Macrolide-resistant mycoplasma genitalium assay with reduced incidence of false-positive reporting

Abstract

Provided are methods, compositions, and systems for detecting nucleic acids of macrolideresistant Mycoplasma genitalium. The illustrated method can involve real-time nucleic acid amplification, and further can involve processing real-time run curve data using a derivative-based analytical method to distinguish macrolide-resistant M. genitalium from macrolide-sensitive M. genitalium. Advantageously, the disclosed technique reduces the incidence of false-positive identification of macrolide resistance.

Description

MACROLIDE-RESISTANT MYCOPLASMA GENITALIUM ASSAY WITH REDUCEDINCIDENCE OF FALSE-POSITIVE REPORTINGRELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 715,498, filed November 1, 2024. The entire disclosure of this related application is hereby incorporated by reference.TECHNICAL FIELD

[0002] The disclosure relates generally to the field of biotechnology. More specifically, the disclosure relates to compositions, methods, kits, and systems for detecting macrolide-resistant Mycoplasma genitalium.BACKGROUND

[0003] Mycoplasmas are small prokaryotic organisms (0.2 to 0.3 pm) belonging to the class Mollicutes, whose members lack a cell wall and have a small genome size. The mollicutes include at least 100 species of Mycoplasma, 13 of which are known to infect humans.

[0004] One Mycoplasma species of clinical relevance is M. genitalium. This organism, which is thought to be a cause of sexually transmitted nongonococcal urethritis (NGU), has been detected to a significantly greater extent in symptomatic males than in asymptomatic males. See Yoshida et al., “Phylogeny-Based Rapid Identification of Mycoplasma and Ureaplasmas from Urethritis Patients,” J. Clin. Microbiol., 40: 105-110 (2002). In addition to NGU, M. genitalium is thought to be involved in pelvic inflammatory disease (which can lead to infertility in women in severe cases), adverse birth outcomes, and increased risk for human immunodeficiency virus (HIV) infection. See Maniloff et al., Mycoplasmas: Molecular Biology and Pathogenesis 417 (ASM 1992); and Manhart et al., supplement to Contemporary OB / GYN (July 2017).

[0005] Significantly, M. genitalium is more common than many other sexually transmitted pathogens. Studies of low-risk individuals estimated the prevalence of M. genitalium among women to be in the range of from 0.8% - 4.1%, and among men to be in the range of from 1.1% - 1.2%. Among the population of women attending an STI clinic, the prevalence of M. genitalium ranged as high as 19% in two major U.S. cities. The prevalence was as high as 15% for men attending the STI clinics. In recent studies, M. genitalium prevalence was higher than all other bacterial sexually transmitted infections.

[0006] The advent and spread of antibiotic-resistant strains of M. genitalium greatly complicates infection control. Current treatment protocols for M. genitalium infection rely on administration of the macrolide antibiotic azithromycin. One study conducted in Australia more than a decade ago revealed evidence for progressive dissemination of M. genitalium bacteria that were resistant to this treatment. The resistance was attributed to adjacent mutations at two positions in the 23S rRNA that could be detected using nucleic acid sequencing or “high resolution melt analysis” techniques. Unfortunately, nucleic acid sequencing approaches do not lend themselves to rapid testing, and melt curve analyses, although effective, had trouble differentiating genotypes (i.e., wild-type and mutants). Benefits of early detection include the opportunity to reduce transmission of resistant M. genitalium strains in the community, and shortening the time to effective second line treatment.(See Twin et al., PUoS ONE 7(4): e35593. Doi: 10.1371 / journal.pone.0035593)

[0007] Sensitive and specific molecular tests for nucleic acids of M. genitalium have been described in U.S. Patent No. 7,345,155, and in published U.S. Patent Application Ser. No. 18 / 015,619, the disclosures of which are incorporated by reference. However, these tests do not detect the macrolide resistance genetic marker. The present disclosure provides supplemental techniques that can be used for detecting the genetic marker of macrolide resistance in M. genitalium.SUMMARY OF THE DISCLOSURE

[0008] Provided herein are the following embodiments.

[0009] Embodiment 1 is a probe reagent for detecting a nucleic acid marker indicative of macrolide-resistance in M. genitalium among a collection of nucleic acid amplification products, the probe reagent including a probe of the base sequence of SEQ ID NO: 15.

[0010] Embodiment 2 is the probe reagent of embodiment 1 , where the probe of the base sequence of SEQ ID NO: 15 is labeled with a fluorophore and a quencher.

[0011] Embodiment 3 is the probe reagent of either embodiment 1 or embodiment 2, further including at least one probe selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 17.

[0012] Embodiment 4 is the probe reagent of embodiment 3, where the probe of the base sequence of SEQ ID NO: 15 and the at least one probe selected from the group consisting of SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 17 are combined with each other and packaged in the same vial or container.

[0013] Embodiment 5 is the probe reagent of any one of embodiments 1 to 4, where all of the probes are labeled with the same fluorophore and the same quencher.

[0014] Embodiment 6 is the probe reagent of embodiment 2, where a fluorescent emission from the fluorophore is detectable in a ROX detection channel of a fluorometer.

[0015] Embodiment 7 is the probe reagent of any one of embodiments 1 to 6, where the probe reagent does not include any probe with the wild-type sequence of SEQ ID NO: 18 or the complement thereof.

[0016] Embodiment 8 is the probe reagent of any one of embodiments 2 to 7, further including an oligonucleotide probe complementary to a sequence present in the 23S rRNA of both macrolidesensitive M. genitalium and macrolide-resistant M. genitalium, and where the oligonucleotide probe includes a fluorophore label that is different from the fluorophore of the probe of the base sequence of SEQ ID NO: 15.

[0017] Embodiment 9 is the probe reagent of any one of embodiments 1 to 8, where each probe is in a dried form and not in a liquid phase.

[0018] Embodiment 10 is the probe reagent of embodiment 9, where the dried form includes a dried cyclodextrin.

[0019] Embodiment 11 is a method of determining macrolide resistance status of M. genitalium in a test sample, the method including the steps of: (a) isolating nucleic acids from M. genitalium in the test sample; (b) amplifying, in a multiplex real-time nucleic acid amplification reaction, a nucleic acid locus or region of M. genitalium 23S rRNA that is characteristic of macrolide resistance using nucleic acids isolated in step (a) as templates, whereby a real-time run curve data set is obtained, where said nucleic acid locus includes either the sequence of SEQ ID NO: 18, which is present in wild-type M. genitalium, or a macrolide resistance marker selected from the group consisting of: 2058C, 2058G, 2058T, 2059C, and 2059G, where the multiplex real-time nucleic acid amplification reaction includes a plurality of probes specific for macrolide resistance markers, but does not include a detectably labeled probe specific for wild-type M. genitalium that includes the sequence of ACGGAAAGACC (SEQ ID NO: 18), and where each of the plurality of probes is detectably labeled with a fluorophore; (c) establishing the presence of M. genitalium nucleic acids among the nucleic acids isolated in step (a) by amplifying and detecting nucleic acids of an M. genitalium detection locus in a nucleic acid amplification reaction, where the M. genitalium detection locus is present in macrolide-sensitive M. genitalium and macrolide-resistant M. genitalium, but not present in nucleic acids of M. pneumoniae, and where the nucleic acid amplification reaction is either the multiplex real-time nucleic acid amplification reaction or a nucleic acid amplification reaction other than the multiplex real-time nucleic acid amplification reaction; (d) processing the real-time ran curve data set from step (b) to (i) calculate a maximum first derivative of the real-time ran curve data set, and (ii) compare the calculated maximum to a cutoff that distinguishes first derivatives ofrun curves characteristic of macrolide-resistant M. genitalium from first derivatives of run curves characteristic of macrolide-sensitive M. genitalium', and (e) determining either that the test sample includes macrolide-resistant M. genitalium if the calculated maximum first derivative exceeds the cutoff, or the test sample does not include macrolide-resistant M. genitalium if the calculated maximum first derivative does not exceed the cutoff.

[0020] Embodiment 12 is the method of embodiment 11 , where the nucleic acid amplification reaction of step (c) includes the nucleic acid amplification reaction other than the multiplex realtime nucleic acid amplification reaction, and where the nucleic acid amplification reaction other than the multiplex real-time nucleic acid amplification reaction includes an isothermal transcription- mediated amplification (TMA) reaction.

[0021] Embodiment 13 is the method of either embodiment 11 or 12, where the nucleic acid amplification reaction of step (c) is the multiplex real-time nucleic acid amplification reaction, and where the multiplex real-time nucleic acid amplification reaction further includes a second pair of primers and a second probe to amplify and detect nucleic acids of the M. genitalium detection locus.

[0022] Embodiment 14 is the method of any one of embodiments 11 to 13, where the cutoff in step (d) is a cutoff established to minimize false-positive and false-negative results using determinations obtained for a plurality of control reactions having known M. genitalium macrolide resistance types.

[0023] Embodiment 15 is the method of any one of embodiments 11 to 14, where the cutoff is a predetermined cutoff provided with a kit used for performing the multiplex real-time nucleic acid amplification reaction.

[0024] Embodiment 16 is the method of any one of embodiments 11 to 15, further including the step of recording the result of step (e) in a non-transient form selected from the group consisting of: printing on paper, and saving on a computer-readable storage medium.

[0025] Embodiment 17 is the method of any one of embodiments 11 to 16, where the fluorophore of each of the plurality of probes in step (b) is the same fluorophore, and where each of the plurality of probes further includes a quencher moiety.

[0026] Embodiment 18 is the method of embodiment 17, where the quencher moiety is the same for each probe of the plurality of probes.

[0027] Embodiment 19 is the method of any one of embodiments 11 to 18, where the test sample is selected from the group consisting of a vaginal swab sample, a penile meatal swab sample, and a urine sample.

[0028] Embodiment 20 is the method of any one of embodiments 11 to 19, where steps (a) and (b) are performed under automated process control using an automated nucleic acid analyzer thatcarries out nucleic acid amplification reactions and monitors synthesis of amplification products while the reaction is occurring.

[0029] Embodiment 21 is the method of any one of embodiments 11 to 20, where the multiplex real-time nucleic acid amplification reaction is a multiplex reverse transcriptase PCR reaction that uses reverse transcription to convert RNA into cDNA, which is then amplified in a PCR reaction.

[0030] Embodiment 22 is a computer programmed with software instructions to determine from results of a real-time nucleic acid amplification reaction whether a test sample of M. genitalium includes macrolide-resistant M. genitalium, nucleic acids isolated from the test sample having been used as templates for amplification of 23 s rRNA sequences in the real-time nucleic acid amplification reaction, the software instructions, when executed by the computer, cause the computer to: (a) receive a run curve data set for the real-time nucleic acid amplification reaction, the run curve data set comprising signal data for amplification of an Mgen Res detection locus in the 23S rRNA that comprises either the wild-type sequence of SEQ ID NO: 18, or a sequence selected from the group consisting of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO: 30, and SEQ ID NO:31; (b) calculate a first derivative of the run curve data set in (a) and determine whether the maximum value of the first derivative exceeds a predetermined cutoff value; and (c) determine either that the test sample includes macrolide-resistant M. genitalium if the maximum value of the first derivative exceeds the predetermined cutoff value, or the test sample does not include macrolide-resistant M. genitalium if the maximum value of the first derivative does not exceed the cutoff.

[0031] Embodiment 23 is the computer of embodiment 22, where the software instructions are executed by the computer if the test sample is known to include M. genitalium, the real-time nucleic acid amplification reaction being a reflex assay.

[0032] Embodiment 24 is the computer of either embodiment 22 or embodiment 23, where the software instructions, when executed by the computer, further cause the computer to generate a non-transient record of the result from (c).

[0033] Embodiment 25 is the computer of any one of embodiments 22 to 24, where the real-time nucleic acid amplification reaction is a real-time PCR reaction, where the run curve data set in (a) includes fluorescent signal data as a function of PCR cycle number, and where the first derivative in (b) has units of RFU / cycle.

[0034] Embodiment 26 is the computer of any one of embodiments 22 to 25, where the predetermined cutoff value is a predetermined constant numerical cutoff value.

[0035] Embodiment 27 is the computer of any one of embodiments 22 to 26, where the predetermined cutoff value is obtained as a component of a kit used to perform the real-time nucleic acid amplification reaction and then programmed into the computer.

[0036] Embodiment 28 is the computer of any one of embodiments 22 to 27, where the computer is in communication with an automated thermal cycling device equipped with a fluorometer.

[0037] Embodiment 29 is the computer of 28, where the computer is an integral component of the automated thermal cycling device.

[0038] Embodiment 30 is a computer programmed with software instructions to determine from results of a real-time nucleic acid amplification reaction whether a test sample of M. genitalium includes macrolide-resistant M. genitalium, nucleic acids isolated from the test sample having been used as templates for amplification of 23s rRNA sequences in the real-time nucleic acid amplification reaction, the software instructions, when executed by the computer, cause the computer to: (a) receive a ran curve data set for the real-time nucleic acid amplification reaction, the run curve data set including signal data for amplification of (i) an M. genitalium detection locus that is present in nucleic acids of both wild-type M. genitalium and macrolide-resistant M. genitalium, but absent from nucleic acids of M. pneumoniae, and (ii) an Mgen Res detection locus in the 23S rRNA that comprises either the wild-type sequence of SEQ ID NO: 18, or a sequence selected from the group consisting of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO: 30, and SEQ ID NO: 31 ; (b) establish from the run curve data set in (a)(i) whether the M. genitalium detection locus was amplified and detected in the real-time nucleic acid amplification reaction; (c) calculate a first derivative of the run curve data set in (a)(ii) and determine whether the maximum value of the first derivative exceeds a predetermined cutoff value; and (d) determine either that the test sample includes macrolide-resistant M. genitalium if it is established in (b) that the M. genitalium detection locus was amplified and detected, and if the maximum value of the first derivative from (c) exceeds the predetermined cutoff value, or the test sample does not include macrolide-resistant M. genitalium if either (i) it is established in (b) that the M. genitalium detection locus was not amplified and detected, or (ii) the maximum value of the first derivative from (c) does not exceed the cutoff.

[0039] Embodiment 31 is the computer of embodiment 30, where the software instructions, when executed by the computer, further cause the computer to generate a non-transient record of the result from (d).

[0040] Embodiment 32 is the computer of either embodiment 30 or 31, where the real-time nucleic acid amplification reaction is a real-time PCR reaction, where the run curve data set in (a) includesfluorescent signal data as a function of PCR cycle number, and where the first derivative in (c) has units of RFU / cycle.

[0041] Embodiment 33 is the computer of any one of embodiments 30 to 32, where the predetermined cutoff value is a predetermined constant numerical cutoff value.

[0042] Embodiment 34 is the computer of any one of embodiments 30 to 33, where the predetermined cutoff value is obtained as a component of a kit used to perform the real-time nucleic acid amplification reaction and then programmed into the computer.

[0043] Embodiment 35 is the computer of any one of embodiments 30 to 34, where the computer is in communication with an automated thermal cycling device equipped with a fluorometer.

[0044] Embodiment 36 is the computer of embodiment 35, where the computer is an integral component of the automated thermal cycling device.BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Fig. 1 is a schematic diagram of the 23S ribosomal RNA (rRNA) of M. genitalium showing two regions or domains that can be amplified and detected in the disclosed multiplex assay. The “Mgen Res Detection Locus” (boxed “Mgen Res”) illustrated in the left portion of the diagram was amplified using a pair of oppositely disposed primers, and then detected using five probes specific for different single nucleotide polymorphisms (SNPs), where each SNP is characteristic of resistance to macrolide antibiotics in M. genitalium (2058G, 2058C, 2058G, 2059C, 2059G, E. coli numbering). The five SNP probes are identified in the diagram as 58C, 58G, 58T, 59C, and 59G. All five probes harbored the same fluorescent label that was detected in the ROX channel of a fluorometer. The “Mgen Detection Locus” (boxed “Mgen”) illustrated in the right portion of the diagram was amplified using a pair of oppositely disposed primers, and then detected using a single probe that hybridized to a nucleic acid sequence common to wild-type (macrolide- sensitive) M. genitalium and macrolide-resistant M. genitalium. This probe was labeled with a fluorophore that was detected in the FAM channel of the fluorometer. Amplification and detection of the target region shown in the right portion of the schematic is also referred to herein as the “MG / MG Res control.”

[0046] Fig. 2 presents a series of line graphs showing Ct values (vertical axis) as a function of input Log Copies / mL of template nucleic acid used in PCR amplification reactions (horizontal axis). Target IVTs included sequences corresponding to the five macrolide resistance markers (58C, 58G, 58T, 59C, and 59G). An IVT harboring the “Mgen Detection Locus” illustrated in Fig. 1 (i.e., serving as the MG / MG Res control) also was used. This latter IVT included a sequence common to macrolide-sensitive M. genitalium and macrolide-resistant M. genitalium.

[0047] Figs. 3A and 3B are graphical plots showing results from real-time amplification reactions. Fig. 3A is a plot of real-time run curves showing magnitudes of measured FAM channel signals (vertical axis) as a function of PCR cycle number (horizontal axis). Here, FAM-labeled probes were specific for wild-type (macrolide-sensitive) M. genitalium detection. Template nucleic acid in the reaction was the wild-type (macrolide-sensitive) M. genitalium IVT. Different run curves represent results obtained using reaction mixtures that included different ROX-labeled probes specific for macrolide-resistant M. genitalium. Run curves crossing the horizontal threshold drawn at 1500 RFU on the vertical axis achieved a measurable level of amplification. Fig. 3B is a plot of real-time run curves showing magnitudes of measured ROX-channel signals (vertical axis) as a function of PCR cycle number (horizontal axis) for a collection of reactions. Templates in the amplification reactions were wild-type (macrolide-sensitive) M. genitalium IVT, and IVT that included the 2058T macrolide resistance marker of macrolide-resistant M. genitalium (two concentrations). Different run curves represent individual reactions that included one of the ROX- labeled probes from Table 10, together with ROX-labeled probes for detecting 58C, 58G, 59C, and 59G (see Table 3). A negative control reaction that omitted template nucleic acid gave essentially no measurable signal. Run curves in this figure are identified by the type of template used in the reaction, but not by the probe sequence. Notably, all curves that included either of the two template types and the collection of ROX-labeled probes exceeded the horizontal threshold drawn at 300 RFU on the vertical axis.

[0048] Fig. 4 is a variability chart for ROX-channel signals measured for reactions that included different probe combinations. The chart shows data points for determined maxima (vertical axis) of ROX channel run curves for results presented in Fig. 3B. Template types (i.e., mutant, negative control, and wild-type) and their respective concentrations used in the different trials are indicated. Each trial had the same parameters of testing except for the Mutant 58T-specific probes design. Mutant 58T-specific probes included in the combinations were (left to right) 58T-23 (SEQ ID NO:22), 58T-22 (SEQ ID NO:21), 58T-21 (SEQ ID NO:15), 58T-26 (SEQ ID NO:25), 58T-24 (SEQ ID NO:23), 58T-25 (SEQ ID NO:24), and 58T-12 ROX (SEQ ID NO:26). Results from trials conducted using the 58T-21 (SEQ ID NO:15) probe are boxed to highlight superior separation between maxima for first derivatives determined for mutant versus wild-type templates.

[0049] Figs. 5A and 5B are graphs presenting real-time run curves showing fluorescence (vertical axis) as a function of cycle number (horizontal axis), where fluorescence was measured in the FAM channel (Fig. 5A) and in the ROX channel (Fig. 5B). Fig. 5A indicates synthesis of amplification products detected by a FAM-labeled probe complementary to the MG / MG Res control sequence common to nucleic acids of wild-type (macrolide-sensitive) M. genitalium and macrolide-resistantM. genitalium. Fig. 5B indicates synthesis of amplification products detected by a collection of five different probes harboring ROX fluorescent labels, each probe being fully complementary to one of the five macrolide resistance markers.

