WO2024028818A1 - Procédé de détection de contamination par mycoplasma - Google Patents

Procédé de détection de contamination par mycoplasma Download PDF

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Publication number
WO2024028818A1
WO2024028818A1 PCT/IB2023/057886 IB2023057886W WO2024028818A1 WO 2024028818 A1 WO2024028818 A1 WO 2024028818A1 IB 2023057886 W IB2023057886 W IB 2023057886W WO 2024028818 A1 WO2024028818 A1 WO 2024028818A1
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sequence
seq
primer
pair
sample
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PCT/IB2023/057886
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English (en)
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Shyam Kumar GUDEY
Kuldip Singh SRA
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Crispr Therapeutics Ag
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Publication of WO2024028818A1 publication Critical patent/WO2024028818A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria

Definitions

  • the Sequence Listing is provided as a file entitled 80EM-341714- WO_SequenceListing, created August 2, 2023, which is 14 kilobytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.
  • BACKGROUND Field [0003] The present disclosure relates generally to methods and compositions for the detection of mycoplasma in a sample. More specifically, the present disclosure relates to detection of M. arginini, M. orale, M. synoviae, M. hyorhinis, A. laidlawii, M. fermentans, M. gallisepticum, M. pneumoniae and S. citri in sample by nucleic acid-based testing methods.
  • Mycoplasma are microscopic bacteria that lack a cell wall and have very small genomes with a low G+C content.
  • Mycoplasmas belong to the class of Mollicutes. Mollicutes are classified into six genera (Acholeplasma, Anaeroplasma, Asteroleplasma, Mycoplasma, Spiroplasma, and Ureaplasma). The phylogenetic classification is based on the 16s rRNA and 23s rRNA sequences and are further sub-divided into five clusters: M. fermentans group, M. pneumoniae group, M. hyorhinis group, M. hominis and M. mycoides.
  • Mycoplasmas are the most common cell culture contaminant during manufacturing processes, therefore, rigorous testing of cell culture material and drug product is an important criterion for lot release of a drug product as per FDA/EMA guidelines.
  • the conventional testing method (culture inoculation) takes a minimum of 28 days for assay completion.
  • a rapid probe-based quantitative PCR method capable of detecting mycoplasma species contamination in a sample, e.g., cell harvest media and drug substance.
  • assays that are designed to detect mycoplasmas belonging to more than one different clusters/genera in a single assay.
  • the method comprises: (a) contacting a sample with a pair of primers capable of hybridizing to a target region of M. arginini, M. orale, M. synoviae, and M. hyorhinis genome, wherein the target region comprises the 16s rRNA gene region of M. arginini, M. orale, M. synoviae, and M.
  • each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2; (b) generating amplicons of the target region of M. arginini, M. orale, M. synoviae, M. hyorhinis, or any combination thereof, from the sample, if the sample comprises one or more of M. arginini, M. orale, M. synoviae, and M. hyorhinis; and (c) determining the presence or amount of one or more amplicons as an indication of the presence of one or more of M. arginini, M. orale, M. synoviae, and M.
  • each primer in the pair of primers comprises the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 1 or SEQ ID NO: 2.
  • determining the presence or amount of the one or more amplicons of the target region comprises contacting the amplicons with an oligonucleotide probe comprising a sequence having at least about 85% identity to the sequence of SEQ ID NO: 3.
  • the oligonucleotide probe comprises the sequence of SEQ ID NO: 3, or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 3.
  • the oligonucleotide probe comprises a fluorescence emitter moiety, a fluorescence quencher moiety, or both.
  • the fluorescence emitter moiety is at the 5’ terminus of the oligonucleotide probe, the 3’ terminus of the oligonucleotide probe, or both.
  • the oligonucleotide probe further comprises a second fluorescence quencher moiety.
  • the second fluorescence quencher moiety is between the ninth and tenth nucleotide of the oligonucleotide probe.
  • the method comprises: (a) contacting a sample with a pair of primers capable of hybridizing to a target region of A. laidlawii genome, wherein the target region comprises the 23s rRNA gene region of A. laidlawii, and wherein each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 4 or SEQ ID NO: 5; (b) generating amplicons of the target region of A.
  • each primer in the pair of primers comprises the sequence of SEQ ID NO: 4 or SEQ ID NO: 5, or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 4 or SEQ ID NO: 5.
  • determining the presence or amount of the amplicons of the target region comprises contacting the amplicons with an oligonucleotide probe comprising a sequence having at least about 85% identity to the sequence of SEQ ID NO: 6.
  • the oligonucleotide probe comprises the sequence of SEQ ID NO: 6, or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 6.
  • the oligonucleotide probe comprises a fluorescence emitter moiety, a fluorescence quencher moiety, or both.
  • each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 7 or SEQ ID NO: 8; (b) generating amplicons of the target region of M. fermentans from the sample, if the sample comprises M. fermentans; and (c) determining the presence or amount of the amplicons of the target region as an indication of the presence of M. fermentans in the sample.
  • Each primer in the pair of primers can comprise the sequence of SEQ ID NO: 7 or SEQ ID NO: 8, or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 7 or SEQ ID NO: 8.
  • Determining the presence or amount of the amplicons of the target region can comprise contacting the amplicons with an oligonucleotide probe comprising a sequence having at least about 85% identity to the sequence of SEQ ID NO: 9.
  • the oligonucleotide probe comprises the sequence of SEQ ID NO: 9, or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 9.
  • the oligonucleotide probe can comprise a fluorescence emitter moiety, a fluorescence quencher moiety, or both.
  • the fluorescence emitter moiety is at the 5’ terminus of the oligonucleotide probe, the 3’ terminus of the oligonucleotide probe, or both.
  • the oligonucleotide probe further comprises a second fluorescence quencher moiety.
  • the second fluorescence quencher moiety is between the ninth and tenth nucleotide of the oligonucleotide probe.
  • the target region comprises the 23s rRNA gene region of M. gallisepticum and M. pneumoniae, and wherein each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 10 or SEQ ID NO: 11; (b) generating amplicons of the target region of M. gallisepticum, M. pneumoniae, or both from the sample, if the sample comprises one or more of M. gallisepticum and M. pneumoniae; and (c) determining the presence or amount of one or more amplicons as an indication of the presence of one or more of M. gallisepticum and M. pneumoniae in the sample.
  • Each primer in the pair of primers can comprise the sequence of SEQ ID NO: 10 or SEQ ID NO: 11, or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 10 or SEQ ID NO: 11.
  • determining the presence or amount of the one or more amplicons of the target region comprises contacting the amplicons with an oligonucleotide probe comprising a sequence having at least about 85% identity to the sequence of SEQ ID NO: 12.
  • the oligonucleotide probe can comprise the sequence of SEQ ID NO: 12, or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 12.
  • the oligonucleotide probe can comprise a fluorescence emitter moiety, a fluorescence quencher moiety, or both.
  • Disclosed herein include methods for detecting S. citri in a sample.
  • the method comprises: (a) contacting a sample with a pair of primers capable of hybridizing to a target region of S. citri genome, wherein the target region comprises the 16s rRNA gene region of S. citri, and wherein each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 13 or SEQ ID NO: 14; (b) generating amplicons of the target region of S. citri from the sample, if the sample comprises S.
  • the oligonucleotide probe comprises the sequence of SEQ ID NO: 15, or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 15.
  • the oligonucleotide probe can comprise a fluorescence emitter moiety, a fluorescence quencher moiety, or both.
  • the fluorescence emitter moiety is at the 5’ terminus of the oligonucleotide probe, 3’ terminus of the oligonucleotide probe, or both.
  • the oligonucleotide probe further comprises a second fluorescence quencher moiety.
  • the second fluorescence quencher moiety is between the ninth and tenth nucleotide of the oligonucleotide probe.
  • Disclosed herein include methods for detecting one or more of M. arginini, M. orale, M. synoviae, M. hyorhinis, A. laidlawii, M. fermentans, M. gallisepticum, M. pneumoniae and S. citri in a sample.
  • the method comprises: a) contacting a sample with a plurality of pairs of primers, wherein the plurality of pairs of primers comprises: a pair of primers capable of hybridizing to the 16s rRNA gene region of M. arginini, M.
  • each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2; a pair of primers capable of hybridizing to the 23s rRNA gene region of A. laidlawii, wherein each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 4 or SEQ ID NO: 5; a pair of primers capable of hybridizing to the 16s rRNA gene region of M.
  • each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 7 or SEQ ID NO: 8; a pair of primers capable of hybridizing to the 23s rRNA gene region of M. gallisepticum and M. pneumoniae, wherein each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 10 or SEQ ID NO: 11; and/or a pair of primers capable of hybridizing to the 16s rRNA gene region of S.
  • each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 13 or SEQ ID NO: 14; b) generating amplicons of the 16s rRNA gene region of M. arginini, the 16s rRNA gene region of M. orale, the 16s rRNA gene region of M. synoviae, the 16s rRNA gene region of M. hyorhinis, the 23s rRNA gene region of A. laidlawii, the 16s rRNA gene region of M. fermentans, the 23s rRNA gene region of M. gallisepticum, the 23s rRNA gene region of M. pneumoniae, the 16s rRNA gene region of S.
  • the sample comprises one or more of M. arginini, M. orale, M. synoviae, M. hyorhinis, A. laidlawii, M. fermentans, M. gallisepticum, M. pneumoniae, and S. citri; and c) determining the presence or amount of one or more amplicons as an indication of the presence of one or more of M. arginini, M. orale, M. synoviae, M. hyorhinis, A. laidlawii, M. fermentans, M. gallisepticum, M. pneumoniae, and S. citri in the sample.
  • the pair of primers capable of hybridizing to the 16s rRNA gene region of M. arginini, M. orale, M. synoviae, and M. hyorhinis comprises (a) a primer having the sequence of SEQ ID NO: 1 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 1 and (b) a primer having the sequence of SEQ ID NO: 2 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 2; the pair of primers capable of hybridizing to the 23s rRNA gene region of A.
  • laidlawii comprises (a) a primer having the sequence of SEQ ID NO: 4 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 4 and (b) a primer having the sequence of SEQ ID NO: 5 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 5; the pair of primers capable of hybridizing to the 16s rRNA gene region of M.
  • fermentans comprises (a) a primer having the sequence of SEQ ID NO: 7 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 7 and (b) a primer having the sequence of SEQ ID NO: 8 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 8; the pair of primers capable of hybridizing to the 23s rRNA gene region of M. gallisepticum and M.
  • pneumoniae comprises (a) a primer having the sequence of SEQ ID NO: 10 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 10 and (b) a primer having the sequence of SEQ ID NO: 11 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 11; and/or the pair of primers capable of hybridizing to the 16s rRNA gene region of S.
  • citri comprises (a) a primer having the sequence of SEQ ID NO: 13 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 13 and (b) a primer having the sequence of SEQ ID NO: 14 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 14.
  • Determining the presence or amount of the one or more amplicons can comprise contacting the amplicons with one or more oligonucleotide probes each comprising a sequence having at least about 85% identity to the sequence of SEQ ID NO: 3, 6, 9, 12, or 15.
  • Each of the one or more oligonucleotide probes can comprise the sequence of SEQ ID NO: 3, 6, 9, 12, or 15; or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 3, 6, 9, 12, or 15.
  • Disclosed herein include methods for detecting one or more of M. arginini, M. orale, M. synoviae, M. hyorhinis, and A. laidlawii in a sample.
  • the method comprises: a) contacting a sample with a plurality of pairs of primers, wherein the plurality of pairs of primers comprises: a pair of primers capable of hybridizing to the 16s rRNA gene region of M.
  • each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2; and a pair of primers capable of hybridizing to the 23s rRNA gene region of A. laidlawii, wherein each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 4 or SEQ ID NO: 5; b) generating amplicons of the 16s rRNA gene region of M. arginini, the 16s rRNA gene region of M. orale, the 16s rRNA gene region of M.
  • orale, M. synoviae, and M. hyorhinis can comprise (a) a primer having the sequence of SEQ ID NO: 1 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 1 and (b) a primer having the sequence of SEQ ID NO: 2 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 2.
  • the pair of primers capable of hybridizing to the 23s rRNA gene region of A.
  • laidlawii comprises (a) a primer having the sequence of SEQ ID NO: 4 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 4 and (b) a primer having the sequence of SEQ ID NO: 5 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 5.
  • Determining the presence or amount of the one or more amplicons can comprise contacting the amplicons with one or more oligonucleotide probes each comprising a sequence having at least about 85% identity to the sequence of SEQ ID NO: 3 or 6.
  • each of the one or more oligonucleotide probes comprises the sequence of SEQ ID NO: 3 or 6; or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 3 or 6.
  • Disclosed herein include methods for detecting one or more of M. fermentans, M. gallisepticum, and M. pneumoniae in a sample.
  • the method comprises: a) contacting a sample with a plurality of pairs of primers, wherein the plurality of pairs of primers comprises: a pair of primers capable of hybridizing to the 16s rRNA gene region of M.
  • each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 7 or SEQ ID NO: 8; and a pair of primers capable of hybridizing to the 23s rRNA gene region of M. gallisepticum and M. pneumoniae, wherein each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 10 or SEQ ID NO: 11; b) generating amplicons of the 16s rRNA gene region of M. fermentans, the 23s rRNA gene region of M. gallisepticum, the 23s rRNA gene region of M. pneumoniae, or any combination thereof, if the sample comprises one or more of M. fermentans, M.
  • the pair of primers capable of hybridizing to the 16s rRNA gene region of M. fermentans can comprise (a) a primer having the sequence of SEQ ID NO: 7 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 7 and (b) a primer having the sequence of SEQ ID NO: 8 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 8.
  • the pair of primers capable of hybridizing to the 23s rRNA gene region of M. gallisepticum and M. pneumoniae comprises (a) a primer having the sequence of SEQ ID NO: 10 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 10 and (b) a primer having the sequence of SEQ ID NO: 11 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 11.
  • Determining the presence or amount of the one or more amplicons can comprise contacting the amplicons with one or more oligonucleotide probes each comprising a sequence having at least about 85% identity to the sequence of SEQ ID NO: 9 or 12.
  • each of the one or more oligonucleotide probes comprises the sequence of SEQ ID NO: 9 or 12; or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 9 or 12.
  • Disclosed herein include methods for detecting one or more of A. laidlawii and S. citri in a sample.
  • the method comprises: a) contacting a sample with a plurality of pairs of primers, wherein the plurality of pairs of primers comprises: a pair of primers capable of hybridizing to the 23s rRNA gene region of A.
  • each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 4 or SEQ ID NO: 5; and a pair of primers capable of hybridizing to the 16s rRNA gene region of S. citri, wherein each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 13 or SEQ ID NO: 14; b) generating amplicons of the 23s rRNA gene region of A. laidlawii, the 16s rRNA gene region of S. citri, or both, if the sample comprises one or more of A. laidlawii and S.
  • the pair of primers capable of hybridizing to the 23s rRNA gene region of A. laidlawii can comprise (a) a primer having the sequence of SEQ ID NO: 4 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 4 and (b) a primer having the sequence of SEQ ID NO: 5 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 5.
  • citri comprises (a) a primer having the sequence of SEQ ID NO: 13 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 13 and (b) a primer having the sequence of SEQ ID NO: 14 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 14.
  • Determining the presence or amount of the one or more amplicons can comprise contacting the amplicons with one or more oligonucleotide probes each comprising a sequence having at least about 85% identity to the sequence of SEQ ID NO: 6 or 15.
  • each of the one or more oligonucleotide probes comprises the sequence of SEQ ID NO: 6 or 15; or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 6 or 15.
  • at least one of the one or more oligonucleotide probes comprises a fluorescence emitter moiety, a fluorescence quencher moiety, or both.
  • the fluorescence emitter moiety is at the 5’ terminus of the oligonucleotide probe, the 3’ terminus of the oligonucleotide probe, or both.
  • At least one of the one or more oligonucleotide probes further comprises a second fluorescence quencher moiety.
  • the second fluorescence quencher moiety is between the ninth and tenth nucleotide of the oligonucleotide probe.
  • the second fluorescence quencher moiety comprises ZEN or TAO.
  • each oligonucleotide probe is flanked by complementary sequences at the 5’ end and 3’ end.
  • one of the complementary sequences comprises a fluorescence emitter moiety and the other complementary sequence comprises a fluorescence quencher moiety.
  • (c) determining the presence or amount of the amplicons comprises using a reference standard curve.
  • the sample comprises, or is suspected to comprise, nucleic acids of one or more of M. arginini, M. orale, M. synoviae, M. hyorhinis, A. laidlawii, M. fermentans, M. gallisepticum, M. pneumoniae, and S. citri.
  • the sample comprises at least about 10 copies to at most about 1 ⁇ 10 6 copies of M. arginini genome, M. orale genome, M. synoviae genome, M. hyorhinis genome, A. laidlawii genome, M. fermentans genome, M.
  • the sample can be, or can be derived from, a biological sample or an environmental sample.
  • the biological sample can be obtained from, e.g., a tissue sample, saliva, blood, plasma, sera, stool, urine, sputum, mucous, lymph, synovial fluid, cerebrospinal fluid, ascites, pleural effusion, seroma, pus, swab of skin or a mucosal membrane surface, cultures thereof, or any combination thereof.
  • the biological sample comprises genetically modified cells.
  • the sample is a food sample, a beverage sample, a paper surface, a fabric surface, a metal surface, a wood surface, a plastic surface, a soil sample, a fresh water sample, a waste water sample, a saline water sample, a gas sample, a clinical sample, a pharmaceutical composition, cultures thereof, or any combination thereof.
  • the amplification can be carried out using a method selected from polymerase chain reaction (PCR), ligase chain reaction (LCR), loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), replicase-mediated amplification, Immuno-amplification, nucleic acid sequence based amplification (NASBA), self- sustained sequence replication (3SR), rolling circle amplification, transcription-mediated amplification (TMA), or any combination thereof.
  • the PCR is quantitative real-time PCR (qPCR).
  • qPCR quantitative real-time PCR
  • the composition comprises: a pair of primers capable of hybridizing to the 16s rRNA gene region of M. arginini, M. orale, M. synoviae, and M. hyorhinis, wherein each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
  • the composition can comprise: an oligonucleotide probe comprising a sequence having at least about 85% identity to the sequence of SEQ ID NO: 3.
  • Disclosed herein include compositions for detecting A. laidlawii in a sample.
  • the composition comprises: a pair of primers capable of hybridizing to the 23s rRNA gene region of A.
  • compositions for detecting M. fermentans in a sample comprising: a pair of primers capable of hybridizing to the 16s rRNA gene region of M. fermentans, wherein each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 7 or SEQ ID NO: 8.
  • the composition can comprise: an oligonucleotide probe comprising a sequence having at least about 85% identity to the sequence of SEQ ID NO: 9.
  • compositions for detecting one or more of M. gallisepticum and M. pneumoniae in a sample comprising: a pair of primers capable of hybridizing to the 23s rRNA gene region of M. gallisepticum and M. pneumoniae, wherein each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 10 or SEQ ID NO: 11.
  • the composition can comprise: an oligonucleotide probe comprising a sequence having at least about 85% identity to the sequence of SEQ ID NO: 12.
  • compositions for detecting S. citri in a sample comprising: a pair of primers capable of hybridizing to the 16s rRNA gene region of S. citri, wherein each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 13 or SEQ ID NO: 14.
  • the composition can comprise: an oligonucleotide probe comprising a sequence having at least about 85% identity to the sequence of SEQ ID NO: 15.
  • the composition comprises: a pair of primers capable of hybridizing to the 16s rRNA gene region of M. arginini, M. orale, M. synoviae, and M. hyorhinis, wherein each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2; a pair of primers capable of hybridizing to the 23s rRNA gene region of A.
  • each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 4 or SEQ ID NO: 5; a pair of primers capable of hybridizing to the 16s rRNA gene region of M. fermentans, wherein each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 7 or SEQ ID NO: 8; a pair of primers capable of hybridizing to the 23s rRNA gene region of M. gallisepticum and M.
  • each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 10 or SEQ ID NO: 11; and/or a pair of primers capable of hybridizing to the 16s rRNA gene region of S. citri, wherein each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 13 or SEQ ID NO: 14.
