Nucleic acid primers and probes for detecting Mycoplasma pneumoniae

FIELD OF THE INVENTION
The present invention relates to Mycoplasma pneumoniae and, in particular, it relates to oligonucleotides for detecting Mycoplasma pneumoniae in a test sample.
BACKGROUND OF THE INVENTION
Bacteria known as Mycoplasmas contain minimum intracellular organelles and are therefore some of the most simple and smallest bacteria. At least one member of the genus Mycoplasma is clinically important because of its ability to cause disease. In humans, Mycoplasma pneumoniae (M. pneumoniae) is responsible for atypical pneumonia and the symptoms of atypical pneumoniae can range from a subclinical infection to bronchopneumonia. Thus, diagnosing a M. pneumoniae infection based upon symptoms alone would be impractical given the range of symptoms that a patient infected with M. pneumoniae might display.
Currently, diagnosing a M. pneumoniae infection typically requires growing the organism in culture or detecting the presence of antibody against M. pneumoniae in a serum sample. M. pneumoniae is a slow growing organism and therefore, culture based methods for detecting M. pneumoniae may take several weeks before a result is obtained. Serological testing requires two samples from an individual suspected of being infected with M. pneumoniae because merely detecting antibody directed against M. pneumoniae is not necessarily diagnostic of a recent M. pneumoniae infection, but a significant increase in antibody titer may indicate a recent M. pneumoniae infection. Since a rise in antibody titer over time is measured, acute and convalescent serum samples are taken, but these samples are often times taken weeks apart. Hence, detecting a M. pneumoniae infection can be a time consuming process. Accordingly, there is a need for methods and reagents capable of detecting M. pneumoniae in a specific and timely manner.
SUMMARY OF THE INVENTION
The present invention provides nucleic acid sequences that can be used to specifically detect M. pneumoniae by using these sequences as probes and/or primers. Such primers or probes are designated SEQ ID NO 2, SEQ ID NO3, SEQ ID NO4, SEQ ID NO5, SEQ ID NO6, SEQ ID NO7, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, and SEQ ID NO 26. Those skilled in the art will recognize that homologs of these sequences and combinations of these sequences can also be employed to detect M. pneumoniae in a test sample. Preferably, the sequences are employed in amplification reactions and can be provided in kits along with other reagents for performing an amplification reaction.
Methods provided by the present invention include hybridization assays as well as amplification based assays. Thus, according to one method, a method of detecting the presence of M. pneumoniae in a test sample may comprise the steps of (a) contacting the test sample with one or more of the sequences listed above, or their homologs; and (b) detecting hybridization between the above sequences and a M. pneumoniae target sequence as an indication of the presence of M. pneumoniae in the test sample.
According to another embodiment, a method for detecting the presence of M. pneumoniae in a test sample may comprise the steps of (a) forming a reaction mixture comprising nucleic acid amplification reagents, a test sample containing a M. pneumoniae target sequence, and at least one primer and one probe oligonucleotide selected from the group consisting of SEQ ID NOs. 2 and 5; SEQ ID NOs. 3 and 4; SEQ ID NOs. 2, 3 and 4; SEQ ID NOs. 2, 3 and 5; SEQ ID NOs. 2, 3, 4 and 5; SEQ ID NOs. 10 and 12; SEQ ID NOs. 10 and 13; SEQ ID NOs. 10 and 14; SEQ ID NOs. 10, 11 and 12; SEQ ID NOs. 10, 11 and 13; SEQ ID NOs. 10, 11 and 14; SEQ ID NOs. 10, 11, 12, and 13; SEQ ID NOs. 10, 11, 12, 13 and 14; SEQ ID NOs. 11 and 17; SEQ ID NOs. 11 and 18; SEQ ID NOs. 11 and 19; SEQ ID NOs. 10, 11 and 17; SEQ ID NOs. 10, 11 and 18; SEQ ID NOs. 10, 11 and 19; SEQ ID NOs. 10, 11, 17, and 18; SEQ ID NOs. 10, 11, 17, 18 and 19; SEQ ID NOs. 21 and 23; SEQ ID NOs. 22 and 26; SEQ ID NOs. 21, 22 and 23; SEQ ID NOs. 21, 22 and 26; and SEQ ID NOs. 21, 22, 23 and 26. (b) subjecting the mixture to hybridization conditions to generate at least one nucleic acid sequence complementary to the target sequence; (c) hybridizing the probe to the nucleic acid sequence complementary to the target sequence, so as to form a complex comprising the probe and the complementary nucleic acid sequence; and (d) detecting the so-formed complex as an indication of the presence of M pneumoniae in the sample.
According to another embodiment, the invention provides kits which comprise a set of oligonucleotide primers and probes, selected from the SEQ ID NOs. listed above, and amplification reagents.
DETAILED DESCRIPTION OF THE INVENTION
As previously mentioned, the present invention provides nucleic acid sequences, methods for using these sequences and kits containing these sequences, all of which can be employed to specifically detect M. pneumoniae. The sequences provided are designated herein as SEQ ID NOs. 2, 3, 4, 5, 6, 7, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 22, 23, 24, 25, 26 and homologs thereof. These sequences are derived from a M. pneumoniae gene encoding a proline rich 65 kDa protein (P65) disclosed in Proft, T., et. al., Journal of Bacteriology, 177(12) p. 3370-3378 (1995); a repeat region designated SDC1 disclosed in Colman, S. D., et. al., Gene, 87 p. 91-96 (1990) and a repeat region designated REP 5 disclosed in Ruland, K., et. al., Journal of Bacteriology, 176(17) p. 5202-5209 (1994).
With respect to the sequences herein provided, the term "homologs" means those sequences sharing about 80% homology with SEQ ID NO2, SEQ ID NO3, SEQ ID NO4, SEQ ID NO5, SEQ ID NO6, SEQ ID NO7, SEQ ID NO10, SEQ ID NO11, SEQ ID NO12, SEQ ID NO13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, and SEQ ID NO 26, and more preferably those sequences that share about 90% homology with SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, and SEQ ID NO 26. Thus, sequences that share about 80% homology with the sequences provided herein and specifically hybridize with M. pneumoniae are intended to be within the scope of the present invention. For example, extensions of the present sequences, sequences that are shorter than the present sequences but contain a subset of the present sequences, and those sequences that deviate from the present sequences by minor base substitutions are contemplated as within the scope of the present invention.
Those skilled in the art will recognize various modifications that can be made to the sequences designated SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO4, SEQ ID NO5, SEQ ID NO6, SEQ ID NO7, SEQ ID NO10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO21, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, and SEQ ID NO 26 without departing from their ability to specifically detect M. pneumoniae and share about 80% homology with these sequences. For example, 3' or 5' extensions of the present sequences with bases that are complementary to succeeding or preceding bases in either of the repeat regions (SDC1 or REP5) or the P65 gene are considered to be homologs of the present sequences when they share about 80% homology with the present sequences and specifically detect M. pneumoniae. Additionally, 3' or 5' extensions of present sequences with bases that are not complementary to succeeding or preceding bases in either of the repeat regions or the P65 gene that share about 80% homology with the present sequences and specifically detect M. pneumoniae are contemplated as within the scope of the present invention. Further, base substitutions can be made to SEQ ID NO2, SEQ ID NO3, SEQ ID NO4, SEQ ID NO5, SEQ ID NO6, SEQ ID NO 7, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO21, SEQ ID NO22, SEQ ID NO23, SEQ ID NO24, SEQ ID NO 25, and SEQ ID NO 26, but these modified sequences will nevertheless maintain the ability to specifically hybridize with M. pneumoniae and share about 80% homology with the sequences provided herein. Sequences that are so-modified, are therefore contemplated as within the scope of the present invention. Moreover, sequences that contain about 80% of the sequences designated SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, and SEQ ID NO 26 but have bases deleted from the 3' or 5' end, are considered to be within the scope of the term homolog.
