNA synthetase 
               
               
                 MCAP_0844 
                   
                 PTS system glucose-specific IIBC component 
               
               
                 MCAP_0849 
                 deoD 
                 purine nucleoside phosphorylase 
               
               
                 MCAP_0856 
                 gidA 
                 glucose inhibited division protein A 
               
               
                 MCAP_0858 
                   
                 pyridine nucleotide-disulphide oxidoreductase 
               
               
                 MCAP_0860 
                   
                 membrane protein, putative 
               
               
                 MCAP_0870 
                 rpmH 
                 50S ribosomal protein L34 
               
               
                   
               
             
          
         
       
     
       Example 1 
       Mycoplasma hyorhinis  Enzymes 
       [0041]    The subset of  Mycoplasma  genes from the  Mycoplasma hyorhinis  genome that were selected for further study are listed in Table 2, below. DNA PCR primers were made for each of these genes and the PCR are shown in  FIG. 5  and  FIG. 6 . 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Hydrolytic enzymes in common among  Mycoplasma  ssp. 
               
             
          
           
               
                   
                 Gene 
                 Symbol 
                 Common Name 
               
               
                   
                   
               
             
          
           
               
                 1 
                 MHP7448_0010 
                 hrcA 
                 heat-inducible transcription repressor 
               
               
                 2 
                 MHP7448_0037 
                 vacB 
                 VACB-like ribonuclease II 
               
               
                 3 
                 MHP7448_0062 
                 nfo 
                 endonuclease IV 
               
               
                 4 
                 MHP7448_0066 
                 uvrC 
                 excinuclease ABC subunit C 
               
               
                 5 
                 MHP7448_0082 
                 nox 
                 NADH oxidase 
               
               
                 6 
                 MHP7448_0091 
                 uvrA 
                 excinuclease ABC subunit A 
               
               
                 7 
                 MHP7448_0097 
                 lgt 
                 prolipoprotein diacylglyceryl transferase 
               
               
                 8 
                 MHP7448_0098 
                 trxB 
                 thioredoxin reductase 
               
               
                 9 
                 MHP7448_0173 
                 map 
                 methionine aminopeptidase 
               
               
                 10 
                 MPH7448_0204 
                 pth 
                 prptidyl-tRNA hydrolase 
               
               
                 11 
                 MHP7448_0398 
                 rnc 
                 ribonuclease III 
               
               
                 12 
                 MHP7448_0464 
                 pepA 
                 leucyl aminopeptidase 
               
               
                 13 
                 MHP7448_0521 
                 pepF 
                 oligoendopeptidase F 
               
               
                 14 
                 MHP7448_0524 
                 lon 
                 heat shock ATP-dependent protease 
               
               
                 15 
                 MHP7448_0581 
                   
                 5′-3′ exonuclease 
               
               
                 16 
                 MHP7448_0635 
                 gcp 
                 O-sialoglycoprotein endopeptidase 
               
               
                 17 
                 MHP7448_0648 
                 uvrB 
                 excinuclease ABC subunit B 
               
               
                 18 
                 MHP7448_0659 
                 pepP 
                 XAA-PRO aminopeptidase 
               
               
                   
               
             
          
         
       
     