  • hyorhinis can comprise (a) a primer having the sequence of SEQ ID NO: 1 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 1 and (b) a primer having the sequence of SEQ ID NO: 2 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 2; the pair of primers capable of hybridizing to the 23s rRNA gene region of A.
  • laidlawii comprises (a) a primer having the sequence of SEQ ID NO: 4 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 4 and (b) a primer having the sequence of SEQ ID NO: 5 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 5; the pair of primers capable of hybridizing to the 16s rRNA gene region of M.
  • fermentans comprises (a) a primer having the sequence of SEQ ID NO: 7 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 7 and (b) a primer having the sequence of SEQ ID NO: 8 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 8; the pair of primers capable of hybridizing to the 23s rRNA gene region of M. gallisepticum and M.
  • pneumoniae comprises (a) a primer having the sequence of SEQ ID NO: 10 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 10 and (b) a primer having the sequence of SEQ ID NO: 11 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 11; and/or the pair of primers capable of hybridizing to the 16s rRNA gene region of S.
  • citri comprises (a) a primer having the sequence of SEQ ID NO: 13 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 13 and (b) a primer having the sequence of SEQ ID NO: 14 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 14.
  • the composition can comprise: one or more oligonucleotide probes each comprising a sequence having at least about 85% identity to the sequence of SEQ ID NO: 3, 6, 9, 12, or 15; for example the sequence of SEQ ID NO: 3, 6, 9, 12, or 15, or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 3, 6, 9, 12, or 15.
  • compositions for detecting one or more of M. arginini, M. orale, M. synoviae, M. hyorhinis, and A. laidlawii in a sample comprises: a pair of primers capable of hybridizing to the 16s rRNA gene region of M. arginini, M. orale, M. synoviae, and M. hyorhinis, wherein each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2; and a pair of primers capable of hybridizing to the 23s rRNA gene region of A.
  • each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 4 or SEQ ID NO: 5.
  • the pair of primers capable of hybridizing to the 16s rRNA gene region of M. arginini, M. orale, M. synoviae, and M. hyorhinis can comprise (a) a primer having the sequence of SEQ ID NO: 1 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 1 and (b) a primer having the sequence of SEQ ID NO: 2 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 2.
  • the pair of primers capable of hybridizing to the 23s rRNA gene region of A. laidlawii comprises (a) a primer having the sequence of SEQ ID NO: 4 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 4 and (b) a primer having the sequence of SEQ ID NO: 5 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 5.
  • the composition can comprise: one or more oligonucleotide probes each comprising a sequence having at least about 85% identity to the sequence of SEQ ID NO: 3 or 6, for example the sequence of SEQ ID NO: 3 or 6; or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 3 or 6.
  • compositions for detecting one or more of M. fermentans, M. gallisepticum, and M. pneumoniae in a sample comprises: a pair of primers capable of hybridizing to the 16s rRNA gene region of M.
  • each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 7 or SEQ ID NO: 8; and a pair of primers capable of hybridizing to the 23s rRNA gene region of M. gallisepticum and M. pneumoniae, wherein each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 10 or SEQ ID NO: 11.
  • fermentans comprises (a) a primer having the sequence of SEQ ID NO: 7 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 7 and (b) a primer having the sequence of SEQ ID NO: 8 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 8.
  • the pair of primers capable of hybridizing to the 23s rRNA gene region of M. gallisepticum and M.
  • pneumoniae comprises (a) a primer having the sequence of SEQ ID NO: 10 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 10 and (b) a primer having the sequence of SEQ ID NO: 11 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 11.
  • the composition can comprise: one or more oligonucleotide probes each comprising a sequence having at least about 85% identity to the sequence of SEQ ID NO: 9 or 12, for example the sequence of SEQ ID NO: 9 or 12; or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 9 or 12.
  • compositions for detecting one or more of A. laidlawii and S. citri in a sample comprising: a pair of primers capable of hybridizing to the 23s rRNA gene region of A. laidlawii, wherein each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 4 or SEQ ID NO: 5; and a pair of primers capable of hybridizing to the 16s rRNA gene region of S. citri, wherein each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 13 or SEQ ID NO: 14.
  • the pair of primers capable of hybridizing to the 23s rRNA gene region of A. laidlawii comprises (a) a primer having the sequence of SEQ ID NO: 4 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 4 and (b) a primer having the sequence of SEQ ID NO: 5 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 5.
  • citri comprises (a) a primer having the sequence of SEQ ID NO: 13 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 13 and (b) a primer having the sequence of SEQ ID NO: 14 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 14.
  • the composition can comprise: one or more oligonucleotide probes each comprising a sequence having at least about 85% identity to the sequence of SEQ ID NO: 6 or 15, for example the sequence of SEQ ID NO: 6 or 15; or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 6 or 15.
  • At least one of the one or more oligonucleotide probes comprises a fluorescence emitter moiety, a fluorescence quencher moiety, or both.
  • the fluorescence emitter moiety is at the 5’ terminus of the oligonucleotide probe, the 3’ terminus of the oligonucleotide probe, or both.
  • at least one of the one or more oligonucleotide probes further comprises a second fluorescence quencher moiety.
  • the second fluorescence quencher moiety is between the ninth and tenth nucleotide of the oligonucleotide probe.
  • the second fluorescence quencher moiety comprises ZEN or TAO.
  • each oligonucleotide probe is flanked by complementary sequences at the 5’ end and 3’ end.
  • one of the complementary sequences comprises a fluorescence emitter moiety and the other complementary sequence comprises a fluorescence quencher moiety.
  • the composition can comprise: one or more compositions described herein and a DNA polymerase.
  • the composition can comprise: one or more of a buffer and MgCl2.
  • the composition can comprise: nucleic acids from M. arginini, M. orale, M. synoviae, M. hyorhinis, A.
  • the nucleic acids comprise an M. arginini DNA, an M. orale DNA, an M. synoviae DNA, an M. hyorhinis DNA, an A. laidlawii DNA, an M. fermentans DNA, an M. gallisepticum DNA, an M. pneumoniae DNA, an S. citri DNA, or any combination thereof.
  • the composition is an amplification reaction mixture. [0050] Disclosed herein include oligonucleotide probes or primers up to about 100 nucleotides in length capable of hybridizing to the 16s rRNA gene region of M.
  • the probe or primer comprises the sequence of any one of SEQ ID NOs: 1-3, or a sequence having at least about 85% identity to any one of SEQ ID NOs: 1-3.
  • Disclosed herein include oligonucleotide probes or primers up to about 100 nucleotides in length capable of hybridizing to the 23s rRNA gene region of A. laidlawii.
  • the probe or primer comprises the sequence of any one of SEQ ID NOs: 4-6, or a sequence having at least about 85% identity to any one of SEQ ID NOs: 4-6.
  • oligonucleotide probes or primers up to about 100 nucleotides in length capable of hybridizing to the 16s rRNA gene region of M. fermentans.
  • the probe or primer comprises the sequence of any one of SEQ ID NOs: 7-9, or a sequence having at least about 85% identity to any one of SEQ ID NOs: 7-9.
  • oligonucleotide probes or primers up to about 100 nucleotides in length capable of hybridizing to the 23s rRNA gene region of M. gallisepticum and M. pneumoniae.
  • the probe or primer comprises the sequence of any one of SEQ ID NOs: 10-12, or a sequence having at least about 85% identity to any one of SEQ ID NOs: 10-12.
  • oligonucleotide probes or primers up to about 100 nucleotides in length capable of hybridizing to the 16s rRNA gene region of S. citri.
  • the probe or primer comprises the sequence of any one of SEQ ID NOs: 13-15, or a sequence having at least about 85% identity to any one of SEQ ID NOs: 13-15.
  • compositions comprising two or more of the oligonucleotide probe or primer described herein.
  • nucleic acid amplifications can be performed to determine the presence, absence, type, and/or level of one or more of M. arginini, M. orale, M. synoviae, M. hyorhinis, A. laidlawii, M. fermentans, M. gallisepticum, M. pneumoniae and S. citri in a sample.
  • the method comprises: (a) contacting a sample with a pair of primers capable of hybridizing to a target region of M. arginini, M. orale, M. synoviae, and M. hyorhinis genome, wherein the target region comprises the 16s rRNA gene region of M. arginini, M. orale, M. synoviae, and M. hyorhinis, and wherein each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2; (b) generating amplicons of the target region of M. arginini, M. orale, M.
  • the method comprises: (a) contacting a sample with a pair of primers capable of hybridizing to a target region of A.
  • each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 4 or SEQ ID NO: 5; (b) generating amplicons of the target region of A. laidlawii from the sample, if the sample comprises A. laidlawii; and (c) determining the presence or amount of the amplicons of the target region as an indication of the presence of A. laidlawii in the sample.
  • Disclosed herein include methods for detecting M. fermentans in a sample.
  • the method comprises: (a) contacting a sample with a pair of primers capable of hybridizing to a target region of M. fermentans genome, wherein the target region comprises the 16s rRNA gene region of M. fermentans, and wherein each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 7 or SEQ ID NO: 8; (b) generating amplicons of the target region of M. fermentans from the sample, if the sample comprises M. fermentans; and (c) determining the presence or amount of the amplicons of the target region as an indication of the presence of M. fermentans in the sample.
  • Disclosed herein include methods for detecting one or more of M. gallisepticum and M.
  • the method comprises: (a) contacting a sample with a pair of primers capable of hybridizing to a target region of M. gallisepticum and M. pneumoniae genome, wherein the target region comprises the 23s rRNA gene region of M. gallisepticum and M. pneumoniae, and wherein each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 10 or SEQ ID NO: 11; (b) generating amplicons of the target region of M. gallisepticum, M. pneumoniae, or both from the sample, if the sample comprises one or more of M. gallisepticum and M.
  • the method comprises: (a) contacting a sample with a pair of primers capable of hybridizing to a target region of S. citri genome, wherein the target region comprises the 16s rRNA gene region of S. citri, and wherein each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 13 or SEQ ID NO: 14; (b) generating amplicons of the target region of S.
  • Disclosed herein include methods for detecting one or more of M. arginini, M. orale, M. synoviae, M. hyorhinis, A. laidlawii, M. fermentans, M. gallisepticum, M. pneumoniae and S. citri in a sample.
  • the method comprises: a) contacting a sample with a plurality of pairs of primers, wherein the plurality of pairs of primers comprises: a pair of primers capable of hybridizing to the 16s rRNA gene region of M. arginini, M. orale, M. synoviae, and M. hyorhinis, wherein each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2; a pair of primers capable of hybridizing to the 23s rRNA gene region of A.
  • each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 4 or SEQ ID NO: 5; a pair of primers capable of hybridizing to the 16s rRNA gene region of M. fermentans, wherein each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 7 or SEQ ID NO: 8; a pair of primers capable of hybridizing to the 23s rRNA gene region of M. gallisepticum and M.
  • each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 10 or SEQ ID NO: 11; and/or a pair of primers capable of hybridizing to the 16s rRNA gene region of S. citri, wherein each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 13 or SEQ ID NO: 14; b) generating amplicons of the 16s rRNA gene region of M. arginini, the 16s rRNA gene region of M. orale, the 16s rRNA gene region of M. synoviae, the 16s rRNA gene region of M.
  • the sample comprises one or more of M. arginini, M. orale, M. synoviae, M. hyorhinis, A. laidlawii, M. fermentans, M. gallisepticum, M. pneumoniae, and S. citri; and c) determining the presence or amount of one or more amplicons as an indication of the presence of one or more of M.
  • the method comprises: a) contacting a sample with a plurality of pairs of primers, wherein the plurality of pairs of primers comprises: a pair of primers capable of hybridizing to the 16s rRNA gene region of M. arginini, M.
  • each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2; and a pair of primers capable of hybridizing to the 23s rRNA gene region of A. laidlawii, wherein each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 4 or SEQ ID NO: 5; b) generating amplicons of the 16s rRNA gene region of M. arginini, the 16s rRNA gene region of M. orale, the 16s rRNA gene region of M.
  • the method comprises: a) contacting a sample with a plurality of pairs of primers, wherein the plurality of pairs of primers comprises: a pair of primers capable of hybridizing to the 16s rRNA gene region of M. fermentans, wherein each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 7 or SEQ ID NO: 8; and a pair of primers capable of hybridizing to the 23s rRNA gene region of M. gallisepticum and M.
  • each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 10 or SEQ ID NO: 11; b) generating amplicons of the 16s rRNA gene region of M. fermentans, the 23s rRNA gene region of M. gallisepticum, the 23s rRNA gene region of M. pneumoniae, or any combination thereof, if the sample comprises one or more of M. fermentans, M. gallisepticum, and M. pneumoniae; and c) determining the presence or amount of one or more amplicons as an indication of the presence of one or more of M. fermentans, M. gallisepticum, and M. pneumoniae in the sample.
  • the method comprises: a) contacting a sample with a plurality of pairs of primers, wherein the plurality of pairs of primers comprises: a pair of primers capable of hybridizing to the 23s rRNA gene region of A. laidlawii, wherein each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 4 or SEQ ID NO: 5; and a pair of primers capable of hybridizing to the 16s rRNA gene region of S.
  • each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 13 or SEQ ID NO: 14; b) generating amplicons of the 23s rRNA gene region of A. laidlawii, the 16s rRNA gene region of S. citri, or both, if the sample comprises one or more of A. laidlawii and S. citri; and c) determining the presence or amount of one or more amplicons as an indication of the presence of one or more of A. laidlawii and S. citri in the sample.
  • the composition can comprise: a pair of primers capable of hybridizing to the 16s rRNA gene region of M. arginini, M. orale, M. synoviae, and M. hyorhinis, wherein each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
  • each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
  • compositions for detecting M. fermentans in a sample can comprise: a pair of primers capable of hybridizing to the 16s rRNA gene region of M. fermentans, wherein each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 7 or SEQ ID NO: 8.
  • compositions for detecting one or more of M. gallisepticum and M. pneumoniae in a sample are disclosed.
  • the composition can comprise: a pair of primers capable of hybridizing to the 23s rRNA gene region of M. gallisepticum and M. pneumoniae, wherein each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 10 or SEQ ID NO: 11.
  • compositions for detecting S. citri in a sample can comprise: a pair of primers capable of hybridizing to the 16s rRNA gene region of S. citri, wherein each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 13 or SEQ ID NO: 14.
  • compositions for detecting one or more of M are examples of a pair of primers capable of hybridizing to the 23s rRNA gene region of M. gallisepticum and M. pneumoniae, wherein each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 10 or SEQ ID NO: 11.
  • the composition comprises: a pair of primers capable of hybridizing to the 16s rRNA gene region of M. arginini, M. orale, M. synoviae, and M. hyorhinis, wherein each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2; a pair of primers capable of hybridizing to the 23s rRNA gene region of A.
  • each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 4 or SEQ ID NO: 5; a pair of primers capable of hybridizing to the 16s rRNA gene region of M. fermentans, wherein each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 7 or SEQ ID NO: 8; a pair of primers capable of hybridizing to the 23s rRNA gene region of M. gallisepticum and M.
  • each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 10 or SEQ ID NO: 11; and/or a pair of primers capable of hybridizing to the 16s rRNA gene region of S. citri, wherein each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 13 or SEQ ID NO: 14.
  • compositions for detecting one or more of M. arginini, M. orale, M. synoviae, M. hyorhinis, and A. laidlawii in a sample are examples of compositions for detecting one or more of M. arginini, M. orale, M. synoviae, M. hyorhinis, and A. laidlawii in a sample.
  • the composition comprises: a pair of primers capable of hybridizing to the 16s rRNA gene region of M. arginini, M. orale, M. synoviae, and M. hyorhinis, wherein each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2; and a pair of primers capable of hybridizing to the 23s rRNA gene region of A. laidlawii, wherein each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 4 or SEQ ID NO: 5.
  • the composition comprises: a pair of primers capable of hybridizing to the 16s rRNA gene region of M. fermentans, wherein each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 7 or SEQ ID NO: 8; and a pair of primers capable of hybridizing to the 23s rRNA gene region of M. gallisepticum and M. pneumoniae, wherein each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 10 or SEQ ID NO: 11.
  • the composition can comprise: a pair of primers capable of hybridizing to the 23s rRNA gene region of A. laidlawii, wherein each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 4 or SEQ ID NO: 5; and a pair of primers capable of hybridizing to the 16s rRNA gene region of S. citri, wherein each primer in the pair of primers comprises a sequence having at least about 85% identity to the sequence of SEQ ID NO: 13 or SEQ ID NO: 14.
  • compositions comprising one or more compositions described herein and a DNA polymerase.
  • oligonucleotide probes or primers up to about 100 nucleotides in length capable of hybridizing to the 16s rRNA gene region of M. arginini, M. orale, M. synoviae, and M. hyorhinis.
  • the probe or primer comprises the sequence of any one of SEQ ID NOs: 1-3, or a sequence having at least about 85% identity to any one of SEQ ID NOs: 1-3.
  • oligonucleotide probes or primers up to about 100 nucleotides in length capable of hybridizing to the 23s rRNA gene region of A. laidlawii.
  • the probe or primer comprises the sequence of any one of SEQ ID NOs: 4-6, or a sequence having at least about 85% identity to any one of SEQ ID NOs: 4-6.
  • Disclosed herein include oligonucleotide probes or primers up to about 100 nucleotides in length capable of hybridizing to the 16s rRNA gene region of M. fermentans.
  • the probe or primer comprises the sequence of any one of SEQ ID NOs: 7-9, or a sequence having at least about 85% identity to any one of SEQ ID NOs: 7-9.
  • oligonucleotide probes or primers up to about 100 nucleotides in length capable of hybridizing to the 23s rRNA gene region of M. gallisepticum and M. pneumoniae.
  • the probe or primer comprises the sequence of any one of SEQ ID NOs: 10-12, or a sequence having at least about 85% identity to any one of SEQ ID NOs: 10-12.
  • oligonucleotide probes or primers up to about 100 nucleotides in length capable of hybridizing to the 16s rRNA gene region of S. citri.
  • the probe or primer comprises the sequence of any one of SEQ ID NOs: 13-15, or a sequence having at least about 85% identity to any one of SEQ ID NOs: 13-15.
  • Dislosed herein include compositions comprising two or more of the oligonucleotide probe or primer described herein. Definitions [0072] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. See, e.g. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J.
  • nucleic acid can refer to a polynucleotide sequence, or fragment thereof.
  • a nucleic acid can comprise nucleotides.
  • a nucleic acid can be exogenous or endogenous to a cell.
  • a nucleic acid can exist in a cell-free environment.
  • a nucleic acid can be a gene or fragment thereof.
  • a nucleic acid can be DNA.
  • a nucleic acid can be RNA.
  • a nucleic acid can comprise one or more analogs (e.g., altered backbone, sugar, or nucleobase).
  • analogs include: 5-bromouracil, peptide nucleic acid, xeno nucleic acid, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to the sugar), thiol containing nucleotides, biotin linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queuosine, and wyosine.
  • nucleic acid can refer to a polymeric compound comprising nucleosides or nucleoside analogs which have nitrogenous heterocyclic bases, or base analogs, linked together by nucleic acid backbone linkages (e.g., phosphodiester bonds) to form a polynucleotide.
  • nucleic acid backbone linkages e.g., phosphodiester bonds
  • the nucleic acid backbone can include a variety of linkages, for example, one or more of sugar-phosphodiester linkages, peptide-nucleic acid bonds, phosphorothioate or methylphosphonate linkages or mixtures of such linkages in a single oligonucleotide.
  • Sugar moieties in the nucleic acid can be either ribose or deoxyribose, or similar compounds with known substitutions.
  • nucleic acid can include only conventional sugars, bases and linkages found in RNA and DNA, or include both conventional components and substitutions (e.g., conventional bases and analogs linked via a methoxy backbone, or conventional bases and one or more base analogs linked via an RNA or DNA backbone).
  • a nucleic acid can comprise one or more modifications (e.g., a base modification, a backbone modification), to provide the nucleic acid with a new or enhanced feature (e.g., improved stability).
  • a nucleic acid can comprise a nucleic acid affinity tag.
  • a nucleoside can be a base-sugar combination. The base portion of the nucleoside can be a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines.