[0050] Fig. 6 is a graph presenting first derivatives (vertical axis) of ran curves from Fig. 5B as a function of reaction cycle number (horizontal axis). A horizontal Curve Maximum Value (“CMValue”) cutoff drawn at 140 RFU / cycle distinguishes first derivatives of ran curves associated with amplification of nucleic acids of macrolide-resistant M. genitalium (having first derivative maxima above cutoff) from first derivatives of ran curves associated with amplification of nucleic acids of wild-type (macrolide- sensitive) M. genitalium (having first derivative maxima below cutoff).

[0051] Figs. 7A and 7B are plots displaying determined maximum first derivative values of ran curves. Individual points on the plot are categorized by genotype on the horizontal axis (2058C, 2058G, 2058T, 2059C, 2059G, and wild-type). Fig. 7A includes horizontal cutoff lines drawn at 110 RFU / cycle and 140 RFU / cycle on the vertical axis to define three regions. Points above the upper bound cutoff corresponded to macrolide-resistant M. genitalium samples. Points below the lower bound cutoff corresponded to samples that were not macrolide-resistant M. genitalium. Genotyping showed these samples represented the wild-type strain. Points between the two cutoffs were considered “not determined. ” Fig. 7B includes a single cutoff drawn at 140 RFU / cycle. Samples harboring macrolide resistance markers (i.e., representing macrolide-resistant M. genitalium) substantially partitioned above the single cutoff, while substantially all samples that did not correspond to macrolide-resistant M. genitalium partitioned below the single cutoff. The numerical cutoff was chosen to minimize false-positive identification of samples as macrolide- resistant M. genitalium.Definitions

[0052] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art pertinent to the methods and compositions described. General definitions may be found in technical books relevant to the art of molecular biology (e.g., Dictionary of Microbiology and Molecular Biology, 2nd ed., Singleton et al., 1994, John Wiley & Sons, New York, NY; or The Harper Collins Dictionary of Biology, Hale & Marham, 1991, Harper Perennial, New York, NY). As used herein, the following terms and phrases have the meanings ascribed to them unless specified otherwise.

[0053] The terms "a," "an," and "the" include plural referents, unless the context clearly indicates otherwise. For example, "a nucleic acid" as used herein is understood to represent one or morenucleic acids. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0054] It will be appreciated that there is an implied "about" prior to the temperatures, concentrations, and times discussed in the present disclosure, such that slight and insubstantial deviations are within the scope of the present teachings. For example, conventional thermocycling instruments reach and maintain temperatures within a tolerance of ± 2°C, or even ± 1°C. Thus, “about” in the context of reaction temperatures means the specified temperature ± 2°C, or more preferably ± 1°C. With respect to reagent and component concentrations, “about” means ± 20%, or more preferably ± 10%. In general, the term "about" indicates insubstantial variation in a quantity of a component of a composition not having any significant effect on the activity or stability of the composition. All ranges are to be interpreted as encompassing the endpoints in the absence of express exclusions such as “not including the endpoints"; thus, for example, "within 10-15" includes the values 10 and 15.

[0055] Unless specifically noted, embodiments in the specification that recite "comprising" various components are also contemplated as "consisting of" or "consisting essentially of" the recited components; embodiments in the specification that recite "consisting of" various components are also contemplated as "comprising" or "consisting essentially of" the recited components; and embodiments in the specification that recite "consisting essentially of" various components are also contemplated as "consisting of' or "comprising" the recited components (this interchangeability does not apply to the use of these terms in the claims). "Consisting essentially of" means that additional component(s), composition(s) or method step(s) that do not materially change the basic and novel characteristics of the compositions and methods described herein may be included in those compositions or methods. Such characteristics include the ability to detect a nucleic acid sequence present in a sample with specificity that distinguishes macrolide-resistant M. genitalium nucleic acid from wild-type (macrolide-sensitive) M. genitalium nucleic acid or other known pathogens, optionally at a sensitivity that can detect the target nucleic acid present in a sample at a concentration of about 50 copies / ml, and, optionally within about 60 minutes and / or within about 40 cycles from the beginning of an amplification reaction when a cycled amplification reaction is used.

[0056] As used herein, the term “sample” refers to a specimen that may contain macrolide-resistant M. genitalium or components thereof (e.g., nucleic acids). Samples may be from any source, such as biological specimens or environmental sources. Biological specimens include any tissue or material derived from a living or dead organism. Examples of biological samples include vaginal swab samples, respiratory tissue, exudates (e.g., bronchoalveolar lavage), biopsy, sputum,peripheral blood, plasma, serum, lymph node, gastrointestinal tissue, feces, urine, or other fluids, tissues or materials. Samples may be processed specimens or materials, such as obtained from treating a sample by using filtration, centrifugation, sedimentation, or adherence to a medium, such as matrix or support. Other processing of samples may include treatments to physically or mechanically disrupt tissue, cellular aggregates, or cells to release intracellular components that include nucleic acids into a solution which may contain other components, such as enzymes, buffers, salts, detergents, and the like. Samples being tested for the presence of an analyte may sometimes be referred to as “test samples.”

[0057] As used herein, a "nucleotide" is a subunit of a nucleic acid consisting of a phosphate group, a 5-carbon sugar, and a nitrogenous base (sometimes referred to as a "nucleobase"). The 5-carbon sugar found in RNA is ribose. In DNA, the 5-carbon sugar is 2’-deoxyribose. The term also includes analogs of such subunits, such as a methoxy group at the 2' position of the ribose (also referred to herein as "2'-0-Me" or "2'-methoxy").

[0058] "Nucleic acid" and "polynucleotide" refer to a multimeric compound comprising nucleosides or nucleoside analogs which have nitrogenous heterocyclic bases or base analogs linked together by a chemical backbone. The terms embrace conventional RNA, DNA, mixed RNA- DNA, and polymers that are analogs thereof. A nucleic acid "backbone" may be made up of a variety of linkages, including one or more of sugar-phosphodiester linkages, peptide-nucleic acid bonds ("peptide nucleic acids" or PNA; PCT Publication No. WO 95 / 32305), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. Sugar moieties of a nucleic acid may be ribose, deoxyribose, or similar compounds with substitutions (e.g., 2' methoxy or 2' halide substitutions). Nitrogenous bases may be conventional bases (A, G, C, T, U), analogs thereof (e.g., inosine or others; see The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11thed., 1992), derivatives of purines or pyrimidines (e.g., N4-methyl deoxyguanosine, deaza- or aza-purines, deaza- or aza-pyrimidines, pyrimidine bases with substituent groups at the 5 or 6 position, purine bases with a substituent at the 2, 6, or 8 positions, 2-amino-6-methylaminopurine, O6- methylguanine, 4-thio-pyrimidines, 4-amino-pyrimidines, 4-dimethylhydrazine-pyrimidines, and O4-alkyl-pyrimidines; U.S. Patent No. 5,378,825 and PCT Publication No. WO 93 / 13121). Nucleic acids may include one or more "abasic" residues where the backbone includes no nitrogenous base for position(s) of the polymer (U.S. Patent No. 5,585,481). A nucleic acid may comprise only conventional RNA or DNA sugars, bases and linkages, or may include both conventional components and substitutions (e.g., conventional bases with 2' methoxy linkages, or polymers containing both conventional bases and one or more base analogs). Nucleic acid includes "locked nucleic acid" (UNA), an analogue containing one or more LNA nucleotide monomers with abicyclic furanose unit locked in an RNA mimicking sugar conformation, which enhance hybridization affinity toward complementary RNA and DNA sequences (Vester and Wengel, 2004, Biochemistry 43(42): 13233-41). Embodiments of oligomers that may affect stability of a hybridization complex include PNA oligomers, oligomers that include 2'-methoxy or 2'-fluoro substituted RNA, or oligomers that affect the overall charge, charge density, or steric associations of a hybridization complex, including oligomers that contain charged linkages (e.g., phosphorothioates) or neutral groups (e.g., methylphosphonates). 5-methylcytosines may be used in conjunction with any of the foregoing backbones / sugars / linkages including RNA or DNA backbones (or mixtures thereof) unless otherwise indicated. Similarly, 5-propynyl-2'-deoxycytidine (sometimes “pdC”) may be used in conjunction with any of the foregoing backbones / sugars / linkages including RNA or DNA backbones (or mixtures thereof) unless otherwise indicated. Likewise, 5-propynyl-2'-deoxyuridine (sometimes “pdU”) can be used as a substitute for “T” bases, and may be used in conjunction with any of the foregoing backbones / sugars / linkages including RNA or DNA backbones (or mixtures thereof) unless otherwise indicated. It is understood that when referring to ranges for the length of an oligonucleotide, amplicon, or other nucleic acid, that the range is inclusive of all whole numbers (e.g., 19-25 contiguous nucleotides in length includes 19, 20, 21, 22, 23, 24, and 25).

[0059] As used herein, an “analyte” is a chemical or biochemical species that is to be detected and / or quantified. For example, a “polynucleotide analyte” refers to a polynucleotide (e.g., a segment of a viral nucleic acid, or of a bacterial ribosomal nucleic acid) that is to be detected or quantified in a test procedure.

[0060] As used herein, an "oligonucleotide" (sometimes “oligomer” or “oligo”) is a molecule comprising two or more nucleotides (e.g., deoxyribonucleotides or ribonucleotides), preferably at least 5 nucleotides, more preferably at least about 10-15 nucleotides and more preferably at least about 15 to 30 nucleotides, or longer (e.g., oligonucleotides are typically less than 200 residues long (e.g., between 15 and 100 nucleotides). The exact size will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide. Oligonucleotides are often referred to by their length. For example, a 24 residue oligonucleotide is referred to as a "24-mer." Oligonucleotides can form secondary and tertiary structures by self-hybridizing or by hybridizing to other polynucleotides. Such structures can include, but are not limited to, duplexes, hairpins, cruciforms, bends, and triplexes. Oligonucleotides may be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, PCR, or a combination thereof.

[0061] By " RNA and DNA equivalents" is meant RNA and DNA molecules having essentially the same complementary base pair hybridization properties. RNA and DNA equivalents have differentsugar moieties (i.e., ribose versus deoxyribose) and may differ by the presence of uracil in RNA and thymine in DNA. The differences between RNA and DNA equivalents do not contribute to differences in homology because the equivalents have the same degree of complementarity to a particular sequence. By "DNA / RNA chimeric" is meant a nucleic acid comprising both DNA and RNA nucleotides. Unless the context clearly dictates otherwise, reference to an M. genitalium nucleic acid includes M. genitalium RNA and DNA equivalents, and DNA / RNA chimerics thereof.

[0062] By "RNA and DNA equivalent bases" is meant nucleotide bases having the same complementary base pair hybridization properties in RNA and DNA. Here the base uracil can be substituted in place of the base thymine, or vice versa, and so uracil and thymine are RNA and DNA equivalent bases. A polynucleotide base sequence 5’-AGCT-3’ that allows for substitution of RNA and DNA equivalent bases would also describe the sequence 5’-AGCU-3’. The differences between RNA and DNA equivalent bases do not contribute to differences in homology because the equivalents have the same degree of complementarity to a particular sequence.

[0063] The term, “complement” refers to a nucleic acid molecule that comprises a contiguous nucleic acid sequence that is complementary to a contiguous nucleic acid sequence of another nucleic acid molecule (for standard nucleotides A:T, A:U, C:G). For example, 5’-AACTGUC-3’ is the complement of 5’-GACAGTT-3’. Two nucleic acid sequences are “sufficiently complementary” when their respective contiguous nucleic acid sequences are at least 70% complementary.

[0064] A "target nucleic acid" as used herein is a nucleic acid comprising a target sequence to be amplified and / or detected. Target nucleic acids may be DNA or RNA, and may be either singlestranded or double- stranded. The target nucleic acid may include other sequences besides the target sequence, which may not be amplified.

[0065] The term "target sequence" as used herein refers to the particular nucleotide sequence of the target nucleic acid that is to be amplified and / or detected. The "target sequence" includes the complexing sequences to which oligonucleotides (e.g., primers) complex during an amplification processes (e.g., PGR, TMA). Where the target nucleic acid is originally single-stranded, the term "target sequence" will also refer to the sequence complementary to the "target sequence" as present in the target nucleic acid. Where the target nucleic acid is originally double-stranded, the term "target sequence" refers to both the sense (+) and antisense (-) strands.

[0066] "Target-hybridizing sequence" or "target-specific sequence" is used herein to refer to the portion of an oligomer that is configured to hybridize with a target nucleic acid sequence. Preferably, the target-hybridizing sequences are configured to specifically hybridize with a target nucleic acid sequence. Target-hybridizing sequences may be 100% complementary to the portionof the target sequence to which they are configured to hybridize, but not necessarily. Targethybridizing sequences may also include inserted, deleted and / or substituted nucleotide residues relative to a target sequence.

[0067] The term "target a sequence," as used herein in reference to a region of M. genitalium nucleic acid, refers to a process whereby an oligonucleotide hybridizes to a target sequence in a manner that allows for amplification and detection as described herein. In one preferred embodiment, the oligonucleotide is complementary to the targeted M. genitalium nucleic acid sequence and contains no mismatches. In another preferred embodiment, the oligonucleotide is complementary but contains 1, 2, 3, 4, or 5 mismatches with the targeted M. genitalium nucleic acid sequence. Preferably, the oligomer specifically hybridizes to the target sequence.

[0068] The term "configured to" denotes an actual arrangement of the polynucleotide sequence configuration of a referenced oligonucleotide target-hybridizing sequence. For example, amplification oligomers that are configured to generate a specified amplicon from a target sequence have polynucleotide sequences that hybridize to the target sequence and can be used in an amplification reaction to generate the amplicon. Also as an example, oligonucleotides that are configured to specifically hybridize to a target sequence have a polynucleotide sequence that specifically hybridizes to the referenced sequence under stringent hybridization conditions.

[0069] The term "configured to specifically hybridize to" as used herein means that the targethybridizing region of an amplification oligonucleotide, detection probe, or other oligonucleotide is designed to have a polynucleotide sequence that could target a sequence of the referenced M. genitalium target region. Such an oligonucleotide is not limited to targeting that sequence only, but is rather useful as a composition, in a kit, or in a method for targeting an M. genitalium target nucleic acid. The oligonucleotide is designed to function as a component of an assay for amplification and detection of M. genitalium from a sample, and therefore is designed to target M. genitalium in the presence of other nucleic acids commonly found in testing samples. "Specifically hybridize to" does not mean exclusively hybridize to, as some small level of hybridization to nontarget nucleic acids may occur. Rather, "specifically hybridize to" means that the oligonucleotide is configured to function in an assay to primarily hybridize the target so that an accurate detection of target nucleic acid in a sample can be determined.

[0070] The term "region," as used herein, refers to a portion of a nucleic acid wherein said portion is smaller than the entire nucleic acid. For example, when the nucleic acid in reference is an amplicon, the term may be used to refer to the smaller nucleotide sequence identified for hybridization by the target-hybridizing sequence of a probe.

[0071] As used herein, the phrase "or its complement, or an RNA equivalent or DNA / RNA chimeric thereof," with reference to a DNA sequence, includes (in addition to the referenced DNA sequence) the complement of the DNA sequence, an RNA equivalent of the referenced DNA sequence, an RNA equivalent of the complement of the referenced DNA sequence, a DNA / RNA chimeric of the referenced DNA sequence, and a DNA / RNA chimeric of the complement of the referenced DNA sequence.

[0072] Similarly, the phrase "or its complement, or a DNA equivalent or DNA / RNA chimeric thereof," with reference to an RNA sequence, includes (in addition to the referenced RNA sequence) the complement of the RNA sequence, a DNA equivalent of the referenced RNA sequence, a DNA equivalent of the complement of the referenced RNA sequence, a DNA / RNA chimeric of the referenced RNA sequence, and a DNA / RNA chimeric of the complement of the referenced RNA sequence.

[0073] As used herein, a "primer" is an oligomer that hybridizes to a template nucleic acid and has a 3' terminal hydroxyl group that can be extended by a polymerase (e.g., a DNA polymerase). A primer may be optionally modified (e.g., by including a 5' region that is non-complementary to the target sequence). Such modification can include functional additions, such as tags, promoters, or other non-target-specific sequences used or useful for manipulating or amplifying the primer or target oligonucleotide.

[0074] The term "amplify" is used in the broad sense to mean creating an amplification product that can be synthesized enzymatically with a DNA or RNA polymerase (including a reverse transcriptase). By “amplification” or “nucleic acid amplification” or “polynucleotide amplification” and the like is meant any known procedure for obtaining multiple copies, allowing for RNA and DNA equivalents, of a target polynucleotide sequence or its complement or fragments thereof. "Multiple copies" mean at least two copies. A "copy" does not necessarily mean perfect sequence complementarity or identity to the template sequence. Methods for amplifying mRNA are generally known in the art, and include reverse transcription PCR (RT-PCR). Another method which may be used is quantitative PCR (or Q-PCR).As used herein, the terms “coamplify” and “coamplifying” and variants thereof refer to a process wherein different target polynucleotide sequences are amplified in a single (i.e., the same) amplification reaction. For example, a nucleic acid analyte and an unrelated internal calibrator nucleic acid are “coamplified” when both nucleic acids are amplified in reactions taking place in a single tube, and when both amplification reactions share at least one reagent (e.g., deoxyribonucleotide triphosphates, enzyme, primer(s), etc.) in common.

[0075] As used herein, the terms "polymerase chain reaction" and "PCR" refer to an enzymatic reaction in which a segment of DNA is replicated from a target nucleic acid in vitro. The reactiongenerally involves extension of a primer on each strand of a target nucleic acid with a template dependent DNA polymerase to produce a complementary copy of a portion of that strand. The chain reaction comprises iterative cycles of denaturation of the DNA strands, for example by heating, followed by cooling to allow primer annealing and extension, resulting in an exponential accumulation of copies of the region of the target nucleic acid that is flanked by and that includes the primer binding sites. When an RNA target nucleic acid is amplified by PCR, it is generally converted to a DNA copy strand with an enzyme capable of reverse transcription. Exemplary enzymes include MMLV reverse transcriptase, AMV reverse transcriptase, as well as other enzymes that will be familiar to those having an ordinary level of skill in the art.

[0076] As used herein, "thermal cycling" refers to repeated changes of temperature, (z.e., increases or decreases of temperature) in a reaction mixture. Samples undergoing thermal cycling may shift from one temperature to another, stabilize at that temperature, transition to a second temperature or return to the starting temperature. The temperature cycle may be repeated as many times as required to study or complete the particular chemical reaction of interest.

[0077] By "amplicon" or "amplification product" is meant a nucleic acid molecule generated in a nucleic acid amplification reaction and which is derived from a target nucleic acid. An amplicon or amplification product contains a target nucleic acid sequence that may be of the same or oppositesense as the target nucleic acid. Preferred amplification products comprise DNA.

[0078] As used herein, a “signal” is a detectable quantity or impulse of energy, such as electromagnetic energy (e.g., light). Emission of light from an appropriately stimulated fluorophore is an example of a fluorescent signal. In some embodiments, “signal” refers to the aggregated energy detected in a single channel of a detection instrament (e.g., a fluorometer).

[0079] As used herein, a “background” signal is the signal e.g., a fluorescent signal) generated under conditions that do not permit a target nucleic acid-specific reaction (e.g., cleavage of a labeled oligonucleotide hydrolysis probe) to take place.

[0080] As used herein, a “nucleic acid analyzer” is an apparatus that amplifies, detects, and optionally quantifies nucleic acid analytes. Certain preferred nucleic acid analyzers include a temperature-controlled incubator (e.g., a block, plate, or chamber), a fluorometer in optical communication with contents of the temperature-controlled incubator, and one or more computers or processors that process data gathered by the fluorometer to quantify a nucleic acid analyte of interest.