The sequences disclosed herein, as well as their homologs, may comprise deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or nucleic acid analogs such as uncharged nucleic acid analogs including but not limited to peptide nucleic acids (PNAs) which are disclosed in International Patent Application WO 92/20702 or morpholino analogs which are described in U.S. Pat. Nos. 5,185,444, 5,034,506, and 5,142,047 all of which are herein incorporated by reference. Such sequences can routinely be synthesized using a variety of techniques currently available. For example, a sequence of DNA can be synthesized using conventional nucleotide phosphoramidite chemistry and the instruments available from Applied Biosystems, Inc, (Foster City, Calif.); DuPont, (Wilmington, Del.); or Milligen, (Bedford, Mass.). Similarly, and when desirable, the sequences can be labeled using methodologies well known in the art such as described in U.S. Pat. Nos. 5,464,746; 5,424,414; and 4,948,882 all of which are herein incorporated by reference.
The term "label" as used herein means a molecule or moiety having a property or characteristic which is capable of detection. A label can be directly detectable, as with, for example, radioisotopes, fluorophores, chemiluminophores, enzymes, colloidal particles, fluorescent microparticles and the like; or a label may be indirectly detectable, as with, for example, specific binding members. It will be understood that directly detectable labels may require additional components such as, for example, substrates, triggering reagents, light, and the like to enable detection of the label. When indirectly detectable labels are used, they are typically used in combination with a "conjugate". A conjugate is typically a specific binding member which has been attached or coupled to a directly detectable label. Coupling chemistries for synthesizing a conjugate are well known in the art and can include, for example, any chemical means and/or physical means that does not destroy the specific binding property of the specific binding member or the detectable property of the label. As used herein, "specific binding member" means a member of a binding pair, i.e., two different molecules where one of the molecules through, for example, chemical or physical means specifically binds to the other molecule. In addition to antigen and antibody specific binding pairs, other specific binding pairs include, but are not intended to be limited to, avidin and biotin; haptens and antibodies specific for haptens; complementary nucleotide sequences; enzyme cofactors or substrates and enzymes; and the like.
Generally, the sequences provided herein can be employed to detect the presence of M. pneumoniae in a test sample by contacting a test sample with at least one of the sequences provided herein under hybridizing conditions and detecting hybridization between the M. pneumoniae target sequence and at least one of the sequences designated herein as SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO6, SEQ ID NO7, SEQ ID NO10, SEQ ID NO11, SEQ ID NO12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, and SEQ ID NO 26. Several well known methods for detecting hybridization can be employed according to the present invention and may include, for example, the use of gels and stains or detecting a label associated with one or more of the sequences provided herein after performing, for example, a dot blot or amplification reaction.
The term "test sample" as used herein, means anything suspected of containing the target sequence. The test sample can be derived from any biological source, such as for example, blood, bronchial alveolar lavage, saliva, throat swabs, ocular lens fluid, cerebral spinal fluid, sweat, sputa, urine, milk, ascites fluid, mucous, synovial fluid, peritoneal fluid, amniotic fluid, tissues such as heart tissue and the like, or fermentation broths, cell cultures, chemical reaction mixtures and the like. The test sample can be used (i) directly as obtained from the source or (ii) following a pre-treatment to modify the character of the sample. Thus, the test sample can be pre-treated prior to use by, for example, preparing plasma from blood, disrupting cells, preparing liquids from solid materials, diluting viscous fluids, filtering liquids, distilling liquids, concentrating liquids, inactivating interfering components, adding reagents, purifying nucleic acids, and the like.
A "target sequence" as used herein means a nucleic acid sequence that is detected, amplified, both amplified and detected or otherwise is complementary to one of the sequences herein provided. Thus, a target sequence generally will be at least about 80% complementary with the sequences provided herein. Additionally, while the term target sequence is sometimes referred to as single stranded, those skilled in the art will recognize that the target sequence may actually be double stranded.
"Hybridization" or "hybridizing" conditions are defined generally as conditions which promote annealing between complementary nucleic acid sequences or annealing and extension of one or more nucleic acid sequences. It is well known in the art that such annealing is dependent in a rather predictable manner on several parameters, including temperature, ionic strength, sequence length, complementarity, and G:C content of the sequences. For example, lowering the temperature in the environment of complementary nucleic acid sequences promotes annealing. For any given set of sequences, melt temperature, or Tm, can be estimated by any of several known methods. Typically, diagnostic applications utilize hybridization temperatures which are close to (i.e. within 10.degree. C.) the melt temperature. Ionic strength or "salt" concentration also impacts the melt temperature, since small cations tend to stabilize the formation of duplexes by negating the negative charge on the phosphodiester backbone. Typical salt concentrations depend on the nature and valency of the cation but are readily understood by those skilled in the art. Similarly, high G:C content and increased sequence length are also known to stabilize duplex formation because G:C pairings involve 3 hydrogen bonds where A:T pairs have just two, and because longer sequences have more hydrogen bonds holding the sequences together. Thus, a high G:C content and longer sequence lengths impact the hybridization conditions by elevating the melt temperature.
Once sequences are selected for a given diagnostic application, the G:C content and length will be known and can be accounted for in determining precisely what hybridization conditions will encompass. Since ionic strength is typically optimized for enzymatic activity, the only parameter left to vary is the temperature. Generally, the hybridization temperature is selected close to or at the Tm of the primers or probe. Thus, obtaining suitable hybridization conditions for a particular primer, probe, or primer and probe set is well within ordinary skill of one practicing this art.
The sequences provided herein also can be used as amplification primers or probes according to amplification procedures well known in the art. Such reactions include, but are not intended to be limited to, the polymerase chain reaction (PCR) described in U.S. Pat. Nos. 4,683,195 and 4,683,202, the ligase chain reaction (LCR) described in EP-A-320 308, and gap LCR (GLCR) described in U.S. Pat. No. 5,427,930 all of which are herein incorporated by reference.
According to a preferred embodiment, the sequences are employed in the "oligonucleotide hybridization PCR" (variably referred to herein as "OH PCR") amplification reaction as described in U.S. patent application Ser. No. 08/514,704, filed Aug. 14, 1995, that is herein incorporated by reference. Briefly, the reagents employed in the preferred method comprise at least one amplification primer and at least one internal hybridization probe, as well as other reagents for performing an amplification reaction.
The primer sequence is employed to prime extension of a copy of a target sequence (or its complement) and is labeled with either a capture label or a detection label. The probe sequence is used to hybridize with the sequence generated by the primer sequence, and typically hybridizes with a sequence that does not include the primer sequence. Similarly to the primer sequence, the probe sequence is also labeled with either a capture label or a detection label with the caveat that when the primer is labeled with a capture label the probe is labeled with a detection label and vice versa. Detection labels have the same definition as "labels" previously defined and "capture labels" are typically used to separate extension products, and probes associated with any such products, from other amplification reactants. Specific binding members (as previously defined) are well suited for this purpose. Also, probes used according to this method are preferably blocked at their 3' ends so that they are not extended under hybridization conditions. Methods for preventing extension of a probe are well known and are a matter of choice for one skilled in the art. Typically, adding a phosphate group to the 3' end of the probe will suffice for purposes of blocking extension of the probe.
