Compositions, methods and kits for determining the presence of Mycoplasma pneumoniae in a test sample

#This application claims the benefit of U.S. Provisional Application No. 60/335,015, filed Nov. 2, 2001, the contents of which are hereby incorporated by reference herein.

#The present invention relates to hybridization assay probes, capture probes, amplification primers, nucleic acid compositions, methods and kits useful for determining the presence of Mycoplasma pneumoniae and/or Mycoplasma genitalium in a test sample.

#All references referred to herein are hereby incorporated by reference in their entirety. The incorporation of these references, standing alone, should not be construed as an assertion or admission by the inventors that any portion of the contents of all of these references, or any particular reference, is considered to be essential material for satisfying any national or regional statutory disclosure requirement for patent applications. Notwithstanding, the inventors reserve the right to rely upon any of such references, where appropriate, for providing material deemed essential to the claimed invention by an examining authority or court. No reference referred to herein is admitted to be prior art to the claimed invention.

#FIGS. 1 and 2 are graphs which were used to determine the differential hydrolysis ratios of two M. pneumoniae probes disclosed by Hammond et al., “Nucleic Acid Hybridization Assay Probes, Helper Probes and Amplification Oligonucleotides Targeted to M. pneumoniae Nucleic Acid,” U.S. Pat. No. 5,656,427. Using the time and signal data set forth in Tables 2-5 of Example 2 infra, these graphs plot the data for hybrids (▪) and controls ([Circlesolid]) as the log of the percentage of time zero chemiluminescence on the y-axis versus time in minutes on the x-axis. Slopes and associated t1/2 values (time required to hydrolyze 50% of the probe associated acridinium ester label) were determined for the controls and hybrids of each probe using standard linear-regression analysis. See Arnold et al., “Assay Formats Involving Acridinium-Ester-Labeled DNA Probes,” Clinical Chemistry, 35:1588-1594 (1989). Based on the t1/2 values determined from these graphs, the differential hydrolysis ratio for each probe was calculated by comparing the t1/2 value of the hybrid to the t. value of the control.

#FIGS. 3 and 4 are graphs which were used to determine the differential hydrolysis ratios of two probes according to the present invention. Using the time and signal data set forth in Tables 7-10 of Example 2 infra, these graphs plot the data for hybrids (▪) and controls ([Circlesolid]) as the log of the percentage of time zero chemiluminescence on the y-axis versus time in minutes on the x-axis. Slopes and associated t., values were determined for the controls and hybrids of each probe using standard linear-regression analysis. Based on the t1/2 values determined from these graphs, the differential hydrolysis ratio for each probe was calculated by comparing the t1/2 value of the hybrid to the t1/2 value of the control.

#FIG. 5 depicts a linking reagent having an extended aminoalkylcarboxy linker arm which can be used to join a detectable label to an oligonucleotide.

#The present invention describes oligonucleotides targeted to nucleic acid derived from Mycoplasma organisms which are useful for determining the presence or absence of M. pneumoniae or M. genitalium in a test sample. The oligonucleotides can aid in detecting M. pneumoniae or M. genitalium in different ways, such as by functioning as hybridization assay probes, capture probes and/or amplification primers. Hybridization assay probes of the present invention can preferentially hybridize to a target nucleic acid sequence present in nucleic acid derived from M. pneumoniae or M. genitalium under stringent hybridization assay conditions to form detectable duplexes which indicate the presence of M. pneumoniae or M genitalium in a test sample. Some of the probes are believed to be capable of distinguishing between the target organism and its known closest phylogenetic neighbors. Capture probes of the present invention can hybridize to a target nucleic acid sequence present in nucleic acid derived from Mycoplasma organisms under stringent hybridization assay conditions and can be used to separate target nucleic acid from clinical specimens. Amplification primers of the present invention can hybridize to a target nucleic acid sequence present in nucleic acid derived from Mycoplasma organisms under amplification conditions and can be used as primers in an amplification reaction to generate M -derived nucleic acid. The probes and amplification primers may be used in assays for the detection and/or quantitation of M. pneumoniae or M. genitalium in a test sample.

#The following terms have the indicated meanings in the specification unless expressly indicated to have a different meaning.

#By “sample” or “test sample” is meant any substance suspected of containing a target organism or nucleic acid derived from the target organism. The substance may be, for example, an unprocessed clinical specimen, such as a sputum or urethral specimen, a buffered medium containing the specimen, a medium containing the specimen and lytic agents for releasing nucleic acid belonging to the target organism, or a medium containing nucleic acid derived from the target organism which has been isolated and/or purified in a reaction receptacle or on a reaction material or device. In the claims, the terms “sample” and “test sample” may refer to. specimen in its raw form or to any stage of processing to release, isolate and purify nucleic acid derived from target organisms in the specimen. Thus, within a method of use claim, each reference to a “sample” or “test sample” may refer to a substance suspected of containing nucleic acid derived from the target organism or organisms at different stages of processing and is not limited to the initial form of the substance in the claim.