[0081] As used herein a “channel” of an energy sensor device, such as a device equipped with an optical energy sensor, refers to a pre-defined band of wavelengths that can be detected or quantified to the exclusion of other bands of wavelengths. For example, one detection channel of afluorometer might be capable of detecting light energy emitted by one or more fluorescent labels over a range of wavelengths as a single event. Light emitted as the result of fluorescence can be quantified as relative fluorescence units (RFU) at a given wavelength, or over a band of wavelengths.

[0082] As used herein, the term "relative fluorescence unit" ("RFU") is a unit of measurement of fluorescence intensity. RFU varies with the characteristics of the detection means used for the measurement, and can be used as a measurement to compare relative intensities between samples and controls.

[0083] As used herein, the term "detection probe" (or simply "probe") refers to an oligomer that hybridizes specifically to a target sequence, including an amplified sequence, under conditions that promote nucleic acid hybridization, for detection of the target nucleic acid. Detection probes may be DNA, RNA, analogs thereof or combinations thereof (e.g., DNA / RNA chimerics), and they may be labeled or unlabeled. Detection probes may further include alternative backbone linkages (e.g. , 2'-O-methyl linkages). A probe's target sequence generally refers to the specific sequence within a larger sequence which the probe hybridizes specifically. A detection probe may include targetspecific sequence(s) and non-target-specific sequence(s). Such non-target-specific sequences can include sequences which will confer a desired secondary or tertiary structure, such as a hairpin structure, which can be used to facilitate detection and / or amplification (see, e.g., U.S. Patent Nos. 5,118,801, 5,312,728, 6,835,542, and 6,849,412). Probes of a defined sequence may be produced by techniques known to those of ordinary skill in the art, such as by chemical synthesis, and by in vitro or in vivo expression from recombinant nucleic acid molecules.

[0084] By "hybridization" or "hybridize" is meant the ability of two completely or partially complementary nucleic acid strands to come together e.g., under specified hybridization assay conditions) in a parallel or antiparallel orientation to form a stable structure having a doublestranded region. The two constituent strands of this double- stranded structure, sometimes called a hybrid, are held together by hydrogen bonds. Although these hydrogen bonds most commonly form between nucleotides containing the bases adenine and thymine or uracil (A and T or U) or cytosine and guanine (C and G) on single nucleic acid strands, base pairing can also form between bases which are not members of these "canonical" pairs. Non-canonical base pairing is well-known in the art. See, e.g., R. L. P. Adams et al., The Biochemistry of the Nucleic Acids (1 1th ed. 1992).

[0085] By "preferentially hybridize" is meant that an amplification or detection probe oligomer can hybridize to its target nucleic acid to form stable oligomerrtarget hybrid, but not form a sufficient number of stable oligomermon-target hybrids. Amplification and detection oligomers that preferentially hybridize to a target nucleic acid are useful to amplify and detect target nucleic acids,but not non-targeted organisms, especially phylogenetically closely related organisms. Thus, the oligomer hybridizes to target nucleic acid to a sufficiently greater extent than to non-target nucleic acid to enable one having ordinary skill in the art to accurately amplify and / or detect the presence (or absence) of nucleic acid derived from the specified target as appropriate. In general, reducing the degree of complementarity between an oligonucleotide sequence and its target sequence will decrease the degree or rate of hybridization of the oligonucleotide to its target region. However, the inclusion of one or more non-complementary nucleosides or nucleobases may facilitate the ability of an oligonucleotide to discriminate against non-target organisms. Preferential hybridization can be measured using techniques known in the art and described herein, such as in the examples provided below. In some embodiments, there is at least a 3-fold difference between target and non- target hybridization signals in a test sample, or at least a 5-fold difference between target and non- target hybridization signals in a test sample, or at least a 10-fold difference between target and non- target hybridization signals in a test sample, or at least a 1 OO-fold difference, or at least a 1 ,000-fold difference. In some embodiments, non-target hybridization signals in a test sample are no more than the background signal level.

[0086] As used herein, "label" or "detectable label" refers to a moiety or compound attached or joined, directly or indirectly, to a probe that is detected or that leads to a detectable signal. Direct joining may use covalent bonds or non-covalent interactions (e.g., hydrogen bonding, hydrophobic or ionic interactions, and chelate or coordination complex formation) whereas indirect joining may use a bridging moiety or linker (e.g., via an antibody or additional oligonucleotide(s)). Any detectable moiety may be used, including a radionuclide, a ligand such as biotin or avidin or even a polynucleotide sequence, an enzyme, an enzyme substrate, a reactive group, a chromophore such as a dye or particle (e.g. , a latex or metal bead) that imparts a detectable color, a luminescent compound (e.g., bioluminescent, phosphorescent, or a chemiluminescent compound), and a fluorescent compound or moiety (i.e., fluorophore). Embodiments of fluorophores include those that absorb light in the range of about 495 to 650 nm and emit light in the range of about 520 to 670 nm, which include those known as FAM™, TET™, CAL FLUOR™ (Orange or Red), and QUASAR™ compounds. Fluorophores may be used in combination with a quencher molecule that absorbs light when in close proximity to the fluorophore to diminish background fluorescence.Such quenchers are well known in the art and include, for example, BLACK HOLE QUENCHER™ (or BHQ™) or TAMRA™ compounds. Quencher moieties modified to include minor groove -binding (sometimes “MGB”) moieties are considered to be quenchers within the context of the disclosure.

[0087] Sequences are "sufficiently complementary" if they allow stable hybridization of two nucleic acid sequences, e.g., stable hybrids of probe and target sequences, although the sequences need not be completely complementary. That is, a "sufficiently complementary" sequence that hybridizes to another sequence by hydrogen bonding between a subset series of complementary nucleotides by using standard base pairing (e.g., G:C, A:T, or A:U), although the two sequences may contain one or more residues (including abasic positions) that are not complementary so long as the entire sequences in appropriate hybridization conditions to form a stable hybridization complex. Sufficiently complementary sequences may be at least about 80%, at least about 90%, or completely complementary in the sequences that hybridize together. Appropriate hybridization conditions are well-known to those skilled in the art, can be predicted based on sequence composition, or can be determined empirically by using routine testing (e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nded. at §§ 1.90-1.91, 7.37-7.57, 9.47-9.51 and 11.47- 11.57, particularly §§ 9.50-9.51, 11.12-11.13, 11.45-11.47 and 11.55-11.57).

[0088] "Sample preparation" refers to any steps or method that treats a sample for subsequent amplification and / or detection of M. genitalium nucleic acids present in the sample. Samples may be complex mixtures of components of which the target nucleic acid is a minority component. Sample preparation may include any known method of concentrating components, such as microbes or nucleic acids, from a larger sample volume, such as by filtration of airborne or waterborne particles from a larger volume sample or by isolation of microbes from a sample by using standard microbiology methods. Sample preparation may include physical disruption and / or chemical lysis of cellular components to release intracellular components into a substantially aqueous or organic phase and removal of debris, such as by using filtration, centrifugation or adsorption. Sample preparation may include use of a nucleic acid oligonucleotide that selectively or non-specifically captures a target nucleic acid and separates it from other sample components (e.g., as described in US Patent No. 6,110,678 and International Patent Application Pub. No. WO 2008 / 016988, each incorporated by reference herein).

[0089] "Separating" (and grammatical equivalents) or "purifying" (and grammatical equivalents) means that one or more components of a sample are removed or separated from other sample components. Sample components include target nucleic acids usually in a generally aqueous solution phase, which may also include cellular fragments, proteins, carbohydrates, lipids, and other nucleic acids. "Separating" or "purifying" does not connote any degree of purification. Typically, separating or purifying removes at least 70%, or at least 80%, or at least 95% of the target nucleic acid from other sample components.

[0090] The term "specificity," in the context of an amplification and / or detection system, is used herein to refer to the characteristic of the system which describes its ability to distinguish between target and non-target sequences dependent on sequence and assay conditions. In terms of nucleic acid amplification, specificity generally refers to the ratio of the number of specific amplicons produced to the number of si de -products (e.g., the signal -to-noise ratio). In terms of detection, specificity generally refers to the ratio of signal produced from target nucleic acids to signal produced from non-target nucleic acids.

[0091] The term "sensitivity" is sometimes used herein to refer to the precision with which a nucleic acid amplification reaction can be detected or quantitated. The sensitivity of an amplification reaction is generally a measure of the smallest copy number of the target nucleic acid that can be reliably detected in the amplification system, and will depend, for example, on the detection assay being employed, and the specificity of the amplification reaction, e.g., the ratio of specific amplicons to side-products.

[0092] A “reaction mixture” is a combination of reagents (e.g., oligonucleotides, target nucleic acids, enzymes, etc.) in a single reaction vessel.

[0093] As used herein, a “multiplex” assay is a type of assay that is able to detect or measure multiple analytes (e.g. , two or more nucleic acid sequences) in a single run of the assay. It is distinguished from procedures that measure one analyte per reaction mixture. A multiplex assay can be carried out by combining into a single reaction vessel the reagents (e.g., probe reagents) for two or more different target sequences. In some embodiments, the same species of fluorescent reporter is detected in each of the assays of the multiplex.

[0094] As used herein, the term "donor" refers to a moiety (e.g., a fluorophore) that absorbs at a first wavelength and emits at a second, longer wavelength. The term "acceptor" refers to a moiety such as a fluorophore, chromophore, or quencher and that can absorb some or most of the emitted energy from the donor when it is near the donor group (e.g., between 1-100 nm). An acceptor may have an absorption spectrum that overlaps the donor's emission spectrum. Generally, if the acceptor is a fluorophore, it then re-emits at a third, still longer wavelength; if it is a chromophore or quencher, it releases the energy absorbed from the donor without emitting a photon. In some preferred embodiments, alteration in energy levels of donor and / or acceptor moieties are detected (e.g., via measuring energy transfer, for example by detecting light emission) between or from donors and / or acceptor moieties). In some preferred embodiments, the emission spectrum of an acceptor moiety is distinct from the emission spectrum of a donor moiety such that emissions (e.g., of light and / or energy) from the moieties can be distinguished (e.g., spectrally resolved) from each other.

[0095] As used herein, “attached” (e.g., two things are “attached”) means chemically bonded together. For example, a fluorophore moiety is “attached” to an oligonucleotide probe when it is chemically bonded to the structure of the oligonucleotide probe.

[0096] As used herein, an “interactive” label pair refers to a donor moiety and an acceptor moiety (e.g., a quencher moiety) being attached to the same oligonucleotide probe, and being in energy transfer relationship (i.e., whether by a FRET or a non- FRET mechanism) with each other. A signal (e.g., a fluorescent signal) can be generated when the donor and acceptor moieties are separated, for example by hybridization and / or cleavage of a labeled oligonucleotide probe.

[0097] As used herein, emission from a donor moiety (e.g., a fluorophore) is “quenched” when the emission of a photon from the donor is prevented because an acceptor moiety (e.g., a quencher) is sufficiently close. For example, emission from a donor moiety is quenched when the donor moiety and the acceptor moiety are both attached to the same oligonucleotide probe.

[0098] As used herein, a “nucleic acid analyzer” is an apparatus or instrument that amplifies, detects, and optionally quantifies nucleic acid analytes. Certain preferred nucleic acid analyzers include a temperature-controlled incubator (e.g. , a block, plate, or chamber), a fluorometer in optical communication with contents of the temperature-controlled incubator, and one or more computers or processors that process data gathered by the fluorometer to quantify a nucleic acid analyte of interest. In some embodiments, preferred nucleic acid analyzers perform enzyme -based reactions that amplify or increase the number of copies of a target nucleic acid that is to be quantified. In other embodiments, “signal amplification” is used to detect and / or quantify the target nucleic acid that is to be quantified. An example signal amplification system is provided by the “serial invasive signal amplification reaction” disclosed by Hall et al., in Proc. Natl. Acad. Sci. USA 97:8272-8277 (2000).

[0099] As used herein, a “run curve” (sometimes “growth curve” herein) refers to the characteristic partem of appearance of a synthetic product, such as an amplicon, in a reaction as a function of time or cycle number (i.e., reaction progress parameters). A run curve is conveniently represented as a two-dimensional plot of time or cycle number (x-axis) against some indicator of product amount, such as a fluorescence measurement (y-axis). Some, but not all, run curves have a sigmoid-shape.

[0100] As used herein, the phrase “threshold-based indicia of amplification” refers to indicia of amplification that measure the time or cycle number when a growth curve signal crosses an arbitrary value or threshold (e.g., a threshold fluorescence value). Cycle threshold (Ct) and TTime values are examples of threshold-based indicia of amplification, while TArc and OTArc determinations are examples of non-threshold-based indicia of amplification.

[0101] As used herein, the phrase “as a function of’ describes the relationship between a dependent variable (z.e., a variable that depends on one or more other variables) and an independent variable (i.e., a variable that may have its value freely chosen without considering the values of any other variables), wherein each input value for the independent variable relates to exactly one output value for the dependent variable. Conventional notation for an equation that relates a y-value (i.e. , the dependent variable) “as a function of’ an x-value (i.e., the independent variable) is y = f(x).

[0102] As used herein, a “computer” is an electronic device capable of receiving and processing input information using software instructions to generate an output. The computer may be a standalone device (e.g., a personal computer), or may be an integrated component of an instrument (e.g., a nucleic acid analyzer that amplifies a target nucleic acid and monitors synthesis of amplification products as a function of reaction cycle number or time). Particularly embraced by the term is an embedded processor resident within an analyzer instrument, and harboring embedded software instructions (sometimes referred to a “firmware”).

[0103] As used herein, a “system” is an arrangement of parts or components organized to cooperate with one another. For example, a system may include an instrument that detects nucleic acids in a sequence-specific manner, and a computer programmed with software to analyze results, where the computer and the instrument are in communication with each other.

[0104] As used herein, “apparatus” generally refers to the collection of equipment (e.g., tools, instruments, etc.) needed for a particular purpose or function.

[0105] As used herein, an “instrument” is a tool, device, or implement for performing a task. In some embodiments, an instrument is a device contained within a single housing or situated on common support structure (e.g., a single chassis).

[0106] The term “wild-type” (also “WT” herein) refers to a gene or gene product that has the characteristics of that gene or gene product when isolated from a common, naturally occurring source. In the context of the present disclosure, wild-type M. genitalium is macrolide-sensitive. The phrase, "wild-type (macrolide- sensitive) M. genitalium” is used to confirm that wild-type M. genitalium is associated with sensitivity to macrolide antibiotics.

[0107] As used herein, a “threshold” or “threshold cutoff’ refers to a quantitative limit used for interpreting experimental results, where results above and below the cutoff lead to opposite conclusions. For example, a measured signal falling below a cutoff may indicate the absence of a particular target, but a measured signal that exceeds the same cutoff may indicate the presence of that target. By convention, a result that meets a cutoff (i.e., has exactly the cutoff value) is given the same interpretation as a result that exceeds the cutoff.

[0108] As used herein, a “threshold cycle number’’ refers to indicia of amplification that measure the time or cycle number when a real-time run curve signal crosses an arbitrary value or threshold. “TTime” and “Ct” determinations are examples of threshold-based indicia of amplification. Other methods involve performing a derivative analysis of the real-time run curve. For this disclosure, TArc and OTArc also can be used to determine when a real-time run curve signal crosses an arbitrary value (e.g., corresponding to a maximum or minimum angle in curvature, respectively). Methods of Time determination are disclosed in U.S. 8,615,368; methods of Ct determination are disclosed in EP 0640828 Bl ; derivative-based methods are disclosed in U.S. 6,303,305; and methods of TArc and OTArc determination are disclosed in U.S. 7,739,054. Those having an ordinary level of skill in the art will be aware of variations that also can be used for determining threshold cycle numbers.

[0109] As used herein, a “reaction vessel” or “reaction receptacle” is a container for holding a reaction mixture. Examples include individual wells of a multi well plate, and plastic tubes (e.g., including individual tubes within a formed linear array of a multi-tube unit, etc.). However, it is to be understood that any suitable container may be used for containing the reaction mixture.

[0110] As used herein, a “vial” is a container, typically cylindrical, for holding liquid or dry (e.g., lyophilized) reagents. Vials commonly are used for packaging oligonucleotide or enzyme reagents into kits. Vials can be made of a variety of materials, such as glass or plastic.

[0111] As used herein, the phrase “a nucleic acid locus of M. genitalium that is characteristic of macrolide sensitivity or macrolide resistance” refers to a region of the M. genitalium 23S rRNA or 23S rDNA sequence containing base positions corresponding to 2058 and 2059 in the E. coli numbering system. The nucleic acid locus characteristic of macrolide sensitivity or macrolide resistance can include, for example, the sequence extending from position 2054 to 2064 of the rRNA in the E. coli numbering system. This range embraces each of SEQ ID NOs:18 (wild-type) where both base positions are occupied by “A” residues, SEQ ID NO:27 (2058T), SEQ ID NO:28 (2058C), SEQ ID NO:29 (2058G), SEQ ID NO:30 (2059C), and SEQ ID NO:31 (2059G). Alternatively, the nucleic acid locus characteristic of macrolide sensitivity or macrolide resistance can include, for example, the sequence extending from position 2049 to 2069 in the E. coli numbering system. This range embraces each of SEQ ID NO:32 (wild-type) where base positions 2058 and 2059 are both occupied by “A” residues, SEQ ID NO:33 (2058T), SEQ ID NO:34 (2058C), SEQ ID NO:35 (2058G), SEQ ID NO:36 (2059C), and SEQ ID NO:37 (2059G).

[0112] As used herein, the phrase “Mgen Res Detection Locus” refers to a region of 23 S rRNA or rDNA of M. genitalium that identifies, or is characteristic of macrolide sensitivity or macrolide resistance. The Mgen Res Detection Locus includes base positions corresponding to positions 2058and 2059 in the E. coli numbering system. Single nucleotide polymorphisms associated with macrolide resistance are identified herein.

[0113] As used herein, the phrase “Mgen Detection Locus” refers to a nucleic acid sequence within the bacterial genome that is useful for determining that a sample included nucleic acids of M. genitalium. The Mgen Detection Locus is present in both macrolide-sensitive M. genitalium and macrolide-resistant M. genitalium, but not present in nucleic acids of M. pneumoniae. The Mgen Detection Locus may be found in the 23S rRNA or rDNA of M. genitalium, but optionally may be found in a different RNA or gene that identifies M. genitalium with specificity.DETAILED DESCRIPTION

[0114] Disclosed herein are oligonucleotides, compositions, kits, and methods that can be used to amplify and detect genetic markers of macrolide resistance in M. genitalium. While nucleic acids of wild-type (macrolide-sensitive) M. genitalium may be amplified, the results of that amplification are substantially separated from amplification of macrolide-resistant M. genitalium by a results- processing procedure prior to determining the presence or absence of macrolide-resistant M. genitalium in a sample undergoing testing.

[0115] The disclosed method can be used for detecting and identifying macrolide-resistant M. genitalium by testing naive samples, but can also be used as a reflex assay that particularly reports the presence or absence of macrolide resistance in a sample already known to contain M. genitalium. The reflex assay approach can yield a superior positive predictive value for the assay. Positive predictive value correlates with prevalence. By testing a reflex sample set, the disclosed assay is used for testing samples known to be positive for M. genitalium, thereby maximizing the positive predictive value of the assay.

[0116] The disclosed technique synthesizes multiple copies of an M. genitalium target nucleic acid and detects the sequences of macrolide-resistant variants. This can involve a pair of oligonucleotides, where one oligonucleotide is configured to hybridize to a sense strand of an M. genitalium nucleic acid and the other is configured to hybridize to an antisense strand of an M. genitalium nucleic acid. Such oligonucleotides include primer pairs for PCR or other forms of amplification. The amplification product (e.g., a PCR product) can include both wild-type sequences and sequences associated with resistance to macrolide antibiotics. In some embodiments, preferred probes that detect sequences indicating macrolide resistance also crosshybridize to sequences associated with macrolide sensitivity, and can detect the wild-type sequences. In preferred embodiments, the presence of M. genitalium 23S ribosomal nucleic acidsequences is determined using a different amplification product from the one used to establish the presence of nucleic acids harboring macrolide resistance markers.