"Other reagents for performing an amplification reaction" or "nucleic acid amplification reagents" include reagents which are well known and may include, but are not limited to, an enzyme having polymerase activity, enzyme cofactors such as magnesium; salts; nicotinamide adenine dinucleotide (NAD); and deoxynucleotide triphosphates (dNTPs) such as for example deoxyadenine triphosphate, deoxyguanine triphosphate, deoxycytosine triphosphate and deoxythymine triphosphate.
The preferred method generally comprises the steps of (a) forming a reaction mixture comprising nucleic acid amplification reagents, at least one hybridization probe, at least one amplification primer and a test sample suspected of containing a target sequence; (b) subjecting the mixture to hybridization conditions to generate at least one copy of a nucleic acid sequence complementary to the target sequence; (c) hybridizing the probe to the nucleic acid sequence complementary to the target sequence, so as to form a hybrid comprising the probe and the nucleic acid sequence complementary to the target sequence; and (d) detecting the hybrid as an indication of the presence of M. pneumoniae in the sample. It will be understood that step (b) of the above method can be repeated several times prior to step (c) by thermal cycling the reaction mixture as is well known in the art.
According to the above method, it is preferable to select primers, probes and reaction conditions such that the probe sequence has a lower melt temperature than the primer sequences so that upon placing the reaction mixture under hybridization conditions copies of the target sequence or its complement are produced at temperature above the Tm of the probe. After such copies are synthesized, they are denatured and the mixture is cooled to enable the formation of hybrids between the probes and any copies of the target or its complement. The rate of temperature reduction from the denaturation temperature down to a temperature at which the probes will bind to single stranded copies is preferably quite rapid (for example 8 to 15 minutes) and particularly through the temperature range in which an enzyme having polymerase activity is active for primer extension. Such a rapid cooling favors copy sequence/probe hybridization rather that primer/copy sequence hybridization and extension.
Upon formation of the copy sequence/probe hybrids, the differential labels (i.e. capture and detection labels) on the copy sequence and probe sequence can be used to separate and detect such hybrids. Preferably, detection is performed according to the protocols used by the commercially available Abbott LCx.RTM. instrumentation Abbott Laboratories; Abbott Park, Ill.).
Thus, keeping the preferred method in mind, the sequences of the present invention are preferably provided in groups of at least two different sequences (i.e. at least one primer sequence and at least one probe sequence complementary to the extension product of the primer). Hence, SEQ ID NOs. 2 and 5; SEQ ID NOs. 3 and 4; SEQ ID NOs. 2, 3 and 4; SEQ ID NOs. 2, 3 and 5; SEQ ID NOs. 2, 3, 4 and 5; SEQ ID NOs. 10 and 12; SEQ ID NOs. 10 and 13; SEQ ID NOs. 10 and 14; SEQ ID NOs. 10, 11 and 12; SEQ ID NOs. 10, 11 and 13; SEQ ID NOs. 10, 11 and 14; SEQ ID NOs. 10, 11, 12, and 13; SEQ ID NOs. 10, 11, 12, 13 and 14; SEQ ID Nos. 11 and 17; SEQ ID NOs. 11 and 18; SEQ ID NOs. 11 and 19; SEQ ID NOs. 10, 11 and 17; SEQ ID NOs. 10, 11 and 18; SEQ ID NOs. 10, 11 and 19; SEQ ID NOs. 10, 11, 17, and 18; SEQ ID NOs. 10, 11, 17, 18 and 19; SEQ ID NOs. 21 and 23; SEQ ID NOs. 22 and 26; SEQ ID NOs. 21, 22 and 23; SEQ ID NOs. 21, 22 and 26; and SEQ ID NOs. ,21, 22, 23 and 26; or homologs of these sequences are preferably provided together.
The sequences of the present invention can be provided as part of a kit useful for detecting M. pneumoniae. The kits comprise one or more suitable containers containing one or more sequences according to the present invention, an enzyme having polymerase activity, and deoxynucleotide triphosphates. Typically, at least one sequence bears a label, but detection is possible without this.
The following examples are provided to further illustrate the present invention and not intended to limit the invention.

EXAMPLES
The following examples demonstrate M. pneumoniae detection using the DNA oligomer primers and probes herein provided. These DNA primers and probes are identified as SEQUENCE ID NO. 2, SEQUENCE ID NO. 3, SEQUENCE ID NO. 4, SEQUENCE ID NO. 5, SEQUENCE ID NO. 10, SEQUENCE ID NO. 11, SEQUENCE ID NO. 12, SEQUENCE ID NO. 13, SEQUENCE ID NO. 14, SEQUENCE ID NO. 21, SEQUENCE ID NO. 22, and SEQUENCE ID NO. 23. SEQUENCE ID NOs. 2, 3, 4 and 5 are specific for the multicopy repetitive element, Rep 5, region in the ORF6 gene of M. pneumoniae. A portion of the Rep 5 sequence is designated as SEQ ID NO. 1. SEQUENCE ID NOs. 10, 11, 12, 13 and 14 are specific for the multicopy repetitive SDC1 element of M. pneumoniae. Regions of the SDC1 repeat element from M. pneumoniae strains M129 and FH are designated herein as SEQ ID NO. 8 and SEQ ID NO. 9 respectively. SEQUENCE ID NOs. 21, 22 and 23 are specific for the gene encoding the proline-rich p65 protein of M. pneumoniae. A portion of this gene is designated herein as SEQ ID NO. 20. In the following examples, SEQUENCE ID NO. 2 and SEQUENCE ID NO. 3 are used as M. pneumoniae amplification primers specific for the Rep 5 region. SEQUENCE ID NO. 4 and SEQUENCE ID NO. 5 are used as internal hybridization probes for the Rep 5 amplification product. SEQUENCE ID NO 10 and SEQUENCE ID NO 11 are used as M. pneumoniae amplification primers specific for the SDC1 region and SEQUENCE ID NO 12, 13 and 14 are used as internal hybridization probes for the SDC1 amplification product. SEQUENCE ID NO. 21 and SEQUENCE ID NO. 22 are used as M. pneumoniae amplification primers specific for the p65 region and SEQUENCE ID NO. 23 is used as an internal hybridization probe for the p65 amplification product.
Example 1
Preparation of M. pneumoniae Primers and Probes
A. Rep 5 Primers and Probes Target-specific primers and probes were designed to detect the M. pneumoniae Rep 5 target sequence by oligonucleotide hybridization PCR. These primers were SEQUENCE ID NO. 2 and SEQUENCE ID NO. 3. Primer sequences were synthesized using standard oligonucleotide synthesis methodology and haptenated with adamantane at their 5' ends using standard cyanoethyl phosphoramidite coupling chemistry as described in U.S. Pat. No. 5,424,414 incorporated herein by reference.