  • Nucleotides can be nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside.
  • the phosphate group can be linked to the 2’, the 3’, or the 5’ hydroxyl moiety of the sugar.
  • the phosphate groups can covalently link adjacent nucleosides to one another to form a linear polymeric compound.
  • the respective ends of this linear polymeric compound can be further joined to form a circular compound; however, linear compounds are generally suitable.
  • linear compounds may have internal nucleotide base complementarity and may therefore fold in a manner as to produce a fully or partially double-stranded compound.
  • the phosphate groups can commonly be referred to as forming the internucleoside backbone of the nucleic acid.
  • the linkage or backbone can be a 3’ to 5’ phosphodiester linkage.
  • a nucleic acid can comprise a modified backbone and/or modified internucleoside linkages. Modified backbones can include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.
  • Suitable modified nucleic acid backbones containing a phosphorus atom therein can include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonate such as 3’-alkylene phosphonates, 5’-alkylene phosphonates, chiral phosphonates, phosphinates, phosphoramidates including 3’-amino phosphoramidate and aminoalkyl phosphoramidates, phosphorodiamidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and boranophosphates having normal 3’-5’ linkages, 2’-5’ linked analogs, and those having inverted polarity wherein one or more internucleotide linkages is a 3’ to 3’, a 5’ to 5
  • a nucleic acid can comprise polynucleotide backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
  • These can include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.
  • siloxane backbones siloxane backbones
  • sulfide, sulfoxide and sulfone backbones formacetyl and thioformacetyl backbones
  • a nucleic acid can comprise a nucleic acid mimetic.
  • the term “mimetic” can be intended to include polynucleotides wherein only the furanose ring or both the furanose ring and the internucleotide linkage are replaced with non-furanose groups, replacement of only the furanose ring can also be referred as being a sugar surrogate.
  • the heterocyclic base moiety or a modified heterocyclic base moiety can be maintained for hybridization with an appropriate target nucleic acid.
  • One such nucleic acid can be a peptide nucleic acid (PNA).
  • the sugar-backbone of a polynucleotide can be replaced with an amide containing backbone, in particular an aminoethylglycine backbone.
  • the nucleotides can be retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone
  • the backbone in PNA compounds can comprise two or more linked aminoethylglycine units which gives PNA an amide containing backbone.
  • the heterocyclic base moieties can be bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • a nucleic acid can comprise a morpholino backbone structure.
  • a nucleic acid can comprise a 6-membered morpholino ring in place of a ribose ring.
  • a phosphorodiamidate or other non-phosphodiester internucleoside linkage can replace a phosphodiester linkage.
  • a nucleic acid can comprise linked morpholino units (e.g., morpholino nucleic acid) having heterocyclic bases attached to the morpholino ring.
  • Linking groups can link the morpholino monomeric units in a morpholino nucleic acid.
  • Non-ionic morpholino-based oligomeric compounds can have less undesired interactions with cellular proteins.
  • Morpholino-based polynucleotides can be nonionic mimics of nucleic acids.
  • a variety of compounds within the morpholino class can be joined using different linking groups.
  • a further class of polynucleotide mimetic can be referred to as cyclohexenyl nucleic acids (CeNA).
  • the furanose ring normally present in a nucleic acid molecule can be replaced with a cyclohexenyl ring.
  • CeNA DMT protected phosphoramidite monomers can be prepared and used for oligomeric compound synthesis using phosphoramidite chemistry. The incorporation of CeNA monomers into a nucleic acid chain can increase the stability of a DNA/RNA hybrid.
  • CeNA oligoadenylates can form complexes with nucleic acid complements with similar stability to the native complexes.
  • a further modification can include Locked Nucleic Acids (LNAs) in which the 2’-hydroxyl group is linked to the 4’ carbon atom of the sugar ring thereby forming a 2’-C, 4’-C-oxymethylene linkage thereby forming a bicyclic sugar moiety.
  • the linkage can be a methylene (-CH2), group bridging the 2’ oxygen atom and the 4’ carbon atom wherein n is 1 or 2.
  • a nucleic acid may also include nucleobase (often referred to simply as “base”) modifications or substitutions.
  • base can include the purine bases, (e.g., adenine (A) and guanine (G)), and the pyrimidine bases, (e.g., thymine (T), cytosine (C) and uracil (U)).
  • Modified nucleobases can include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g., 9-(2-aminoethoxy)-H-pyrimido(5,4-(b) (1,4)benzoxazin- 2(3H)-one), phenothiazine cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G- clamps such as a substituted phenoxazine cytidine (e.g., 9-(2-aminoethoxy)-
  • isolated nucleic acids can refer to the purification of nucleic acids from one or more cellular components.
  • samples processed to “isolate nucleic acids” therefrom can include components and impurities other than nucleic acids.
  • Samples that comprise isolated nucleic acids can be prepared from specimens using any acceptable method known in the art. For example, cells can be lysed using known lysis agents, and nucleic acids can be purified or partially purified from other cellular components.
  • the extracted nucleic acid solution can be added directly to a reagents (e.g., either in liquid, bound to a substrate, in lyophilized form, or the like, as discussed in further detail below), required to perform a test according to the embodiments disclosed herein.
  • template can refer to all or part of a polynucleotide containing at least one target nucleotide sequence.
  • a “primer” can refer to a polynucleotide that can serve to initiate a nucleic acid chain extension reaction.
  • the length of a primer can vary, for example, from about 5 to about 100 nucleotides, from about 10 to about 50 nucleotides, from about 15 to about 40 nucleotides, or from about 20 to about 30 nucleotides.
  • the length of a primer can be about 10 nucleotides, about 20 nucleotides, about 25 nucleotides, about 30 nucleotides, about 35 nucleotides, about 40 nucleotides, about 50 nucleotides, about 75 nucleotides, about 100 nucleotides, or a range between any two of these values.
  • the primer has a length of 10 to about 50 nucleotides, i.e., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 2526, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more nucleotides. In some embodiments, the primer has a length of 18 to 32 nucleotides.
  • a “probe” can refer to an polynucleotide that can hybridize (e.g., specifically) to a target sequence in a nucleic acid, under conditions that allow hybridization, thereby allowing detection of the target sequence or amplified nucleic acid.
  • a probe generally refers to a sequence within or a subset of an amplified nucleic acid sequence which hybridizes specifically to at least a portion of a probe oligomer by standard hydrogen bonding (i.e., base pairing).
  • a probe may comprise target-specific sequences and other sequences that contribute to three-dimensional conformation of the probe.
  • Sequences are “sufficiently complementary” if they allow stable hybridization in appropriate hybridization conditions of a probe oligomer to a target sequence that is not completely complementary to the probe's target-specific sequence.
  • the length of a probe can vary, for example, from about 5 to about 100 nucleotides, from about 10 to about 50 nucleotides, from about 15 to about 40 nucleotides, or from about 20 to about 30 nucleotides.
  • the length of a probe can be about 10 nucleotides, about 20 nucleotides, about 25 nucleotides, about 30 nucleotides, about 35 nucleotides, about 40 nucleotides, about 50 nucleotides, about 100 nucleotides, or a range between any two of these values.
  • the probe has a length of 10 to about 50 nucleotides.
  • the primers and or probes can be at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 2526, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more nucleotides.
  • the probe can be non- sequence specific.
  • the primers and/or probes can be between 8 and 45 nucleotides in length.
  • the primers and/or probes can be at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, or more nucleotides in length.
  • the primer and probe can be modified to contain additional nucleotides at the 5' or the 3' terminus, or both.
  • additional bases to the 3' terminus of amplification primers are generally complementary to the template sequence.
  • the primer and probe sequences can also be modified to remove nucleotides at the 5' or the 3' terminus.
  • the primers or probes will be of a minimum length and annealing temperature as disclosed herein.
  • Primers and probes can bind to their targets at an annealing temperature, which is a temperature less than the melting temperature (Tm).
  • Tm melting temperature
  • melting temperature are interchangeable terms which refer to the temperature at which 50% of a population of double-stranded polynucleotide molecules becomes dissociated into single strands. The formulae for calculating the Tm of polynucleotides are well known in the art.
  • the T m of a hybrid polynucleotide may also be estimated using a formula adopted from hybridization assays in 1 M salt, and commonly used for calculating Tm for PCR primers: [(number of A+T) ⁇ 2 o C + (number of G+C) x 4 o C]. See, e.g., C. R. Newton et al. PCR, 2nd ed., Springer-Verlag (New York: 1997), p.24 (incorporated by reference in its entirety, herein).
  • the melting temperature of an oligonucleotide can depend on complementarity between the oligonucleotide primer or probe and the binding sequence, and on salt conditions.
  • an oligonucleotide primer or probe provided herein has a T m of less than about 90 ⁇ C in 50mM KCl, 10 mM Tris-HCl buffer, for example about 89 ⁇ C, 88, 87, 86, 85, 84, 83, 82, 81, 8079, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39 ⁇ C, or less, including ranges between any two of the listed values.
  • the primers disclosed herein can be provided as an amplification primer pair, e.g., comprising a forward primer and a reverse primer (first amplification primer and second amplification primer).
  • the forward and reverse primers have T m ’s that do not differ by more than 10 o C, e.g., that differ by less than 10 o C, less than 9 o C, less than 8 o C, less than 7 o C, less than 6 o C, less than 5 o C, less than 4 o C, less than 3 o C, less than 2 o C, or less than 1 o C.
  • the primer and probe sequences may be modified by having nucleotide substitutions (relative to the target sequence) within the oligonucleotide sequence, provided that the oligonucleotide contains enough complementarity to hybridize specifically to the target nucleic acid sequence. In this manner, at least 1, 2, 3, 4, or up to about 5 nucleotides can be substituted.
  • the term “complementary” can refer to sequence complementarity between regions of two polynucleotide strands or between two regions of the same polynucleotide strand.
  • a first region of a polynucleotide is complementary to a second region of the same or a different polynucleotide if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide of the first region is capable of base pairing with a base of the second region. Therefore, it is not required for two complementary polynucleotides to base pair at every nucleotide position.
  • “Fully complementary” can refer to a first polynucleotide that is 100% or “fully complementary” to a second polynucleotide and thus forms a base pair at every nucleotide position.
  • Partially complementary also can refer to a first polynucleotide that is not 100% complementary (e.g., 90%, or 80% or 70% complementary) and contains mismatched nucleotides at one or more nucleotide positions.
  • an oligonucleotide includes a universal base.
  • an “exogenous nucleotide sequence” can refer to a sequence introduced by primers or probes used for amplification, such that amplification products will contain exogenous nucleotide sequence and target nucleotide sequence in an arrangement not found in the original template from which the target nucleotide sequence was copied.
  • sequence identity or “percent identical” as applied to nucleic acid molecules can refer to the percentage of nucleic acid residues in a candidate nucleic acid molecule sequence that are identical with a subject nucleic acid molecule sequence, after aligning the sequences to achieve the maximum percent identity, and not considering any nucleic acid residue substitutions as part of the sequence identity.
  • Nucleic acid sequence identity can be determined using any method known in the art, for example CLUSTAL OMEGA, T- COFFEE, BLASTN.
  • the term “sufficiently complementary” can refer to a contiguous nucleic acid base sequence that is capable of hybridizing to another base sequence by hydrogen bonding between a series of complementary bases.
  • Complementary base sequences can be complementary at each position in the oligomer sequence by using standard base pairing (e.g., G:C, A:T or A:U) or can contain one or more residues that are not complementary (including abasic positions), but in which the entire complementary base sequence is capable of specifically hybridizing with another base sequence in appropriate hybridization conditions.
  • Contiguous bases can be at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100% complementary to a sequence to which an oligomer is intended to hybridize.
  • Substantially complementary sequences can refer to sequences ranging in percent identity from 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 75, 70 or less, or any number in between, compared to the reference sequence.
  • a skilled artisan can readily choose appropriate hybridization conditions which can be predicted based on base sequence composition, or be determined by using routine testing (see e.g., Green and Sambrook, Molecular Cloning, A Laboratory Manual, 4th ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2012)).
  • multiplex PCR refers to a type of PCR where more than one set of primers is included in a reaction allowing one single target, or two or more different targets, to be amplified in a single reaction vessel (e.g., tube).
  • the multiplex PCR can be, for example, a real-time PCR.
  • Mycoplasmas are microscopic bacteria that lack a cell wall and have very small genomes with a low G+C content. Mycoplasmas belong to the class of Mollicutes. Mollicutes are classified into six genera (Acholeplasma, Anaeroplasma, Asteroleplasma, Mycoplasma, Spiroplasma, and Ureaplasma).
  • the phylogenetic classification is based on the 16s rRNA and 23s rRNA sequences and are further sub-divided into five clusters.
  • M. fermentans group M. pneumoniae group, M. hyorhinis group, M. hominis and M. mycoides.
  • Mycoplasmas are the most common cell culture contaminant during manufacturing processes, therefore rigorous testing of cell culture material and drug product is an important criterion for lot release as per FDA/EMA guidelines.
  • the conventional mycoplasma testing method (culture inoculation) takes a minimum of 28 days for assay completion and requires a large sample volume; therefore, this methodology does not allow fast turnaround times and is very laborious.
  • CMOS complementary metal-oxide-semiconductor
  • shelf-life data as well as production volume can be limited, making it essential to be able to administer the product to patients within a short time span.
  • a fast, simple, and reliable method to detect mycoplasma contamination is required.
  • Disclosed herein includes a rapid probe-based quantitative PCR method for detecting mycoplasma species contamination in, for example cell harvest media (CHM) and drug substance (DS), including cell-based therapeutics.
  • CHM cell harvest media
  • DS drug substance
  • the assays can detect mycoplasmas belonging to at least two different clusters/genera in a single assay.
  • nucleic acid amplifications can be performed to determine the presence, absence, type, and/or level of one or more of M. arginini, M. orale, M. synoviae, M. hyorhinis, A. laidlawii, M. fermentans, M. gallisepticum, M. pneumoniae and S. citri in a sample.
  • citri is determined by detecting one or more target gene regions of the target organism(s) using methods known in the art, such as nucleic acid (e.g., DNA) amplifications.
  • an individual amplification reaction e.g., PCR
  • M. arginini M. orale, M. synoviae, M. hyorhinis, A. laidlawii, M. fermentans, M. gallisepticum, M. pneumoniae and S. citri in a sample.
  • M. arginini M. orale, M. synoviae, M. hyorhinis, A. laidlawii, M. fermentans, M. gallisepticum, M. pneumoniae and S. citri in a sample.
  • the presence, absence and/or level of A is amplification reaction that detect the presence, absence and/or level of A.
  • laidlawii is determined by an amplification reaction assay separate from the amplification reaction assay(s) used for other mycoplasma species.
  • a multiplex PCR is performed to detect the presence, absence or level of two or more (e.g., two or three) of M. arginini, M. orale, M. synoviae, M. hyorhinis, A. laidlawii, M. fermentans, M. gallisepticum, M. pneumoniae and S. citri mycoplasma in a sample.
  • an amplification reaction assay can be used to detect simultaneously the presence, absence and/or level of A. laidlawii and S. citri mycoplasmas.
  • real-time PCR Polymerase Chain Reaction
  • primers and probes combinations as well as detection methods for identification of one or more of M. arginini, M. orale, M. synoviae, M. hyorhinis, A. laidlawii, M. fermentans, M. gallisepticum, M. pneumoniae and S. citri in a sample.
  • methods e.g., qPCR assays
  • compositions e.g., primers and probes
  • a single assay can be used to detect the presence or absence of more than one species of mycoplasma. In some embodiments, a single assay can be used to detect the presence or absence of one or more of M. arginini, M. orale, M. synoviae, M. hyorhinis, and A. laidlawii in a sample. In some embodiments, a single assay can be used to detect the presence or absence of one or more of M. fermentans, M. gallisepticum, and M. pneumoniae in a sample.
  • a single assay can be used to detect the presence or absence of one or more of A. laidlawii and S. citri in a sample. In some embodiments, a single assay can be used to detect the presence or absence of one or more of M. arginini, M. orale, M. synoviae, M. hyorhinis, A. laidlawii, M. fermentans, M. gallisepticum, M. pneumoniae and S. citri in a sample.
  • compositions e.g., reagents utilizing fluorogenic sequence-specific hybridization probes
  • the methods provided herein can comprise: subjecting the nucleic acid from a sample or culture suspected of containing one or more of M. arginini, M. orale, M. synoviae, M. hyorhinis, A. laidlawii, M. fermentans, M. gallisepticum, M. pneumoniae and S.
  • probe-based e.g., TaqMan probe-based
  • real-time PCR compositions e.g., reagents
  • methods e.g., assays
  • the primers/probes provided herein can achieve high linearity, precision, accuracy, robustness, and specificity. Moreover, the disclosed methods are both fast and easy to perform, particularly in comparison to methods such as culturing.
  • Oligonucleotides e.g., amplification primers and probes
  • Oligonucleotides that are capable of specifically hybridizing (e.g., under standard nucleic acid amplification conditions, e.g., standard PCR conditions, and/or stringent hybridization conditions) to a target gene region, or complement thereof, in M. arginini, M. orale, M. synoviae, M. hyorhinis, A.
  • the target gene region of a mycoplasma under detection can vary.
  • the target region comprises a 16s rRNA gene region.
  • the target region comprises a 23s rRNA gene region.
  • the target gene region of M. arginini comprises, or is, the 16s rRNA gene region.
  • the target gene region of M. orale can comprise, or can be, the 16s rRNA gene region.
  • the target gene region of M. synoviae comprises, or is, the 16s rRNA gene region.
  • the target gene region of M. hyorhinis comprises, or is, the 16s rRNA gene region.
  • the target gene region of A. laidlawii comprises, or is, the 23s rRNA gene region.
  • the target gene region of M. fermentans comprises, or is, the 16s rRNA gene region.
  • the target gene region of M. gallisepticum comprises, or is, the 23s rRNA gene region.
  • the target gene region of M. pneumoniae comprises, or is, the 23s rRNA gene region.
  • citri comprises, or is, the 16s rRNA gene region.
  • nucleic acid amplification e.g., PCR
  • M. arginini, M. orale, M. synoviae, and M. hyorhinis can be used for generating amplicons of the 16s rRNA gene region of M. arginini, M. orale, M. synoviae, and M. hyorhinis for detecting one or more of M. arginini, M. orale, M. synoviae, and M. hyorhinis (e.g., the absence, presence and/or amount of one or more of M. arginini, M. orale, M. synoviae, and M. hyorhinis) in a sample.
  • oligonucleotides e.g., amplification primers and probes
  • oligonucleotides that are capable of specifically hybridizing (e.g., under standard nucleic acid amplification conditions, e.g., standard PCR conditions, and/or stringent hybridization conditions) to the 16s rRNA gene region in M. arginini, M. orale, M. synoviae, and M. hyorhinis
  • primers and probes that can specifically bind to the 16s rRNA gene region in M. arginini, M. orale, M. synoviae, and M. hyorhinis are used in detection of the presence, absence and/or level of one or more of M.
  • oligonucleotides capable of specifically hybridizing to the 16s rRNA gene region in M. arginini, M. orale, M. synoviae, and M. hyorhinis include, but are not limited to, SEQ ID NOs: 1-3 as provided in Table 7 and sequences that exhibit at least about 85% identity to a sequence selected from the group consisting of SEQ ID NOs: 1-3.
  • nucleic acid amplification e.g., PCR
  • oligonucleotides e.g., amplification primers and probes
  • oligonucleotides that are capable of specifically hybridizing (e.g., under standard nucleic acid amplification conditions, including standard PCR conditions, and/or stringent hybridization conditions) to the 23s rRNA gene region of A. laidlawii are provided.
  • the 23s rRNA gene region is used as the target gene region for the DNA amplification to detect the presence, absence and/or level of A. laidlawii in the sample.
  • primers and probes that can specifically bind to the 23s rRNA gene region of A. laidlawii are used in detection of the presence, absence and/or amount of A. laidlawii in a biological sample.
  • oligonucleotides capable of specifically hybridizing to the 23s rRNA gene region of A. laidlawii include, but are not limited to, SEQ ID NOs: 4-6 as provided in Table 7 and sequences that exhibit at least about 85% identity to a sequence selected from the group consisting of SEQ ID NOs: 4-6.