       Purification of Total RNA 
       [0042]    Total RNA was isolated from  Mycoplasma hyorhinis  grown in BHK-21 and Swiss 3T3 tissue culture cells ( FIG. 4A ). RNA was purified both from the infected tissue culture media and culture cells (BHK-21 &amp; Swiss 3T3) from the infected dish. RNA was purified using either the Invitrogen Triazol® Max bacterial RNA isolation kit or an acid phenol-guanidium thiocyanate and chloroform extraction procedure. Although the acid phenol-guanidium thiocyanate and chloroform extraction procedure had better quality RNA as judged by gel electrophoresis in a 1.5% agarose gel, the RNA from the Triazol® Max bacterial RNA kit had more mycoplasma RNA as judged by PCR of the p37 control gene. As a positive control for the RT-PCR reactions, we amplified the p37 gene from the total infected BHK-21 and 3T3 cell media. As a negative control, we also isolated total RNA from uninfected tissue culture media and uninfected BHK-21 or 3T3 cells and then performed RT-PCR using the p37 primer set. RT PCR was performed in a iCycler iQ PCR Detection System (Bio-Rad) using the SYBR Green One-Step Quantitative RT-PCR kit. Alternatively, for the preliminary studies RT PCR was performed with the Ambion Ag-Path kit.  FIG. 4A  shows an exemplary result of agarose gel electrophoresis of the RNA samples used in RT-PCR. The lanes are: a) 1 kB DNA Ladder, b) BHK-21 cells infected with  Mycoplasma hyorhinis  as a monolayer, c) the pellet of BHK-21 cells medium infected with  Mycoplasma hyorhinis , and d) the pellet of  Mycoplasma hyorhinis  from mycoplasma enrichment broth (not from tissue culture cells). 
         [0043]    The results of preliminary studies indicate that the following genes vacB, trxB, map, pepA, lon, and uvrB are transcribed at a high level.  FIG. 4B  is a graphical representation of the expression of several  Mycoplasma hyorhinis  genes under conditions A-J: A) lon, 3T3 cells growing as a monolayer, B) lon, BHK-21 cells growing in DMEM, C) map, 3T3 cells growing as a monolayer, D) map, BHK-21 cells growing in DMEM, E) pepA, 3T3 cells growing as a monolayer, F) pepA, BHK-21 cells growing in DMEM, G) trxB, 3T3 cells growing as a monolayer, H) trxB, BHK-21 cells growing in DMEM, I) vacB, 3T3 cells growing as a monolayer, J) vacB, BHK-21 cells growing in DMEM. The level of expression from strongest to weakest for these abundant mRNA is trxB&gt;pepA&gt;map&gt;vacB&gt;lon&gt;uvrB. 
         [0044]    Substrates for the hydrolytic enzymes corresponding to these putative abundant mRNAs from  Mycoplasma  were identified using both literature and patent searches. Certain selected examples are provided in Table 3, below. 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                   Mycoplasma  Hydrolase Substrates 
               
             
          
           
               
                 Gene Symbol 
                 Substrates 
                 References 
               
               
                   
               
               
                 vacB 
                 ds RNAse activity 
                 J. Cell. Physiol. 143(3) 416-419 
               
               
                 trxB 
                 DTNB 
                 Cayman Chemical Company 
               
               
                 Map 
                 Rhodamine based fluorogenic 
                 J. Biomol. Screening 7(6) 
               
               
                   
                 substrates 
                 531-540, 2002 
               
               
                 pepA 
                 Leucine at N-terminal of a peptide 
                 Curr. Microbiol. 48(1)32-38, 2004 
               
               
                 lon 
                 Glutaryl-Ala-Ala-Phe- 
                 J. Biol. Chem. 260(22) 1 1985 
               
               
                   
                 methoxynaphthylamine + ATP 
               
               
                 uvrB 
                 (UvrA)2(UvrB)1 com 
                 Proc. Natl Soc. USA 86(14) 
               
               
                   
                   
                 5237-5241, 1985. 
               
               
                   
               
             
          
         
       
     
         [0045]    The genes that were determined by quantitative RT-PCR results to be highly expressed in  Mycoplasma hyorhinis  when infecting 3T3 cells or BHK-21 cells: trxB, pepA, lon, vacB, map, and uvrB. The substrates for the enzymes produced by these gene products is reported in Table 4, below. 
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 
                   Mycoplasma hyorhinis 
                 
               
               
                 Gene Encoding Enzymes and Enzyme Substrates 
               
             
          
           
               
                   
                 Gene 
                 Enzyme 
                 Substrate 
               
               
                   
                   
               
               
                   
                 trxB 
                 Thioredoxin reductase 
                 DTNB 
               
               
                   
                 pepA 
                 Leucine aminopeptidase 
                 leu-MCA 
               
               
                   
                 lon 
                 ATP-dependent protease 
                 glt-ala-ala-phe-MCA 
               
               
                   
                 map 
                 Methionine aminopeptidase 
                 met-MCA 
               
               
                   
                 vacB 
                 exoribonuclease 
                 dsRNA 
               
               
                   
                 uvrB 
                 excinuclease 
                 dsDNA 
               
               
                   
                   
               
             
          
         
       