[0117] As indicated above, the disclosed method or assay can be used as a reflex test to a positive result from a different assay that detects M. genitalium to determine if an infection with this organism is resistant to azithromycin (a macrolide antibiotic). Stated differently, the disclosed method can be used for testing samples already known to contain M. genitalium bacteria. Patients identified as having azithromycin-resistant infections can be diverted to treatment with fluoroquinolones, the last known antibiotic class that is effective against M. genitalium.

[0118] The assay method can be carried out according to different assay formats. In preferred embodiments, synthesis of M. genitalium-specific amplification products can be monitored periodically as the amplification reaction is taking place. This is sometimes referred to as a “realtime” formatted assay.

[0119] In some embodiments, one or more oligonucleotides, such as a primer set (defined as at least two primers configured to generate or detect an amplicon from a target sequence) or a primer set and an additional oligonucleotide (e.g., a probe) which is optionally non-extendible and / or labeled, are configured to hybridize to an amplification product of M. genitalium 23S ribosomal nucleic acid. In some embodiments, the primer set includes at least one reverse primer configured to hybridize to the 23S rRNA of M. genitalium, and at least one forward primer configured to hybridize to an extension product of the reverse primer using the ribosomal nucleic acid of M. genitalium as the template. When present, the additional oligonucleotide (e.g., a probe oligonucleotide) can be configured to hybridize to an amplicon produced by the primer set.

[0120] In some embodiments, a plurality of oligonucleotides, optionally non-extendible and / or labeled, are provided which collectively hybridize to one or more sequences within an M. genitalium nucleic acid amplification product. In some embodiments, a plurality of oligonucleotides, such as a plurality of primers or a plurality of primers and probes are provided which collectively hybridize to opposite strands of a double- stranded amplification product. In some embodiments, amplification or detection of the sequence indicative of M. genitalium discriminates the presence of M. genitalium from many other Mycoplasma species. Optionally, amplification or detection of the sequence indicative of M. genitalium can be highly specific for M. genitalium, so that nucleic acids from no other known organisms are detected.

[0121] In some embodiments, one or more oligonucleotides in a set, kit, composition, or reaction mixture include one or more methylated cytosine (e.g., 5 -methylcytosine or sometimes “C5M”) residues. In some embodiments, at least about half of the cytosines in an oligonucleotide are methylated. In some embodiments, all or substantially all (e.g., all but one or two) of the cytosinesin an oligonucleotide are methylated. For example, one or more cytosines at the 3 ’-end or within 2, 3, 4, or 5 bases of the 3’-end can be methylated. Alternatively, one or more cytosines at the 3’-end or within 2, 3, 4, or 5 bases of the 3’-end can be unmethylated.

[0122] In some embodiments, one or more oligonucleotides in a set, kit, composition, or reaction mixture include one or more 5-propynyl-modified cytidine (e.g., 5-propynyl-2’ -deoxycytidine) residues. In some embodiments, at least about half of the cytidnes in an oligonucleotide are 5- propynylcytidine analogs. In some embodiments, all or substantially all (e.g., all but one or two) of the cytidines in an oligonucleotide are 5-propynylcytidine analogs. For example, one or more cytidines at the 3’-end or within 2, 3, 4, or 5 bases of the 3’-end can be 5-propynylcytidine analogs.

[0123] In some embodiments, one or more oligonucleotides in a set, kit, composition, or reaction mixture include one or more 5-propynyl-2’-deoxyuridine residues (sometimes “propyne dU” or “Tdu”) as substitutes for thymidine (“T”). In some embodiments, at least about half of the thymidines in an oligonucleotide are 5-propynyl-2’ -deoxyuridine analogs. In some embodiments, all or substantially all (e.g., all but one or two) of the thymidines in an oligonucleotide are 5- propynyl-2’ -deoxyuridine analogs. For example, one or more thymidines at the 3 ’-end or within 2, 3, 4, or 5 bases of the 3’-end can be 5-propynyl-2’-deoxyuridine analogs.

[0124] M. genitalium macrolide resistance can be assessed using reverse-transcription PCR of M. genitalium 23S rRNA, with hybridization or hydrolysis probe-based detection to permit real-time monitoring of amplicon synthesis. To detect mutations at either of base locations 2058 or 2059 (E. coli numbering in region V of the 23S rRNA), which have been shown to be associated with M. genitalium macrolide resistance (see Couldwell et al., Infect. Drug Resist. 8:147-161 (2015)), a collection of probes was used. Macrolide resistance is indicated when there is a C or G at position 2059. Alternatively, macrolide resistance is indicated when the naturally occurring A residue at position 2058 is replaced by any of G, C, or T. Either of these conditions (i.e., mutation at one of two adjacent nucleotide positions) can result in macrolide resistance, and it is unnecessary for both positions to be mutated simultaneously to produce the drug -resistant condition. Optionally, each different base change indicative of macrolide resistance is detected using a different hybridization probe (e.g., a hydrolysis probe, useful in a TaqMan- formatted assay, labeled with each of a fluorophore and a quencher), where the detectable label is the same (e.g., the same fluorophore chemical species) for all probes. By this approach, macrolide resistance can be detected without identifying the position or identity of the base change leading to the antibiotic-resistant phenotype. In this way any genotype being associated with macrolide resistance can be indicated by a single type of fluorescent signal (e.g., detected in the ROX channel of a fluorometer).

[0125] In some embodiments, an oligonucleotide is provided that includes a label and / or is non- extendable. Such an oligonucleotide can be used as a probe or as part of a probe system. In some embodiments, the label is a non-nucleotide label. Example labels include compounds that emit a detectable light signal, such as fluorophores or luminescent (e.g., chemiluminescent) compounds that can be detected in a homogeneous mixture. More than one label, and more than one type of label, can be present on a particular probe, or detection can rely on using a mixture of probes in which each probe is labeled with a compound that produces a detectable signal (see e.g., U.S. Pat. Nos. 6,180,340 and 6,350,579). Labels can be attached to a probe by various means including covalent linkages, chelation, and ionic interactions. In some embodiments the label is covalently attached. For example, in some embodiments, a detection probe has an attached chemiluminescent label such as, for example, an acridinium ester (AE) compound (see e.g., U.S. Pal. Nos. 5,185,439; 5,639,604; 5,585,481; and 5,656,744). A label, such as a fluorescent or chemiluminescent label, can be attached to the probe by a non-nucleotide linker (see e.g., U.S. Pat. Nos. 5,585,481; 5,656,744; and 5,639,604).

[0126] In some embodiments, a probe can harbor two different labels (i.e., “first” and “second” labels), where the two labels interact with each other in an energy transfer relationship. These probes are sometimes referred to as “dual-label” probes. In one example, the first label can be a fluorescent moiety, and the second label can be a quencher moiety. Such probes can be used where hybridization of the probe to a target or amplicon followed by nucleolysis (i.e., hydrolysis of nucleic acid) by a polymerase including 5 ’-3’ exonuclease activity results in liberation of the fluorescent label and thereby increased fluorescence. This embraces the well known TaqMan™ assay format.

[0127] Examples of interacting donor / acceptor label pairs that can be used in connection with the disclosure include fluorescein / tetramethylrhodamine, lAEDANS / fluororescein, EDANS / DABCYL, coumarin / DABCYL, fluorescein / fluorescein, BODIPY® FL / BODIPY® FL, fluorescein / DABCYL, lucifer yellow / DABCYL, BODIPY® / DABCYL, eosine / DABCYL, erythrosine / DABCYL, tetramethylrhodamine / DABCYL, Texas Red / DABCYL, CY5 / BHQ1®, CY5 / BHQ2®, CY3 / BIIQ1®, CY3 / BIIQ2® and fluorescein / QSY7® dye. Those having an ordinary level of skill in the art will understand that when donor and acceptor dyes are different, energy transfer can be detected by the appearance of sensitized fluorescence of the acceptor or by quenching of donor fluorescence. Non-fluorescent acceptors such as DABCYL and the QSY7® dyes advantageously eliminate the potential problem of background fluorescence resulting from direct (i.e., nonsensitized) acceptor excitation. Exemplary fluorophore moieties that can be used as one member of a donor- acceptor pair include fluorescein, HEX, ROX, and the CY dyes (such as CY5). Exemplaryquencher moieties that can be used as another member of a donor- acceptor pair include DAB CYL BLACKBERRY QUENCHER® which are available from Berry and Associates (Dexter, MI) , and the BLACK HOLE QUENCHER® moieties which are available from Biosearch Technologies, Inc., (Novato, Calif). One of ordinary skill in the art will be able to use appropriate pairings of donor and acceptor labels for use in various detection formats (e.g., FRET, TaqMan™, Invader®, etc.). Exemplified herein is the combination of a CalRed610 fluorescent donor moiety and a BHQ2® quencher moiety (Biosearch Technologies, Inc.; Petaluma, CA); and a fluorescein (FAM) fluorescent donor moiety and a BHQ1® quencher moiety (Biosearch Technologies, Inc.; Petaluma, CA).

[0128] Optionally, a probe oligonucleotide may be non-extendable. The oligonucleotide can be rendered non-extendable by the presence of a 3’-adduct (e.g., 3 ’-phosphorylation or 3 ’-alkanediol), having a 3’-terminal 3 ’-deoxynucleotide (e.g., a terminal 2 ’,3 ’-dideoxy nucleotide), having a d’terminal inverted nucleotide (e.g., in which the last nucleotide is inverted such that it is joined to the penultimate nucleotide by a 3’ to 3’ phosphodiester linkage or analog thereof, such as a phosphorothioate), or having an attached fluorophore, quencher, or other label that interferes with extension (possibly but not necessarily attached via the 3’ position of the terminal nucleotide). In some embodiments, the 3’-terminal nucleotide is not methylated. In some embodiments, a detection oligonucleotide includes a 3’-terminal adduct such as a 3’-alkanediol (e.g., hexanediol).

[0129] In some embodiments, an oligonucleotide, such as a probe, is configured to specifically hybridize to an M. genitalium amplicon. The oligonucleotide can include or consist of a targethybridizing sequence sufficiently complementary to the amplicon for specific hybridization. Optionally, the target-hybridizing sequence can be joined at its 5 ’-end to a nucleotide sequence that is not complementary to the amplicon being detected.

[0130] Also provided are kits for performing the methods described herein. “Kits” refer to packaged products that can be provided to an end-user, and typically will include one or more vials or containers holding one type of oligonucleotide, or a combination of different oligonucleotides or other reagents. A kit in accordance with the present disclosure can include at least one or more of the following: an amplification oligonucleotide (e.g., a primer), or oligonucleotide combination (e.g., a pair of primers) capable of amplifying an M. genitalium 23S ribosomal nucleic acid, or at least one detection probe as for determining the presence or absence of one or more macrolide resistance markers in an M. genitalium amplification product. In some embodiments, any oligonucleotide, or combination of oligonucleotides, described herein is present in the kit. The kits can further include a number of optional components such as, for example, capture probes (e.g., poly-(k) capture probes as described in US 2013 / 0209992), as well as a delectably labeled probe(e.g., a dual-labeled probe) that detects a wild-type M. genitalium sequence in an amplicon produced in the same reaction that amplified the macrolide resistance marker(s). To be clear, kits can include individual oligonucleotides or combinations of oligonucleotides in a single vial. Probe oligonucleotides, optionally including a detectable label (such as a fluorescent label), can be packaged individually, or can be packaged in combination with each other. Vials containing individual probes (e.g., each vial containing a different probe) optionally can be packaged into a single container, such as a box. Alternatively, kits can include one or more vials, where an individual vial contains a mixture of two or more different oligonucleotides (e.g., either primers and / or probes).

[0131] Other reagents that can be present in the kits include reagents suitable for performing in vitro amplification such as, for example, buffers, salt solutions, appropriate nucleotide triphosphates (e.g. , dATP, dCTP, dGTP, and one or both of dTTP or dUTP; and / or ATP, CTP, GTP and UTP), and / or enzymes (e.g., a thermostable DNA polymerase, and / or reverse transcriptase and / or RNA polymerase and / or FEN enzyme), and will typically include test sample components, in which an M. genitalium target nucleic acid may or may not be present. In addition, for a kit that includes a detection probe together with an amplification oligonucleotide combination, selection of amplification oligonucleotides and detection probe oligonucleotides for a reaction mixture are linked by a common target region (i.e., the reaction mixture will include a probe that hybridizes to a sequence amplifiable by an amplification oligonucleotide combination of the reaction mixture). In certain embodiments, the kit further includes a set of instructions for practicing methods in accordance with the present disclosure, where the instructions can be associated with a package insert and / or the packaging of the kit or the components thereof.

[0132] Any method disclosed herein is also to be understood as a disclosure of corresponding uses of materials involved in the method directed to the purpose of the method. Any of the oligonucleotides including an M. genitalium sequence and any combinations (e.g., kits and compositions, including but not limited to reaction mixtures) including such an oligonucleotide are to be understood as also disclosed for use in detecting or quantifying macrolide-resistant M. genitalium, and for use in the preparation of a composition for detecting macrolide-resistant M. genitalium.

[0133] Broadly speaking, methods can employ one or more of the following elements: target capture, in which M. genitalium nucleic acid (e.g., from a sample, such as a clinical sample) is annealed to a capture oligonucleotide (e.g., a specific or nonspecific capture oligonucleotide); isolation (e.g., washing, to remove material not associated with a capture oligonucleotide); amplification; and amplicon detection, which for example can be performed in real-time withamplification. Certain embodiments involve each of the foregoing steps. Certain embodiments involve exponential amplification, optionally with a preceding linear amplification step. Certain embodiments involve exponential amplification and amplicon detection. Certain embodiments involve any two of the components listed above. Certain embodiments involve any two elements listed adjacently above (e.g., washing and amplification, or amplification and detection).

[0134] In some embodiments, amplification includes (1) contacting a nucleic acid sample with at least two oligonucleotides for amplifying a region of M. genitalium 23S ribosomal nucleic acid, where the amplified region includes positions corresponding to positions 2058 and 2059 of region V in E. colt 23S rRNA. The oligonucleotides can include at least two amplification oligonucleotides (e.g., one oriented in the sense direction and one oriented in the antisense direction for exponential amplification); (2) performing an in vitro nucleic acid amplification reaction, where any M. genitalium 23 S ribosomal nucleic acid target present in the sample is used as a template for generating an amplification product; and (3) detecting the presence or absence of markers of macrolide resistance in the amplification product, thereby determining the presence or absence of macrolide-resistant M. genitalium in the sample. The markers of macrolide resistance include a C or G at position 2059, or a change from A to any of G, C, or T at position 2058.

[0135] Methods in accordance with the present disclosure can further include the step of obtaining the sample to be subjected to subsequent steps of the method. In certain embodiments, “obtaining” or “providing” or “receiving” (including grammatical variants thereof) a sample to be used includes, for example, receiving the sample at a testing facility or other location where one or more steps of the method are performed, and / or retrieving the sample from a location (e.g., from storage or other depository) within a facility where one or more steps of the method are performed. Alternatively, the step of obtaining can involve lysing M. genitalium cells to release nucleic acids. Optionally, a target capture step for enhancement of M. genitalium rRNA can be included as a component of the obtaining step.

[0136] Exponentially amplifying a target sequence can utilize an in vitro amplification reaction using at least two amplification oligonucleotides that flank a target region to be amplified. In some embodiments, at least two amplification oligonucleotides as described herein are provided. The amplification reaction can be temperature-cycled or isothermal. Suitable amplification methods include, for example, replicase-mediated amplification, polymerase chain reaction (PGR), ligase chain reaction (LCR), strand-displacement amplification (SDA), and transcription-mediated amplification (TMA).

[0137] A detection step can be performed using any of a variety of known techniques to detect a signal specifically associated with the amplified target sequence, such as by hybridizing theamplification product with a labeled detection probe and detecting a signal resulting from the labeled probe (including from label released from the probe following hybridization). In some embodiments, the labeled probe includes a second moiety, such as a quencher or other moiety that interacts with the first label, as discussed above. The detection step can also provide additional information on the amplified sequence, such as all or a portion of its nucleic acid sequence. Detection can be performed after the amplification reaction is completed, but preferably is performed simultaneously with amplifying the target region (e.g., in real-time). In one embodiment, the detection step allows homogeneous detection (e.g., detection of the hybridized probe without removal of unhybridized probe from the mixture). In some embodiments, the nucleic acids are associated with a surface that results in a physical change, such as a detectable electrical change. Amplified nucleic acids can be detected by concentrating them in or on a matrix and detecting the nucleic acids or dyes associated with them (e.g., an intercalating agent such as ethidium bromide or SYBR® dye), or detecting an increase in dye associated with nucleic acid in solution phase. Other methods of detection can use nucleic acid detection probes configured to hybridize to a sequence in the amplified product and detecting the presence of the probe:product complex, or by using a complex of probes that can amplify the detectable signal associated with the amplified products (e.g., U.S. Pat. Nos. 5,424,413; 5,451,503; and 5,849,481; each incorporated by reference herein). Directly or indirectly labeled probes that specifically associate with the amplified product provide a detectable signal that indicates the presence of the target nucleic acid in the sample. In particular, the amplified product will contain a target sequence in or complementary to a sequence in the M. genitalium chromosome, and a probe will bind directly or indirectly to a sequence contained in the amplified product to indicate the presence of macrolide-resistant M. genitalium nucleic acid in the tested sample.

[0138] The disclosed assay can employ a target capture step as part of a procedure to obtain and isolate 23S rRNA from M. genitalium, then reverse transcription PCR with real-time detection to amplify and detect DNA copies of the 23S rRNA harboring marker(s) of macrolide resistance. A mixture of dual-labeled probes (e.g., labeled with a fluorophore and a quencher) can be used to interrogate base positions 2058 and 2059, which are mutated in M. genitalium that is resistant to macrolide antibiotics (e.g., azithromycin). Preferably, dual-labeled probes that produce signals indicating the presence of nucleic acid markers of macrolide resistance do not also produce signals indicating the presence of wild-type nucleic acids associated with macrolide-sensitive M. genitalium.

[0139] In some embodiments, a single fluorophore species produces signals indicating the presence of any of the macrolide resistance markers. Importantly, the disclosed technique can be used fordetecting the genetic markers of macrolide resistance, even though nucleic acids of wild-type (macrolide-sensitive) M. genitalium may be present in a mixed infection, and even though the wildtype sequence may be detected by cross-hybridization to one or more probes used to detect the nucleic acid associated with macrolide resistance.

[0140] Briefly, the target capture method used in the presently disclosed assay can employ an oligonucleotide probe immobilized directly to a magnetically attractable solid support (i.e., the “immobilized probe”) and a “capture probe” (or sometimes “target capture probe” or “target capture oligonucleotide”) that bridged the immobilized probe and the 23S M. genitalium target ribosomal nucleic acid to form a hybridization complex that could be separated from other components in the mixture. An illustrative instrument workstation that can be used to carry out such a purification step is disclosed by Acosta et al., in U.S. Patent No. 6,254,826, the disclosure of which is incorporated by reference. The capture probe is preferably designed so that the melting temperature of the capture probe:target nucleic acid hybrid is greater than the melting temperature of the capture probe:immobilized probe hybrid. In this way, different sets of hybridization assay conditions can be employed to facilitate hybridization of the capture probe to the target nucleic acid prior to hybridization of the capture probe to the immobilized oligonucleotide, thereby maximizing the concentration of free probe and providing favorable liquid phase hybridization kinetics. This “two- step” target capture method is disclosed by Weisburg et al., U.S. Patent No. 6,110,678. In some embodiments, the 23 S M. genitalium target ribosomal nucleic acid is captured onto the solid support by direct interaction (e.g., hybridization) with the immobilized probe, and there is no requirement for a target capture probe. Other target capture schemes readily adaptable to the present technique are well known in the art and include, without limitation, those disclosed by the following: Dunn et al., Methods in Enzymology, “Mapping viral mRNAs by sandwich hybridization,” 65(l):468-478 (1980); Ranki et al., U.S. Patent No. 4,486,539; Stabinsky, U.S. Patent No. 4,751,177; and Becker et al., U.S. Patent No. 6,130,038.