  • nucleic acid amplification e.g., PCR
  • oligonucleotides e.g., amplification primers and probes
  • oligonucleotides that are capable of specifically hybridizing (e.g., under standard nucleic acid amplification conditions, e.g., standard PCR conditions, and/or stringent hybridization conditions) to the 16s rRNA gene region of M. fermentans are provided.
  • the 16s rRNA gene region is used as the target gene region for the DNA amplification to detect the presence, absence and/or level of M. fermentans in the sample.
  • primers and probes that can specifically bind to t the 16s rRNA gene region of M. fermentans are used in detection of the presence, absence and/or level of M. fermentans in a biological sample.
  • oligonucleotides capable of specifically hybridizing to the 16s rRNA gene region of M. fermentans include, but are not limited to, SEQ ID NOs: 7-9 as provided in Table 7 and sequences that exhibit at least about 85% identity to a sequence selected from the group consisting of SEQ ID NOs: 7-9.
  • nucleic acid amplification e.g., PCR
  • oligonucleotides e.g., amplification primers and probes
  • oligonucleotides that are capable of specifically hybridizing (e.g., under standard nucleic acid amplification conditions, e.g., standard PCR conditions, and/or stringent hybridization conditions) to the 23s rRNA gene region of M. gallisepticum and M. pneumoniae are provided.
  • the 23s rRNA gene region is used as the target gene region for the DNA amplification to detect the presence, absence and/or level of M.
  • primers and probes that can specifically bind to the 23s rRNA gene region of M. gallisepticum and M. pneumoniae are used in detection of the presence, absence and/or level of one or more of M. gallisepticum and M. pneumoniae in a biological sample.
  • oligonucleotides capable of specifically hybridizing to the 23s rRNA gene region of M. gallisepticum and M. pneumoniae include, but are not limited to, SEQ ID NOs: 10-12 as provided in Table 7 and sequences that exhibit at least about 85% identity to a sequence selected from the group consisting of SEQ ID NOs: 10-12.
  • nucleic acid amplification e.g., PCR
  • PCR nucleic acid amplification
  • oligonucleotides e.g., amplification primers and probes
  • amplification primers and probes that are capable of specifically hybridizing (e.g., under standard nucleic acid amplification conditions, e.g., standard PCR conditions, and/or stringent hybridization conditions) to the 16s rRNA gene region of S. citri are provided.
  • the 16s rRNA gene region is used as the target gene region for the DNA amplification to detect the presence, absence and/or level of S. citri in the sample.
  • primers and probes that can specifically bind to the 16s rRNA gene region of S. citri are used in detection of the presence, absence and/or level of S. citri in a biological sample.
  • oligonucleotides capable of specifically hybridizing to the 16s rRNA gene region of S. citri include, but are not limited to, SEQ ID NOs: 13-15 as provided in Table 7 and sequences that exhibit at least about 85% identity to a sequence selected from the group consisting of SEQ ID NOs: 13-15.
  • a primer/probe combination can comprise a forward primer, a reverse primer, and a probe (e.g., A. laidlawii 23s rRNA Primer 1, A. laidlawii 23s rRNA Primer 2, and A. laidlawii 23s rRNA Probe in tandem).
  • the compositions and methods provided herein can comprise one or more of the primer/probe combinations provided in Table 7.
  • a method or composition can comprise a primer/probe combination (e.g., A. laidlawii 23s rRNA Primer 1, A. laidlawii 23s rRNA Primer 2, and A. laidlawii 23s rRNA Probe in tandem).
  • a method or composition can comprise multiple primer/probe combinations (e.g., M. hyorhinis Primer 1, M. hyorhinis Primer 2, and M. hyorhinis Probe; A. laidlawii 23s rRNA Primer 1, A. laidlawii 23s rRNA Primer 2, and A. laidlawii 23s rRNA Probe; M. fermentans Primer 1, M. fermentans Primer 2, and M. fermentans Probe; M. pneumoniae Primer 1, M. pneumoniae Primer 2, and M.
  • kits comprising: (1) one or more primer/probe combinations capable of specifically hybridizing to the sequence of the 16s rRNA gene region, or a complement thereof, of one or more of M. arginini, M. orale, M. synoviae, and M. hyorhinis (e.g., M. hyorhinis Primer 1, M. hyorhinis Primer 2, and M.
  • hyorhinis Probe described in Table 7 (2) one or more primer/probe combinations capable of specifically hybridizing to the sequence of the 23s rRNA gene region, or a complement thereof, of A. laidlawii (e.g., A. laidlawii 23s rRNA Primer 1, A. laidlawii 23s rRNA Primer 2, and A. laidlawii 23s rRNA Probe described in Table 7); (3) one or more primer/probe combinations capable of specifically hybridizing to the sequence of the 16s rRNA gene region, or a complement thereof, of M. fermentans (e.g., M. fermentans Primer 1, M. fermentans Primer 2, and M.
  • M. fermentans e.g., M. fermentans Primer 1, M. fermentans Primer 2, and M.
  • the nucleic acids provided herein can be in various forms.
  • nucleic acids are dissolved (either alone or in combination with various other nucleic acids) in solution, for example buffer.
  • nucleic acids are provided, either alone or in combination with other isolated nucleic acids, as a salt.
  • nucleic acids are provided in a lyophilized form that can be reconstituted.
  • the isolated nucleic acids disclosed herein can be provided in a lyophilized pellet alone, or in a lyophilized pellet with other isolated nucleic acids.
  • nucleic acids are provided affixed to a solid substance, such as a bead, a membrane, or the like.
  • nucleic acids are provided in a host cell, for example a cell line carrying a plasmid, or a cell line carrying a stably integrated sequence.
  • the composition, reaction mixture, and kit comprise one or more pairs of amplification primers capable of specifically hybridizing to the sequence of the 16s rRNA gene region of M. arginini, the 16s rRNA gene region of M. orale, the 16s rRNA gene region of M. synoviae, the 16s rRNA gene region of M. hyorhinis, the 23s rRNA gene region of A. laidlawii, the 16s rRNA gene region of M. fermentans, the 23s rRNA gene region of M.
  • the composition, reaction mixture, and kit comprise one or more probes capable of specifically hybridizing to the 16s rRNA gene region of M. arginini, the 16s rRNA gene region of M. orale, the 16s rRNA gene region of M. synoviae, the 16s rRNA gene region of M. hyorhinis, the 23s rRNA gene region of A. laidlawii, the 16s rRNA gene region of M. fermentans, the 23s rRNA gene region of M. gallisepticum, the 23s rRNA gene region of M.
  • oligonucleotide probes or primers up to about 100 nucleotides in length capable of hybridizing to the 16s rRNA gene region of M. arginini, M. orale, M. synoviae, and M. hyorhinis.
  • the probe or primer comprises, or consists of, the sequence of any one of SEQ ID NOs: 1-3, or a sequence having at least about 85% identity, at least about 90% identity, at least about 95% identity, at least about 98% identity, or at least about 99% identity to any one of SEQ ID NOs: 1-3.
  • the probe or primer comprises, or consists of, a sequence selected from SEQ ID NOs: 1-3.
  • Disclosed herein include oligonucleotide probes or primers up to about 100 nucleotides in length capable of hybridizing to the 23s rRNA gene region of A. laidlawii.
  • the probe or primer comprises, or consists of, the sequence of any one of SEQ ID NOs: 4-6, or a sequence having at least about 85% identity, at least about 90% identity, at least about 95% identity, at least about 98% identity, or at least about 99% identity to any one of SEQ ID NOs: 4-6.
  • the probe or primer comprises, or consists of, a sequence selected from SEQ ID NOs: 4-6.
  • Disclosed herein include oligonucleotide probes or primers up to about 100 nucleotides in length capable of hybridizing to the 16s rRNA gene region of M. fermentans.
  • the probe or primer comprises, or consists of, the sequence of any one of SEQ ID NOs: 7-9, or a sequence having at least about 85% identity, at least about 90% identity, at least about 95% identity, at least about 98% identity, or at least about 99% identity to any one of SEQ ID NOs: 7-9.
  • the probe or primer comprises, or consists of, a sequence selected from SEQ ID NOs: 7-9.
  • Disclosed herein include oligonucleotide probes or primers up to about 100 nucleotides in length capable of hybridizing to the 23s rRNA gene region of M. gallisepticum and M. pneumoniae.
  • the probe or primer comprises, or consists of, the sequence of any one of SEQ ID NOs: 10-12, or a sequence having at least about 85% identity, at least about 90% identity, at least about 95% identity, at least about 98% identity, or at least about 99% identity to any one of SEQ ID NOs: 10-12.
  • the probe or primer comprises, or consists of, a sequence selected from SEQ ID NOs: 10-12.
  • Disclosed herein include oligonucleotide probes or primers up to about 100 nucleotides in length capable of hybridizing to the 16s rRNA gene region of S. citri.
  • the probe or primer comprises, or consists of, the sequence of any one of SEQ ID NOs: 13-15, or a sequence having at least about 85% identity, at least about 90% identity, at least about 95% identity, at least about 98% identity, or at least about 99% identity to any one of SEQ ID NOs: 13-15.
  • the probe or primer comprises, or consists of, a sequence selected from SEQ ID NOs: 13-15.
  • compositions comprising one or more, or two or more, of the oligonucleotide probes and/or primers disclosed herein.
  • Oligonucleotide probes can, in some embodiments, include a detectable moiety.
  • the oligonucleotide probes disclosed herein can comprise a radioactive label.
  • radioactive labels include 3 H, 14 C, 32 P, and 35 S.
  • oligonucleotide probes can include one or more non-radioactive detectable markers or moieties, including but not limited to ligands, fluorophores, chemiluminescent agents, enzymes, and antibodies.
  • Other detectable markers for use with probes which can enable an increase in sensitivity of the method of the invention, include biotin and radio-nucleotides. It will become evident to the person of ordinary skill that the choice of a particular label dictates the manner in which it is bound to the probe. For example, oligonucleotide probes labeled with one or more dyes, such that upon hybridization to a template nucleic acid, a detectable change in fluorescence is generated.
  • sequence-specific probes can provide more accurate measurements of amplification.
  • One configuration of sequence-specific probe can include one end of the probe tethered to a fluorophore, and the other end of the probe tethered to a quencher. When the probe is unhybridized, it can maintain a stem-loop configuration, in which the fluorophore is quenched by the quencher, thus preventing the fluorophore from fluorescing. When the probe is hybridized to a template nucleic sequence, it is linearized, distancing the fluorophore from the quencher, and thus permitting the fluorophore to fluoresce.
  • sequence-specific probe can include a first probe tethered to a first fluorophore of a FRET pair, and a second probe tethered to a second fluorophore of a FRET pair.
  • the first probe and second probe can be configured to hybridize to sequences of an amplicon that are within sufficient proximity to permit energy transfer by FRET when the first probe and second probe are hybridized to the same amplicon.
  • the probe can be, for example, a TaqMan probe.
  • TaqMan probes can comprise a fluorophore and a quencher.
  • the quencher molecule can quench the fluorescence emitted by the fluorophore when excited by the cycler’s light source via Förster resonance energy transfer (FRET). As long as the fluorophore and the quencher are in proximity, quenching can inhibit any detectable (e.g., fluorescence) signals.
  • FRET Förster resonance energy transfer
  • TaqMan probes provided herein can designed such that they anneal within a DNA region amplified by primers provided herein.
  • a PCR polymerase e.g., Taq
  • the 5' to 3' exonuclease activity of the PCR polymerase degrades the probe that has annealed to the template. Degradation of the probe can release the fluorophore from it and break the proximity to the quencher, thereby relieving the quenching effect and allowing fluorescence of the fluorophore.
  • fluorescence detected in the quantitative PCR thermal cycler can, in some embodiments, be directly proportional to the fluorophore released and the amount of DNA template present in the PCR.
  • the sequence specific probe comprises an oligonucleotide as disclosed herein conjugated to a fluorophore.
  • the probe is conjugated to two or more fluorophores.
  • fluorophores include: xanthene dyes, e.g., fluorescein and rhodamine dyes, such as fluorescein isothiocyanate (FITC), 2-[ethylamino)-3-(ethylimino)-2-7-dimethyl-3H-xanthen-9-yl]benzoic acid ethyl ester monohydrochloride (R6G)(emits a response radiation in the wavelength that ranges from about 500 to 560 nm), 1,1,3,3,3',3'-Hexamethylindodicarbocyanine iodide (HIDC) (emits a response radiation in the wavelength that ranged from about 600 to 660 nm), 6-carboxyfluor
  • Cy3, Cy5 and Cy7 dyes include coumarins, e.g., umbelliferone; benzimide dyes, e.g. Hoechst 33258; phenanthridine dyes, e.g. Texas Red; ethidium dyes; acridine dyes; carbazole dyes; phenoxazine dyes; porphyrin dyes; polymethine dyes, e.g. cyanine dyes such as Cy3 (emits a response radiation in the wavelength that ranges from about 540 to 580 nm), Cy5 (emits a response radiation in the wavelength that ranges from about 640 to 680 nm), etc; BODIPY dyes and quinoline dyes.
  • Cy3 emits a response radiation in the wavelength that ranges from about 540 to 580 nm
  • Cy5 emits a response radiation in the wavelength that ranges from about 640 to 680 nm
  • fluorophores of interest include: Pyrene, Coumarin, Diethylaminocoumarin, FAM, Fluorescein Chlorotriazinyl, Fluorescein, R110, Eosin, JOE, R6G, HIDC, Tetramethylrhodamine, TAMRA, Lissamine, ROX, Napthofluorescein, Texas Red, Napthofluorescein, Cy3, and Cy5, CAL fluor orange, and the like.
  • fluorescein dyes include 6-carboxyfluorescein (6-FAM), 2′,4′,1,4,- tetrachlorofluorescein (TET), 2′,4′,5′,7′,1,4-hexachlorofluorescein (HEX), 2′,7′-dimethoxy-4′,5′- dichloro-6-carboxyrhodamine (JOE), 2′-chloro-5′-fluoro-7′,8′-fused phenyl-1,4-dichloro-6- carboxyfluorescein (NED), and 2′-chloro-7′-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC).
  • 6-FAM 6-carboxyfluorescein
  • TET 2′,4′,1,4,- tetrachlorofluorescein
  • HEX 2′,4′,5′,7′,1,4-hexachlorofluorescein
  • Probes can comprise SpC6, or functional equivalents and derivatives thereof. Probes can comprise a spacer moiety. A spacer moiety can comprise an alkyl group of at least 2 carbons to about 12 carbons. A probe can comprise a spacer comprising an abasic unit. A probe can comprise a spacer selected from the group comprising of idSp, iSp9, iS18, iSpC3, iSpC6, iSpC12, or any combination thereof. [0118] In some embodiments, the probe is conjugated to a quencher (e.g., in addition to a fluorophore). A quencher can absorb electromagnetic radiation and dissipate it as heat, thus remaining dark.
  • a quencher e.g., in addition to a fluorophore
  • Example quenchers include Dabcyl, NFQ’s, such as BHQ-1 or BHQ-2 (Biosearch), IOWA BLACK FQ (IDT), and IOWA BLACK RQ (IDT).
  • the quencher is selected to pair with a fluorophore so as to absorb electromagnetic radiation emitted by the fluorophore.
  • Fluorophore/quencher pairs useful in the compositions and methods disclosed herein are well-known in the art, and can be found, e.g., described in Marras, “Selection of Fluorophore and Quencher Pairs for Fluorescent Nucleic Acid Hybridization Probes” available at www.molecular-beacons.org/download/marras,mmb06%28335%293.pdf.
  • quencher moieties include, but are not limited to: a dark quencher, a Black Hole Quencher® (BHQ®) (e.g., BHQ-0, BHQ-1, BHQ-2, BHQ-3), a Qxl quencher, an ATTO quencher (e.g., ATTO 540Q, ATTO 580Q, and ATTO 612Q), dimethylaminoazobenzenesulfonic acid (Dabsyl), Iowa Black RQ, Iowa Black FQ, IRDye QC- 1, a QSY dye (e.g., QSY 7, QSY 9, QSY 21), AbsoluteQuencher, Eclipse, and metal clusters such as gold nanoparticles, and the like.
  • BHQ® Black Hole Quencher®
  • BHQ® Black Hole Quencher®
  • ATTO quencher e.g., ATTO 540Q, ATTO 580Q, and ATTO 612Q
  • Dabsyl dimethylaminoazobenzen
  • an ATTO quencher examples include, but are not limited to: ATTO 540Q, ATTO 580Q, and ATTO 612Q.
  • Examples of a Black Hole Quencher® (BHQ®) include, but are not limited to: BHQ-0 (493 nm), BHQ-1 (534 nm), BHQ-2 (579 nm) and BHQ-3 (672 nm).
  • the oligonucleotide probe further comprises a second fluorescence quencher moiety.
  • the second fluorescence quencher moiety can comprise a ZEN quencher or TAO quencher.
  • the detectable label can be a fluorescent label selected from: an Alexa Fluor® dye (e.g., Alexa Fluor® 350, Alexa Fluor® 405, Alexa Fluor® 430, Alexa Fluor® 488, Alexa Fluor® 500, Alexa Fluor® 514, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 610, Alexa Fluor® 633, Alexa Fluor® 635, Alexa Fluor® 647, Alexa Fluor® 660, Alexa Fluor® 680, Alexa Fluor® 700, Alexa Fluor® 750, Alexa Fluor® 790), an ATTO dye (e.g., ATTO 390, ATTO 425, ATTO 465, ATTO 488, ATTO 495, ATTO 514, ATTO 520, ATTO 532, ATTO Rho6G, ATTO 542, ATTO 550, ATTO 5
  • the fluorophore can be attached to a first end of the probe, and a quencher is attached to a second end of the probe.
  • the probe can comprise two or more fluorophores.
  • a probe can comprise two or more quencher moieties.
  • a probe can comprise one or more quencher moieties and/or one or more fluorophores.
  • a quencher moiety or a fluorophore can be attached to any portion of a probe (e.g., on the 5’ end, on the 3’ end, in the middle of the probe).
  • Any probe nucleotide can comprise a fluorophore or a quencher moiety, such as, for example, BHQ1dT.
  • Attachment can include covalent bonding, and can optionally include at least one linker molecule positioned between the probe and the fluorophore or quencher.
  • a fluorophore is attached to a 5’ end of a probe, and a quencher is attached to a 3’ end of a probe.
  • the fluorophore is attached to a 3’ end of a probe, and a quencher is attached to a 5’ end of a probe.
  • the second fluorescence quencher moiety can be between the ninth and tenth nucleotide of the oligonucleotide probe.
  • probes that can be used in quantitative nucleic acid amplification include molecular beacons, SCORPIONTM probes (Sigma), TAQMANTM probes (Life Technologies) and the like.
  • Other nucleic acid detection technologies that are useful in the embodiments disclosed herein include, but are not limited to nanoparticle probe technology (See e.g., Elghanian, et al. (1997) Science 277:1078-1081.) and Amplifluor probe technology (See, U.S. Pat. Nos: 5,866,366; 6,090,592; 6,117,635; and 6,117,986).
  • the composition comprises: a pair of primers capable of hybridizing to the 16s rRNA gene region of M. arginini, M. orale, M. synoviae, and M. hyorhinis, wherein each primer in the pair of primers comprises a sequence having at least about 85% identity (or a sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
  • the pair of primers capable of hybridizing to the 16s rRNA gene region of M. arginini, M. orale, M. synoviae, and M. hyorhinis comprises a primer comprising the sequence of SEQ ID NO: 1 (or a sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or a number or a range between any two of these values to the sequence of SEQ ID NO: 1) and another primer comprising the sequence of SEQ ID NO: 2 (or a sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or a number or a range between any two of these values to the sequence of SEQ ID NO: 2).
  • the composition can comprise: an oligonucleotide probe comprising a sequence having at least about 85% identity (or a sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 3.
  • the oligonucleotide probe comprises the sequence of SEQ ID NO: 3.
  • the composition comprises: a pair of primers capable of hybridizing to the 23s rRNA gene region of A. laidlawii, wherein each primer in the pair of primers comprises a sequence having at least about 85% identity (or a sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 4 or SEQ ID NO: 5.