The detection probes were designed to hybridize with the amplified M. pneumoniae Rep 5 target sequence by oligonucleotide hybridization. These probes are SEQUENCE ID NO 4 and SEQUENCE ID NO 5. The probe sequences were synthesized using standard oligonucleotide synthesis methodology and haptenated with 2 carbazoles at the 5' end using standard cyanoethyl phosphoramidite coupling chemistry as described in U.S. Pat. No. 5,464,746 (herein incorporated by reference), and blocked with phosphate at the 3' end. Reactivity was assessed against a known standard of M. pneumoniae DNA.
B. SDC1 Primers and Probes Target-specific primers and probes were designed to detect the M. pneumoniae SDC1 target sequence by oligonucleotide hybridization PCR. These primers were SEQUENCE ID NO. 10 and SEQUENCE ID NO.11. Primer sequences were synthesized using standard oligonucleotide synthesis methodology and haptenated with adamantane at their 5' ends using standard cyanoethyl phosphoramidite coupling chemistry (as above).
The detection probes were designed to hybridize with the amplified M. pneumoniae SDC1 target sequence by oligonucleotide hybridization. These probes are SEQUENCE ID NOs. 12, 13 and 14. The probe sequences were synthesized using standard oligonucleotide synthesis methodology and haptenated with 2 carbazoles at the 5' end using standard cyanoethyl phosphoramidite coupling chemistry (as above), and blocked with phosphate at the 3' end. Reactivity was assessed against a known standard of M. pneumoniae DNA.
C. p65 Primers and Probe Target-specific primers and probes were designed to detect the M. pneumoniae p65 target sequence by oligonucleotide hybridization PCR. These primers were SEQUENCE ID NO. 21 and SEQUENCE ID NO. 22. Primer sequences were synthesized using standard oligonucleotide synthesis methodology and haptenated with adamantane at their 5' ends using standard cyanoethyl phosphoramidite coupling chemistry (as above).
The detection probe was designed to hybridize with the amplified M. pneumoniae p65 target sequence by oligonucleotide hybridization. This probe is SEQUENCE ID NO. 23. The probe sequence was synthesized using standard oligonucleotide synthesis methodology and haptenated with 2 carbazoles at the 5' end using standard cyanoethyl phosphoramidite coupling chemistry and blocked with phosphate at the 3' end (as above). Reactivity was assessed against a known standard of M. pneumoniae DNA.
Example 2
Sensitivity of M. pneumoniae Detection
The M. pneumoniae standards used were obtained from American Type Culture Collection, Rockville, Md.; the FH strain of M. pneumoniae was from ATCC #15531 and the M129 strain of M. pneumoniae was from ATCC #29342. ATCC #15293 (M. pneumoniae, strain not indicated) was used in instances where knowledge of the particular strain was not necessary.
DNA was extracted and purified from the cell cultures by lysing the cells with detergent, digesting RNA and proteins with RNase and Proteinase K respectively, heating to inactivate, then isolating DNA using phenol/chloroform extraction and ethanol precipitation. DNA was quantitated by taking an absorbance reading at 260 nm using a spectrophotometer.
A. M. pneumoniae Rep 5 detection Dilutions of the DNA purified from the M. pneumoniae standard cell lines were PCR amplified and detected using the REP 5 primers (SEQ ID NOs. 2 and 3) with each of the REP 5 detection probes (SEQ ID NO. 4 or SEQ ID NO. 5) described in Example 1. The detection probes were used in separate reactions to detect the amplification products produced by the REP 5 primers. Taq polymerase was used at a concentration of 2.5 units and the final concentration of the nucleotides was 0.2 mM each in a total reaction volume of 0.2 ml. PCR extension was performed using 10.times. PCR buffer (Perkin Elmer, Foster City, Calif.) which consists of 100 mM Tris-HCl, pH 8.3, 500 mM KCl, at a final concentration of 1.times.. The reaction mixtures containing 5 nM of SEQ ID NO. 4 used primers at a concentration of 0.2 .mu.M each, with a final concentration of 1.5 mM MgCl.sub.2. The reaction mixtures containing 10 nM of SEQ ID NO. 5 used primers at a concentration on 0.5 .mu.M each, with a final concentration of 1.0 mM MgCl.sub.2. Testing was done in triplicate with calf thymus DNA as a negative control.
Reaction mixtures were amplified in a Perkin-Elmer 480 Thermal Cycler. For reaction mixtures employing SEQ ID NO. 4 as a probe, the following cycling conditions were used: 95.degree. C. for 5 minutes followed by cycling at 95.degree. C. for 1 minute/68.degree. C. for 1 minute/72.degree. C. for 1 minute for 40 cycles. For reaction mixtures employing SEQ ID NO. 5 as a probe, the following cycling conditions were used: 95.degree. C. for 5 minutes followed by cycling at 95.degree. C. for 1 minute/65.degree. C. for 1 minute/72.degree. C. for 1 minute for 40 cycles. After the reaction mixtures were thermal cycled, the mixtures were maintained at 97.degree. C. for 5 minutes and probe oligo hybridization was accomplished by lowering the temperature to 15.degree. C. Following probe hybridization, samples were either assayed immediately or held at 2.degree. C.-25.degree. C., for up to 24 hours before being tested.
Reaction products were detected on the Abbott LCx.RTM. system (available from Abbott Laboratories, Abbott Park, Ill.). A suspension of anti-carbazole antibody coated microparticles and an anti-adamantane antibody/alkaline phosphatase conjugate (all of which are commercially available from Abbott Laboratories, Abbott Park, Ill.) were used in conjunction with the LCx.RTM. to capture and detect the reaction products. The average values from this experiment (calculated as counts/second/second; c/s/s) are presented in TABLE 1 and show detection of both strains of M. pneumoniae by both probe sequences at concentrations as low as 1 molecule of M. pneumoniae genomic DNA.
TABLE 1______________________________________ Molecules of M. Probe SEQ Probe SEQ pneumoniae ID NO 4 ID NO 5 Genomic LCx .RTM. rate LCx .RTM. rateSample DNA (c/s/s) (c/s/s)______________________________________M. pneumoniae, FH 100 1285 1379" 10 1149 1321" 1 708 1099Negative Control 0 16 20M. pneumoniae, M129 1000 1370 1448" 100 1358 1382" 10 1175 1238" 1 1072 1110Negative Control 0 18 18______________________________________
Additional testing was performed comparing dilutions of M. pneumoniae strain M129 in either human placental DNA (tested in triplicate) or calf thymus DNA (tested in duplicate). Oligonucleotide hybridization PCR with SEQ ID NOs. 2, 3, 4, and 5 was performed with the individual probes as above. The results, shown as averages in Table 2, again indicate detection of M. pneumoniae at concentrations as low as 1 molecule M. pneumoniae genomic DNA.
TABLE 2______________________________________ Probe SEQ ID Probe SEQMolecules of M. NO 4 ID NO 5pneumoniae Background LCx .RTM. rate LCx .RTM. rateGenomic DNA DNA (c/s/s) (c/s/s)______________________________________1000 Human placental 1468 1430100 " 1498 140110 " 1531 11451 " 1254 6110 " 20 201000 Calf Thymus 1486 1486100 " 1500 143410 " 1541 13121 " 1167 10000 " 21 21______________________________________
B. M. pneumoniae SDC1 detection Dilutions of the DNA purified from the FH strain of the M. pneumoniae standard cell line were PCR amplified and detected using the SDC1 primers (SEQ ID NOs. 10 and 11) with each of the SDC1 detection probes (SEQ ID NOs. 12, 13 or 14) in separate reactions. Testing was done in duplicate. Taq polymerase was used at a concentration of 2.5 units and the final concentration of the nucleotides was 0.2 mM each in a total reaction volume of 0.2 ml. PCR extension was performed using 10.times. PCR buffer (Perkin Elmer, Foster City, Calif.) which consists of 100 mM Tris-HCl, pH 8.3, 500 mM KCl, at a final concentration of 1.times.. The primers were used at a concentration of 0.25 .mu.M each, and probes were used at a concentration of 10 nM. The final concentration of MgCl.sub.2 was 1 mM.