[0141] Isolation can follow capture, wherein the complex on the solid support is separated from other sample components. Isolation can be accomplished by any appropriate technique (e.g., washing a support associated with the M. genitalium target sequence one or more times (e.g., 2 or 3 times) to remove other sample components and / or unbound oligonucleotide). In embodiments using a particulate solid support, such as paramagnetic beads, particles associated with the M. genitalium- target can be suspended in a washing solution and retrieved from the washing solution, in some embodiments by using magnetic attraction. To limit the number of handling steps, the M. genitalium target nucleic acid can be amplified by simply mixing the M. genitalium target sequencein the complex on the support with amplification oligonucleotides and proceeding with amplification steps.Preferred Systems and Apparatus

[0142] The methods disclosed herein are conveniently implemented using a computer or similar processing device (“computer” hereafter). In different preferred embodiments, software or machine-executable instructions can be loaded or otherwise held in a memory component of a freestanding computer, or in a memory component of a computer linked to a device used for monitoring, preferably as a function of a reaction progress parameter (e.g., either reaction time or reaction cycle number), the amount of a product undergoing analysis. In a highly preferred embodiment, software for executing the disclosed procedure is held in a memory component of a computer that is linked to, or that is an integral part of a device or apparatus capable of monitoring the amount of an amplicon present in a reaction mixture as a function of reaction cycle number. This includes a processing device component on an electronic circuit board (e.g., embedded software) of an automated nucleic acid analyzer. Generally speaking, the computer is said to be “in communication with” the apparatus that detects and / or quantifies a target nucleic acid when information from the nucleic acid analyzer is transferred from the apparatus to the computer, by any means. In some embodiments, results generated by the computer can be delivered to an output device that displays or records a result of a calculation or comparison. Exemplary output devices include a video monitor and a printer. In some embodiments, the output device is a recording device that produces a “non-transient” record (e.g., a “tangible” record). The non-transient record may be printed on paper, or stored electronically (such as on a computer hard drive or flash drive, magnetic tape or other computer-readable media, etc.).

[0143] In some embodiments, the computer can be in communication with, either by wired or wireless means, a fluorometer that detects fluorescent signals, where the fluorometer is arranged or configured to monitor fluorescent signals generated in one or more reaction vessels contained within a temperature-controlled incubator. The incubator can be a temperature-controlled block (e.g., a metal block configured for receiving and containing one or more tubes, or even a multi-well plate), or a chamber that exposes one or more reaction vessels to controlled temperature conditions.

[0144] In some embodiments, either or both of a controller system for controlling a real-time amplification device and / or the detection system of the real-time amplification device can be coupled to an appropriately programmed computer that functions to instruct the operation of these instruments in accordance with preprogrammed or user input instructions. The computer preferablyalso can receive data and information from these instruments, and interpret, manipulate, and report this information to the user.

[0145] In some embodiments, the computer also can include appropriate software for receiving user instructions, either in the form of user input into a set of parameter fields, or in the form of preprogrammed instructions (e.g., preprogrammed for a variety of different specific operations). The software then converts these instructions to appropriate language for instructing the operation of the real-time amplification controller to carry out the desired operation. Preferably, the computer also is capable of receiving data from one or more sensors or detectors included within the system, and interpreting the data in accordance with the programming. The system preferably includes software that correlates a feature of a growth curve representing the quantity of amplified copies of the nucleic acid of interest as a function of time, as detected by the detector, to the number of copies of the nucleic acid of interest present in a test sample.

[0146] Preferably, when the computer used for executing the disclosed technique is an integral component of an apparatus for performing and analyzing real-time nucleic acid amplification reactions, the apparatus preferably comprises a temperature-controlled incubator, a detection device for collecting signals (e.g., a fluorometer), and an analyzing device (e.g., a computer or processor) for analyzing signals. The apparatus optionally can further include an output device for displaying data obtained or generated. The analyzing device may be connected to the temperature-controlled incubator through an input device known in the art, and / or connected to an output device known in the art for data display. In one embodiment, the temperature-controlled incubator is capable of temperature cycling, and may be configured as a block for receiving one or more tubes, or reaction receptacles (e.g., multi-tube units).

[0147] Generally speaking, the various components of an apparatus for performing the real-time nucleic acid amplification useful in connection with the disclosed methods will be conventional components that will be familiar to those having an ordinary level of skill in the art. The temperature-controlled incubator used to perform and analyze real-time nucleic acid amplification may be of a conventional design which can hold a plurality of reaction tubes, or reaction samples in a temperature-controlled block in standard amplification reaction tubes or in wells of a multiwell plate. In one aspect, the detection system is suitable for detecting optical signals from one or more fluorescent labels. The output of the detection system (e.g., signals corresponding to those generated during the amplification reaction) can be fed to the computer for data storage and manipulation. In one embodiment, the system detects multiple different types of optical signals, such as multiple different types of fluorescent labels and has the capabilities of a microplate fluorescence reader. The detection system is preferably a multiplexed fluorimeter containing anexcitation light source, which may be a visible light laser or an ultraviolet lamp or a halogen lamp, a multiplexer device for distributing the excitation light to the individual reaction tubes and for receiving fluorescent light from the reaction tubes, a filtering means for separating the fluorescence light from the excitation light by their wavelengths, and a detection means for measuring the fluorescence light intensity. Preferably, the detection system of the temperature-controlled incubator provides a broad detection range that allows flexibility of fluorophore choice, high sensitivity and excellent signal-to-noise ratio. Optical signals received by the detection system are generally converted into signals which can be operated on by the computer or processor to provide data which can be viewed by a user on a display of a user device in communication with the computer or processor. The user device may comprise a user interface or may be a conventional commercially available computer system with a keyboard and video monitor. Examples of data which can be displayed by the user device include amplification plots, scatter plots, sample value screens for all the tubes or reaction vessels in the assembly and for all labels used, an optical signal intensity screen (e.g., fluorescent signal intensity screen), final call results, text reports, and the like.Computer Program Products

[0148] Included within the scope of the disclosure are software-based products (e.g., tangible embodiments of software for instructing a computer to execute various procedural steps) that can be used for performing the data processing method. These include software instructions stored on a computer or computer-readable media, such as magnetic media, optical media, “flash” memory devices, and computer networks or cloud storage.

[0149] The disclosure further embraces a system or an apparatus that amplifies nucleic acids, detects nucleic acid amplification products, and processes results to indicate a quantitative result for target in a test sample. Although the various components of the apparatus preferably function in a cooperative fashion, there is no requirement for the components to be part of an integrated assembly (e.g., on a single chassis). However, in a preferred embodiment, components of the apparatus are connected together. Included within the meaning of “connected” are connections via wired and wireless connections.

[0150] Particularly falling within the scope of the disclosure is an apparatus or system that includes a computer linked to a device that amplifies nucleic acids and monitors amplicon synthesis as a function of cycle number or time, where the computer is programmed to execute the algorithmic steps disclosed herein. An exemplary system in accordance with the disclosure will include a temperature-controlled incubator, and a fluorometer capable of monitoring and distinguishing atleast two wavelengths of fluorescent emissions. These emissions may be used to indicate target amplicon synthesis, and internal control or internal calibrator amplicon synthesis.

[0151] In connection with computer-implemented or software-implemented embodiments of the disclosure, a result can be recorded or stored in a “non-transient” format where it can be accessed for reference at a later time than when the data analysis to be recorded was carried out or performed. For example, a computed result can be recorded in a non-transient format by printing on paper, or by storing on a computer-readable memory device (e.g., a hard drive, flash memory device, file in cloud storage, etc.).Methods of Treatment and Changing Treatments

[0152] In some embodiments, the assays disclosed herein can be selected or ordered from a menu of testing options available to a healthcare professional caring for a human patient. For example, a physician may place an order using an electronic, paper, or other ordering system so that a sample obtained from the human patient will be subjected to the various steps needed to determine the presence or absence of macrolide-resistant M. genitalium associated with particular SNPs. In this regard, the individual placing the order or request can be said to “direct” or “have” certain steps performed for the purpose of making the determination regarding the presence or absence of the M. genitalium organism (e.g., the macrolide-resistant organism). For example, there can be a step for obtaining, or “having” obtained the sample to be used for testing, etc. Simply stated, the individual requesting an assay need not perform all of the procedural steps themselves. Of course, this might be considered relevant not only for initiating the sequence of events needed to obtain the molecular diagnostic result, but also relevant for automated systems, or data processing systems where data analysis is performed at a remote site.

[0153] In some embodiments, the molecular diagnostic assay is useful for detecting the presence of wildtype M. genitalium, and of determining the macrolide-resistance status of the organism, if present in the test sample. For example, a single test may combine detection of genetic markers for M. genitalium (e.g., the wildtype organism) and for macrolide-resistance. In a different embodiment, the assay for detecting macrolide-resistance can be performed on a test sample that previously was determined by independent testing to contain M. genitalium. This latter approach is sometimes referred to as a “reflex” test.

[0154] If it is determined that an M. genitalium-contaming test sample obtained from a patient either includes or does not include macrolide-resistant M. genitalium, then a course of action can be implemented or changed to treat the patient for an improved outcome. If it is determined that the sample obtained from the patient includes macrolide-resistant M. genitalium, then the patient can betreated with a course of one or more antibiotics other than a macrolide antibiotic (e.g., azithromycin). For example, the treating healthcare professional may elect to prescribe, recommend, or treat with a fluoroquinolone antibiotic, or another agent effective against macrolide- resistant M. genitalium. Alternatively, if it is determined that the patient sample includes nucleic acids of M. genitalium, but does not include nucleic acids of macrolide-resistant M. genitalium, then a course of antibiotics other than fluoroquinolones may be prescribed or recommended. For example, a patient harboring an infection with M. genitalium that is not macrolide-resistant M. genitalium may be treated with a macrolide antibiotic (e.g., azithromycin) or another antibiotic effective against M. genitalium. Yet a different possibility is that a patient may have been treated with a course of fluoroquinolone antibiotics that will have been effective at controlling or eliminating an infection with macrolide-resistant M. genitalium. A subsequent test result indicating the absence of macrolide-resistant M. genitalium nucleic acid in a sample obtained following the initial treatment may guide the healthcare professional to change the treatment plan by discontinuing administration of the fluoroquinolone antibiotic (e.g., because it is no longer necessary).Exemplary Multiplex Assay

[0155] Disclosed herein is a multiplex real-time PCR assay that detects nucleic acids of antibioticresistant Mycoplasma genitalium. In preferred embodiments, the assay detects two specific nonoverlapping regions of the 23S ribosomal RNA (23S rRNA). In certain preferred embodiments, the two specific non-overlapping regions can be detected using two different optical channels of a fluorometer. For example, the two specific non-overlapping regions can be detected using only two different optical channels of the fluorometer. In certain preferred embodiments, the fluorometer is a component of an automated nucleic acid analyzer that isolates nucleic acids from biological or clinical samples, and then amplifies the isolated nucleic acids and detects amplification products. The illustrated assay detected amplified M. genitalium target or template nucleic acids in the FAM channel of a fluorometer without distinguishing between macrolide-sensitive and macrolide- resistant template types. Amplified target or template nucleic acids from any of five M. genitalium mutants resistant to macrolide antibiotics were detected in the ROX channel of the fluorometer in the illustrated assay. Nucleic acids templates used in the assay preferably are isolated and purified from preserved or stabilized clinical specimens, and then tested and determined to include nucleic acids of M. genitalium by an independent assay, such as the APTIMA® Mycoplasma genitalium Assay (Hologic, Inc.; CA) before testing with the presently disclosed multiplex assay. This pretesting ensures that samples tested with the disclosed macrolide resistance assay include M.genitalium. The independent M. genitalium assay used for pretesting preferably does not detect macrolide resistance markers. In this type of application, the disclosed multiplex assay is sometimes referred to as a “reflex” assay. Oligonucleotide reagents that amplified and detected a first locus harboring macrolide resistance markers, and a second locus that was common to wildtype (macrolide-sensitive) M. genitalium and macrolide-resistant M. genitalium were included in the multiplex reaction. Notably, the second locus is not identically present in nucleic acids of Mycoplasma pneumoniae. Oligonucleotide reagents that amplified and detected an added or exogenous RNA Internal Control (“IC”) also were included in the multiplex reaction. This IC is sometimes referred to as the “Process Control IC” or simply “Process Control.” Detection of amplified Process Control IC confirmed operational integrity of the amplified assay. The nucleic acid sequence of the Process Control IC can be unique, and preferably is not found in the genome of the M. genitalium organism. Amplification of the Process Control IC can be monitored in a third optical channel of the fluorometer.

[0156] An illustrative multiplex assay for detecting macrolide-resistant M. genitalium included six probes and four primers in addition to the primers and probe used to amplify and detect the Process Control IC. Details of the Process Control IC amplification have been omitted because they are considered relevant only to confirming proper operation of PCR assays and are not particularly relevant to detection of specific pathogens. Arrangement of the two amplified regions of the 23S rRNA is illustrated in Fig. 1.Nucleic Acid Templates and Oligonucleotide Reagents

[0157] Certain procedures described below employed in vitro transcripts (IVTs) as amplification templates having known sequences. It is to be understood that IVTs are RNA molecules produced by in vitro transcription of DNA templates containing a sequence of interest. Here the IVTs were either: wild-type (macrolide-sensitive) M. genitalium template; a template common to all M. genitalium strains (i.e., macrolide-sensitive M. genitalium, including wild-type, and macrolide- resistant M. genitalium)-, or one of the five templates for macrolide-resistant M. genitalium).Nucleic acid IVTs used for demonstrating the disclosed assay included sequences described in Table 1. Underlining in the tabulated sequences indicates portions of the sequence complementary to oligonucleotide primers and probes.Table 1Description of in vitro Transcripts

[0158] Table 2 presents exemplary forward and reverse primer sequences that were used for amplifying each of: (1) the region of the M. genitalium 23S rRNA template nucleic acid that includes the five nucleic acid markers for macrolide resistance (“Mgen Res” in Fig. 1), and (2) a target region of the 23 S rRNA template nucleic acid (“Mgen” in Fig. 1) that is common to both wild-type (macrolide-sensitive) and macrolide-resistant M. genitalium, but which is not present in M. pneumoniae.Table 2Forward and Reverse Primers1C5M= 5 ’-methyl-2’ -deoxycytosine2Tdu= Propyne dU

[0159] Probe sequences are presented in Table 3.Table 3Probe Oligonucleotides1Tdu= Propyne dU2C5M= 5 ’-methyl-2’ -deoxycytosine

[0160] Base sequences of four of the five probes specific for nucleic acids of macrolide-resistant M. genitalium presented in Table 3 are closely related to base sequences disclosed in commonly assigned U.S. patent application Sr. No. 18 / 015,619, the disclosure of which is hereby incorporated by reference. Probes identified as 58C, 58G, 59C, and 59 G in Table 3 included particular nucleotide analogs to impart desirable properties in the multiplex amplification reaction. A fifth probe, identified as 58T in Table 3, also included nucleotide analogs and was included in the multiplex reaction mixture to obtain superior differentiation from results obtained using nucleic acids of wild-type (macrolide- sensitive) M. genitalium (see Example 5).Validity Controls

[0161] Two internal controls used in the multiplex assay served different purposes.

[0162] First there was a “Process Control” to verify integrity of the amplification and detection system. Each reaction mixture included an exogenous RNA template (e.g., an added “Process Control IC” template), together with a pair of primers to amplify the Process Control IC template, and a probe complementary to amplicons synthesized in the reaction mixture using the pair of primers. Detecting Process Control amplicons during an amplification reaction confirmed that all necessary components had been included in the reaction mixtures and that temperature cycling and fluorescence monitoring were operational. Preferably, the base sequence detected by the probe that hybridized to the amplified Process Control template is not found in the sequence of the M.genitalium genome. Preferably, the probe used for detecting amplicons from the Process Control template does not detect any other amplicon produced in the multiplex reaction. Examples of exogenous internal controls are well known in the art.

[0163] Second, there was the “MG / MG Res control” to verify the presence of M. genitalium nucleic acid in samples undergoing testing. A region of the 23S rRNA template that was shared in common between wild-type (macrolide-sensitive) M. genitalium and macrolide-resistant M. genitalium - but not Mycoplasma pneumoniae - served as this additional control. The MG / MG Res control, illustrated as the amplified region in the right portion of Fig. 1 (labeled “Mgen Detection Locus”), provided valuable information for multiple reasons. Because certain preferred sample types used for testing with the multiplex assay would already have been identified as including nucleic acids of M. genitalium (i.e., at least one of macrolide-sensitive M. genitalium and macrolide-resistant M. genitalium), the MG / MG Res control was useful for confirming the presence of the intended sample type in the reaction mixture. As well, and following from the discovery that M. pneumoniae template nucleic acids could be amplified and detected in the multiplex assay, simply detecting amplification products in the ROX channel, in the absence of further information or analysis, ambiguously could indicate the presence of either nucleic acids of macrolide-resistant M. genitalium or M. pneumoniae nucleic acids. Considering the result of the MG / MG Res control amplification reaction (FAM channel) could resolve this ambiguity, as determination of the presence of nucleic acids of macrolide-resistant M. genitalium preferably required detection of the MG / MG Res control. In some embodiments, detection of a macrolide resistance marker in the multiplex reaction could be paired with detection of the MG / MG Res control to confirm the presence of macrolide-resistant M. genitalium nucleic acids in the reaction mixture. Still further, because a mathematical algorithm described herein could be used to effectively remove contributions of fluorescent signals arising from amplification of wild-type sequences when making determinations about the presence or absence of macrolide resistance markers in amplification products, an approach was needed for confirming the presence of M. genitalium nucleic acid in the reaction mixture. Detection of MG / MG Res control amplicons addressed this need.

[0164] There is flexibility in the choice of oligonucleotide reagents, and target nucleic acid regions that can be amplified in useful MG / MG Res control amplification and detection systems. The MG / MG Res control probe is fully complementary to, and detects, a sequence shared between nucleic acids of wild-type (macrolide- sensitive) M. genitalium and macrolide-resistant M. genitalium. Examples of hybridization probes useful in this respect are disclosed in U.S. Pat. No. 7,345,155 under Example 8. The disclosure of this U.S. patent is incorporated by reference. In preferred embodiments, the shared sequence is contained within the sequence of the 23S rRNA ofM. genitalium (see Fig. 1). Generally speaking, the MG / MG Res control assay does not distinguish between nucleic acids of wild-type (macrolide-sensitive) M. genitalium and macrolide-resistant M. genitalium. In some preferred embodiments, the amplicon serving as a target for hybridization of the probe that detects MG / MG Res control amplicons is different from the amplicon serving as a target for hybridization of the probes that detect the macrolide resistance markers in macrolide- resistant M. genitalium nucleic acids (see Fig. 1).