  • laidlawii comprises a primer comprising the sequence of SEQ ID NO: 4 (or a sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or a number or a range between any two of these values to the sequence of SEQ ID NO: 4) and another primer comprising the sequence of SEQ ID NO: 5 (or a sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or a number or a range between any two of these values to the sequence of SEQ ID NO: 5).
  • the composition can comprise: an oligonucleotide probe comprising a sequence having at least about 85% identity (or a sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 6.
  • the oligonucleotide probe comprises the sequence of SEQ ID NO: 6.
  • Disclosed herein include compositions for detecting M. fermentans in a sample.
  • the composition comprises: a pair of primers capable of hybridizing to the 16s rRNA gene region of M.
  • each primer in the pair of primers comprises a sequence having at least about 85% identity (or a sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 7 or SEQ ID NO: 8.
  • the pair of primers capable of hybridizing to the 16s rRNA gene region of M.
  • fermentans comprises a primer comprising the sequence of SEQ ID NO: 7 (or a sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or a number or a range between any two of these values to the sequence of SEQ ID NO: 7) and another primer comprising the sequence of SEQ ID NO: 8 (or a sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or a number or a range between any two of these values to the sequence of SEQ ID NO: 8).
  • the composition can comprise: an oligonucleotide probe comprising a sequence having at least about 85% identity (or a sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 9.
  • the oligonucleotide probe comprises the sequence of SEQ ID NO: 9.
  • the composition comprises: a pair of primers capable of hybridizing to the 23s rRNA gene region of M. gallisepticum and M. pneumoniae, wherein each primer in the pair of primers comprises a sequence having at least about 85% identity (or a sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity, or a number or a range between any two of these values)to the sequence of SEQ ID NO: 10 or SEQ ID NO: 11.
  • pneumoniae comprises a primer comprising the sequence of SEQ ID NO: 10 (or a sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or a number or a range between any two of these values to the sequence of SEQ ID NO: 10) and another primer comprising the sequence of SEQ ID NO: 11 (or a sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or a number or a range between any two of these values to the sequence of SEQ ID NO: 11).
  • the composition can comprise: an oligonucleotide probe comprising a sequence having at least about 85% identity (or a sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 12.
  • the oligonucleotide probe comprises the sequence of SEQ ID NO: 12.
  • each primer in the pair of primers comprises a sequence having at least about 85% identity (or a sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 13 or SEQ ID NO: 14.
  • the pair of primers capable of hybridizing to the 16s rRNA gene region of S.
  • citri comprises a primer comprising the sequence of SEQ ID NO: 13 (or a sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or a number or a range between any two of these values to the sequence of SEQ ID NO: 13) and another primer comprising the sequence of SEQ ID NO: 14 (or a sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity or a number or a range between any two of these values to the sequence of SEQ ID NO: 14).
  • the composition can comprise: an oligonucleotide probe comprising a sequence having at least about 85% identity to the sequence of SEQ ID NO: 15 (or a sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity, or a number or a range between any two of these values).
  • the oligonucleotide probe comprises the sequence of SEQ ID NO: 15.
  • compositions comprising two or more primers and/or probes (e.g., for multiplexed reactions). Disclosed herein include compositions for detecting one or more of M.
  • the composition comprises: a pair of primers capable of hybridizing to the 16s rRNA gene region of M. arginini, M. orale, M. synoviae, and M.
  • each primer in the pair of primers comprises a sequence having at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2; a pair of primers capable of hybridizing to the 23s rRNA gene region of A.
  • 85% identity e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values
  • each primer in the pair of primers comprises a sequence having at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 4 or SEQ ID NO: 5; a pair of primers capable of hybridizing to the 16s rRNA gene region of M.
  • 85% identity e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values
  • each primer in the pair of primers comprises a sequence having at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 7 or SEQ ID NO: 8; a pair of primers capable of hybridizing to the 23s rRNA gene region of M. gallisepticum and M.
  • each primer in the pair of primers comprises a sequence having at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 10 or SEQ ID NO: 11; and/or a pair of primers capable of hybridizing to the 16s rRNA gene region of S.
  • 85% identity e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values
  • each primer in the pair of primers comprises a sequence having at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 13 or SEQ ID NO: 14.
  • the pair of primers capable of hybridizing to the 16s rRNA gene region of M. arginini, M. orale, M. synoviae, and M.
  • hyorhinis can comprise (a) a primer having the sequence of SEQ ID NO: 1 or a sequence differing by one, two, three, four, five, six, or more nucleotide mismatches relative to SEQ ID NO: 1 and (b) a primer having the sequence of SEQ ID NO: 2 or a sequence differing by one, two, three, four, five, six, or more nucleotide mismatches relative to SEQ ID NO: 2; the pair of primers capable of hybridizing to the 23s rRNA gene region of A.
  • laidlawii comprises (a) a primer having the sequence of SEQ ID NO: 4 or a sequence differing by one, two, three, four, five, six, or more nucleotide mismatches relative to SEQ ID NO: 4 and (b) a primer having the sequence of SEQ ID NO: 5 or a sequence differing by one, two, three, four, five, six, or more nucleotide mismatches relative to SEQ ID NO: 5; the pair of primers capable of hybridizing to the 16s rRNA gene region of M.
  • fermentans comprises (a) a primer having the sequence of SEQ ID NO: 7 or a sequence differing by one, two, three, four, five, six, or more nucleotide mismatches relative to SEQ ID NO: 7 and (b) a primer having the sequence of SEQ ID NO: 8 or a sequence differing by one, two, three, four, five, six, or more nucleotide mismatches relative to SEQ ID NO: 8; the pair of primers capable of hybridizing to the 23s rRNA gene region of M. gallisepticum and M.
  • pneumoniae comprises (a) a primer having the sequence of SEQ ID NO: 10 or a sequence differing by one, two, three, four, five, six, or more nucleotide mismatches relative to SEQ ID NO: 10 and (b) a primer having the sequence of SEQ ID NO: 11 or a sequence differing by one, two, three, four, five, six, or more nucleotide mismatches relative to SEQ ID NO: 11; and/or the pair of primers capable of hybridizing to the 16s rRNA gene region of S.
  • citri comprises (a) a primer having the sequence of SEQ ID NO: 13 or a sequence differing by one, two, three, four, five, six, or more nucleotide mismatches relative to SEQ ID NO: 13 and (b) a primer having the sequence of SEQ ID NO: 14 or a sequence differing by one, two, three, four, five, six, or more nucleotide mismatches relative to SEQ ID NO: 14.
  • the composition can comprise: one or more oligonucleotide probes each comprising a sequence having at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 3, 6, 9, 12, or 15.
  • Each of the one or more oligonucleotide probes can comprise the sequence of SEQ ID NO: 3, 6, 9, 12, or 15, or a sequence differing by one, two, three, four, five, six, or more nucleotide mismatches relative to SEQ ID NO: 3, 6, 9, 12, or 15.
  • each of the one or more oligonucleotide probes consists of a sequence selected from SEQ ID NO: 3, 6, 9, 12, or 15. At least one of the one or more oligonucleotide probes can comprise a fluorescence emitter moiety, a fluorescence quencher moiety, or both.
  • Disclosed herein include compositions for detecting one or more of M. arginini, M. orale, M. synoviae, M. hyorhinis, and A. laidlawii in a sample.
  • the composition comprises: a pair of primers capable of hybridizing to the 16s rRNA gene region of M. arginini, M. orale, M.
  • each primer in the pair of primers comprises a sequence having at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2; and a pair of primers capable of hybridizing to the 23s rRNA gene region of A.
  • 85% identity e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values
  • each primer in the pair of primers comprises a sequence having at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 4 or SEQ ID NO: 5.
  • the pair of primers capable of hybridizing to the 16s rRNA gene region of M. arginini, M. orale, M. synoviae, and M.
  • hyorhinis can comprise (a) a primer having the sequence of SEQ ID NO: 1 or a sequence differing by one, two, three, four, five, six, or more nucleotide mismatches relative to SEQ ID NO: 1 and (b) a primer having the sequence of SEQ ID NO: 2 or a sequence differing by one, two, three, four, five, six, or more nucleotide mismatches relative to SEQ ID NO: 2.
  • the pair of primers capable of hybridizing to the 23s rRNA gene region of A.
  • laidlawii can comprise (a) a primer having the sequence of SEQ ID NO: 4 or a sequence differing by one, two, three, four, five, six, or more nucleotide mismatches relative to SEQ ID NO: 4 and (b) a primer having the sequence of SEQ ID NO: 5 or a sequence differing by one, two, three, four, five, six, or more nucleotide mismatches relative to SEQ ID NO: 5.
  • the composition can comprise: one or more oligonucleotide probes each comprising a sequence having at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 3 or 6.
  • Each of the one or more oligonucleotide probes can comprise the sequence of SEQ ID NO: 3 or 6; or a sequence differing by one, two, three, four, five, six, or more nucleotide mismatches relative to SEQ ID NO: 3 or 6.
  • each of the one or more oligonucleotide probes consists of a sequence selected from SEQ ID NO: 3 or 6. At least one of the one or more oligonucleotide probes can comprise a fluorescence emitter moiety, a fluorescence quencher moiety, or both.
  • Disclosed herein include compositions for detecting one or more of M. fermentans, M. gallisepticum, and M. pneumoniae in a sample.
  • the composition comprises: a pair of primers capable of hybridizing to the 16s rRNA gene region of M.
  • each primer in the pair of primers comprises a sequence having at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 7 or SEQ ID NO: 8; and a pair of primers capable of hybridizing to the 23s rRNA gene region of M. gallisepticum and M.
  • each primer in the pair of primers comprises a sequence having at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 10 or SEQ ID NO: 11.
  • the pair of primers capable of hybridizing to the 16s rRNA gene region of M.
  • fermentans can comprise (a) a primer having the sequence of SEQ ID NO: 7 or a sequence differing by one, two, three, four, five, six, or more nucleotide mismatches relative to SEQ ID NO: 7 and (b) a primer having the sequence of SEQ ID NO: 8 or a sequence differing by one, two, three, four, five, six, or more nucleotide mismatches relative to SEQ ID NO: 8.
  • the pair of primers capable of hybridizing to the 23s rRNA gene region of M. gallisepticum and M.
  • pneumoniae can comprise (a) a primer having the sequence of SEQ ID NO: 10 or a sequence differing by one, two, three, four, five, six, or more nucleotide mismatches relative to SEQ ID NO: 10 and (b) a primer having the sequence of SEQ ID NO: 11 or a sequence differing by one, two, three, four, five, six, or more nucleotide mismatches relative to SEQ ID NO: 11.
  • the composition can comprise: one or more oligonucleotide probes each comprising a sequence having at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 9 or 12.
  • Each of the one or more oligonucleotide probes can comprise the sequence of SEQ ID NO: 9 or 12; or a sequence differing by one, two, three, four, five, six, or more nucleotide mismatches relative to SEQ ID NO: 9 or 12.
  • each of the one or more oligonucleotide probes consists of a sequence selected from SEQ ID NO: 9 or 12. At least one of the one or more oligonucleotide probes can comprise a fluorescence emitter moiety, a fluorescence quencher moiety, or both.
  • Disclosed herein include compositions for detecting one or more of A. laidlawii and S. citri in a sample.
  • the composition comprises: a pair of primers capable of hybridizing to the 23s rRNA gene region of A.
  • each primer in the pair of primers comprises a sequence having at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 4 or SEQ ID NO: 5; and a pair of primers capable of hybridizing to the 16s rRNA gene region of S.
  • 85% identity e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values
  • each primer in the pair of primers comprises a sequence having at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 13 or SEQ ID NO: 14.
  • the pair of primers capable of hybridizing to the 23s rRNA gene region of A.
  • laidlawii can comprise (a) a primer having the sequence of SEQ ID NO: 4 or a sequence differing by one, two, three, four, five, six, or more nucleotide mismatches relative to SEQ ID NO: 4 and (b) a primer having the sequence of SEQ ID NO: 5 or a sequence differing by one, two, three, four, five, six, or more nucleotide mismatches relative to SEQ ID NO: 5.
  • the pair of primers capable of hybridizing to the 16s rRNA gene region of S.
  • citri can comprise (a) a primer having the sequence of SEQ ID NO: 13 or a sequence differing by one, two, three, four, five, six, or more nucleotide mismatches relative to SEQ ID NO: 13 and (b) a primer having the sequence of SEQ ID NO: 14 or a sequence differing by one, two, three, four, five, six, or more nucleotide mismatches relative to SEQ ID NO: 14.
  • the composition can comprise: one or more oligonucleotide probes each comprising a sequence having at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 6 or 15.
  • Each of the one or more oligonucleotide probes can comprise the sequence of SEQ ID NO: 6 or 15; or a sequence differing by one, two, three, four, five, six, or more nucleotide mismatches relative to SEQ ID NO: 6 or 15.
  • each of the one or more oligonucleotide probes consists of a sequence selected from SEQ ID NO: 6 or 15.
  • At least one of the one or more oligonucleotide probes can comprise a fluorescence emitter moiety, a fluorescence quencher moiety, or both.
  • the fluorescence emitter moiety can be at the 5’ terminus of the oligonucleotide probe, the 3’ terminus of the oligonucleotide probe, or both.
  • at least one of the one or more oligonucleotide probes further comprises a second fluorescence quencher moiety.
  • the second fluorescence quencher moiety can be between the ninth and tenth nucleotide of the oligonucleotide probe.
  • the second fluorescence quencher moiety can comprise ZEN or TAO.
  • Each oligonucleotide probe can be flanked by complementary sequences at the 5’ end and 3’ end.
  • One of the complementary sequences can comprise a fluorescence emitter moiety and the other complementary sequence can comprise a fluorescence quencher moiety.
  • the composition comprises a DNA polymerase.
  • the composition can comprise: one or more of a buffer and MgCl 2.
  • the composition can comprise: nucleic acids from M. arginini, M. orale, M. synoviae, M.
  • the nucleic acids can comprise an M. arginini DNA, an M. orale DNA, an M. synoviae DNA, an M. hyorhinis DNA, an A. laidlawii DNA, an M. fermentans DNA, an M. gallisepticum DNA, an M. pneumoniae DNA, an S. citri DNA, or any combination thereof.
  • the composition can be, for example, an amplification reaction mixture. Samples [0147] The methods and compositions disclosed herein are suitable for detecting one or more of M. arginini, M. orale, M.
  • sample can refer to any type of material of biological origin.
  • the sample can comprise, for example, fluids, tissues or cells.
  • the sample can comprise a biological material taken directly from a subject, or cultured cells or tissues, or any fraction or products produced from or derived from biological materials.
  • a sample can be purified, partially purified, unpurified, enriched, or amplified.
  • the sample comprises genetically modified cells.
  • the sample is or comprises a therapeutical or pharmaceutical composition.
  • the sample can be a biological sample, for example a clinical sample.
  • the sample is taken from a biological source, such as vagina, urethra, penis, anus, throat, cervix, fermentation broths, cell cultures, and the like.
  • the biological sample can be used (i) directly as obtained from the subject or source, or (ii) following a pre-treatment to modify the character of the sample.
  • the test sample can be pre-treated prior to use, for example, by disrupting cells or viral particles, preparing liquids from solid materials, diluting viscous fluids, filtering liquids, concentrating liquids, inactivating interfering components, adding reagents, purifying nucleic acids, and the like.
  • a “biological sample” as used herein includes nucleic acids (DNA, RNA or total nucleic acids) extracted from a clinical or biological specimen.
  • Sample preparation can also include using a solution that contains buffers, salts, detergents, and/or the like which are used to prepare the sample for analysis.
  • the sample is processed before molecular testing.
  • the sample is analyzed directly, and is not pre-processed prior to testing.
  • a sample to be tested is processed prior to performing the methods disclosed herein.
  • the sample can be isolated, concentrated, or subjected to various other processing steps prior to performing the methods disclosed herein.
  • the sample can be processed to isolate nucleic acids from the sample prior to contacting the sample with the oligonucleotides, as disclosed herein.
  • the methods disclosed herein are performed on the sample without culturing the sample in vitro.
  • the methods disclosed herein are performed on the sample without isolating nucleic acids from the sample prior to contacting the sample with oligonucleotides as disclosed herein.
  • a sample can comprise one or more nucleic acids (e.g., a plurality of nucleic acids).
  • the term “plurality” as used herein can refer to two or more.
  • a sample includes two or more (e.g., 3 or more, 5 or more, 10 or more, 20 or more, 50 or more, 100 or more, 500 or more, 1,000 or more, or 5,000 or more) nucleic acids (e.g., gDNA, RNA).
  • a disclosed method can be used as a very sensitive way to detect a target nucleic acid (e.g., the 23s rRNA gene region of A. laidlawii) present in a sample (e.g., in a complex mixture of nucleic acids such as gDNAs).
  • the sample includes 5 or more nucleic acids (e.g., 10 or more, 20 or more, 50 or more, 100 or more, 500 or more, 1,000 or more, or 5,000 or more nucleic acids) that differ from one another in sequence.
  • the sample includes 10 or more, 20 or more, 50 or more, 100 or more, 500 or more, 10 3 or more, 5 x 10 3 or more, 10 4 or more, 5 x 10 4 or more, 10 5 or more, 5 x 10 5 or more, 10 6 or more 5 x 10 6 or more, or 10 7 or more, nucleic acids.
  • the sample can be, or comprise, a pharmaceutical or therapeutical product, for example cell therapy or gene therapy product.
  • the sample is, or comprise, a composition for use in a CAR-T or gene- editing therapy [0151]
  • the sample comprises from 10 to 20, from 20 to 50, from 50 to 100, from 100 to 500, from 500 to 10 3 , from 10 3 to 5 x 10 3 , from 5 x 10 3 to 10 4 , from 10 4 to 5 x 10 4 , from 5 x 10 4 to 10 5 , from 10 5 to 5 x 10 5 , from 5 x 10 5 to 10 6 , from 10 6 to 5 x 10 6 , or from 5 x 10 6 to 10 7 , or more than 10 7 , copies of target nucleic acids.
  • the sample comprises from 5 to 10 7 nucleic acids (e.g., that differ from one another in sequence)(e.g., from 5 to 10 6 , from 5 to 10 5 , from 5 to 50,000, from 5 to 30,000, from 10 to 10 6 , from 10 to 10 5 , from 10 to 50,000, from 10 to 30,000, from 20 to 10 6 , from 20 to 10 5 , from 20 to 50,000, or from 20 to 30,000 nucleic acids, or a number or a range between any two of these values).
  • the sample includes 20 or more nucleic acids that differ from one another in sequence.
  • the sample can be derived from any source, e.g., the sample can be a synthetic combination of purified nucleic acids; the sample can be a cell lysate, a DNA-enriched cell lysate, or nucleic acids isolated and/or purified from a cell lysate.
  • the sample can be from a patient (e.g., for the purpose of diagnosis).
  • the sample can be from permeabilized cells.
  • the sample can be from crosslinked cells.
  • the sample can be in tissue sections.
  • the sample can be from tissues prepared by crosslinking followed by delipidation and adjustment to make a uniform refractive index.
  • a sample can include a target nucleic acid (e.g., the 23s rRNA gene region of A.
  • the target nucleic acid is present in the sample at one copy per 10 non-target nucleic acids, one copy per 20 non- target nucleic acids, one copy per 25 non-target nucleic acids, one copy per 50 non-target nucleic acids, one copy per 100 non-target nucleic acids, one copy per 500 non-target nucleic acids, one copy per 10 3 non-target nucleic acids, one copy per 5 x 10 3 non-target nucleic acids, one copy per 10 4 non-target nucleic acids, one copy per 5 x 10 4 non-target nucleic acids, one copy per 10 5 non-target nucleic acids, one copy per 5 x 10 5 non-target nucleic acids, one copy per 10 6 non-target nucleic acids, less than one copy per 10 6 non-target nucleic acids, or a number or a range between any two of these values.