Reaction mixtures were amplified in a Perkin-Elmer 480 Thermal Cycler under the following cycling conditions: 95.degree. C. for 5 minutes followed by cycling at 95.degree. C. for 1 minute/65.degree. C. for 1 minute/72.degree. C. for 1 minute for 40 cycles. After maintaining the reaction mixture at 97.degree. C. for 5 minutes, probe oligo hybridization was accomplished by lowering the temperature to 15.degree. C. Following probe hybridization, samples were either assayed immediately or held at 2.degree. C.-25.degree. C. for up to 24 hours before being tested.
Reaction products were detected on the Abbott LCx.RTM. system as above in Example 2.A. Results, shown in Table 3 as averages of the duplicate values indicate detection of M. pneumoniae by all 3 probe sequences at concentrations as low as 1 molecule of M. pneumoniae genomic DNA.
TABLE 3______________________________________ Probe SEQ ID Probe SEQ ID Probe SEQ IDMolecules of M. NO 12 NO 13 NO 14pneumoniae LCx .RTM. rate LCx .RTM. rate LCx .RTM. rateDNA (c/s/s) (c/s/s) (c/s/s)______________________________________100 1807 1656 1181 10 1638 1485 656 1 495 619 1470 (Neg. Control) 96 52 32______________________________________
Dilutions of both strains of M. pneumoniae DNA were then tested using the SEQ ID NO 12 probe only. In this experiment primers were used at a concentration of 0.35 .mu.M each, probes were used at a concentration of 12.5 nM and the final concentration of MgCl.sub.2 was 1.5 mM. Samples were tested in replicates of four, and calf thymus DNA was used as a negative control (NC).
Reaction mixtures were amplified in a Perkin-Elmer 480 Thermal Cycler under the following cycling conditions: 95.degree. C. for 2 minutes followed by cycling at 95.degree. C. for 1 minute/67.degree. C. for 1 minute/72.degree. C. for 1 minute for 40 cycles. Probe hybridization and detection was accomplished on the Abbott LCx.RTM. system as in Example 2.A. The average values from this experiment are presented in TABLE 4 and show detection of both strains of M. pneumoniae at concentrations as low as 1 molecule of M. pneumoniae genomic DNA.
TABLE 4______________________________________ Probe SEQ ID NO Molecules of M. 12Sample pneumoniae DNA LCx .RTM. rate (c/s/s)______________________________________M. pneumoniae, M129 1000 1795" 100 1792" 10 1568" 1 982M. pneumoniae, FH 1 892calf thymus DNA NC 0 18______________________________________
C. M. pneumoniae detection with p65 primers and probes. Dilutions of both strains of M. pneumoniae DNA were PCR amplified and detected using the p65 primers (SEQ ID NOs. 21 and 22) and the p65 detection probe (SEQ ID NO. 23) described in Example 1. Taq polymerase was used at a concentration of 2.5 units and the final concentration of the nucleotides was 0.2 mM each in a total reaction volume of 0.2 ml. PCR extension was performed using 10.times. PCR buffer (Perkin Elmer, Foster City, Calif.) which consists of 100 mM Tris-HCl, pH 8.3, 500 mM KCl, at a final concentration of 1.times.. Primers were used at a concentration of 0.35 .mu.M each, probes were used at a concentration of 12.5 nM and the final concentration of MgCl.sub.2 was 1.5 mM. Samples were tested in replicates of four, and calf thymus DNA was used as a negative control (NC).
Reaction mixtures were amplified in a Perkin-Elmer 480 Thermal Cycler under the following cycling conditions: 95.degree. C. for 5 minutes followed by cycling at 95.degree. C. for 1 minute/67.degree. C. for 1 minute/72.degree. C. for 1 minute for 40 cycles. After maintaining the reaction mixture at 97.degree. C. for 5 minutes, probe oligo hybridization was accomplished by lowering the temperature to 15.degree. C. Following probe hybridization, samples were either assayed immediately or held at 2.degree. C.-25.degree. C. for up to 24 hours before being tested.
Reaction products were detected on the Abbott LCx.RTM. system as in Example 2.A. Results, with averages shown in Table 5, show detection of both strains of M. pneumoniae at concentrations as low as 10 molecules of M. pneumoniae genomic DNA.
TABLE 5______________________________________ Molecules of M. p65Sample pneumoniae DNA LCx .RTM. rate (c/s/s)______________________________________M. pneumoniae, M129 1000 1555" 100 1486" 10 1323M. pneumoniae, FH 100 1545" 10 1233calf thymus DNA (NC) 0 17______________________________________
Additional testing was performed with both strains in triplicate at concentrations below 20 molecules of DNA. Calf thymus DNA was used as the negative control (NC) and tested in quadruplicate. The results shown in Table 6 indicate detection of M. pneumoniae at concentrations as low as 5 molecules of M. pneumoniae genomic DNA.
TABLE 6______________________________________ Molecules of M. p65Sample pneumoniae DNA LCx .RTM. rate (c/s/s)______________________________________M. pneumoniae, M129 10 1423" 5 1435" 1 25*M. pneumoniae, FH 10 1527" 5 1200*" 1 25*calf thymus DNA (NC) 0 34______________________________________ *One aberrant value not included in calculation of average.
Example 3
Specificity of M. pneumoniae Detection
Three other members of the genus Mycoplasma, M. genitalium, M. orale and M. salivarium (ATCC #33530, 23714 and 23064 respectively) were purchased from Advanced Biotechnologies, Inc. (Columbia, Md.). Organisms were quantified by titration and plating out to determine the CFU titer (the number of colony forming units/ml). They were then lysed and diluted to 10.sup.3 to 10.sup.5 CFU/reaction. Alternatively DNA was extracted, purified and quantified as in Example 2., except that RNase digestion was done following DNA extraction and purification, with phenol/chloroform extraction and ethanol precipitation repeated after RNase digestion. Samples were then diluted to 10.sup.6 to 10.sup.8 molecules of DNA/reaction and assayed side by side with the purified M. pneumoniae DNA from Example 2., as described below.
A. Specific detection of M. pneumoniae using the REP 5 primers and probes The REP 5 primers (SEQ ID NO 2 and SEQ ID NO 3) and REP 5 detection probes (SEQ ID NO 4 or SEQUENCE ID NO 5) described in Example 1 were used to amplify and detect four samples from the genus Mycoplasma by the method previously described in Example 2.A. above for the SEQ ID NO. 5 probe. Samples were tested in duplicate with water as a negative control. The data from this experiment is presented in TABLE 7 and shows specific amplification and detection of M. pneumoniae only, with the 3 other Mycoplasma genus samples being non-reactive.