[0165] The following look-up table illustrates example approaches for interpreting results obtained using detection of macrolide resistance markers (2058C, 2058G, 2058T, 2059C, and 2059G) and two controls. In this table, “(+)” indicates detection of the target nucleic acid, and “(-)” indicates the target nucleic acid was not detected. Detection can be by any approach employing the disclosed multiplex assay. Criteria for gauging positivity include, for example: achievement of a level of fluorescence, for example by a certain cycle number; by determined Ct values; and by derivativebased analysis, including magnitude of the derivative of a run curve.Table 4Exemplary Look-Up Table Integrating Two Controls

[0166] Table 4 illustrates certain approaches for determining the nature of samples undergoing testing using results from three component assays of the disclosed multiplex assay. Validity of assay results (col. 4) was established (i.e., “Valid”) when at least one of the three component assays (cols. 1-3) of the multiplex yielded a positive result for amplification and detection. Validity established that amplification and detection systems were operational (e.g., that reagents and enzymes were present and active). The multiplex assay was considered “Invalid” only if none of the three component assays yielded positive amplicon detection in the reaction (row 8). This would be consistent with failure of either the amplification or detection reaction. Column 5 presents example interpretations of results when the multiplex assay is used as a reflex test following detection of M. genitalium nucleic acids in a separate assay. Determination that a test sample included nucleicacids of macrolide-resistant M. genitalium followed from positive detection (“POS”) of the macrolide resistance marker (row 1-4). Here the result of the Process Control and MG / MG Res control amplification optionally could be ignored. If the macrolide resistance marker and MG / MG Res control were not detected (row 7), then it could not be determined that nucleic acids of macrolide-resistant M. genitalium were present in the multiplex amplification reaction mixture. This result, shown as “Indeterminate” in the table, optionally can lead to retesting of the sample. In the absence of positive detection of a macrolide resistance marker and detection of the MG / MG Res control (rows 5-6), it was determined that the reaction mixture did not include nucleic acids of macrolide-resistant M. genitalium (“Neg”). Column 6 presents example interpretations of results when the multiplex assay is used as a stand-alone test for detecting macrolide-resistant M. genitalium. Here it is essential to have positive results indicating detection of macrolide resistance markers (ROX channel) in combination with positive detection of the MG / MG Res control (FAM channel) to conclude macrolide-resistant M. genitalium is present in the sample undergoing testing (rows 1-2). If a positive result is not obtained in the FAM channel, then a positive signal in the ROX channel leads to a conclusion of “Indeterminate” (rows 3-4). Absent detection of macrolide resistance markers, there would be no evidence for the presence of macrolide-resistant M. genitalium (rows 5-7).Analysis of Real-Time Run Curve Data

[0167] Two different approaches were used to process real-time run curve data to determine the presence or absence of amplified nucleic acids of macrolide-resistant M. genitalium in a test sample. The two approaches can be understood with reference to Figs. 5A-5B and Fig. 6, which present results obtained in the procedures of Example 6. These approaches could be used individually or in combination with each other.

[0168] To distinguish the results-processing procedures illustrated in Figs. 5A-5B and Fig. 6 from each other, the criteria used in the analyses were given different names. “Threshold” is used with reference to the defined criterion lines drawn at 1,500 RFU (FAM channel) in Fig, 5A and at 500 RFU (ROX channel) in Fig. 5B. Figs. 5A and 5B present real-time run curves for amplification reactions monitored in different channels of the fluorometer onboard the real-time instrument. A Ct or “cycle threshold” indicates the point during an amplification reaction when a run curve crosses the threshold level of fluorescence, and so serves as an indicator that a certain level of amplification has occurred. “Cutoff' is used with reference to the criterion line drawn at 140 RFU / cycle in Fig.6, which presents first derivative transforms of real-time run curves of ROX channel amplification reactions shown in Fig. 5B. This represents signals generated by probes specific for macrolideresistance markers. A maximum first derivative value greater than the cutoff indicates the presence of amplified macrolide-resistant M. genitalium nucleic acid, while a maximum first derivative value below the cutoff indicates the absence of amplified macrolide-resistant M. genitalium nucleic acid.

[0169] The disclosed multiplex assay can be used either for testing naive test samples, or for testing samples already known to include M. genitalium nucleic acids. In both instances, there can be optional processing of real-time run curves to identify Ct values by any approach that will be familiar to those having an ordinary level of skill in the art. As demonstrated by the results presented in Fig. 3B, determination of a Ct value for run curves measured using the five probes specific for macrolide resistance markers was not sufficient to establish the presence or absence of amplified macrolide-resistant M. genitalium nucleic acids. This was because run curves representing amplification reactions primed with nucleic acid templates of wild-type (macrolidesensitive) M. genitalium uniformly exceeded the horizontal threshold for establishing a level of amplification needed to indicate the presence of amplified nucleic acid measured in the ROX channel (i.e., representing macrolide-resistant M. genitalium nucleic acids).Direct Assessment of Real-Time Run Curves (e.g., Determination of Ct Values)

[0170] Further discussion of real-time run curve analysis will be with reference to results monitored in the ROX channel and shown in Fig. 5B and Fig. 6. Fig. 5B shows how amplicons synthesized from all tested clinical samples using the disclosed multiplex assay yielded run curves that exceeded the horizontal threshold used to indicate that amplification had taken place, where amplicons were detected using the ROX-labeled probes specific for the macrolide resistance markers. Using a threshold-based criterion forjudging that amplification took place, any run curve crossing a defined threshold (e.g., drawn at 500 RFU in the figure) was judged positive for amplification of M. genitalium nucleic acids. Those having an ordinary level of skill in the art will appreciate that Ct values indicate when a specified level of amplification has occurred in a nucleic acid amplification reaction, and may be established by different approaches as discussed elsewhere herein. For example, Ct values can be established as the point during an amplification reaction when a fluorescence run curve crossed the static threshold represented by the horizontal line in the figure. Notably, at about 42 cycles on the horizontal axis all run curves exceeded the threshold for positivity - even though some samples did not include nucleic acids of macrolide-resistant M. genitalium (see Example 6). Thus, use of threshold-based criteria forjudging detection of macrolide-resistant M. genitalium was contraindicated.

[0171] Even though none of the five probes used in the detection procedure leading to the results shown in Fig. 5B (see Table 3) was fully complementary to wild-type M. genitalium sequences thatmay have amplified in the multiplex reaction, wild-type amplicons could be detected at higher cycle numbers (e.g., greater than about 32 cycles in Fig. 5B) by cross-hybridization to the collection of five labeled probes. This cross-hybridization of probes specific for the five macrolide resistance markers to amplification products synthesized from wild-type templates meant that fluorescent signals detected at higher cycle numbers ambiguously indicated the presence of either wild-type template or templates that included one of the macrolide resistance markers. Thus, probe crosshybridization created ambiguity since it was unclear whether detected signals leading to Ct determinations indicated the presence of nucleic acids from macrolide-resistant M. genitalium or wild-type (macrolide-sensitive) M. genitalium.

[0172] This ambiguity was resolved using a computer-implemented algorithm that distinguished run curves arising from amplification of the different template types. Run curves characteristic of amplification of templates that included a macrolide resistance marker exhibited sigmoid shapes and robust amplification profiles characterized by a period of rapid rise in fluorescence.Conversely, run curves characteristic of amplified wild-type templates and detected by undesired cross-hybridization of the probes in Table 3 exhibited shallow sigmoid curves that were missing periods of rapid increase in fluorescence. The features of both curve types will be apparent from inspection of results presented in Fig. 5B.Derivative-Based Assessment of Real-Time Run Curves

[0173] In accordance with the disclosed technique, fluorescent signals resulting from crosshybridization of probes specific for macrolide resistance markers (see Table 3) to amplified templates from wild-type (macrolide-sensitive) M. genitalium was substantially eliminated before the results were used to determine the presence or absence of nucleic acid comprising any of the macrolide resistance markers. Although the U.S. patent application identified by S / N 18 / 015,619 teaches that wild-type (macrolide-sensitive) M. genitalium nucleic acid was not detected using the primers and probes disclosed therein, a different result was obtained using modified primers and closely related probes of the present assay. Thus, it was important to be able to identify and distinguish results from probe cross-hybridization.

[0174] Fig. 6 illustrates use of the run curve data from Fig. 5B to demonstrate an alternative data processing approach based on derivative analysis. Maxima of calculated first derivatives of amplification run curves from Fig. 5B were compared to a cutoff value (shown as a horizontal line drawn at 140 RFU / cycle in Fig. 6). Only amplification of macrolide-resistant M. genitalium nucleic acids led to first derivative transforms of run curves where the maximum values exceeded a cutoff needed to indicate that macrolide-resistant M. genitalium nucleic acids had been amplified. Runcurves representing amplification of wild-type (macrolide-sensitive) M. genitalium nucleic acid templates did not yield first derivative transforms having maxima that exceeded the cutoff. Thus, determining the maximum of a first derivative transform of a run curve and comparing the result to a defined cutoff could be used to distinguish true-positive results (macrolide-resistant M. genitalium)' from false-positive results that were actually due to detection of wild-type (macrolidesensitive) M. genitalium nucleic acid. Ambiguity between identification of macrolide-resistant M. genitalium and wild-type (macrolide-sensitive) M. genitalium was resolved by implementing a mathematical algorithm that distinguished between these possibilities using first derivative transforms of run curves and comparing to a cutoff.

[0175] As will be clear from inspection of Fig. 5B and Fig. 6, the derivative-based approach advantageously distinguished amplified nucleic acids of macrolide-resistant M. genitalium from amplified nucleic acids of wild-type (macrolide-sensitive) M. genitalium that were detected by cross-hybridization to probes specific for the macrolide resistance markers. Preferably, the approach used to establish the presence of amplified macrolide-resistant M. genitalium nucleic acids involves determining whether maxima of first derivative transforms of real-time run curves exceed a cutoff. In some embodiments, the approach does not rely on the cycle number at which the first derivative maximum occurs.

[0176] In summary, it was discovered that first derivative transforms of real-time nan curves could distinguish wild-type run curves from those representing amplification of the macrolide resistance markers at all reaction cycles. The first derivative transforms were compared to a cutoff value to determine the presence or absence of amplified sequences of macrolide-resistant M. genitalium in the reaction mixture. In this procedure the cutoff value of the comparing step was a pre-determined, fixed or constant cutoff that was established to achieve a balance between sensitivity and specificity for detection of SNP-containing nucleic acid targets obtained from macrolide-resistant M. genitalium (see discussion under Example 7). Those having an ordinary level of skill in the art will appreciate that such cutoffs conventionally are determined using results from a plurality of control reactions conducted using known templates and concentrations thereof. Cutoffs were selected to maximize correct results (e.g., true-positives and true-negatives) while minimizing incorrect results (e.g., false-positives and false-negatives) for the control reactions. Of course, cutoffs will be assayspecific and may depend on platforms (e.g., PCR, TMA, etc.) used for testing. This approach established a cutoff that was used for subsequent analyses, where the cutoff was termed a “predetermined cutoff' in the subsequent assays. It will be apparent from inspection of Fig. 6 that one or more alternative cutoffs could have been selected and used with equally good results. For example, a cutoff of between about 130 RFU / cycle and about 250 RFU / cycle, or even betweenabout 130 RFU / cycle and about 300 RFU / cycle would have distinguished the two types of run curves in the experimental results and permitted identification of reactions that amplified nucleic acids of macrolide-resistant M. genitalium. In some embodiments, the cutoff value can be established by the manufacturer of a kit to be used by a customer or other end-user. Only first derivative maxima that exceeded the predetermined cutoff value indicated the presence of SNP- containing nucleic acid targets characteristic of macrolide resistance.

[0177] Some of the real-time run curve data collected in the procedure described in the Examples herein was processed by calculating first derivatives and comparing maxima of the calculated results to a cutoff. This procedure reduced the impact of probe cross-hybridization on determinations of macrolide-resistant M. genitalium. More particularly, the technique reduced the incidence of false-positive results arising from detection of signals produced by hybridization of probes specific for macrolide resistance markers to non-target nucleic acid amplification products (e.g., amplicons resulting from amplification of wild-type (macrolide-sensitive) M. genitalium nucleic acids). Details of the relevant algorithm are disclosed in commonly assigned U.S. provisional patent application identified by S / N 63 / 500,475, now PCT application PCT / US24 / 27803, the entire disclosure of which is hereby incorporated by reference.

[0178] The method of determining whether a test sample includes a target nucleic acid indicative of macrolide resistance involved steps automated using a computer programmed with software. In certain preferred embodiments the computer was in communication with an automated nucleic acid analyzer that amplified nucleic acid templates and monitored synthesis of amplification products as amplification was taking place. In some embodiments, the computer is an integral component of an automated nucleic acid analyzer that amplifies template nucleic acids and monitors amplicon synthesis as the reaction is occurring (i.e., real-time monitoring). The method begins with the step of acquiring a real-time run curve data set that includes signal data representing synthesis of an amplification product in the multiplex amplification reaction as a function of a reaction progress parameter (e.g., time or cycle number), where the nucleic acid amplification reaction uses any of the macrolide-resistant M. genitalium target nucleic acid and the macrolide-sensitive M. genitalium nucleic acid that may have been present in the test sample as templates to produce the amplification product, and where fluorescent hybridization probes (e.g., hydrolysis probes labeled with a fluorophore and a quencher) are specific for macrolide-resistant M. genitalium nucleic acids (see Table 3). There also is the step of calculating the first derivative of run curves among the real-time run curve data set, including calculating magnitude values of the first derivatives. There also is the step of comparing the calculated magnitude values of the first derivatives with a predetermined cutoff value that distinguished amplification of nucleic acids of macrolide-resistant M. genitaliumfrom amplification of nucleic acids of wild-type (macrolide-sensitive) M. genitalium. There also is the step of determining either that: (i) the test sample includes nucleic acid of macrolide-resistant M. genitalium if any calculated magnitude value of the first derivative met or exceeded the cutoff value, or (ii) the test sample does not include nucleic acid of macrolide-resistant M. genitalium if no calculated magnitude value of the first derivative met or exceeded the cutoff value.

[0179] In some embodiments, determining the presence or absence of wild-type (macrolidesensitive) M. genitalium nucleic acid may be of somewhat less interest than determining the presence or absence of nucleic acid including one of the five macrolide resistance markers discussed herein. In some embodiments, only determination of the presence or absence of nucleic acid of macrolide-resistant M. genitalium (i.e., including one of the five macrolide resistance markers) is reported for assay results. In some embodiments of results reporting, the presence or absence of wild-type M. genitalium nucleic acid is ignored or not reported.Overview of Data Processing Approaches

[0180] Procedures for processing real-time run curve data to establish the presence or absence of amplifying nucleic acids of macrolide-resistant M. genitalium preferably include determining the first derivative of the run curve, including determination of the maximum value of the first derivative. The maximum value of the first derivative is then compared to a cutoff value (e.g., a predetermined cutoff) to establish whether the maximum value exceeds the cutoff. If the maximum value of the first derivative exceeds the cutoff, then it is determined that nucleic acids of macrolide- resistant M. genitalium were present in the sample undergoing testing. If the maximum value of the first derivative does not exceed the cutoff, then it is determined that nucleic acids of macrolide- resistant M. genitalium were not present in the sample undergoing testing.

[0181] Optionally, there can be included in the steps for making these determinations the consideration of additional information to confirm the conclusions. In some embodiments, the disclosed multiplex assay can be used as a reflex test to a positive result with an independent or different assay that detects nucleic acids of M. genitalium. This independent assay can confirm the presence of M. genitalium in the sample undergoing testing with the disclosed multiplex assay. In some other embodiments, results from the MG / MG Res control of the multiplex assay can provide similar information, thereby confirming the presence of M. genitalium in the sample undergoing testing. In yet some other embodiments, Ct values determined for run curves that cross a fluorescence threshold can indicate the presence of M. genitalium in the sample undergoing testing.

[0182] Results confirming the presence of at least one of macrolide-sensitive M. genitalium and macrolide-resistant M. genitalium in the sample undergoing testing can provide added functionalityto the disclosed assay. For example, if it is known (e.g., from detection of the MG / MG Res control in an amplification reaction) that the test sample includes M. genitalium, and if the maximum of a calculated first derivative of a run curve for real-time amplification reaction employing the five probes specific for macrolide resistance markers presented in Table 3 exceeds a cutoff, then it can be determined that macrolide-resistant M. genitalium is present in the test sample. Conversely, if it is known that the test sample includes M. genitalium, and if the maximum of a calculated first derivative of a run curve for real-time amplification reaction employing the five probes specific for the macrolide resistance markers presented in Table 3 does not exceed the cutoff, in some embodiments it can be determined that macrolide-sensitive M. genitalium is present in the test sample. An alternative nomenclature identifies this organism as “M. genitalium - not macrolide- resistant” instead of “wild-type."Illustrative Examples

[0183] The following Examples describe real-time nucleic acid amplification assays capable of detecting any of five single base mutations or polymorphisms (SNPs) in the M. genitalium 23S ribosomal nucleic acid (e.g., rRNA), where each of the five SNPs is associated with resistance to macrolide antibiotics. The assays can be used for determining the presence or absence of macrolide-resistant M. genitalium in a test sample.

[0184] All five markers of macrolide resistance were detected using different amplified templates and a collection of SNP-specific hydrolysis probes labeled with a single fluorophore species. None of the probes used in the assay harbored the wild-type sequence of ACGGAAAGACC (SEQ ID NO: 18) or the complement thereof. Thus, the assay did not employ any probe fully complementary to amplified wild- type (i.e., macrolide-sensitive) M. genitalium 23 S rRNA in the Mgen Res Detection Locus (see Fig. 1), yet the assay was capable of detecting the wild-type target nucleic acid.

[0185] It was discovered that PCR amplification of wild-type M. genitalium target nucleic acid (i.e., not including any of the five macrolide resistance markers disclosed herein) was detected in the multiplex reaction after extended cycling. This was undesired because detection of fluorescent signals in an amplification reaction primed with only wild-type templates conceivably could lead to a false-positive determination that templates harboring the macrolide resistance markers were present in a test sample. To eliminate this undesired possibility, a data processing algorithm was applied to the run curve data obtained using probes specific for the macrolide resistance markers. The algorithm used for this purpose has been disclosed in commonly owned International Patent Application No. PCT / US2024 / 027803, the disclosure of which is hereby incorporated by reference.Potential ambiguities that might confound results interpretation were resolved by processing realtime run curves to exclude or remove contributions of fluorescent signals arising from crosshybridization of probes specific for the five macrolide resistance markers to amplified wild-type (macrolide-sensitive) M. genitalium nucleic acids. As a result, determination of the presence of macrolide resistance markers could be made without interference from the detection of nucleic acids from the wild-type organism.

[0186] Two different amplified sequences from within the 23S ribosomal nucleic acid were used to detect: (1) sequences that included macrolide resistance markers (i.e., five SNPs); and (2) sequences common to both wild-type (macrolide- sensitive) M. genitalium and macrolide-resistant M. genitalium. This second amplified sequence is referred to herein as the “MG / MG Res control.” Detection of a first amplicon (left portion of Fig. 1), after data processing to exclude contributions from amplified wild-type templates, was used for indicating the presence of macrolide-resistant M. genitalium nucleic acids in a sample undergoing testing. Detection of a sequence in a second amplicon (right portion of Fig. 1) was used for indicating the presence of M. genitalium (i.e., either macrolide-resistant M. genitalium or macrolide-sensitive M. genitalium), while excluding closely related species (e.g., Mycoplasma pneumoniae). To be clear, detecting fluorescent signals indicating synthesis of the “Mgen Detection Locus” (i.e., the MG / MG Res control) in Fig. 1 did not imply the detected nucleic acid was derived from wild-type M. genitalium. Instead, detecting the sequence at this second locus indicated the amplification reaction included at least one of macrolide-resistant M. genitalium and macrolide-sensitive M. genitalium. Since detection of the second amplicon (right portion of Fig. 1) was specific for M. genitalium, and not related species of mycoplasma, this control assay was useful for ensuring the presence of M. genitalium in the test sample.

[0187] Finally, an added or exogenous internal control template was amplified and detected in the real-time amplification reaction as a “Process Control,” or an additional validity control, for the assay, again using a unique primer and probe set. The purpose of the Process Control was to confirm integrity or operability of the amplification reaction. For example, this control was useful for confirming that all reagents were included in the reaction mixture, and that temperature cycling was appropriate.