  • the target nucleic acid is present in the sample at from one copy per 10 non-target nucleic acids to 1 copy per 20 non- target nucleic acids, from 1 copy per 20 non-target nucleic acids to 1 copy per 50 non target nucleic acids, from 1 copy per 50 non-target nucleic acids to 1 copy per 100 non-target nucleic acids, from 1 copy per 100 non-target nucleic acids to 1 copy per 500 non-target nucleic acids, from 1 copy per 500 non target nucleic acids to 1 copy per 10 3 non-target nucleic acids, from 1 copy per 10 3 non-target nucleic acids to 1 copy per 5 x 10 3 non-target nucleic acids, from 1 copy per 5 x 10 3 non-target nucleic acids to 1 copy per 10 4 non target nucleic acids, from 1 copy per 10 4 non-target nucleic acids to 1 copy per 10 5 non-target nucleic acids, from 1 copy per 10 5 non-target nucleic acids to 1 copy per 10 6 non-target nucleic acids, or
  • Suitable samples include but are not limited to saliva, blood, serum, plasma, urine, aspirate, cerebral spinal fluid (CSF) and biopsy samples.
  • sample with respect to a patient encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof.
  • the definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents; washed; or enrichment for certain cell populations, such as cancer cells.
  • Cells can be genetically modified (e.g., by a CRISPR-Cas9 gene editing system).
  • the sample can be a sample that have been enriched for particular types of molecules, e.g., nucleic acids.
  • a sample for use in the methods disclosed herein can include any conventional biological sample obtained from an organism or a part thereof, such as animal.
  • the biological sample is obtained from an animal subject, such as a human subject.
  • a sample can also be a sample obtained from any organ or tissue (including a biopsy or autopsy specimen, such as a tumor biopsy) or can include a cell (whether a primary cell or cultured cell) or medium conditioned by any cell, tissue or organ.
  • Exemplary samples include, without limitation, cells, cell lysates, blood smears, cytocentrifuge preparations, cytology smears, bodily fluids (e.g., blood, plasma, serum, saliva, sputum, urine, bronchoalveolar lavage, semen, etc.), tissue biopsies (e.g., tumor biopsies), fine-needle aspirates, and/or tissue sections (e.g., cryostat tissue sections and/or paraffin-embedded tissue sections).
  • the sample includes circulating tumor cells (which can be identified by cell surface markers).
  • samples are used directly (e.g., fresh or frozen), or can be manipulated prior to use, for example, by fixation (e.g., using formalin) and/or embedding in wax (such as formalin-fixed paraffin-embedded (FFPE) tissue samples).
  • the sample can be, or can comprise, an environmental sample, such as water, soil, or a surface such as industrial or medical surface. Owing to the increased sensitivity of the embodiments disclosed herein, in certain example embodiments, the assays and methods may be run on crude samples or samples where the target molecules to be detected are not further fractionated or purified from the sample.
  • Cell lysis procedures and reagents are known in the art and may be performed by chemical (e.g., detergent, hypotonic solutions, enzymatic procedures, and the like, or combination thereof), physical (e.g., French press, sonication, and the like), or electrolytic lysis methods. Any suitable lysis procedure can be utilized.
  • chemical methods generally employ lysing agents to disrupt cells and extract nucleic acids from the cells, followed by treatment with chaotropic salts.
  • cell lysis comprises use of detergents (e.g., ionic, nonionic, anionic, zwitterionic).
  • cell lysis comprises use of ionic detergents (e.g., SDS, sodium lauryl sulfate (SLS), deoxycholate, cholate, sarkosyl). Physical methods such as freeze/thaw followed by grinding, the use of cell presses and the like also may be useful. High salt lysis procedures also may be used. For example, an alkaline lysis procedure may be utilized. The latter procedure traditionally incorporates the use of phenol- chloroform solutions, and an alternative phenol-chloroform-free procedure involving three solutions may be utilized. [0157] In some sample extractions, cells are lysed by mechanical shearing with glass beads as described in US Patent No.
  • SDS sodium lauryl sulfate
  • deoxycholate cholate
  • sarkosyl sarkosyl
  • High salt lysis procedures also may be used. For example, an alkaline lysis procedure may be utilized. The latter procedure traditionally incorporates the use of phenol- chloroform solutions, and an alternative phenol-ch
  • nucleic acid testing can include, for example, nucleic acid testing.
  • the test can include testing for target nucleic acid sequences in a sample.
  • nucleic acid testing can be used in the embodiments disclosed herein, including but not limited to, testing that involves nucleic acid amplification.
  • a target nucleic acid e.g., bacterial DNA
  • the source of the target nucleic acid can be any source (e.g., any sample).
  • the target nucleic acid is a bacterial nucleic acid (e.g., bacterial DNA).
  • the compositions and methods provided herein can be employed for detecting the presence of a mycoplasma nucleic acid amongst a population of nucleic acids (e.g., in a sample).
  • compositions and methods for detecting a target nucleic acid e.g., the 16s rRNA gene region of M. arginini, the 16s rRNA gene region of M. orale, the 16s rRNA gene region of M. synoviae, the 16s rRNA gene region of M. hyorhinis, the 23s rRNA gene region of A. laidlawii, the 16s rRNA gene region of M. fermentans, the 23s rRNA gene region of M. gallisepticum, the 23s rRNA gene region of M. pneumoniae, the 16s rRNA gene region of S. citri) in a sample that can detect said target nucleic acid with a high degree of sensitivity.
  • a target nucleic acid e.g., the 16s rRNA gene region of M. arginini, the 16s rRNA gene region of M. orale, the 16s rRNA gene region of M. synoviae, the 16s rRNA gene region of M
  • the compositions and methods provided herein can be used to detect a target nucleic acid present in a sample comprising a plurality of nucleic acids (including the target nucleic acid and a plurality of non-target nucleic acids).
  • the target nucleic acid can, for example, be present at one or more copies per 10 7 non-target nucleic acids, one or more copies per 10 6 non-target nucleic acids, one or more copies per 10 5 non-target nucleic acids, one or more copies per 10 4 non-target nucleic acids, one or more copies per 10 3 non-target nucleic acids, one or more copies per 10 2 non-target nucleic acids, one or more copies per 50 non-target nucleic acids, one or more copies per 20 non-target nucleic acids, one or more copies per 10 non-target nucleic acids, one or more copies per 5 non-target nucleic acids, or a number or any range between two of these values.
  • the disclosed methods can be used to detect a target nucleic acid present in a sample comprising a plurality of nucleic acids (including the target nucleic acid and a plurality of non-target nucleic acids), wherein the target nucleic acid is present at one or more copies per 10 18 non-target nucleic acids, per 10 15 non-target nucleic acids, per 10 12 non-target nucleic acids, per 10 9 non-target nucleic acids, per 10 6 non- target nucleic acids, per 10 5 non-target nucleic acids, per 10 4 non-target nucleic acids, per 10 3 non-target nucleic acids, per 10 2 non-target nucleic acids, per 50 non-target nucleic acids, per 20 non-target nucleic acids, per 10 non-target nucleic acids, or per 5 non-target nucleic acids, or a number or a range between any two of these values.
  • the concentration at which a target nucleic acid can be detected comprises a range from about 10 copies of target nucleic acid sequence per sample to about 1 ⁇ 10 8 copies of target nucleic acid sequence per sample (e.g., about 10, 1 ⁇ 10 2 , 1 ⁇ 10 3 , 1 ⁇ 10 4 , 1 ⁇ 10 5 , 1 ⁇ 10 6 , 1 ⁇ 10 7 , 1 ⁇ 10 8 copies per sample, or a number or a range between any two of these values).
  • the concentration at which a target nucleic acid can be detected comprises a range from about 10 copies of target nucleic acid sequence per sample to about 1 ⁇ 10 6 copies of target nucleic acid sequence per sample (e.g., about 10, 1 ⁇ 10 2 , 1 ⁇ 10 3 , 1 ⁇ 10 4 , 1 ⁇ 10 5 , 1 ⁇ 10 6 , or a number or a range between any two of these values).
  • nucleic acid amplification can refer to any known procedure for obtaining multiple copies of a target nucleic acid sequence or its complement or fragments thereof, using sequence-specific methods.
  • amplification methods include, but are not limited to, polymerase chain reaction (PCR), ligase chain reaction (LCR), loop- mediated isothermal amplification (LAMP), strand displacement amplification (SDA) (e.g., multiple displacement amplification (MDA)), replicase-mediated amplification, immuno- amplification, nucleic acid sequence based amplification (NASBA), self-sustained sequence replication (3SR), rolling circle amplification, and transcription-mediated amplification (TMA).
  • PCR polymerase chain reaction
  • LCR loop- mediated isothermal amplification
  • SDA strand displacement amplification
  • MDA multiple displacement amplification
  • replicase-mediated amplification e.g., multiple displacement amplification (MDA)
  • immuno- amplification e.g., nucleic acid sequence based amplification (NASBA), self-sustained sequence replication (3SR), rolling circle amplification, and transcription-mediated amplification (TMA).
  • NASBA nucle
  • LCR amplification uses at least four separate oligonucleotides to amplify a target and its complementary strand by using multiple cycles of hybridization, ligation, and denaturation.
  • SDA amplifies by using a primer that contains a recognition site for a restriction endonuclease which nicks one strand of a hemimodified DNA duplex that includes the target sequence, followed by amplification in a series of primer extension and strand displacement steps.
  • PCR is a method well-known in the art for amplification of nucleic acids. PCR involves amplification of a target sequence using two or more extendable sequence- specific oligonucleotide primers that flank the target sequence.
  • the nucleic acid containing the target sequence of interest is subjected to a program of multiple rounds of thermal cycling (denaturation, annealing and extension) in the presence of the primers, a thermostable DNA polymerase (e.g., Taq polymerase) and various dNTPs, resulting in amplification of the target sequence.
  • PCR uses multiple rounds of primer extension reactions in which complementary strands of a defined region of a DNA molecule are simultaneously synthesized by a thermostable DNA polymerase. At the end of each cycle, each newly synthesized DNA molecule acts as a template for the next cycle.
  • PCR can generate double-stranded amplification products suitable for post- amplification processing. If desired, amplification products can be detected by visualization with agarose gel electrophoresis, by an enzyme immunoassay format using probe-based colorimetric detection, by fluorescence emission technology, or by other detection means known to one of skill in the art. [0164] A wide variety of PCR methods are known in the art.
  • PCR method examples include, but not limited to, Real-Time PCR, End-Point PCR, Amplified fragment length polymorphism PCR (AFLP-PCR), Alu-PCR, Asymmetric PCR, Colony PCR, DD-PCR, Degenerate PCR, Hot-start PCR, In situ PCR, Inverse PCR Long-PCR, Multiplex PCR, Nested PCR, PCR-ELISA, PCR-RFLP, PCR-single strand conformation polymorphism (PCR-SSCP), quantitative competitive PCR (QC-PCR), rapid amplification of cDNA ends-PCR (RACE-PCR), Random Amplification of Polymorphic DNA-PCR (RAPD-PCR), Real-Time PCR, Repetitive extragenic palindromic-PCR (Rep-PCR), reverse transcriptase PCR (RT-PCR), TAIL-PCR, Touchdown PCR and Vectorette PCR.
  • AFLP-PCR Amplified fragment length polymorphism PCR
  • Alu-PCR Asymmetric PCR
  • Real-time PCR such as quantitative real time PCR (qPCR) can be used to simultaneously quantify and amplify a specific part of a given nucleic acid molecule. It can be used to determine whether a specific sequence is present in the sample; and if it is present, the number of copies of the sequence that are present.
  • the term “real-time” can refer to periodic monitoring during PCR. Certain systems such as the ABI 7700 and 7900HT Sequence Detection Systems (Applied Biosystems, Foster City, Calif.) conduct monitoring during each thermal cycle at a pre-determined or user-defined point. Real-time analysis of PCR with fluorescence resonance energy transfer (FRET) probes measures fluorescent dye signal changes from cycle-to-cycle, preferably minus any internal control signals.
  • FRET fluorescence resonance energy transfer
  • the real-time procedure follows the general pattern of PCR, but the nucleic acid is quantified after each round of amplification.
  • Two examples of methods of quantification are the use of fluorescent dyes (e.g., SYBRGreen) that intercalate into double-stranded DNA, and modified DNA oligonucleotide probes that fluoresce when hybridized with a complementary DNA.
  • Intercalating agents have a relatively low fluorescence when unbound, and a relatively high fluorescence upon binding to double-stranded nucleic acids. As such, intercalating agents can be used to monitor the accumulation of double strained nucleic acids during a nucleic acid amplification reaction.
  • non-specific dyes useful in the embodiments disclosed herein include intercalating agents such as SYBR Green I (Molecular Probes), propidium iodide, ethidium bromide, and the like.
  • Samples can be infected and/or contaminated with multiple organisms.
  • the disclosed primers and probes are tolerant to mixed infections of a sample. Because of the specific target sequences, primers and probes, the methods and compositions disclosed herein can be used to detect the presence/absence or level of one or more of M. arginini, M. orale, M. synoviae, M. hyorhinis, A. laidlawii, M. fermentans, M. gallisepticum, M. pneumoniae and S.
  • the primers disclosed herein can be paired with additional PCR systems using a uniform chemistry and thermal PCR profile to provide a panel of assays for the detection of one or more of M. arginini, M. orale, M. synoviae, M. hyorhinis, A. laidlawii, M. fermentans, M. gallisepticum, M. pneumoniae and S. citri to, for example, improve overall assay sensitivity and robustness.
  • multiplex PCR is performed to amplify and detect, e.g., by direct or indirect means, the presence or absence of one or more of M. arginini, M.
  • the presence or absence of one or more of M. arginini, M. orale, M. synoviae, M. hyorhinis can be determined by amplifying and detecting the presence or absence of the M. arginini, M. orale, M. synoviae, M. hyorhinis, 16s rRNA gene; the presence or absence of A.
  • laidlawii can be determined by amplifying and detecting the presence or absence of the A. laidlawii 23s rRNA gene; the presence or absence of M. fermentans can be determined by amplifying and detecting the presence or absence of the M. fermentans 16s rRNA gene; the presence or absence of M. gallisepticum and/or M. pneumoniae can be determined by amplifying and detecting the presence or absence of the M. gallisepticum and/or M. pneumoniae 23s rRNA gene; the presence or absence of S. citri can be determined by amplifying and detecting the presence or absence of the S. citri 16s rRNA gene. [0169] Accordingly, some embodiments for the detection and/or identification of M. arginini, M.
  • orale, M. synoviae, M. hyorhinis, A. laidlawii, M. fermentans, M. gallisepticum, M. pneumoniae and S. citri in a sample include the steps of providing a test sample; and contacting the sample with oligonucleotide primers that can specifically hybridize and amplify (1) the 16s rRNA gene region of M. arginini, M. orale, M. synoviae, M. hyorhinis; (2) the 23s rRNA gene region of A. laidlawii; (3) the 16s rRNA gene region of M. fermentans; (4) the 23s rRNA gene region of M. gallisepticum and M.
  • the 16s rRNA gene region of S. citri and oligonucleotide probes that can specifically hybridize to (1) the 16s rRNA gene region of M. arginini, M. orale, M. synoviae, M. hyorhinis; (2) the 23s rRNA gene region of A. laidlawii; (3) the 16s rRNA gene region of M. fermentans; (4) the 23s rRNA gene region of M. gallisepticum and M. pneumoniae; and/or (5) the 16s rRNA gene region of S. citri under standard nucleic acid amplification conditions and/or stringent hybridization conditions.
  • the sample can be contacted with all of the primers and probes at once, or can be contacted with some of the primers and probes first and subsequently contacted by the remainder of the primers and probes. In some embodiments, the sample is contacted with one primer/probe set.
  • Duplex PCR can be performed to amplify and detect, e.g., by direct or indirect means, the presence or absence of one or more of M. arginini, M. orale, M. synoviae, M. hyorhinis, and A. laidlawii to allow identification and determination of, e.g., mycoplasma contamination of a sample in one test.
  • the presence or absence of one or more of M. arginini, M. orale, M. synoviae, M. hyorhinis can be determined by amplifying and detecting the presence or absence of the M. arginini, M. orale, M. synoviae, M. hyorhinis, 16s rRNA gene and/or the presence or absence of A. laidlawii can be determined by amplifying and detecting the presence or absence of the A. laidlawii 23s rRNA gene.
  • laidlawii in a sample include the steps of providing a test sample; and contacting the sample with oligonucleotide primers that can specifically hybridize and amplify (1) the 16s rRNA gene region of M. arginini, M. orale, M. synoviae, M. hyorhinis and/or (2) the 23s rRNA gene region of A. laidlawii, and oligonucleotide probes that can specifically hybridize to (1) the 16s rRNA gene region of M. arginini, M. orale, M. synoviae, M. hyorhinis and/or (2) the 23s rRNA gene region of A.
  • the sample can be contacted with all of the primers and probes at once, or can be contacted with some of the primers and probes first and subsequently contacted by the remainder of the primers and probes.
  • the sample is contacted with one primer/probe set.
  • duplex PCR is performed to amplify and detect, e.g., by direct or indirect means, the presence or absence of one or more of one or more of M. fermentans, M. gallisepticum, and M. pneumoniae to allow identification and determination of, e.g., mycoplasma contamination of a sample in one test.
  • the presence or absence of M. fermentans can be determined by amplifying and detecting the presence or absence of the M. fermentans 16s rRNA gene and the presence or absence of M. gallisepticum and/or M. pneumoniae can be determined by amplifying and detecting the presence or absence of the M. gallisepticum and/or M. pneumoniae 23s rRNA gene.
  • some embodiments for the detection and/or identification of M. fermentans, M. gallisepticum, and M. pneumoniae in a sample include the steps of providing a test sample; and contacting the sample with oligonucleotide primers that can specifically hybridize and amplify (1) the 16s rRNA gene region of M.
  • the sample can be contacted with all of the primers and probes at once, or can be contacted with some of the primers and probes first and subsequently contacted by the remainder of the primers and probes. In some embodiments, the sample is contacted with one primer/probe set.
  • duplex PCR is performed to amplify and detect, e.g., by direct or indirect means, the presence or absence of one or more of A. laidlawii and S. citri to allow identification and determination of, e.g., mycoplasma contamination of a sample in one test.
  • the presence or absence of one or more of A. laidlawii can be determined by amplifying and detecting the presence or absence of the A. laidlawii 23s rRNA gene and the presence or absence of S. citri can be determined by amplifying and detecting the presence or absence of the S. citri 16s rRNA gene.
  • some embodiments for the detection and/or identification of A. laidlawii and S. citri in a sample include the steps of providing a test sample; and contacting the sample with oligonucleotide primers that can specifically hybridize and amplify (1) the 23s rRNA gene region of A. laidlawii and/or (2) the 16s rRNA gene region of S. citri, and oligonucleotide probes that can specifically hybridize to (1) the 23s rRNA gene region of A. laidlawii and/or (2) the 16s rRNA gene region of S. citri under standard nucleic acid amplification conditions and/or stringent hybridization conditions.
  • the sample can be contacted with all of the primers and probes at once, or can be contacted with some of the primers and probes first and subsequently contacted by the remainder of the primers and probes.
  • the sample is contacted with one primer/probe set.
  • the oligonucleotide probe can be, for example, between about 10 and about 45 nucleotides in length, and comprise a detectable moiety (e.g., a signal moiety, a detectable label).
  • the contacting is performed under conditions allowing for the specific hybridization of the primers to the corresponding targeted gene region if the target organism is present in the sample.
  • the presence and/or amount of probe that is specifically bound to the corresponding targeted gene region can be determined, wherein bound probe is indicative of the presence of the corresponding target organism in the sample. In some embodiments, the amount of bound probe is used to determine the amount of the corresponding target organism in the sample.
  • the determining step can be achieved using any methods known to those skilled in the art, including but not limited to, in situ hybridization, following the contacting step.
  • the detection of hybrid duplexes i.e., of a probe specifically bound to the targeted gene region
  • hybridization duplexes are separated from unhybridized nucleic acids and the labels bound to the duplexes are then detected.
  • Such labels refer to radioactive, fluorescent, biological or enzymatic tags or labels of standard use in the art.
  • a label can be conjugated to either the oligonucleotide probes or the nucleic acids derived from the biological sample.
  • wash steps may be employed to wash away excess sample/target nucleic acids or oligonucleotide probes (as well as unbound conjugate, where applicable).