TABLE 7______________________________________ Probe SEQ ID Probe SEQ ID NO 4 NO 5 Concentration/ LCx .RTM. rate LCx .RTM. rateSample Reaction (c/s/s) (c/s/s)______________________________________Water (Neg. Ctl.) 15 19M. genitalium 10.sup.5 CFU 17 20" 10.sup.4 CFU 13 20" 10.sup.3 CFU 14 19M. orale 10.sup.5 CFU 14 20" 10.sup.4 CFU 14 19" 10.sup.3 CFU 13 18M. salivarium 10.sup.5 CFU 16 19" 10.sup.4 CFU 13 19" 10.sup.3 CFU 13 19M. pneumoniae 10.sup.4 mol of DNA 1491 1812M. genitalium 10.sup.8 mol of DNA 17 22" 10.sup.7 mol of DNA 16 19" 10.sup.6 mol of DNA 15 18M. orale 10.sup.8 mol of DNA 13 17" 10.sup.7 mol of DNA 47 17" 10.sup.6 mol of DNA 18 18M. salivarium 10.sup.8 mol of DNA 18 25" 10.sup.7 mol of DNA 14 19" 10.sup.6 mol of DNA 13 17M. pneumoniae 10.sup.3 mol of DNA 1463 1756______________________________________ (CFU = colony forming units; mol of DNA = molecules of DNA)
B. Specific detection of M. pneumoniae using SDC1 primers and probes The SDC1 primers (SEQ ID NO 10 and SEQ ID NO 11) and SDC1 detection probe (SEQ ID NO 14) described in Example 1 were used to amplify and detect four samples from the genus Mycoplasma by the method described in Example 2.B. Samples were tested in triplicate. The data from this experiment is presented in TABLE 8 and shows specific amplification and detection of M. pneumoniae only, with the 3 other Mycoplasma genus samples being non-reactive.
TABLE 8______________________________________ Molecules of Probe SEQ ID NO 14Sample Mycoplasma DNA LCx .RTM. rate (c/s/s)______________________________________Negative Control 0 33M. genitalium 10.sup.8 21M. orale 10.sup.8 30M. salivarium 10.sup.8 23M. pneumoniae, FH 10.sup.4 1082______________________________________
C. Specific detection of M. pneumoniae using p65 primers and probes The p65 primers (SEQ ID NO 21 and SEQ ID NO 22) and p65 detection probe (SEQ ID NO 23) described in Example 1 were used to amplify and detect four samples from the genus Mycoplasma by the method described in Example 2.C. except that the probe concentration was 14 nM. Samples were tested in triplicate. The data from this experiment is presented in TABLE 9 and shows specific amplification and detection of M. pneumoniae only, with the 3 other Mycoplasma genus samples being non-reactive.
TABLE 9______________________________________ Molecules of p65Sample Mycoplasma DNA LCx .RTM. rate (c/s/s)______________________________________Negative Control 0 21M. genitalium 10.sup.7 19M. orale 10.sup.7 20M. salivarium 10.sup.7 24M. pneumoniae, M129 5 1194" 10 1380" 100 1388" 1000 1426______________________________________
Example 4
Specificity of M. pneumoniae Rep 5 Primers and Probe
A panel of organisms (Table 10) which had been quantified by titration and plating out to determine the CFU titer (the number of colony forming units/ml) was obtained from the ATCC. DNA was isolated from the organisms in the panel using the QIAgen nucleic acid purification method (QIAgen, Inc., Chatsworth, Calif.) and amplified and detected using the REP 5 primers (SEQ ID NO 2 and SEQ ID NO 3) and REP 5 detection probe (SEQ ID NO 5) described in Example 1 by the Rep 5 method described for the SEQ ID NO 5 probe in Example 2.A. above, except that the final concentration of MgCl.sub.2 was 1.5 mM. Purified M. pneumoniae from the FH strain was used as a positive control and calf thymus DNA was used as a negative control (NC); all controls were tested in replicates of twelve. The data from this experiment is presented in TABLE 10 and shows specific amplification and detection of M. pneumoniae only, with all other organisms being non-reactive.
TABLE 10______________________________________ Rep 5 Concentration/ LCx .RTM. rateOrganism ATCC No. Reaction (c/s/s)______________________________________Nocardia asteroides 23825 8.9 .times. 10e5 CFU 24.7Nocardia brasiliensis 19296 2.1 .times. 10e7 CFU 21.2Nocardia 14629 7.7 .times. 10e6 CFU 24.9otitidiscaviarumCorynebacterium 15677 4.9 .times. 10e7 CFU 23.1kutscheriiDermatophilus 14637 2.0 .times. 10e6 CFU 25.1congolensisRhodococcus 4227 6.5 .times. 10e6 CFU 25.8erythropolisN. dassonvillei 21944 2.1 .times. 10e7 CFU 27.8subspecies dassonvilleiStreptococcus mitis 9811 3.7 .times. 10e6 CFU 22.4Streptococcus 6303 5.8 .times. 10e5 CFU 21.5pneumoniaeNocardia asteroides 19247 7.6 .times. 10e6 CFU 21.8Pseudomonas aeruginosa 142 3.8 .times. 10e6 CFU 23.3Candida albicans 18304 1.2 .times. 10e6 CFU 23.3Lactobacillus 25536 1.7 .times. 10e6 CFU 21.6catenaformisListeria monocytogenes 984 7.0 .times. 10e5 CFU 20.6Legionella micdadei 33204 1.5 .times. 10e5 CFU 24.1Streptomyces griseus 3343 1.9 .times. 10e6 CFU 22.7subspecies griseusCorynebacterium 19410 3.5 .times. 10e7 CFU 21.9pseudotuberculosisCorynebacterium 296 2.3 .times. 10e7 CFU 25.9diphtheriaeProprionibacterium 6921 5.4 .times. 10e6 CFU 24.2acnesAchromobacter sp. 14648 2.7 .times. 10e6 CFU 25.2Mycobacteria 15293 4.4 .times. 10e8 CFU 1535.5pneumoniaeB. thetaiotamicron 29148 5.7 .times. 10e6 CFU 25.8Actnomyces israeli 10048 2.1 .times. 10e6 CFU 21.4serotype 1Staphylococcus aureus 12598 7.8 .times. 10e7 CFU 26.0protein AAcinetobacter 14987 2.5 .times. 10e6 CFU 24.9calcoaceticus(A. baumanii)Clostridium perfringens 9081 2.3 .times. 10e6 CFU 22.8Enterococcus faecalis 376 9.0 .times. 10e6 CFU 23.3Salmonella cholerasuis 167 3.0 .times. 10e7 CFU 24.7serotype typhiEnterococcus faecium 349 7.6 .times. 10e6 CFU 23.1Streptococcus 624 3.2 .times. 10e7 CFU 23.7agalactiaeProvidencia stuartii 25825 1.7 .times. 10e7 CFU 30.3Stretococcus bovis 9809 1.3 .times. 10e7 CFU 21.9Citrobacter freundii 6750 2.8 .times. 10e7 CFU 29.8Aeromonas hydrophilia 7966 3.3 .times. 10e6 CFU 29.9Porphyromonas 25260 9.8 .times. 10e6 CFU 24.1asaccharolyticaVeillonella sp. 10790 6.5 .times. 10e6 CFU 23.9Peptostreptococcus 9321 1.1 .times. 10e6 CFU 23.5prevotiActinomyces israeli 10049 1.9 .times. 10e6 CFU 36.3Listena monocytogenes 13932 2.2 .times. 10e5 CFU 25.1Enterobacter aerogenes 13048 2.9 .times. 10e6 CFU 28.1Mobiluncus curtisii 35241 2.4 .times. 10e6 CFU 27.7subspecies curtisiiSerratia marcescens 60 2.8 .times. 