[0188] Example 1 describes procedures used to determine whether a sample undergoing testing included macrolide-resistant M. genitalium.Example 1Multiplex Amplification and Detection of Sequences Characteristic of Macrolide Resistance in M. genitalium

[0189] The Panther Fusion® system for automated nucleic acid analysis (Hologic, Inc.; San Diego, CA) was used to isolate and then amplify 23S ribosomal nucleic acid sequences of M. genitalium present in a test sample. Amplification was by the polymerase chain reaction (PCR). Monitoring of amplicon production was performed in real-time as reaction cycles were occurring. In vitro transcripts (IVTs) corresponding to either a sequence shared in common between wild-type (macrolide-sensitive) M. genitalium and macrolide-resistant M. genitalium of (SEQ ID NO:7), or one of the five macrolide-resistant M. genitalium 23S rRNA sequences (SEQ ID NOs:2-6) (see Table 1) served as sources of amplifiable templates, where input IVT concentrations ranged from 1 x 103to IxlO6copies / mL. An exogenous (i.e., added) internal control RNA template (i.e., the “Process Control IC” template), together with a probe labeled with the QUASAR™ 705 fluorophore (Biosearch Technologies; UK) and BHQ2 quencher (Biosearch Technologies; UK), and primers for amplifying and detecting the Process Control IC template also were included in all amplification reaction mixtures. Template nucleic acids contained in the test sample were enriched by target capture onto magnetic beads and then combined with a thermostable reverse transcriptase enzyme having both RNA-dependent and DNA-dependent DNA polymerase activities, dNTPs, and cofactors in reaction mixtures that supported reverse transcription and PCR amplification, as will be familiar to those having an ordinary level of skill in the art. Multiplex reaction mixtures for amplifying nucleic acids of M. genitalium i.e., a sequence shared by macrolide-sensitive M. genitalium and macrolide-resistant M. genitalium'), and Process Control IC included the forward and reverse primers shown in Table 2, together with all oligonucleotide probes shown in Table 3. The collection of probes for detecting macrolide resistance markers were labeled with a CalRed610 fluorescent dye (Biosearch Technologies, Inc.; Petaluma, CA) at the 5 ’-end, and with a BHQ-2 quencher moiety (Biosearch Technologies, Inc.; Petaluma, CA) at the 3’ -end. Reaction conditions included: a reverse transcription step to synthesize cDNA; a brief 95°C polymerase activation step; and 45 cycles of 95 °C to denature double-stranded nucleic acids and 60°C for primer annealing and extension. Reactions were performed in replicates of three for each level of input template.Amplicon synthesis indicative of macrolide resistance was monitored by detecting fluorescence signals in the ROX channel of the automated nucleic acid analyzer as a function of cycle number. Amplicon synthesis indicating the presence of the MG / MG Res control sequence was monitored by detecting fluorescence in the FAM channel of the automated nucleic acid analyzer as a function of cycle number. Finally, amplicon synthesis indicating the presence of the Process Control templatewas monitored by detecting fluorescence signals from the QUASAR 705 dye in a third channel of the fluorometer onboard the automated nucleic acid analyzer. Detection of Process Control amplicons in all reactions confirmed the integrity of reaction mixtures (i.e., that all reagents were included, and that temperature cycling was appropriate). Ct values for all amplified nucleic acid species were determined in accordance with methods that will be familiar to those having an ordinary level of skill in the art. Reactions were judged positive (z.e., indicating that measurable amplification occurred) if the ROX channel fluorescence run curve met or exceeded a predetermined threshold value. Ct values were determined as the cycle number during the amplification reaction at which the run curve met or exceeded the threshold value following standard background value adjustments.

[0190] Fig. 2 graphically illustrates how the different markers of macrolide resistance in M. genitalium were detectable with substantially equal efficiency. At an input level of 1 x 106copies / mL, all IVTs containing the macrolide resistance marker were detected with 100% of positive calls at a Ct between 25.9 and 29.4. At an input level of 1 x 103copies / mL, all IVT containing the macrolide resistance marker were detected with 100% of positive calls at a Ct between 36.2 and 38.6. Results obtained in the procedures indicated trials performed using IVTs at input levels of 1 x 102copies / mL did not report 100% positive calls, and so were omitted from the plot shown in Fig. 2. Table 5 summarizes the results presented in Fig. 2. Notably, PCR amplification efficiencies for all five IVTs that included macrolide resistance markers were substantially similar (i.e., balanced). This was an advantage because all macrolide resistance markers were detected using the same fluorophore, and so a single fluorescence indicator threshold could be used to indicate positivity of any of the five different macrolide resistance markers.Table 5Linearity and PCR Efficiency Results

[0191] Summarized results appearing in Table 5 confirmed that PCR efficiency readings determined using different templates harboring macrolide resistance markers were all within about 5% of each other.

[0192] Example 2 describes procedures that established analytical sensitivity for the disclosed multiplex assay.Example 2Establishing Assay Sensitivity

[0193] Analytical sensitivity of the multiplex assay was evaluated using IVTs corresponding to IVT Mgen (SEQ ID NO:7) target nucleic acid, and known quantities of IVTs that included the five different M. genitalium macrolide resistance markers (SEQ ID NOs:2-6). All IVTs had been diluted in a phosphate-buffered solution (pH 6.6 to pH 6.8) that included 3% (w / v) lithium lauryl sulfate. In addition to promoting cell lysis, this solution (sometimes specimen transport medium, or “STM”) protects released nucleic acids by inhibiting the activity of nuclease enzymes that may be present in the sample. Alternatively, STM can be substituted by a lysis reagent that includes 0.4 N LiOH and 10% LLS. Sample processing to isolate nucleic acids from the samples, together with amplification of isolated nucleic acids was again performed using the Panther Fusion® system for automated nucleic acid analysis. Amplification reactions were performed to determine the incidence of positive detection at target input levels of about 500 copies / mL and below. Reactions included the primers from Table 2 and the probes from Table 3. Mathematical analysis (e.g., Probit 4P analysis, 4-parameter logistic analysis, or Weibull distribution analysis) was applied to estimate the Limit of Detection (LoD) based on detection at the concentrations tested. Five concentrations were tested in replicates of ten for the IVT Mgen (SEQ ID NO:7) (total of 50 PCR reactions), and in replicates of twenty for the M. genitalium IVTs that harbored one of the macrolide resistance markers (total of 100 PCR reactions per target). Again, signals indicating amplification of the five macrolide-resistance markers of M. genitalium were detected in the ROX channel of the fluorometer during the amplification reaction. Signals indicating detection of the amplified MG / MG Res control sequence were detected in the FAM channel of the fluorometer during the amplification reactions.

[0194] Table 6 presents summarized results indicating % positive calls (z.<?., confirmed determination that target was detected) at various target input concentrations.Table 6Determining Limits of Detection (Lol) )

[0195] Table 7 summarizes the 95% LoD probabilities for different target nucleic acids in the disclosed multiplex assay. Curve fitting models used to make the determinations are listed in the table.Table 7Quantifying Limits of Detection

[0196] Example 3 describes procedures that assessed sensitivity of target detection using cultured strains of M. genitalium instead of IVTs. Again, nucleic acid isolation and amplification was performed using the automated nucleic acid analyzer platform.Example 3Sensitivity Testing Using Cellular Samples

[0197] Samples of macrolide-resistant M. genitalium cells were prepared at three-fold serial dilutions in STM. Six dilution points were tested in replicates of ten for each strain of M. genitalium that harbored a different macrolide resistance marker. A strain harboring the 2059C mutation was not available for testing and so was not included in the procedure. Four strains tested in the procedure were identified as Mega216 (2058C), Megal082 (2058G), M6926 (2058T), and Megal272 (2059G).

[0198] Nucleic acids were isolated and amplified essentially as described above using the Panther Fusion® system for automated nucleic acid analysis. Primers had the sequences presented in Table 2, and probes had the sequences presented in Table 3. Amplicon production was monitored by taking fluorescence readings as thermal cycling amplification reactions were occurring. Again, this multiplex assay amplified and detected: (1) the 23S rRNA locus that included five markers characteristic of macrolide resistance; (2) the MG / MG Res control locus of the 23 S rRNA that was common to nucleic acids of wild-type and macrolide-resistant M. genitalium, but not M. pneumoniae', and (3) an exogenous internal control (i.e., the Process Control IC). Amplification and detection of the MG / MG Res control sequence was important as a validity control. Characterized differently, this “common” locus was detected using a general probe that did not detect nucleic acids of wild-type (i.e., macrolide- sensitive) M. genitalium without also detecting nucleic acids of macrolide-resistant M. genitalium, and vice versa. Real time run curves measuredin the ROX channel and FAM channel of the fluorometer were compared to a threshold to determine whether amplification and detection had occurred.

[0199] Results were scored as the number of positive trials out of ten. Parallel amplification and detection reactions used for comparison were carried out using the APTIMA® Mycoplasma genitalium Assay (Hologic, inc., CA). This latter assay operates under isothermal conditions by a transcription mediated amplification (TMA) mechanism to detect M. genitalium sequences in a ribosomal target nucleic acid, but does not detect any target sequence indicative of resistance to macrolide antibiotics. Input titers of M. genitalium in the table are given in genome equivalents / mL (“GE / mL”), which equates roughly to measures of cells / mL. Sample results from the procedure are presented in Table 8.Table 8Mega216 (A2058C) Fusion vs TMA Sensitivity Comparison

[0200] Results presented in Table 8 illustrate how the multiplex PCR-based assay for detecting the MG / MG Res control and nucleic acids that included macrolide resistance markers, and the TMA- based APTIMA® Mycoplasma genitalium Assay both detected the nucleic acid of the Mega216 strain of M. genitalium with substantially similar sensitivities (e.g., down to less than 1 GE / mL). The multiplex PCR-based assay that detected any of the five nucleic acid markers characteristic of macrolide resistance (i.e., 2058G, 2059G, 2059C, 2058T, and 2058C) was possibly very slightly less sensitive in this testing. However, the multiplex assay was capable of detecting a single M. genitalium cell per mL harboring the A2058C mutation indicative of macrolide resistance.

[0201] Notably, quantitative results obtained using sample input levels below 1 GE / mL are not considered reliable because statistical assessment of small numbers is subject to error. None of the tested strains that harbored a macrolide resistance marker required more than 15 GE / mL input concentration to achieve 100% detection in the multiplex assay. All of the tested strains gave 100% detection at 15 GE / mL and higher.

[0202] In silico analysis identified organisms other than macrolide-resistant M. genitalium having nucleic acids potentially detectable in the multiplex assay. Oligonucleotides used in the multiplex assay were compared against several target databases, including GenBank divisions viral, bacterial, human genome, plants, and invertebrate. Mycoplasma pneumoniae was the only bacterium known to be relevant to the human host, and considered potentially a target for amplification with primer hybridization and detection by probe hybridization.

[0203] Example 4 illustrates how detection of macrolide resistance marker(s) in the multiplex assay could be verified to originate from an organism other than macrolide-resistant M. genitalium.Example 4Validity Control Eliminates False-Positive Assignment of Macrolide- Resistant M. genitalium

[0204] Cultured samples of Mycoplasma pneumoniae spiked in STM at IxltP CFU / mL, IxlO4CFU / mL, and IxlO3CFU / mL were processed to isolate, amplify, and detect nucleic acids using the Panther Fusion® system for automated nucleic acid analysis. Procedures for detecting amplified macrolide-resistant M. genitalium nucleic acids were essentially as described above. The forward and reverse primers from Table 2, and the probes from Table 3 were employed in the multiplex reaction. The reaction was capable of detecting the segment of 23S rRNA that included the five macrolide resistance markers.Table 9Validity Control

[0205] Results presented in Table 9 showed that macrolide resistance markers were detected in the multiplex assay (ROX channel), but that the MG / MG Res control assay component of the multiplex assay (FAM channel) was uniformly negative. This indicated the absence of M. genitalium nucleic acids. Accordingly, signals detected in the ROX channel were not due to the presence of macrolide-resistant M. genitalium nucleic acids because the MG / MG Res control was not detected.

[0206] Two different approaches were used for establishing or confirming the presence of M. genitalium nucleic acids in reaction mixtures being tested for the presence of macrolide-resistant M.genitalium nucleic acid. First, the multiplex assay could include a control reaction to identify the presence of an M. genitalium sequence characteristic of both macrolide-sensitive and macrolide- resistant M. genitalium (e.g., the MG / MG Res control). Second, pretesting with an assay specific for M. genitalium (e.g., the APTIMA® Mycoplasma genitalium Assay) could identify the presence of M. genitalium nucleic acids in a sample, but could not specifically identify the presence of macrolide-resistant M. genitalium nucleic acid. In this respect, this assay specific for M. genitalium served the same purpose as the MG / MG Res control. This confirms that useful MG / MG Res control targets and amplification reactions are not limited to the one amplified by the primers presented in Table 2 and the probes presented in Table 3. Alternatives clearly are available, and can be incorporated into the disclosed multiplex assay in substitute for the MG / MG Res control assay illustrated in the Examples herein. Results processing employing a look-up table identified the presence of macrolide-resistant M. genitalium nucleic acid when amplification was detected for both the macrolide resistance markers (e.g., ROX channel signals in the illustrated examples) and for the MG / MG Res control. The absence of macrolide-resistant M. genitalium nucleic acid could be indicated by failure to detect the MG / MG Res control, or by a pretest result indicating the absence of M. genitalium.

[0207] The following Example addresses the impact of probe cross-hybridization on detection of amplified wild-type sequences in the “Mgen Res Detection Locus” (see left portion of Fig. 1). Results shown below demonstrated detection of amplified wild-type (macrolide- sensitive) M. genitalium sequences by cross-hybridization to labeled probes specific for macrolide resistance markers of macrolide-resistant M. genitalium. There also is demonstrated a method of processing real-time run curve results to better distinguish detection of the two amplified species using only probes specific for the macrolide resistance markers. Still further, the procedure illustrates selection of a probe that mitigated the effects of probe cross-hybridization. Rather than employing the “MG / MG Res control,” the procedure below used a FAM-labeled probe that was specific for the sequence of wild-type (macrolide-sensitive) M. genitalium, and that was fully complementary to a sequence within the “Mgen Res Detection Locus.” The FAM-labeled probe served to confirm the presence of M. genitalium sequences in the amplification reactions. This represented one approach for detecting and confirming the presence of wild-type sequences in the multiplex reaction.

[0208] Example 5 illustrates how labeled probes specific for macrolide resistance markers detected amplified wild-type nucleic acids. Two independent approaches were used to control the impact of probe cross-hybridization. In one approach, the procedure involved processing real-time run curve results using the derivative-based assessment described herein. In another approach, a probe was selected to enhance detection of nucleic acids of macrolide-resistant M. genitalium whileminimizing detection of nucleic acids from the closely related wild-type (macrolide-sensitive) M. genitalium that differed in sequence by only a single nucleotide. The procedure focuses particularly on selection of a probe that detected the amplified 2058T template nucleic acid.Example 5Selection of the SNP-Specific Probe to Detect Amplified Nucleic Acids of 2058T Macrolide-Resistant M. genitalium

[0209] The Panther Fusion® system for automated nucleic acid analysis was used to identify a probe that gave superior differentiation between detection of wild-type (macrolide-sensitive) M. genitalium and macrolide-resistant M. genitalium. Multiplex reactions included the RP25 reverse primer of SEQ ID NO:11 in combination with new forward primer FP10 having the sequence 5’- ACACCCGTTAGGCGCAA-3’ (SEQ ID NO:19). This forward primer, which was used instead of forward primer FP11 (SEQ ID NO: 10), was shifted by three bases relative to the primer of FP11 (SEQ ID NO: 10) thereby confirming flexibility among primers that could be used for amplifying the target sequence. The primer combination efficiently amplified the “Mgen Res Detection Locus” (see Fig. 1) in the multiplex reactions. A FAM-labeled probe for detecting amplified wild-type sequence had the sequence of 5’-FAM-ACGGAAAGACCCCGTG-BHQl-3’ (SEQ ID NO:20). Probes specific for macrolide resistance markers were from Table 3, except that the listed probe for detecting the 2058T target of macrolide-resistant M. genitalium was varied. More specifically, the probes from Table 10 were individually substituted in place of the 58T probe of Table 3.Templates used in the amplification reactions were IVTs that included either the wild-type (macrolide-sensitive) M. genitalium sequence (SEQ ID NO:1), or the 2058T SNP characteristic of macrolide resistance in M. genitalium (SEQ ID NO:4). One set of reactions included the wild-type (macrolide- sensitive) M. genitalium IVT at a high input level of 8.33 x 104copies / mL, while two other sets of reactions included the IVT corresponding to the macrolide-resistant M. genitalium (i.e., 2058T) at input levels of either 8.33 x IO2or 8.33 x IO3copies / mL. Real-time run curve data sets were collected for each different amplification reaction by monitoring cycle-dependent fluorescence indicating the presence of the macrolide resistance marker (ROX channel) and the amplified wildtype sequence (FAM channel). Amplification of a Process Control also was detected in each reported reaction (data not shown). Real-time run curve results were processed to determine Ct values (FAM channel and ROX channel); and to determine first derivatives of run curves (ROX channel). The first derivatives of the ROX channel ran curves were further processed to determine maxima.Table 10Probes Specific for Amplified 2058T Macrolide-Resistant M. genitalium Template

[0210] Fig. 3A presents results from monitoring the FAM channel for different reactions that included wild-type (macrolide-sensitive) IVTs, a FAM-labeled probe specific for the amplified wild-type sequence, and one of the probes specific for the amplified 58T SNP characteristic of macrolide resistance (see Table 10). It will be apparent that the wild-type sequence was amplified in all reactions, as expected. All run curves exceeded the threshold drawn at 1,500 RFU at essentially the same time. Thus, results shown in this figure confirmed that the presence of different probes specific for 58T macrolide resistance markers did not substantially impact amplification and detection of the wild-type template.

[0211] Fig. 3B presents results from monitoring the collection of amplification reactions in the ROX channel. Again, reactions were primed either with an IVT for wild-type (macrolide-sensitive) M. genitalium or an IVT harboring the 2058T macrolide resistance marker (two input concentrations). Run curves from reactions that included different ROX-labeled probes from Table 10 are not distinguished from each other in this figure. A horizontal threshold for judging a reaction amplified sufficiently to yield a Ct value is shown at 300 RFU. A negative control reaction that omitted template nucleic acid did not yield detectable amplification, as expected. All reactions that included the IVT template harboring the 2058T macrolide resistance marker yielded robust amplification profiles. Reactions primed with wild-type (macrolide-sensitive) M. genitalium template nucleic acid also yielded detectable signals in the ROX channel, thereby indicating that ROX-labeled probes specific for the 2058 T macrolide resistance marker cross-hybridized to amplification products synthesized using wild-type (macrolide- sensitive) M. genitalium template nucleic acids. Again, the wild-type amplification product was non-complementary to the collection of ROX-labeled probes at only a single nucleotide position. Resulting Ct values ambiguouslyindicated amplification of macrolide-resistant M. genitalium or wild-type (macrolide- sensitive) M. genitalium.

[0212] Fig. 4 presents first derivative maxima calculated for run curves appearing in Fig. 3B plotted for each different candidate probe that detected the 2058T macrolide resistance marker. In all instances, negative control reactions that omitted amplifiable template nucleic acid gave essentially undetectable signals. Run curves for amplified wild-type templates gave low first derivative maxima values. Probes specific for the 2058T macrolide resistance marker yielded a range of maximum derivative values. The most desirable probe specific for the 2058T macrolide resistance marker showed high maximum derivative values with low variability while also having good separation from the maximum derivative value observed for the run curve of amplified wildtype (macrolide-sensitive) M. genitalium. It will be apparent from inspection of Fig. 4 that the probe of 58T-21 (SEQ ID NO: 15) (shown boxed) satisfied this requirement. Accordingly, multiplex amplification reactions described in the following Examples were earned out using this probe for detection of the 2058T amplification product.