  • standard heterogeneous assay formats are suitable for detecting the hybrids using the labels present on the oligonucleotide primers and probes. Determining the presence or amount of one or more amplicons can comprise contacting said amplicons with a plurality of oligonucleotide probes.
  • determining the presence or amount of one or more amplicons comprises measuring a detectable signal, such as, for example, a detectable signal from a probe.
  • determining the presence or amount of one or more amplicons comprises measuring a detectable signal, such as, for example, a detectable signal from a probe (e.g., after cleavage of the probe by the 5 ⁇ –3 ⁇ exonuclease activity of a PCR polymerase (e.g., Taq)).
  • the measuring can in some embodiments be quantitative, e.g., in the sense that the amount of signal detected can be used to determine the amount of target nucleic acid (e.g., the 23s rRNA gene region of A.
  • the measuring can in some embodiments be qualitative, e.g., in the sense that the presence or absence of detectable signal can indicate the presence or absence of targeted nucleic acid (e.g., bacteria).
  • a detectable signal will not be present (e.g., above a given threshold level) unless the targeted nucleic acid (e.g., bacteria) is present above a particular threshold concentration.
  • a disclosed method can be used to determine the amount of a target nucleic acid (e.g., the 23s rRNA gene region of A. laidlawii) in a sample (e.g., a sample comprising the target nucleic acid and a plurality of non-target nucleic acids).
  • the reference standard curve can be generated based on serial dilutions of the standard dilution curve of mycoplasma genomes (See, Example 1 below).
  • a detectable signal is produced by the fluorescence- emitting dye pair of a probe.
  • a disclosed method includes contacting amplicons with a probe comprising a fluorescence resonance energy transfer (FRET) pair or a quencher/fluor pair, or both.
  • a disclosed method includes contacting amplicons with a probe comprising a FRET pair.
  • a disclosed method includes contacting amplicons with a probe comprising a fluor/quencher pair.
  • Fluorescence-emitting dye pairs comprise a FRET pair or a quencher/fluor pair. In both embodiments of a FRET pair and a quencher/fluor pair, the emission spectrum of one of the dyes overlaps a region of the absorption spectrum of the other dye in the pair.
  • fluorescence-emitting dye pair is a generic term used to encompass both a “FRET pair” and a “quencher/fluor pair,” both of which terms are discussed in more detail below.
  • fluorescence-emitting dye pair is used interchangeably with the phrase “a FRET pair and/or a quencher/fluor pair.”
  • At least one of the plurality of oligonucleotide probes provided herein can comprise a fluorescence emitter moiety and a fluorescence quencher moiety.
  • at least one of the one or more oligonucleotide probes further comprises a second fluorescence quencher moiety.
  • the second fluorescence quencher moiety can be between the ninth and tenth nucleotide of the oligonucleotide probe.
  • the probe produces an amount of detectable signal prior to being cleaved, and the amount of detectable signal that is measured is reduced when the probe is cleaved.
  • the probe produces a first detectable signal prior to being cleaved (e.g., from a FRET pair) and a second detectable signal when the probe is cleaved (e.g., from a quencher/fluor pair).
  • the probe comprises a FRET pair and a quencher/fluor pair.
  • the probe comprises a FRET pair.
  • FRET is a process by which radiationless transfer of energy occurs from an excited state fluorophore to a second chromophore in close proximity. The range over which the energy transfer can take place is limited to approximately 10 nanometers (100 angstroms), and the efficiency of transfer is extremely sensitive to the separation distance between fluorophores.
  • FRET fluorescence resonance energy transfer
  • a FRET signal serves as a proximity gauge of the donor and acceptor; only when they are in close proximity to one another is a signal generated.
  • the FRET donor moiety e.g., donor fluorophore
  • FRET acceptor moiety e.g., acceptor fluorophore
  • the donor-acceptor pair is referred to herein as a “FRET pair” or a “signal FRET pair.”
  • a probe includes two signal partners (a signal pair), when one signal partner is a FRET donor moiety and the other signal partner is a FRET acceptor moiety.
  • a probe that includes such a FRET pair (a FRET donor moiety and a FRET acceptor moiety) will thus exhibit a detectable signal (a FRET signal) when the signal partners are in close proximity (e.g., while on the same RNA molecule), but the signal will be reduced (or absent) when the partners are separated (e.g., after cleavage of the probe by the 5 ⁇ –3 ⁇ exonuclease activity of a PCR polymerase (e.g., Taq)).
  • FRET donor and acceptor moieties FRET pairs
  • any convenient FRET pair e.g., any convenient donor and acceptor moiety pair
  • one signal partner of a signal quenching pair produces a detectable signal and the other signal partner is a quencher moiety that quenches the detectable signal of the first signal partner (e.g., the quencher moiety quenches the signal of the signal moiety such that the signal from the signal moiety is reduced (quenched) when the signal partners are in proximity to one another, e.g., when the signal partners of the signal pair are in close proximity).
  • an amount of detectable signal increases when the probe is cleaved.
  • the signal exhibited by one signal partner is quenched by the other signal partner (a quencher signal moiety, a fluorescence quencher moiety), e.g., when both are present on the same ssDNA molecule prior to cleavage by the 5 ⁇ –3 ⁇ exonuclease activity of a PCR polymerase (e.g., Taq).
  • a PCR polymerase e.g., Taq
  • one signal partner e.g., the first signal partner
  • quenching pair is a signal moiety that produces a detectable signal that is quenched by the second signal partner (e.g., a quencher moiety).
  • the signal partners of such a quencher/fluor pair will thus produce a detectable signal when the partners are separated (e.g., after cleavage of the probe by the 5 ⁇ –3 ⁇ exonuclease activity of a PCR polymerase (e.g., Taq)), but the signal will be quenched when the partners are in close proximity (e.g., prior to cleavage of the probe by the 5 ⁇ –3 ⁇ exonuclease activity of a PCR polymerase (e.g., Taq)).
  • a PCR polymerase e.g., Taq
  • a quencher moiety can quench a signal from the signal moiety (e.g., prior to cleavage of the probe by the 5 ⁇ –3 ⁇ exonuclease activity of a PCR polymerase (e.g., Taq)) to various degrees.
  • a quencher moiety quenches the signal from the signal moiety where the signal detected in the presence of the quencher moiety (when the signal partners are in proximity to one another) is 95% or less of the signal detected in the absence of the quencher moiety (when the signal partners are separated).
  • the signal detected in the presence of the quencher moiety can be 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 15% or less, 10% or less, or 5% or less of the signal detected in the absence of the quencher moiety.
  • no signal e.g., above background is detected in the presence of the quencher moiety.
  • the signal detected in the absence of the quencher moiety (when the signal partners are separated) is at least 1.2 fold greater (e.g., at least l.3fold, at least 1.5 fold, at least 1.7 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 5 fold, at least 7 fold, at least 10 fold, at least 20 fold, or at least 50 fold greater, or a number or a range between any two of these values) than the signal detected in the presence of the quencher moiety (when the signal partners are in proximity to one another).
  • the signal moiety can be a fluorescent label.
  • the quencher moiety quenches the signal (e.g., the light signal) from the fluorescent label (e.g., by absorbing energy in the emission spectra of the label).
  • the quencher moiety when the quencher moiety is not in proximity with the signal moiety, the emission (the signal) from the fluorescent label can be detectable because the signal is not absorbed by the quencher moiety.
  • Any convenient donor acceptor pair (signal moiety /quencher moiety pair) can be used and many suitable pairs are known in the art.
  • the quencher moiety absorbs energy from the signal moiety (also referred to herein as a “detectable label” or a “detectable moiety”) and then emits a signal (e.g., light at a different wavelength).
  • the quencher moiety is itself a signal moiety (e.g., a signal moiety can be 6-carboxyfluorescein while the quencher moiety can be 6-carboxy-tetramethylrhodamine), and in some such embodiments, the pair can also be a FRET pair.
  • a quencher moiety is a dark quencher.
  • a dark quencher can absorb excitation energy and dissipate the energy in a different way (e.g., as heat). Thus, a dark quencher has minimal to no fluorescence of its own (does not emit fluorescence).
  • Cleavage of a probe can be detected by measuring a colorimetric read-out. For example, the liberation of a fluorophore (e.g., liberation from a FRET pair, liberation from a quencher/fluor pair, and the like) can result in a wavelength shift (and thus color shift) of a detectable signal. Thus, in some embodiments, cleavage of a probe can be detected by a color- shift.
  • Such a shift can be expressed as a loss of an amount of signal of one color (wavelength), a gain in the amount of another color, a change in the ration of one color to another, and the like.
  • Disclosed herein include methods for detecting one or more of M. arginini, M. orale, M. synoviae, and M. hyorhinis in a sample.
  • the method comprises: (a) contacting a sample with a pair of primers capable of hybridizing to a target region of M. arginini, M. orale, M. synoviae, and M. hyorhinis genome, wherein the target region comprises the 16s rRNA gene region of M. arginini, M.
  • each primer in the pair of primers comprises a sequence having at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2; (b) generating amplicons of the target region of M. arginini, M. orale, M. synoviae, M. hyorhinis, or any combination thereof, from the sample, if the sample comprises one or more of M.
  • Each primer in the pair of primers can comprise the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 1 or SEQ ID NO: 2.
  • Determining the presence or amount of the one or more amplicons of the target region can comprise contacting the amplicons with an oligonucleotide probe comprising a sequence having at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 3.
  • the oligonucleotide probe can comprise the sequence of SEQ ID NO: 3, or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 3.
  • the oligonucleotide probe can comprise a fluorescence emitter moiety, a fluorescence quencher moiety, or both.
  • the fluorescence emitter moiety can be at the 5’ terminus of the oligonucleotide probe, the 3’ terminus of the oligonucleotide probe, or both.
  • the oligonucleotide probe further comprises a second fluorescence quencher moiety.
  • the second fluorescence quencher moiety can be between the ninth and tenth nucleotide of the oligonucleotide probe.
  • the method comprises: (a) contacting a sample with a pair of primers capable of hybridizing to a target region of A. laidlawii genome, wherein the target region comprises the 23s rRNA gene region of A. laidlawii, and wherein each primer in the pair of primers comprises a sequence having at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 4 or SEQ ID NO: 5; (b) generating amplicons of the target region of A.
  • 85% identity e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values
  • Each primer in the pair of primers can comprise the sequence of SEQ ID NO: 4 or SEQ ID NO: 5, or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 4 or SEQ ID NO: 5.
  • Determining the presence or amount of the amplicons of the target region can comprise contacting the amplicons with an oligonucleotide probe comprising a sequence having at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 6.
  • the oligonucleotide probe can comprise the sequence of SEQ ID NO: 6, or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 6.
  • the oligonucleotide probe can comprise a fluorescence emitter moiety, a fluorescence quencher moiety, or both.
  • Disclosed herein include methods for detecting M. fermentans in a sample.
  • the method comprises: (a) contacting a sample with a pair of primers capable of hybridizing to a target region of M. fermentans genome, wherein the target region comprises the 16s rRNA gene region of M.
  • each primer in the pair of primers comprises a sequence having at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 7 or SEQ ID NO: 8; (b) generating amplicons of the target region of M. fermentans from the sample, if the sample comprises M. fermentans; and (c) determining the presence or amount of the amplicons of the target region as an indication of the presence of M. fermentans in the sample.
  • 85% identity e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values
  • Each primer in the pair of primers can comprise the sequence of SEQ ID NO: 7 or SEQ ID NO: 8, or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 7 or SEQ ID NO: 8.
  • Determining the presence or amount of the amplicons of the target region can comprise contacting the amplicons with an oligonucleotide probe comprising a sequence having at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 9.
  • the oligonucleotide probe can comprise the sequence of SEQ ID NO: 9, or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 9.
  • the oligonucleotide probe can comprise a fluorescence emitter moiety, a fluorescence quencher moiety, or both.
  • the fluorescence emitter moiety can be at the 5’ terminus of the oligonucleotide probe, the 3’ terminus of the oligonucleotide probe, or both.
  • the oligonucleotide probe further comprises a second fluorescence quencher moiety.
  • the second fluorescence quencher moiety can be between the ninth and tenth nucleotide of the oligonucleotide probe.
  • Disclosed herein include methods for detecting one or more of M. gallisepticum and M. pneumoniae in a sample.
  • the method comprises: (a) contacting a sample with a pair of primers capable of hybridizing to a target region of M. gallisepticum and M. pneumoniae genome, wherein the target region comprises the 23s rRNA gene region of M. gallisepticum and M.
  • each primer in the pair of primers comprises a sequence having at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 10 or SEQ ID NO: 11; (b) generating amplicons of the target region of M. gallisepticum, M. pneumoniae, or both from the sample, if the sample comprises one or more of M. gallisepticum and M. pneumoniae; and (c) determining the presence or amount of one or more amplicons as an indication of the presence of one or more of M.
  • 85% identity e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two
  • Each primer in the pair of primers can comprise the sequence of SEQ ID NO: 10 or SEQ ID NO: 11, or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 10 or SEQ ID NO: 11.
  • Determining the presence or amount of the one or more amplicons of the target region can comprise contacting the amplicons with an oligonucleotide probe comprising a sequence having at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 12.
  • the oligonucleotide probe can comprise the sequence of SEQ ID NO: 12, or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 12.
  • the oligonucleotide probe can comprise a fluorescence emitter moiety, a fluorescence quencher moiety, or both.
  • Disclosed herein include methods for detecting S. citri in a sample.
  • the method can comprise: (a) contacting a sample with a pair of primers capable of hybridizing to a target region of S. citri genome, wherein the target region comprises the 16s rRNA gene region of S.
  • each primer in the pair of primers comprises a sequence having at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 13 or SEQ ID NO: 14; (b) generating amplicons of the target region of S. citri from the sample, if the sample comprises S. citri; and (c) determining the presence or amount of the amplicons of the target region as an indication of the presence of S. citri in the sample.
  • 85% identity e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values
  • Each primer in the pair of primers can comprise the sequence of SEQ ID NO: 13 or SEQ ID NO: 14, or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 13 or SEQ ID NO: 14.
  • Determining the presence or amount of the amplicons of the target region can comprise contacting the amplicons with an oligonucleotide probe comprising a sequence having at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 15.
  • the oligonucleotide probe can comprise the sequence of SEQ ID NO: 15, or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 15.
  • the oligonucleotide probe can comprise a fluorescence emitter moiety, a fluorescence quencher moiety, or both.
  • the fluorescence emitter moiety can be at the 5’ terminus of the oligonucleotide probe, 3’ terminus of the oligonucleotide probe, or both.
  • the oligonucleotide probe further comprises a second fluorescence quencher moiety.
  • the second fluorescence quencher moiety can be between the ninth and tenth nucleotide of the oligonucleotide probe.
  • Some embodiments provide methods and compositions for multiplex real- time PCR capable of simultaneously detecting two gene targets across eight mycoplasma species, for example detecting one or more of M. arginini, M. orale, M. synoviae, M. hyorhinis, A. laidlawii, M. fermentans, M. gallisepticum, M. pneumoniae and S. citri in a sample.
  • Disclosed herein include methods for detecting one or more of M. arginini, M. orale, M. synoviae, M. hyorhinis, A.
  • the method comprises: a) contacting a sample with a plurality of pairs of primers, wherein the plurality of pairs of primers comprises: a pair of primers capable of hybridizing to the 16s rRNA gene region of M. arginini, M. orale, M. synoviae, and M.
  • each primer in the pair of primers comprises a sequence having at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2; a pair of primers capable of hybridizing to the 23s rRNA gene region of A.
  • 85% identity e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values
  • each primer in the pair of primers comprises a sequence having at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 4 or SEQ ID NO: 5; a pair of primers capable of hybridizing to the 16s rRNA gene region of M.
  • 85% identity e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values
  • each primer in the pair of primers comprises a sequence having at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 7 or SEQ ID NO: 8; a pair of primers capable of hybridizing to the 23s rRNA gene region of M. gallisepticum and M.
  • each primer in the pair of primers comprises a sequence having at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 10 or SEQ ID NO: 11; and/or a pair of primers capable of hybridizing to the 16s rRNA gene region of S.
  • 85% identity e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values
  • each primer in the pair of primers comprises a sequence having at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 13 or SEQ ID NO: 14; b) generating amplicons of the 16s rRNA gene region of M. arginini, the 16s rRNA gene region of M. orale, the 16s rRNA gene region of M. synoviae, the 16s rRNA gene region of M.
  • the sample comprises one or more of M. arginini, M. orale, M. synoviae, M. hyorhinis, A. laidlawii, M. fermentans, M. gallisepticum, M. pneumoniae, and S. citri; and c) determining the presence or amount of one or more amplicons as an indication of the presence of one or more of M.
  • hyorhinis comprises (a) a primer having the sequence of SEQ ID NO: 1 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 1 and (b) a primer having the sequence of SEQ ID NO: 2 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 2; the pair of primers capable of hybridizing to the 23s rRNA gene region of A.
  • laidlawii comprises (a) a primer having the sequence of SEQ ID NO: 4 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 4 and (b) a primer having the sequence of SEQ ID NO: 5 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 5; the pair of primers capable of hybridizing to the 16s rRNA gene region of M.
  • fermentans comprises (a) a primer having the sequence of SEQ ID NO: 7 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 7 and (b) a primer having the sequence of SEQ ID NO: 8 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 8; the pair of primers capable of hybridizing to the 23s rRNA gene region of M. gallisepticum and M.
  • pneumoniae comprises (a) a primer having the sequence of SEQ ID NO: 10 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 10 and (b) a primer having the sequence of SEQ ID NO: 11 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 11; and/or the pair of primers capable of hybridizing to the 16s rRNA gene region of S.
  • citri comprises (a) a primer having the sequence of SEQ ID NO: 13 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 13 and (b) a primer having the sequence of SEQ ID NO: 14 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 14.
  • Determining the presence or amount of the one or more amplicons can comprise contacting the amplicons with one or more oligonucleotide probes each comprising a sequence having at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 3, 6, 9, 12, or 15.
  • Each of the one or more oligonucleotide probes can comprise the sequence of SEQ ID NO: 3, 6, 9, 12, or 15; or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 3, 6, 9, 12, or 15.
  • Disclosed herein include methods for detecting one or more of M. arginini, M. orale, M. synoviae, M. hyorhinis, and A. laidlawii in a sample.
  • the method comprises: a) contacting a sample with a plurality of pairs of primers, wherein the plurality of pairs of primers comprises: a pair of primers capable of hybridizing to the 16s rRNA gene region of M.
  • each primer in the pair of primers comprises a sequence having at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2; and a pair of primers capable of hybridizing to the 23s rRNA gene region of A.
  • 85% identity e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values
  • each primer in the pair of primers comprises a sequence having at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 4 or SEQ ID NO: 5; b) generating amplicons of the 16s rRNA gene region of M. arginini, the 16s rRNA gene region of M. orale, the 16s rRNA gene region of M. synoviae, the 16s rRNA gene region of M.
  • the 23s rRNA gene region of A. laidlawii or any combination thereof, if the sample comprises one or more of M. arginini, M. orale, M. synoviae, M. hyorhinis, and A. laidlawii; and c) determining the presence or amount of one or more amplicons as an indication of the presence of one or more of M. arginini, M. orale, M. synoviae, M. hyorhinis, and A. laidlawii in the sample.
  • the pair of primers capable of hybridizing to the 16s rRNA gene region of M. arginini, M. orale, M. synoviae, and M.
  • hyorhinis can comprise (a) a primer having the sequence of SEQ ID NO: 1 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 1 and (b) a primer having the sequence of SEQ ID NO: 2 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 2.
  • the pair of primers capable of hybridizing to the 23s rRNA gene region of A.
  • laidlawii comprises (a) a primer having the sequence of SEQ ID NO: 4 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 4 and (b) a primer having the sequence of SEQ ID NO: 5 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 5.
  • Determining the presence or amount of the one or more amplicons can comprise contacting the amplicons with one or more oligonucleotide probes each comprising a sequence having at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 3 or 6.
  • Each of the one or more oligonucleotide probes can comprise the sequence of SEQ ID NO: 3 or 6; or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 3 or 6.
  • the method comprises: a) contacting a sample with a plurality of pairs of primers, wherein the plurality of pairs of primers comprises: a pair of primers capable of hybridizing to the 16s rRNA gene region of M.