     
         [0046]    We examined the nuclease activities of VacB and UvrB by challenging extracts from  M. hyorhinis  isolated from a cell culture infection or medium from  Mycoplasma -infected or uninfected cell culture with either double stranded (ds) RNA or dsDNA. The reaction was incubated for 30 minutes at 37° C. and the products were then analyzed by agarose gel electrophoresis, as shown in  FIG. 8 . 
         [0047]      FIG. 8  shows an agarose gel loaded with double stranded DNA (dsDNA, lanes b-i) or double stranded RNA (ribosomal RNA, lanes j-q) treated with  M. hyorhinis  extract from a cell culture infection or supernatants from infected or uninfected cell cultures. The respective lanes contain: a) 1 kb DNA ladder, b) dsDNA exposed to an aliquot of  M. hyorhinis  extract for 30 minutes, c) dsDNA exposed to an aliquot of the supernatant of an infected cell culture for 30 minutes, d) dsDNA exposed to an aliquot of the supernatant of an uninfected cell culture for 30 minutes, e) dsDNA exposed to H 2 O for 30 minutes, f) dsDNA exposed to an aliquot of  M. hyorhinis  extract for 0 minutes, g) dsDNA exposed to an aliquot of the supernatant of an infected cell culture for 0 minutes, h) dsDNA exposed to an aliquot of the supernatant of an uninfected cell culture for 0 minutes, i) dsDNA exposed to H 2 O for 0 minutes, j) ribosomal RNA exposed to an aliquot of  M. hyorhinis  extract for 30 minutes, k) ribosomal RNA exposed to an aliquot of the supernatant of an uninfected cell culture for 30 minutes, l) ribosomal RNA exposed to an aliquot of the supernatant of an uninfected cell culture for 30 minutes, m) ribosomal RNA exposed to exposed to H 2 O 30 minutes, n) ribosomal RNA exposed to an aliquot of  M. hyorhinis  extract for 0 minutes, o) ribosomal RNA exposed to an aliquot of the supernatant of an infected cell culture for 0 minutes, p) ribosomal RNA exposed to an aliquot of the supernatant of an uninfected cell culture for 0 minutes, and q) ribosomal RNA exposed to an aliquot of H 2 O for 0 minutes. 
         [0048]    There was no detectable dsDNAse activity (lanes b &amp; c) in the  M. hyorhinis  extract or  M. hyorhinis -infected medium suggesting that there is insufficient urvB activity for this enzyme to serve as a suitable basis for a diagnostic test for  Mycoplasma  contamination. Although dsRNAse activity was observed in the  M. hyorhinis  extract and  M. hyorhinis -infected medium (lanes j and k), the uninfected tissue culture media control also had appreciable dsRNAse activity. This finding indicates that vacB activity would not be a suitable basis for a  Mycoplasma  diagnostic test due to cross-reactivity from ribonucleases present in the culture medium of uninfected cells. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 
                   Mycoplasma hyorhinis 
                 
               
               
                 Assays for Proteolytic Activity 
               
             
          
           
               
                 Gene 
                 Enzyme 
                 Substrate 
                 Condition 
                 Vmax 
               
               
                   
               
             
          
           
               
                 lon 
                 ATP-dependent protease 
                 glt-ala-ala-phe-MCA 
                 Buffer 
                 55.8 
               
               
                   
                   
                   
                 Uninfected medium 
                 1460 
               
               
                   
                   
                   
                 Infected medium 
                 1802 
               
               
                   
                   
                   
                 
                   Mycoplasma 
                 
                 3354 
               
               
                   
                   
                   
                 positive control 
               
               
                 pepA 
                 Leucine aminopeptidase 
                 leu-MCA 
                 Buffer A   
                 2874 
               
               
                   
                   
                   
                 Uninfected medium 
                 454 
               
               
                   
                   
                   
                 Infected medium 
                 906 
               
               
                   
                   
                   
                 
                   Mycoplasma 
                 
                 522 
               
               
                   
                   
                   
                 positive control 
               
               
                 map 
                 Methionine aminopeptidase 
                 met-MCA 
                 Buffer 
                 19.4 
               
               
                   
                   
                   
                 Uninfected medium 
                 430 
               
               
                   
                   
                   
                 Infected medium 
                 627 
               
               
                   
                   
                   
                 
                   Mycoplasma 
                 
                 733 
               
               
                   
                   
                   
                 positive control 
               
               
                   
               
               
                   A This reading is probably artefactually high, and may indicate a bubble in the well. Subsequent experiments showed that Vmax in buffer was essentially zero. 
               
             
          
         
       