[0213] The following Example illustrates how the disclosed multiplex assay could be used to identify or determine the presence of nucleic acids of macrolide-resistant M. genitalium without interference from wild-type (macrolide-sensitive) M. genitalium. This was true even though probes that hybridized amplification products harboring sequences (e.g., SNPs) characteristic of macrolide resistance cross-hybridized to amplified nucleic acids of wild-type (macrolide-sensitive) M. genitalium.Example 6Distinguishing Amplified Nucleic Acids of Wild-Type (Macrolide-Sensitive) M. genitalium and Macrolide- Resistant M. genitalium

[0214] Nucleic acids were isolated from a collection of 26 clinical samples for testing using the Panther Fusion® system for automated nucleic acid analysis. Samples used for this purpose were vaginal swab samples (7), penile meatal swab samples (4), endocervical swab samples (3), urethral swab samples (4), or urine samples (8). Isolated nucleic acids were subjected to amplification and detection of M. genitalium nucleic acids by two different approaches using the Panther Fusion® system for automated nucleic acid analysis. First, samples were tested using the FDA-cleared APTIMA® Mycoplasma genitalium Assay (Hologic, Inc.; San Diego, CA) that detected nucleic acid of M. genitalium without informing on macrolide-resistance status. All tested samples included M. genitalium nucleic acids according to this assay. Second, samples were tested usingthe presently disclosed real-time multiplex assay for detecting nucleic acids of macrolide-resistantM. genitalium. Oligonucleotide primers and probes used in the procedure are given in Table 11.Table 11Oligonucleotide Reagents1C5M= 5 ’-methyl-2’ -deoxycytosine2Tdu= Propyne dU

[0215] Results of the procedure are presented in Figs. 5A, 5B, and 6. Collected fluorescence run curve data for different probe types is graphically displayed in Figs. 5A-5B. Although not presented graphically, all reactions also amplified an exogenous Process Control RNA template that was used to confirm validity of the PCR reactions. Fig. 5A (FAM channel data) presents real-time run curve data from amplification and detection of the “Mgen Detection Locus” in Fig. 1. The results confirmed the presence of nucleic acids of M. genitalium in the reaction mixtures without identifying whether the template nucleic acid originated from macrolide-resistant M. genitalium or from wild-type (macrolide-sensitive) M. genitalium. Fig. 5B graphically displays ROX channel data for detection of the “Mgen Res Detection Locus” in Fig. 1. In certain preferred embodiments, detection of this locus establishes or determines that nucleic acids of macrolide-resistant M. genitalium were present in a sample or reaction mixture. Importantly, results presented in Fig. 5Bshow that all of the ROX channel run curves exceeded a horizontal threshold line that indicated positive amplification and Ct values. If taken in isolation, this positive result would erroneously suggest that all reaction trials included nucleic acids of macrolide-resistant M. genitalium. Fig. 6 is a plot of first derivatives calculated for the ran curves shown in Fig. 5B, again representing signals due to hybridization and hydrolysis of the collection of probes specific for macrolide resistance markers in the “Mgen Res Detection Locus.” The figure has been labeled to show how a horizontal cutoff could be established to partition or differentiate detection of macrolide-resistant M. genitalium (first derivative maxima greater than cutoff) and wild-type (macrolide-sensitive) M. genitalium (first derivative maxima below cutoff). Except for a single trial that was invalid in the APTIMA® Mycoplasma genitalium Assay, but identified as macrolide-resistant in the multiplex assay, there was 100% agreement between the results for the two different assays. According to results from the multiplex assay, 16 samples were identified as macrolide-resistant M. genitalium (i.e., having first derivative maxima greater than the indicated cutoff), and 10 samples were identified as wild-type (macrolide-sensitive) M. genitalium (i.e., having first derivative maxima below the indicated cutoff). Samples categorized as macrolide- resistant included 5 urine samples and 11 swab samples. Samples categorized as wild-type included 3 urine and 7 swab samples. Trials associated with ran curves that exceeded the threshold in Fig. 5B, but that fell below the cutoff in Fig. 6 were potentially false-positive macrolide-resistant identifications before implementation of the disclosed technique. Thus, comparing the determined maximum first derivatives of ran curves against a cutoff served as a criterion that dramatically reduced falsepositive identifications.

[0216] Example 7 describes procedures that again illustrated how the derivative-based analytical method distinguished macrolide-resistant M. genitalium from wild-type (macrolide-sensitive) M. genitalium using the real-time multiplex nucleic acid amplification assay.Example 7Distinguishing Macrolide-Resistant M. genitalium from Wild-Type (Macrolide-Sensitive) M, genitalium

[0217] Procedures essentially as described under the preceding Example were followed using a larger collection of different samples and data from integration studies. More particularly, 6894 samples (reps included) of macrolide-resistant M. genitalium strains and IVTs were spiked into different matrices at concentrations close to the LoD and above; and 390 samples (reps included) of wild-type (macrolide-sensitive) M. genitalium strains were spiked into different matrices at concentrations up to 1 x 106GE / mL. Matrices used in the procedure included male and femaleurine, penile meatal swabs, vaginal swabs, and simulated vaginal fluids. Real-time PCR amplification reactions were carried out using the Panther Fusion® system for automated nucleic acid analysis, and run curve data was processed as described under the preceding Example to identify first derivative maxima for the ROX channel run curves. Primers and probes used in the reactions are presented in Table 11. Parallel testing using the APTIMA® Mycoplasma genitalium Assay confirmed the presence of M. genitalium in each sample. Nucleic acids from all samples used in the procedure were subjected to nucleic acid sequencing of the “Mgen Res Detection Locus” (see Fig. 1) to confirm genotypes of the samples undergoing testing. Maximum values of calculated first derivatives of run curves determined using ROX channel signals are plotted in Fig. 7.

[0218] Results presented in Figs. 7A and 7B demonstrated that maxima of first derivatives for realtime run curves from each of five genotypes of macrolide-resistant M. genitalium partitioned separate from corresponding maxima of first derivatives for real-time run curves resulting from amplification of wild-type (macrolide-sensitive) M. genitalium nucleic acids. More particularly, first derivative maxima associated with macrolide-resistant samples partitioned above a cutoff, and first derivative maxima associated with wild-type samples partitioned below a cutoff.

[0219] Figs. 7A and 7B illustrate how one or more cutoffs can be used to partition results and identify or determine the presence of macrolide-resistant M. genitalium in a test sample. The vertical axis in both figures presents “Curve Max Value” (sometimes “CMValue”), which is equivalent to the maximum value of first derivative of a run curve. Fig. 7A shows how two cutoffs (an upper bound cutoff, and a lower bound cutoff) were used to partition results of determined maximum first derivative values into three categories. By this approach, there would be no falsepositive determinations (e.g., where wild-type (macrolide-sensitive) samples would be misidentified as macrolide-resistant) above the upper bound. Likewise, there would be no false-negative determinations (e.g., where macrolide-resistant samples would be misidentified as wild-type (macrolide sensitive) M. genitalium) below the lower bound. Sample results falling between the upper and lower bound cutoffs can be considered “not determined” because there was a recognized level of expected error. Samples falling in this latter category optionally could be subjected to retesting. Fig. 7B shows how a single cutoff was used to partition results of determined maximum first derivatives into two categories. Trials above the cutoff were identified as macrolide-resistant M. genitalium. In this instance, the priority was to minimize false-positive results, so that wild-type samples would not be misidentified as macrolide-resistant. Stated differently, “specificity” was the priority in selecting the cutoff appearing in Fig. 7B. As a tradeoff, there were a small number of false-negative results, where some macrolide-resistant samples partitioned below the cutoff andwere misidentified as being negative for the macrolide resistance marker(s).

[0220] Notably, a cutoff identified using a plurality of samples representing wild-type and different macrolide-resistant genotypes can be used as a predetermined cutoff for subsequent analyses. For example, a predetermined cutoff can be established by a kit manufacturer on a particular real-time amplification platform or instrument and then provided to an end-user of the kit for performing the assay. The selection of cutoffs will depend on the arbitrary desire for balancing specificity and sensitivity in the assay for which the cutoff is chosen. Using results from Fig. 7B as an illustration, a selected numerical cutoff value (e.g., 140 RFU / cycle) can be identified using one or more nucleic acid analyzers at a kit manufacturer’s site (one or more “first” instruments), and then provided to an end-user of the kit for use on a “second” instrument to determine the presence or absence of macrolide-resistant M. genitalium in a test sample at the end-user’s site.

[0221] The following table presents sequences of illustrative oligonucleotide reagents and in vitro transcripts used in the above procedures.Table 12Illustrative Nucleic Acid Sequences

[0222] All patents, patent applications, and publications mentioned in the specification are indicative of the levels of those of ordinary skill in the art to which the disclosure pertains. All patents, patent applications, and publications are herein incorporated by reference in their entirety for all purposes and to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference in its entirety for any and all purposes.

[0223] All of the compositions, kits, and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the disclosure describes preferred embodiments, it will be apparent to those of skill in the art that variations may be applied without departing from the spirit and scope of the disclosure. All such variations and equivalents apparent to those skilled in the art, whether now existing or later developed, are deemed to be within the spirit and scope of the disclosure.

Claims

1. WHAT IS CLAIMED IS:1 . A probe reagent for detecting a nucleic acid marker indicative of macrolide- resistance in M. genitalium among a collection of nucleic acid amplification products, the probe reagent including a probe of the base sequence of SEQ ID NO: 15.

2. The probe reagent of claim 1 , wherein the probe of the base sequence of SEQ ID NO: 15 is labeled with a fluorophore and a quencher.

3. The probe reagent of either claim 1 or claim 2, further comprising at least one probe selected from the group consisting of SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO:16, and SEQ ID NO: 17.

4. The probe reagent of claim 3, wherein the probe of the base sequence of SEQ ID NO: 15 and the at least one probe selected from the group consisting of SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 16, and SEQ ID NO:17 are combined with each other and packaged in the same vial or container.

5. The probe reagent of any one of claims 1 to 4, wherein all of the probes are labeled with the same fluorophore and the same quencher.

6. The probe reagent of claim 2, wherein a fluorescent emission from the fluorophore is detectable in a ROX detection channel of a fluorometer.

7. The probe reagent of any one of claims 1 to 6, wherein the probe reagent does not comprise any probe with the wild-type sequence of SEQ ID NO: 18 or the complement thereof.

8. The probe reagent of any one of claims 2 to 7, further comprising an oligonucleotide probe complementary to a sequence present in the 23S rRNA of both macrolide-sensitive M. genitalium and macrolide-resistant M. genitalium, and wherein the oligonucleotide probe comprises a fluorophore label that is different from the fluorophore of the probe of the base sequence of SEQ ID NO: 15.

9. The probe reagent of any one of claims 1 to 8, wherein each probe is in a dried form and not in a liquid phase.

10. The probe reagent of claim 9, wherein the dried form comprises a dried cyclodextrin.

11. A method of determining macrolide resistance status of M. genitalium in a test sample, the method comprising the steps of:(a) isolating nucleic acids from M. genitalium in the test sample;(b) amplifying, in a multiplex real-time nucleic acid amplification reaction, a nucleic acid locus of M. genitalium 23 S rRNA that is characteristic of macrolide sensitivity or macrolide resistance using nucleic acids isolated in step (a) as templates, whereby a real-time run curve data set is obtained, wherein said nucleic acid locus comprises either the sequence of SEQ ID NO: 18, which is present in macrolide-sensitive M. genitalium, or a macrolide resistance marker selected from the group consisting of: 2058C, 2058G, 2058T, 2059C, and 2059G, wherein the multiplex real-time nucleic acid amplification reaction comprises a plurality of probes specific for macrolide resistance markers, but does not comprise a delectably labeled probe specific for macrolide-sensitive M. genitalium that includes the sequence of ACGGAAAGACC (SEQ ID NO: 18), and wherein each of the plurality of probes is delectably labeled with a fluorophore;(c) establishing the presence of M. genitalium nucleic acids among the nucleic acids isolated in step (a) by amplifying and detecting nucleic acids of an M. genitalium detection locus in a nucleic acid amplification reaction, wherein the M. genitalium detection locus is present in macrolide-sensitive M. genitalium and macrolide-resistant M. genitalium, but not present in nucleic acids of M. pneumoniae, and wherein the nucleic acid amplification reaction is either the multiplex real-time nucleic acid amplification reaction or a nucleic acid amplification reaction other than the multiplex real-time nucleic acid amplification reaction;(d) processing the real-time run curve data set from step (b) to(i) calculate a maximum first derivative of the real-time run curve data set, and(ii) compare the calculated maximum to a cutoff that distinguishes first derivatives of run curves characteristic of macrolide-resistant M. genitalium fromfirst derivatives of run curves that are not characteristic of macrolide-resistant M. genitalium', and(e) determining either that the test sample comprises macrolide-resistant M. genitalium if the calculated maximum first derivative meets or exceeds the cutoff, or the test sample does not comprise macrolide-resistant M. genitalium if the calculated maximum first derivative does not meet or exceed the cutoff.

12. The method of claim 11 , wherein the nucleic acid amplification reaction of step (c) comprises the nucleic acid amplification reaction other than the multiplex real-time nucleic acid amplification reaction, and wherein the nucleic acid amplification reaction other than the multiplex real-time nucleic acid amplification reaction comprises an isothermal transcription-mediated amplification (TMA) reaction.

13. The method of either claim 11 or 12, wherein the nucleic acid amplification reaction of step (c) is the multiplex real-time nucleic acid amplification reaction, and wherein the multiplex real-time nucleic acid amplification reaction further comprises a second pair of primers and a second probe to amplify and detect nucleic acids of the M. genitalium detection locus.

14. The method of any one of claims 11 to 13, wherein the cutoff in step (d) is a cutoff established to minimize false-positive and false-negative results using determinations obtained for a plurality of control reactions having known M. genitalium macrolide resistance types.

15. The method of any one of claims 11 to 14, wherein the cutoff is a predetermined cutoff provided with a kit used for performing the multiplex real-time nucleic acid amplification reaction.

16. The method of any one of claims 11 to 15, further comprising the step of recording the result of step (e) in a non-transient form selected from the group consisting of: printing on paper, and saving on a computer-readable storage medium.

17. The method of any one of claims 11 to 16, wherein the fluorophore of each of the plurality of probes in step (b) is the same fluorophore, and wherein each of the plurality of probes further comprises a quencher moiety.

18. The method of claim 17, wherein the quencher moiety is the same for each probe of the plurality of probes.

19. The method of any one of claims 11 to 18, wherein the test sample is selected from the group consisting of a vaginal swab sample, a urethral swab sample, an endocervical swab sample, a penile meatal swab sample, and a urine sample.

20. The method of any one of claims 11 to 19, wherein steps (a) and (b) are performed under automated process control using an automated nucleic acid analyzer that carries out nucleic acid amplification reactions and monitors synthesis of amplification products while the reaction is occurring.

21. The method of any one of claims 11 to 20, wherein the multiplex real-time nucleic acid amplification reaction is a multiplex reverse transcriptase PCR reaction that uses reverse transcription to convert RNA into cDNA, which is then amplified in a PCR reaction.

22. A computer programmed with software instructions to determine from results of a real-time nucleic acid amplification reaction whether a test sample of M. genitalium includes macrolide-resistant M. genitalium, nucleic acids isolated from the test sample having been used as templates for amplification of 23S rRNA sequences in the real-time nucleic acid amplification reaction, the software instructions, when executed by the computer, cause the computer to:(a) receive a run curve data set for the real-time nucleic acid amplification reaction, the run curve data set comprising signal data for amplification of an Mgen Res detection locus in the 23S rRNA that comprises either the wild-type sequence of SEQ ID NO: 18, or a sequence selected from the group consisting of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO: 30, and SEQ ID NO:31;(b) calculate a first derivative of the run curve data set in (a) and determine whether the maximum value of the first derivative meets or exceeds a predetermined cutoff value; and(c) determine either that the test sample includes macrolide-resistant M. genitalium if the maximum value of the first derivative meets or exceeds the predetermined cutoff value, or the test sample does not include macrolide -resistant M. genitalium if the maximum value of the first derivative does not meet or exceed the cutoff.

23. The computer of claim 22, wherein the software instructions are executed by the computer if the test sample is known to include M. genitalium, the real-time nucleic acid amplification reaction being a reflex assay.

24. The computer of either claim 22 or claim 23, wherein the software instructions, when executed by the computer, further cause the computer to generate a non-transient record of the result from (c).

25. The computer of any one of claims 22 to 24, wherein the real-time nucleic acid amplification reaction is a real-time PCR reaction, wherein the run curve data set in (a) comprises fluorescent signal data as a function of PCR cycle number, and wherein the first derivative in (b) has units of RFU / cycle.

26. The computer of any one of claims 22 to 25, wherein the predetermined cutoff value is a predetermined constant numerical cutoff value.

27. The computer of any one of claims 22 to 26, wherein the predetermined cutoff value is obtained as a component of a kit used to perform the real-time nucleic acid amplification reaction and then programmed into the computer.

28. The computer of any one of claims 22 to 27, wherein the computer is in communication with an automated thermal cycling device equipped with a fluorometer.

29. The computer of 28, wherein the computer is an integral component of the automated thermal cycling device.

30. A computer programmed with software instructions to determine from results of a real-time nucleic acid amplification reaction whether a test sample of M. genitalium includes macrolide-resistant M. genitalium, nucleic acids isolated from the test sample having been used as templates for amplification of 23S rRNA sequences in the real-time nucleic acid amplification reaction, the software instructions, when executed by the computer, cause the computer to:(a) receive a run curve data set for the real-time nucleic acid amplification reaction, the run curve data set comprising signal data for amplification of(i) an M. genitalium detection locus that is present in nucleic acids of both macrolide-sensitive M. genitalium and macrolide-resistant M. genitalium, but absent from nucleic acids of M. pneumoniae, and(ii) an Mgen Res detection locus in the 23S rRNA that comprises either the wildtype sequence of SEQ ID NO: 18, or a sequence selected from the group consisting of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO: 30, and SEQ ID NO:31;(b) establish from the run curve data set in (a)(i) whether the M. genitalium detection locus was amplified and detected in the real-time nucleic acid amplification reaction;(c) calculate a first derivative of the ran curve data set in (a)(ii) and determine whether the maximum value of the first derivative meets or exceeds a predetermined cutoff value; and(d) determine either that the test sample includes macrolide-resistant M. genitalium if it is established in (b) that the M. genitalium detection locus was amplified and detected, and if the maximum value of the first derivative from (c) meets or exceeds the predetermined cutoff value, or the test sample does not include macrolide-resistant M. genitalium if either(i) it is established in (b) that the M. genitalium detection locus was not amplified and detected, or(ii) the maximum value of the first derivative from (c) does not meet or exceed the cutoff.31 . The computer of claim 30, wherein the software instructions, when executed by the computer, further cause the computer to generate a non- transient record of the result from (d).

32. The computer of either claim 30 or 31 , wherein the real-time nucleic acid amplification reaction is a real-time PCR reaction, wherein the ran curve data set in (a) comprises fluorescent signal data as a function of PCR cycle number, and wherein the first derivative in (c) has units of RFU / cycle.

33. The computer of any one of claims 30 to 32, wherein the predetermined cutoff value is a predetermined constant numerical cutoff value.

34. The computer of any one of claims 30 to 33, wherein the predetermined cutoff value is obtained as a component of a kit used to perform the real-time nucleic acid amplification reaction and then programmed into the computer.

35. The computer of any one of claims 30 to 34, wherein the computer is in communication with an automated thermal cycling device equipped with a fluorometer.

36. The computer of 35, wherein the computer is an integral component of the automated thermal cycling device.

Images