  • each primer in the pair of primers comprises a sequence having at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 7 or SEQ ID NO: 8; and a pair of primers capable of hybridizing to the 23s rRNA gene region of M. gallisepticum and M.
  • each primer in the pair of primers comprises a sequence having at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 10 or SEQ ID NO: 11; b) generating amplicons of the 16s rRNA gene region of M. fermentans, the 23s rRNA gene region of M. gallisepticum, the 23s rRNA gene region of M. pneumoniae, or any combination thereof, if the sample comprises one or more of M. fermentans, M. gallisepticum, and M.
  • 85% identity e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two
  • the pair of primers capable of hybridizing to the 16s rRNA gene region of M. fermentans can comprise (a) a primer having the sequence of SEQ ID NO: 7 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 7 and (b) a primer having the sequence of SEQ ID NO: 8 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 8.
  • the pair of primers capable of hybridizing to the 23s rRNA gene region of M. gallisepticum and M. pneumoniae comprises (a) a primer having the sequence of SEQ ID NO: 10 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 10 and (b) a primer having the sequence of SEQ ID NO: 11 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 11.
  • Determining the presence or amount of the one or more amplicons can comprise contacting the amplicons with one or more oligonucleotide probes each comprising a sequence having at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 9 or 12.
  • Each of the one or more oligonucleotide probes can comprise the sequence of SEQ ID NO: 9 or 12; or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 9 or 12.
  • the method comprises: a) contacting a sample with a plurality of pairs of primers, wherein the plurality of pairs of primers comprises: a pair of primers capable of hybridizing to the 23s rRNA gene region of A.
  • each primer in the pair of primers comprises a sequence having at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 4 or SEQ ID NO: 5; and a pair of primers capable of hybridizing to the 16s rRNA gene region of S.
  • 85% identity e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values
  • each primer in the pair of primers comprises a sequence having at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 13 or SEQ ID NO: 14; b) generating amplicons of the 23s rRNA gene region of A. laidlawii, the 16s rRNA gene region of S. citri, or both, if the sample comprises one or more of A. laidlawii and S.
  • the pair of primers capable of hybridizing to the 23s rRNA gene region of A. laidlawii comprises (a) a primer having the sequence of SEQ ID NO: 4 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 4 and (b) a primer having the sequence of SEQ ID NO: 5 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 5.
  • the pair of primers capable of hybridizing to the 16s rRNA gene region of S. citri comprises (a) a primer having the sequence of SEQ ID NO: 13 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 13 and (b) a primer having the sequence of SEQ ID NO: 14 or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 14.
  • Determining the presence or amount of the one or more amplicons can comprise contacting the amplicons with one or more oligonucleotide probes each comprising a sequence having at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values) to the sequence of SEQ ID NO: 6 or 15.
  • Each of the one or more oligonucleotide probes can comprise the sequence of SEQ ID NO: 6 or 15; or a sequence differing by one or two nucleotide mismatches relative to SEQ ID NO: 6 or 15.
  • At least one of the one or more oligonucleotide probes can comprise a fluorescence emitter moiety, a fluorescence quencher moiety, or both.
  • the fluorescence emitter moiety can be at the 5’ terminus of the oligonucleotide probe, the 3’ terminus of the oligonucleotide probe, or both.
  • at least one of the one or more oligonucleotide probes further comprises a second fluorescence quencher moiety.
  • the second fluorescence quencher moiety can be between the ninth and tenth nucleotide of the oligonucleotide probe.
  • the second fluorescence quencher moiety can comprise ZEN or TAO.
  • Each oligonucleotide probe can be flanked by complementary sequences at the 5’ end and 3’ end.
  • One of the complementary sequences can comprise a fluorescence emitter moiety and the other complementary sequence comprises a fluorescence quencher moiety. Determining the presence or amount of the amplicons can comprise using a reference standard curve.
  • the sample comprises, or is suspected to comprise, nucleic acids of one or more of M. arginini, M. orale, M. synoviae, M. hyorhinis, A. laidlawii, M. fermentans, M. gallisepticum, M. pneumoniae, and S. citri.
  • the sample can comprise at least about 10 copies to at most about 1 ⁇ 10 6 (e.g., about 10, 1 ⁇ 10 2 , 1 ⁇ 10 3 , 1 ⁇ 10 4 , 1 ⁇ 10 5 , 1 ⁇ 10 6 copies per sample, or a number or a range between any two of these values) of M. arginini genome, M. orale genome, M. synoviae genome, M. hyorhinis genome, A. laidlawii genome, M. fermentans genome, M. gallisepticum genome, M. pneumoniae genome, S. citri genome, or any combination thereof.
  • the sample is, or is derived from, a biological sample or an environmental sample.
  • the biological sample can be obtained from a tissue sample, saliva, blood, plasma, sera, stool, urine, sputum, mucous, lymph, synovial fluid, cerebrospinal fluid, ascites, pleural effusion, seroma, pus, swab of skin or a mucosal membrane surface, cultures thereof, or any combination thereof.
  • the biological sample can comprise genetically modified cells.
  • the sample can be a food sample, a beverage sample, a paper surface, a fabric surface, a metal surface, a wood surface, a plastic surface, a soil sample, a fresh water sample, a waste water sample, a saline water sample, a gas sample, a clinical sample, a pharmaceutical composition, cultures thereof, or any combination thereof.
  • the amplification can be carried out using PCR, ligase chain reaction (LCR), LAMP, SDA, replicase-mediated amplification, Immuno-amplification, NASBA, self-sustained sequence replication (3SR), rolling circle amplification, TMA, or any combination thereof.
  • LCR ligase chain reaction
  • SDA replicase-mediated amplification
  • NASBA self-sustained sequence replication
  • TMA rolling circle amplification
  • the PCR can be quantitative real-time PCR (qPCR).
  • Example 1 Qualification Protocol for Detection of Mycoplasma contamination by qPCR
  • samples e.g., drug substance and/or cell harvest media (CHM)
  • the assay for the detection of mycoplasma species in drug substance is a quantitative assay and the assay for detection of mycoplasma species contamination in cell harvest media is a qualitative assay.
  • Materials and Methods Preparation of Samples for testing [0220] In some embodiments, collected samples were processed and separated into the different testing materials before DNA extraction.
  • the protocol for processing and separating the testing materials was as follows: (1) The sample was centrifuged at 300 x g, 4°C for 10 minutes to pellet the cells, (2) The supernatant was removed (minimum 13 mL) and transferred into a clean 15 mL falcon tube (supernatant was stored on ice when DNA extraction was done the same day or stored at -80°C for later use).
  • the protocol was: (1) Dulbecco’s phosphate buffered saline (DPBS) was added to the cells on ice to dilute to an appropriate concentration (minimum 2 ⁇ 10 6 cells/ mL) and the pellet was dissolved by pipetting up and down, (2) An appropriate volume of cell suspension was aliquoted into tubes, (3) The samples were centrifuged at 300 x g, 4°C for 10 minutes to pellet the cells and the DPBS was removed, and (4) The cell pellets were stored at -80°C.
  • DPBS Dulbecco’s phosphate buffered saline
  • DNA extraction was performed at room temperature (including centrifugation steps). DNA was extracted using the Microsart® AMP Extraction Kit essentially according to manufacturer’s instructions. Before cell harvest media (CHM) DNA extraction, Vivaspin 2 columns were rinsed by adding 2 mL molecular grade water and centrifuged for 1 minute at 4,000 x g, and water was removed.
  • CHM cell harvest media
  • a DNA extraction control (provided in the kit) was added to samples.
  • lyophilized DNA extraction control was spun at 9600 x g (10,000 rpm) for 30 seconds, and the DNA extraction control was resuspended in 600 ⁇ L template preparation buffer (provided in the kit), mixed by pipetting, vortexed for 30 seconds and spun down at 9600 x g (10,000 rpm) for 30 seconds.20 ⁇ L single use aliquots were stored at -20°C until needed.
  • sample preparation with spike [0226] The appropriate amount of cell suspension was added to a new tube (minimum 180 ⁇ L, 11.1 ⁇ 10 6 cells/ mL). The volume was adjusted to 180 ⁇ L with DPBS if necessary.20 ⁇ L of Std 5 from the Std dilution series was added to the sample.
  • DS sample preparation without spike An appropriate amount of cell suspension was added to a new tube (minimum 180 ⁇ L, 11.1 ⁇ 10 6 cells/ mL). The volume was adjusted to 200 ⁇ L with DPBS if necessary.
  • CHM sample preparation [0228] Depending on when the sample(s) were received, the aspirated cell harvest media (CHM) stored on ice was used or stored, frozen CHM was thawed on ice for 15–30 minutes after removing it from -80 °C. CHM sample preparation with spike [0229] 1 mL of CHM was added to each validation standard tube.
  • CHM sample preparation with spike 1 mL of CHM was added to each validation standard tube.
  • E nterococcus faecalis (Andrewes and Horder) [0234] Human genomic DNA was used as an inhibition control. For the inhibition testing, 45 ⁇ L of the human genomic DNA was spiked with 5 ⁇ L of Std 5. A non-spiked sample of human genomic DNA was also used in the assay. Master Mix alone or water was used as non- template or negative controls. Preparation of reaction mix [0235] Assays and the corresponding primers are described in Table 7. Primer/probe mixes were ordered from Integrated DNA Technologies (IDT) and lyophilized Primer/probe mix aliquots were centrifuged for 10 seconds and then dissolved in the correct volume (Table 3) of IDTE buffer.
  • IDTT Integrated DNA Technologies
  • Table 4 Components And Volumes Needed For Reaction Mix For Standard Curve & Controls
  • Table 5 Components And Volumes Needed For Reaction Mix For Ds/Chm Samples S Plate setup [0237] Each standard was mixed by vortexing for 5 seconds before pipetting, then 10 ⁇ L of each Std (1-6) was added into the wells.12 ⁇ L (standard & controls) or 13 ⁇ L (samples) master mix to was added to the wells.12 ⁇ L of master mix was added to 3 wells as non-template control. Samples were run using the conditions shown below in Table 6. Table 6: Run Method For Duplex Assays.
  • Non-spiked samples [0241] The test sample must have shown undetectable signal in at least 3/3 replicates per target. If 2/3 replicates showed detectable signal (Ct value less than Negative control), the sample was considered positive. If 1/3 replicate was positive, the sample was considered indeterminate. One replicate out of 3 may have been removed for each standard if the Ct value of the observed replicate was greater or lower than the mean Ct value by 1.5 Ct. That replicate was then considered as an outlier.
  • Spiked samples [0243] The values of the spiked samples acceptable ranges was established during qualification. During qualification, the target genes must have been detected in at least 2 of 3 replicates across spike samples tested.
  • the acceptable ranges for the spiked samples were calculated as Mean of all replicates ⁇ 3SD, from at least 2 out of the 3 replicates. % CV of Ct values of all replicates was less than 15%. Ranges were established using Mean ⁇ 3SD. One replicate out of 3 may have been removed for each standard if the Ct value of the observed replicate was greater or lower than the mean Ct value by 1.5 Ct. That replicate was then considered as an outlier.
  • Controls [0245] The non-template control sample or negative control (water) must have shown undetectable signal in at least 3/3 replicates per target. The Specificity sample must have shown undetectable signal in 3/3 replicates per target (or less than 1 copy/rxn will be considered as outlier).
  • the positive control sample must have shown detectable signal in at least 2/3 replicates per target.
  • DNA extraction control must have shown detectable signal of VIC in 2/3 replicates per target.
  • one replicate out of 3 may be removed for each standard if the Ct value of the observed replicate is greater or lower than the mean Ct value by 1.5 Ct; that replicate was then considered as an outlier.
  • Other Parameters (Specific to the test)
  • the R 2 of the standard curve must have been ⁇ 0.995. All reported Ct values must have been derived from the linear phase of the exponential amplification when viewed using the log view of the amplification ( ⁇ Rn vs. Cycle). Results were reported as Ct values or log transformed copies/well (quantity values).
  • Mycoplasma contamination was assayed on gDNA extracted from samples to amplify the genes targeting the 16S/23S ribosomal RNA genes in mycoplasma (Table 7-Table 8) using the disclosed method.
  • Standards were prepared by diluting the mycoplasma gDNA ten- fold for up to 5 dilutions ranging from 1 ⁇ 10 6 to 10 copies/reaction (Table 1).
  • Table 7 List Of Assays And Corresponding Primer Sequences D D 1 1 1 1, 1, 1, 1, 2 2 2 2 2 2 3 3 3 *Indicates an internal ZEN quencher T bl 8 R n M th d F r D l x A d M M.
  • the spike study samples were prepared by spiking known concentrations ((10 copies) (for, e.g., cell samples) or 10 CFU/mL (e.g., cell harvest media)) of mycoplasma species into the drug product samples before DNA extraction. An unspiked sample and standards spiked into reconstitution buffer were also be used in the assay. Samples from three different sample lots were used in the assay. [0254] The parameters that were assessed during qualification included: Linearity, Precision (Intraassay and Interassay), Accuracy, Specificity, Stability and Robustness.
  • the assay for the detection of mycoplasma species in drug product is a quantitative assay and the assay for the detection of mycoplasma species in cell harvest media is a qualitative assay.
  • Linearity [0255] The Standard curve was generated using the calibration reagents. A 10-fold serial dilution was assayed to generate 6 standard points starting from 1 ⁇ 10 6 copies/rxn down to 10 copies/rxn to construct a standard curve. Linearity was determined by evaluating the standard curve from the first six qualification runs performed by multiple operators across multiple days. Acceptance criteria included: (1) The %CV of the mean observed value of the CT values must have been ⁇ 15% and (2) The PCR efficiency must have been within 90%-110%.
  • LLOQ/ULOQ Limits of Quantification
  • the results of the linearity were evaluated to determine lower limit of quantification (LLOQ).
  • the upper limit of quantification was defined as the highest concentration (e.g., the log-transformed copy/reaction value) that returned a result within ⁇ 20% of the expected concentration and that also showed a precision with %CV of ⁇ 15%.
  • the LLOQ was defined as the lowest concentration that returned a result within ⁇ 30% of the expected concentration and %CV of ⁇ 20%.
  • Intraassay Precision was ascertained by determining the coefficient of variation of the measured Ct value or concentration (e.g., Log-transformed copies/rxn) obtained between six replicates of DNA extracted from samples spiked with Std 5 to the level of Std 6 (10 copies/rxn) or CHM spiked with 10 CFU/ml in a single assay. [0260] Acceptance criteria for these experiments are described below. For samples: The %CV of the mean observed value of the Log-transformed copies/rxn must have been ⁇ ⁇ 30%.
  • Interassay Precision Intermediate precision was evaluated by determining the coefficient of variation from measured Ct value or concentration (e.g., Log-transformed copies/rxn) obtained between replicates of DNA extracted from drug product spiked with Std 5 to the level of Std 6 (10 copies/rxn) or CHM spiked with 10 CFU/ml performed by multiple operators across multiple days in at least three independent assays. Acceptance criteria for these experiments is described below: For DS: The %CV of the mean observed value of the Log-transformed copies/ rxn must have been ⁇ ⁇ 30%. Results are shown in Table 45-Table 51 (samples).
  • Spent Media e.g., CHM
  • Sample were subjected to 4°C and Room temperature conditions. DNA was extracted after these conditions and spike in recovery was performed where the drug product spiked with Std 5 to the level of Std 6 (10 copies/rxn) or media spiked with 10 CFU/ml within the range of the standard curve was tested. 4°C stability [0266] DNA extracted from samples and/or media was tested at baseline and after storage at 4°C for 24 hrs.
  • RT Stability DNA extracted from samples and/or media was tested at baseline and after storage at RT for 24 hrs. [0268] The acceptance criteria for these experiments are: For cell samples: The %RE of the mean observed value of the Log-transformed copies/ rxn must have been ⁇ ⁇ 30% to the baseline. For media: The %RE of the mean observed value of the Ct values/ rxn must have been ⁇ ⁇ 20% to the baseline. Stability data are shown in Table 66-Table 72. Specificity/ Inhibition Control [0269] To assess the specificity, a different template was used to test the specificity of the primer probe sets of mycoplasma species for off-target amplification in at least 3 independent runs.
  • Species which are closely related and not related to mollicutes were tested. Species with close phylogenetic relationship to mollicutes include: Clostridium sporogenes, Lactobacillus acidophilus, Streptococcus pneumoniae. Non mollicutes species include: Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa. Inhibition Control used was human gDNA. Human gDNA was spiked with Std 6 (10 copies/rxn) of relevant mycoplasma species and was used in the assay.
  • Limit of detection (LOD) of the assay was determined by using at least two serial dilutions of the standard curve beginning with the lowest standard in buffer and tested in at least three replicates each over at least 3 independent assays. LOD was defined as the concentration that returns results above the limit of blank in at least 33% of the replicates in each independent run. Results are shown in Table 59-Table 65.
  • Acceptance/Rejection Criteria for each parameter assessed during this qualification process are listed under each section and for each qualification run and can be defined as described below: (1) %CV for each standard Ct value is ⁇ 15%, (2) R2 value for the standard curves must be ⁇ 0.995, (3) The expected PCR efficiency of the standards is 90-110%, (4) All reported Ct values must be derived from linear phase of the exponential amplification when viewed using the log view of the amplification plot ( ⁇ Rn vs. Cycle), (5) The NTC (mastermix only or water) must show undetectable signal in 3/3 replicates.
  • Spike Samples [0279] The values of the spike samples acceptable ranges were established during qualification. During qualification, the target genes must have been detected in at least 2 of 3 replicates across spike samples tested. The acceptable ranges for the spike samples were calculated as mean of all replicates ⁇ 3SD, from at least 2 out of the 3 replicates. %CV of Ct values of all replicates was less than 15%, and ranges were, in some embodiments, established using Mean ⁇ 3SD.
  • the assay was considered valid if 2 out of the 3 replicates for each standard or spike sample was amplified and produced a measurable Ct value. (One replicate may have been removed for each set of standards/spike sample replicates if the Ct value of the observed replicate had a greater or lower than the mean Ct value by 1.5 Ct; that replicate was considered as an outlier).
  • Test Samples [0280] The test sample (unspiked) must have shown undetectable signal in 3/3 replicates per target. If 2/3 replicates showed detectable signal (Ct value lower than LOD of the assay), the sample was considered positive. If 1/3 replicate is positive, the results are considered indeterminate. Assay is invalid.
  • Table 20 A. Laidlawii And M. Orale - Linearity Standard Average Ct Values g T S 9 3 5 1 8 4 g T g p 1 3 g S 3*SD D M.O Std1 1000000 16.3 16.4 16.0 16.5 16.2 16.0 16.2 0.2 1.1 15.7 16.8 3 6 3 1 1
  • Table 21 A. Laidlawii And M.
  • Orale Accuracy – Spiked In Diluent n n %Recovery 84 100 100 Tbl 34b A Lidl ii And M Orl A r Sikd In Dil nt n . . . Run 3 1083 3.0 99 2.0 10 1.0 Avg 1086 3.0 103 2.0 11 1.0 n Table 35a: A. Laidlawii And M. Arginini Accuracy – Spiked In Diluent ) xn ) xn %Recovery 83 100 101 Table 35b: A Laidlawii And M Ar inini Accurac – Siked In Diluent ) xn . . .
  • Table 36a A. Laidlawii And M. Synoviae Accuracy – Spiked In Diluent AL n n % Recovery 84 100 101 Table 36b: A Laidlawii And M Synoviae Accuracy – Spiked In Diluent n . . . %CV 1 1 1 %Recovery 102 102 104 n Table 37a: A. Laidlawii And S.Citri Accuracy – Spiked In Diluent A L n n %Recovery 83 00 0 Table 37b: A.

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Abstract

L'invention concerne des procédés, des compositions et des kits pour la détection d'un ou plusieurs parmi M. arginini, M. orale, M. synoviae, M. hyorhinis, A. laidlawii, M. fermentans, M. gallisepticum, M. pneumoniae et S. citri.
PCT/IB2023/057886 2022-08-04 2023-08-04 Procédé de détection de contamination par mycoplasma WO2024028818A1 (fr)

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