     
         [0049]    Further studies examined the suitability of the aminopeptidases map or lon or the protease pepA for use in a diagnostic test. These studies used several fluorogenic substrates consisting of small chain amino acids coupled to a methoxy coumarin fluorescent probe (MCA, (7-methoxycoumarin-4-yl)acetyl). The results of the initial studies are provided in Table 5, above. The test conditions were “buffer,” phosphate-buffered saline (PBS), “uninfected medium,” medium from cell cultures not infected with  Mycoplasma , “infected medium,” medium from cell cultures infected with  Mycoplasma , and a  Mycoplasma  positive control derived from a  Mycoplasma  culture. In each case the uninfected media control had substantial background proteolytic activity, with pepA being the most candidate with a signal-to-noise ratio (S/N) of about 2, where S/N=(Vmax infected medium)/(Vmax uninfected medium). 
       Example 2 
     Detection of  Mycoplasma  Using trxB 
       [0050]    In contrast to the protease and the double stranded nuclease markers, thioredoxin reductase (trxB) had significant activity specific to tissue culture cells co-infected with  Mycoplasma hyorhinis . In earlier studies, we demonstrated that  Mycoplasma  thioredoxin reductase activity was measured in infected culture medium using the substrate DTNB (5,5′-Dithio-bis-(2-nitrobenzoic acid), also known as Ellman&#39;s reagent. 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    Other suitable fluorogenic thioredoxin reductase substrates have been reported in the literature. 
         [0051]    Since thioredoxin reductases are widely distributed in eukaryotes and prokaryotic cells, there is a possibility that thioredoxin reductases from other microbes may cross-react with this assay to give a false positive result. One possible approach would be to use gentle lysis buffers that disrupt  Mycoplasma  cells, which do not have a cell wall, but do not appreciably lyse other bacteria that possess a cell wall. Studies demonstrated that there was no appreciable hydrolysis of DTNB by up to 10 6  CFU/ml  E. coli  or  S. aureus  ( FIG. 9 ). This result indicates that the thioredoxin reductases of other gram-positive and gram-negative bacteria, represented by  E. coli  and  S. aureus , are either minor enzymatic components or have a much lower specific activity than that of  M. hyorhinis . This finding is consistent with the previous demonstration that Mollicutes such as  Mycoplasma  have a very highly active thioredoxin reductase system (NTS) (0.09-0.25 SA units) in the presence of NADPH. This high NTS activity is presumed to be useful for  Mycoplasma  for the detoxification of reactive oxygen compounds, since the Mollicutes have simple genomes that lack the genes encoding enzymes such as catalase, peroxidase and oxygen dismutase that function to remove H 2 O 2  and other oxygen radicals in other bacteria (Gibson, D. G., et al., Complete chemical synthesis, assembly, and cloning of a  Mycoplasma genitalium  genome. Science. 319(5867):1215-1220 (2008)). 
         [0052]    Although initial attempts to detect trxB with DTNB were successful and the DTNB did not cross react with  Staphylococcus aureus  or  Escherichia coli  ( FIG. 9 ), the sensitivity of the assay was unacceptable (&gt;10 6  CFU/ml). Other fluorescent probes such as BODIPY®FL L-cystine and the 2′,7′-difluoro-4′-(2-(5-((dimethyl amino phenyl)azo)pyridyl)dithiopropionyl aminomethyl)fluorescein (abbreviated as DFDMAP-fluorescein) were studied to improve the sensitivity and the signal-to-noise ratio of the assay. Assays were performed with 40 mM Tris pH 7.2, 100 mM NaCl±detergent. 
         [0053]    A detergent lysis buffer procedure that would hydrolyze the simple  Mycoplasma  cell membranes but not lyse the tissue culture cells was needed. Methyl-6-O—(N-heptylcarbamoyl)-α-D-glucopyranoside (HECAMEG) is a preferred detergent. We have found that 0.5% HECAMEG was sufficient to lyse  Mycoplasma  cells while not disrupting the membranes of the cells grown in the culture medium. In contrast, 0.25% Triton X-100°, 0.4% BriJ 35®, and digitonin resulted in either significant increase in the background or loss in the true level of trxB activity. The activity of the fluorescent substrates is low compared to reduction with DTT suggesting that they may not be ideal or specific for trxB ( FIG. 11 ). As expected, 0.1 mM NADPH enhanced the activity of trxB ( FIG. 12 ). As a substrate for trxB, Bodipy® FL L-cystine provided a signal that was 7500 times that of the buffer control, however this signal was an artifact of the digitonin. DFDMAP fluorescein had a moderately improved detection level of 10,000-20,000 RFU at 10 7 -10 8  CFU/ml. 
         [0054]    An alternative substrate to produce the thioredoxin reductase signal was to measure the release of horseradish peroxidase (HRP) from a chromatography bead tethered with the heterobifunctional crosslinking reagent 3,3′-dithiobis[sulfosuccinimidylpropionate] (DTSSP). 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    The HRP-DTSSP-BEAD conjugate is shown schematically in  FIG. 10A . 
         [0055]    It was expected that frxB would be able to reduce the disulfide bridge of DTSSP, thereby releasing the HRP to react with its substrate tetramethylbenzidine (TMB), and in the presence of  Mycoplasma , produce a blue color. However, it was found that the HRP-DTSSP-BEAD conjugate also cross-reacted to the tissue culture uninfected medium sample. 
         [0056]    Other alternative substrates can use fluorescence energy transfer (FRET) with a disulfide bridge between EDANS and DABSYL, as shown below: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    Alternatively, DFDMAP and BODIPY® FL L-cysteine moieties could be coupled to the thioredoxin peptide or the central Gly-Ala residues to enhance the specificity of these fluorescent probes. Initial studies of these approaches have not shown improved sensitivity or reduction of background of the uninfected media control. 
       Example 3 
     Detection of  Mycoplasma  Using Proteases 
       [0057]    The proteases pepA, lon, and map were evaluated for use in the detection of  Mycoplasma . pepA and lon were found to be expressed at a higher level than map based on RT-qPCR results ( FIG. 5  and  FIG. 6 ). Arginine amino peptidase activity has also been reported in  Mycoplasma  species. 
         [0058]    A presently preferred substrate is leu-MCA that had significant activity above the uninfected media control under the gentle conditions used to lyse the  Mycoplasma  (0.05% HECAMEG, 1 mM MgCl 2 , 100 mM NaCl, 40 mM Tris buffer, pH 8.5). The MCA-Leu substrate produces a signal level of 500 mOD in 30 minutes with  M. hyorhinis , while  M. hyorhinis  has weak activity for arg-MCA. Results are provided in Table 6, below. 
         [0000]                                                                                  TABLE 6                   Vmax Measured Using MCA Labeled Substrates       Under Different Conditions                Vmax            Condition   Arg-MCA   Leu-MCA   Met-MCA   Leu/Met   All 3                    Buffer   0   236   125   0   193       Medium   676   2118   1731   2024   1443       Uninfected   29034   17422   19004   14937   25770       Medium       Infected   60903   415190   302082   373456   271170       Medium       Signal/Noise   2.10   23.83   15.90   25.00   10.52                    
In further studies, we determined that the background of the uninfected cells could be reduced even further by adjusting the pH, with an optimum at pH 8.5.  FIG. 13  is a graph showing the effect of acid pH levels on the leu-MCA assay.  FIG. 14  is a graph showing the effect of basic pH levels on the leu-MCA assay.
 