10e5 CFU 31.0Xanthomonas maltophilia 12741 2.2 .times. 10e6 CFU 25.3(P. maltophilia)Streptomyces griseus 19437 1.5 .times. 10e6 CFU 22.4subspecies somaliensisPasteurella multocida 6529 5.4 .times. 10e6 CFU 24.2Corynebacterium 43734 1.7 .times. 10e7 CFU 22.1jeikeiumStaphylococcus 146 7.2 .times. 10e6 CFU 22.2epidermidisCorynebacterium renale 19412 7.0 .times. 10e7 CFU 25.7F. nucleatum 25586 1.1 .times. 10e6 CFU 22.2Salmonella cholerasius 4931 1.7 .times. 10e7 CFU 27.8serotype enteritidisStaphylococcus aureus 4012 7.0 .times. 10e5 CFU 21.9subspecies aureusGardnerella vaginalis 14018 5.4 .times. 10e6 CFU 24.2B. fragilis 25285 3.2 .times. 10e6 CFU 21.8Proteus vulgaris 6059 9.8 .times. 10e6 CFU 20.2Klebsiella pneumoniae 132 1.7 .times. 10e7 CFU 26.3subspecies pneumoniaePrevotella denticola 33184 7.4 .times. 10e6 CFU 26.3Corynebacterium renale 10848 2.0 .times. 10e6 CFU 26.7Escherichia coli 26 2.5 .times. 10e7 CFU 24.8C. neoformans 14116 1.1 .times. 10e6 CFU 20.6Fusobacterium 25286 8.7 .times. 10e5 CFU 21.4necrophorumStreptococcus pyogenes 4543 2.8 .times. 10e7 CFU 23.0Haemophilus 7901 2.7 .times. 10e7 CFU 20.5parainfluenzaeNeisseria gonorrhoeae 19424 7.5 .times. 10e6 CFU 215.4Actinomyces meyeri 35568 2.2 .times. 10e6 CFU 20.9Klebsiella pneumoniae 11296 4.0 .times. 10e6 CFU 26.4subspecies ozaenaeEnterobacter cloacae 222 3.3 .times. 10e6 CFU 29.1Prevotella buccalis 35310 9.8 .times. 10e5 CFU 23.4(ID is P. oralis)Peptostreptococcus 14955 2.2 .times. 10e6 CFU 21.7magnusLegionella pneumophilia 33152 1.2 .times. 10e6 CFU 21.2Legionella ND 1.0 .times. 10e7 22.5pneumophiha* DNA copies/reactionChlamydia pneumoniae* ND 7.6 .times. 10e6 24.7 DNA copies/reactionChlamydia trachomatis* ND ND 21.3Neisseria gonorrhoeae* ND ND 22.2Calf Thymus DNA* NA 150 ng DNA 46.1.about.(Negative Control)M. pneumoniae, FH* 15531 10 molecules of 1404.7(Positive Control) DNA______________________________________ *DNA purified as in Example 2; ND = Not Determined; NA = Not Applicable; .about.3 aberrant values not included in calculation of average.
Example 5
Comparison of M. pneumoniae Detection by OH-PCR and Culture
A. Detection of M. pneumoniae in Throat Swab and Nasopharyngeal Swab Samples Using the Rep 5 Primer/Probe Set and Culture Eighteen paired throat swab and nasopharyngeal swab samples obtained from patients were tested for M. pneumoniae by traditional culture methodology and compared to M. pneumoniae detection using Rep 5 primers (SEQ ID NO 2 and SEQ ID NO 3) and Rep 5 detection probe (SEQ ID NO 5) as described in Example 1. Two throat swabs and two nasopharyngeal swabs were done per patient; one swab was used for culture and the other swab for OH-PCR; only throat swabs were cultured for M. pneumoniae. Sample DNA was isolated using the QIAgen nucleic acid purification method (QIAgen, Inc., Chatsworth, Calif.) and amplified and detected by the Rep 5 method described for the SEQ ID NO 5 probe in Example 2.A. above. Results are shown in Table 11.
TABLE 11______________________________________ Rep 5 LCx .RTM. rateSample ID # Cultrue (c/s/s)______________________________________1A - 132A + for M. pneumoniae 12/1383*3A - 124A - 95A + for M. genitalium 116A - 107A - 118A - 119A - 1110A - 1111A - 11812A - 1113A - 1114A C. pneumoniae PC 1115A C. pneumoniae PC 8316A C. pneumoniae PC 1317A M. pneumoniae PC 111518A M. pneumornae PC 129319A + 91420A - 1221A - 1122A - 111B ND 122B ND 103B ND 164B ND 115B ND 146B ND 157B ND 128B ND 149B ND 1310B ND 1711B ND 1612B ND 1713B ND 1214B C. pneumoniae PC 1115B C. pneumoniae PC 1116B M. pneumoniae PC 135817B M. pneumoniae PC 137118B M. pneumoniae PC 129119B ND 40320B ND 1121B ND 1222B ND 12______________________________________ (PC = Positive Control; ND = Not Determined; *Retest of culture swab.)
In Table 11 above, `A` samples were from throat swabs and `B` samples were from nasal swabs. Samples 14A/B, 15A/B and 16A were ten-fold serial dilutions of 1.25 ng/ml of Chlamydia pneumoniae DNA, with 14A being the initial dilution, followed by 14B, 15A, 15B and 16A. None of these were detected using the M. pneumoniae Rep 5 primer/probe set. As further indication of the specificity of these primers and probes for M. pneumoniae, patient 5 was found to be culture positive for M. genitalium and was not detected as positive by the M. pneumoniae primer/probe set.
Samples 16B, 17A/B and 18A/B were ten-fold serial dilutions of 1.25 ng/ml of M. pneumoniae DNA, with 16B being the initial dilution, followed by 17A, 17B, 18A and 18B. All five of these samples were detected using the M. pneumoniae Rep 5 primer/probe set. Patient 19 was culture positive for M. pneumoniae and was also detected as positive by OH-PCR. The throat swab from patient 2 was culture positive for M. pneumoniae but the swab used for PCR testing gave a negative result. However, when the swab that had given the positive culture result was tested by OH-PCR, M. pneumoniae was detected. Therefore, the initial discrepant result was due to differences in swab sampling and was not a false negative result by the OH-PCR test. Thus the results using the Rep 5 M. pneumoniae primer/probe set showed concordance with standard culture for detection of M. pneumoniae in all patient samples tested.
B. OH-PCR and Culture Detection of M. pneumoniae in Patient Samples Four negative patient samples were spiked with M. pneumoniae DNA and tested with paired throat and nasopharyngeal swab samples from one patient found to be culture positive for M. pneumoniae using Rep 5 primers (SEQ ID NO 2 and SEQ ID NO 3) and Rep 5 detection probe (SEQ ID NO 5) or SDC1 primers (SEQ ID NO 10 and SEQ ID NO 11) and SDC1 detection probe (SEQ ID NO 12) as described in Example 1. Sample DNA was isolated using the QIAgen nucleic acid purification method and amplified and detected by the Rep 5 and SDC1 methods as in Example 2.A. for the SEQ ID NO 5 probe and Example 2.B. for the SEQ ID NO 12 probe respectively. Purified M. pneumoniae from strain M129 was used as a positive control and calf thymus DNA was used as a negative control (NC); all controls were tested in triplicate. Results are shown in Table 12.