         [0059]    Detergent lysis of  M. hyorhinis  with HECAMEG gives better signal than sonication. Manganese or magnesium also improves the signal to noise ratio. In the studies on the effects of divalent cations, 1 mM MgCl 2  of the standard mixture was replaced by 1 mM MnCl 2 , 1 mM MgSO 4  or 1 mM EDTA, as indicated in Table 7, below. 
         [0000]                                                                TABLE 7                   Effect of Divalent Cations       Vmax, MCA-Leu Substrate            Condition   EDTA   MnCl 2     MgSO 4     MnCl 2                      Buffer   0   0   483   84       Medium   1710   1703   —   —       Uninfected   3873   3819   5125   3032       Infected   45241   89467   90365   95120       Signal/Noise   11.68   23.43   17.63   31.37                    
Using the leu-MCA substrate, a sensitivity of 10 5  CFU/ml can be achieved ( FIG. 15 ). Further increases in sensitivity may be obtained using a bis-leu rhodamine 110 labeled substrate, or using luciferase-leucine-bead complex, as shown schematically in  FIG. 10B .
 
         [0060]    The sensitivity of the present assay using the leu-MCA substrate was compared to two commercially available  Mycoplasma  detection tests: a  Mycoplasma PCR ELISA test (Roche cat # 11 663 925 910), and the MycoAlert Sample Kit, (Lonza cat #LT37-618). The results are presented in Table 8, below. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 8 
               
             
             
               
                   
               
               
                 Test Sensitivity Comparison 
               
             
          
           
               
                   
                 Present leu-MCA 
                 PCR ELISA 
                 MycoAlert 
               
               
                   
                 Assay 
                 test 
                 Sample Kit 
               
               
                   
                   
               
             
          
           
               
                 
                   M. hyorhinis 
                 
                 10 5   
                 80 
                 10 6   
               
               
                 (CFU/ml) 
               
               
                 Time Required 
                 20 minutes 
                 2 Days 
                 20 minutes 
               
               
                 For Test 
               
               
                   
               
             
          
         
       
     
         [0061]    The cross reactivity of the present assay using the leu-MCA substrate was evaluated with the following microorganisms: two bacteria ( S. aureus, E. coli ) and three species of fungus ( Candida albicans, Aspergillis niger, Saccharomyces cerevisiae ). Only in the case of a completely turbid cultures was there weak low cross reactivity with the present assay using the leu-MCA substrate.