TABLE 12______________________________________ Rep 5 LCx .RTM. SDC1 LCx .RTM.Sample # rate (c/s/s) rate (c/s/s)______________________________________17 1033 172634 1347 190635 1372 193236 510 134359 1264 185063 767 910Calf Thymus DNA (NC) 25 19M. pneumoniae, M129 1165 1118(1 mol of DNA)M. pneumoniae, M129 1203 1658(10 mol of DNA)______________________________________ (mol of DNA = molecules of DNA)
Samples 17, 34, 35 and 36 were from patients previously found negative for M. pneumoniae into which M. pneumoniae DNA was added. Sample 59 was an M. pneumoniae culture positive throat swab and sample 63 an M. pneumoniae nasal swab from the same patient. All samples were correctly identified as positive for M. pneumoniae by both Rep 5 and SCD1 M. pneumoniae primer/probe sets using OH-PCR on the LCx.
While the invention has been described in detail and with reference to specific embodiments, it will be apparent to one skilled in the art that various changes and modifications may be made to such embodiments without departing from the spirit and scope of the invention.
__________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 26(2) INFORMATION FOR SEQ ID NO:1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 165 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: genomic DNA (M. pneumoniae)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:CTCACCCCCTACACGCCCTTTACGACACCGCTCAATGGGGGGCTGGATGT50CGTGCGCGCCGCCCATTTACACCCCTCATACGAACTCGTGGACTGAAAGC100GGGTGGGGGATACCAAGTTGGTGGCGCTGGTCCGCTCAGCGTTGGTCAGG150GTGAAATTCCAGGAC165(2) INFORMATION FOR SEQ ID NO:2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: synthetic DNA(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:ACACGCCCTTTACGACACCG20(2) INFORMATION FOR SEQ ID NO:3:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: synthetic DNA(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:TTCACCCTGACCAACGCTGA20(2) INFORMATION FOR SEQ ID NO: 4:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: synthetic DNA(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:ATTTACACCCCTCATACGAA20(2) INFORMATION FOR SEQ ID NO:5:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: synthetic DNA(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:TTCGTATGAGGGGTGTAAAT20(2) INFORMATION FOR SEQ ID NO:6:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: synthetic DNA(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:CGGTGTCGTAAAGGGCGTGT20(2) INFORMATION FOR SEQ ID NO:7:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: synthetic DNA(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:TCAGCGTTGGTCAGGGTGAA20(2) INFORMATION FOR SEQ ID NO:8:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 149 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: genomic DNA (M. pneumoniae)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:CGTTGTGGCCGACCACCTCGTCTTTGCGGCCTTTAAAGCGGGCGCGGTGG50GGTATGATATGACGACTGATTCGAGCGCTTCGACCTACAACCAAGCACTC100GCCTGGTCGACCACGGCCGGGTTGGACAGTGATGGGGGGTACAAGGCCT149(2) INFORMATION FOR SEQ ID NO:9:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 149 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: genomic DNA (M. pneumoniae)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:CGTTGTCCAAGACCACCTCGTCTTTGCGGCCTTCAAAGCGGGCGCGGTGG50GGTATGATATGACGACTGATTCGAACGCTTCCACCAAAGACCAAGCGCTC100GCCTGGTCGACCACGGCCGGGTTGGACAGTGATGGGGGGTACAAGGCCT149(2) INFORMATION FOR SEQ ID NO:10:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: synthetic DNA(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:GACCACCTCGTCTTTGCGGC20(2) INFORMATION FOR SEQ ID NO:11:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 21 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: synthetic DNA(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:CCCCCCATCACTGTCCAACCC21(2) INFORMATION FOR SEQ ID NO:12:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 19 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: synthetic DNA(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:GTCATATCATACCCCACCG19(2) INFORMATION FOR SEQ ID NO:13:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 18 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: synthetic DNA(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:GTCATATCATACCCCACC18(2) INFORMATION FOR SEQ ID NO:14:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 16 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: synthetic DNA(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:TCATATCATACCCCAC16(2) INFORMATION FOR SEQ ID NO:15:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: synthetic DNA(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:GCCGCAAAGACGAGGTGGTC20(2) INFORMATION FOR SEQ ID NO:16:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 21 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: synthetic DNA(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:GGGTTGGACAGTGATGGGGGG21(2) INFORMATION FOR SEQ ID NO:17:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 19 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: synthetic DNA(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:CGGTGGGGTATGATATGAC19(2) INFORMATION FOR SEQ ID NO:18:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 18 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: synthetic DNA(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:GGTGGGGTATGATATGAC18(2) INFORMATION FOR SEQ ID NO:19:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 16 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: synthetic DNA(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:GTGGGGTATGATATGA16(2) INFORMATION FOR SEQ ID NO:20:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 122 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: genomic DNA (M. pneumoniae)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:CGGCTTATTACGGCTATGGTCAGGATGGTCAAGCATATCCACAAGACTAT50GCTCAAGATCCCAACCAAGCGTATTATGCCGATCCCAATGCTTATCAGGA100CCCAAACGCTTACACCGATCCT122(2) INFORMATION FOR SEQ ID NO:21:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 24 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: synthetic DNA(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:CGGCTATGGTCAGGATGGTCAAGC24(2) INFORMATION FOR SEQ ID NO:22:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 24 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: synthetic DNA(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:AGGATCGGTGTAAGCGTTTGGGTC24(2) INFORMATION FOR SEQ ID NO:23:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: synthetic DNA(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:TTGGGATCTTGAGCATAGTC20(2) INFORMATION FOR SEQ ID NO:24:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 24 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: synthetic DNA(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:GCTTGACCATCCTGACCATAGCCG24(2) INFORMATION FOR SEQ ID NO:25:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 24 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: synthetic DNA(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:GACCCAAACGCTTACACCGATCCT24(2) INFORMATION FOR SEQ ID NO:26:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: synthetic DNA(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:GACTATGCTCAAGATCCCAA20__________________________________________________________________________

Number	Date	Country
0264067	Apr 1988	EPX
0305145	Mar 1989	EPX
0532167	Mar 1993	EPX
0576743	Jan 1994	EPX
9309250	May 1993	WOX
9402634	Feb 1994	WOX
9606949	Mar 1996	WOX
9707235	Feb 1997	WOX

Nucleic acid primers and probes for detecting Mycoplasma pneumoniae

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Proft et al, Journal of Bacteriology (1995) 177: 3370-3378.
Colman et al, Gene (1990), vol. 87: 91-96.
Ruland et al, Journal of Bacteriology (1994) 176: 5202-5209.
Colman, S.D., et al., "Prevalence of novel repeat sequences in and around the P1 operon in the genome of Mycoplasma pneumoniae", Gene, 87:91-96 (1990).
Proft, T., et al., "The Proline-Rich P65 Protein of Mycoplasma pneumoniae Is a Component of the Triton X-100-Insoluble Fraction and Exhibits Size Polymorphism in the Strains M129 and FH", Journal of Bacteriology, 177 (12): 3370-3378 (1995).
Ruland, K., et al., "Sequence Divergence of the ORF6 Gene of Mycoplasma penumoniae", Journal of Bacteriology, 176(17):5202-5209 (1994).