WO2014088088A1 - Procédé pour détecter et identifier des bactéries appartenant au genre mycoplasma et ureaplasma - Google Patents

Procédé pour détecter et identifier des bactéries appartenant au genre mycoplasma et ureaplasma Download PDF

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WO2014088088A1
WO2014088088A1 PCT/JP2013/082779 JP2013082779W WO2014088088A1 WO 2014088088 A1 WO2014088088 A1 WO 2014088088A1 JP 2013082779 W JP2013082779 W JP 2013082779W WO 2014088088 A1 WO2014088088 A1 WO 2014088088A1
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seq
oligonucleotide probe
probe
represented
invader
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PCT/JP2013/082779
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Japanese (ja)
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玲 柳原
真樹 高梨
橋本　修
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三菱化学メディエンス株式会社
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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
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes

Definitions

  • the present invention relates to a method for detecting and identifying Mycoplasma and Ureaplasma bacteria, and a reagent kit.
  • Urinary tract infections are caused by the engraftment and propagation of pathogens from the kidneys through the ureters and bladders to the urethral passages, and exhibit the same pathology and pathophysiology regardless of the type of infectious bacteria. .
  • Bacterial infections of the urethra are caused by bacteria that have retrograde to the urethra propagate acutely or chronically in the upper and lower urinary tracts of men and in the periurethral glands present throughout the female urethra.
  • urinary tract infections in sexually transmitted diseases that are transmitted through sexual negotiations are generally asymptomatic in women, but urethritis is common in men. Most obvious thing pathogenic significance as pathogenic bacteria male urethritis was gonococcal (N.
  • C. trachomatis Chlamydia trachomatis
  • Urethitis is divided into gonococcal urethritis and non-gonococcal urethritis, and non-gonococcal urethritis is further classified into chlamydial urethritis and non-chlamydial urethritis.
  • Trachomatis is detected in about 30 to 40%, and it was not clear what the symptoms originated from.
  • Mycoplasma (genus Mycoplasma ) and Ureaplasma (genus Ureaplasma ) bacteria are distinguished from other bacteria due to the lack of cell walls and the like, and are the smallest microorganisms having a self-replicating function. From humans, detection of 13 species in the genus Mycoplasma and 2 species in the genus Ureaplasma have been reported. Among them, Mycoplasma pneumoniae ( M.
  • pneumoniae which is a pneumonia- causing fungus
  • M. Detection of genitalium has been reported and is being watched as a non-gonococcal non-chlamydial causative fungus.
  • Mycoplasma hominis M. hominis
  • urea plasma ureaity cam U. urealyticum
  • urea plasma parvum U. parvum
  • M.M. genitalium is a causative bacterium of non-gonococcal urethritis due to a significantly higher detection rate than healthy individuals as a result of inoculation experiments with primates since isolation from patients with non-gonococcal urethritis was reported It is strongly suggested.
  • M.M. genitalium is C.I. trachomatis infection and N. Detected in 20-30% of male urethritis patients denied gonorrhoeae infection and in less than 10% of female cervicitis patients, which are significantly higher than the detection rate of asymptomatic controls.
  • ureallyticum is associated with important and significant human morbidity and mortality but can also be found in asymptomatic healthy individuals as well. M.M. hominis and U.I. There is also a report that ureallyticum has no significant difference in the detection rate from healthy individuals, and there are still many unclear points about its pathogenic role. U. It has been suggested that parvum may cause inflammation of the placental amniotic sac in the early stages of pregnancy leading to infertility. As described above, it is desired to clarify these roles in nongonococcal urethritis, and a reliable and highly sensitive detection method from clinical specimens is important.
  • M there is generally no difference between chlamydial non-gonococcal urethritis and non-chlamydia non-gonococcal urethritis, but M.
  • M The possibility of involvement of genitalium has been pointed out.
  • the sensitivity of genitalium to various antibacterial agents is C.I. Slightly different from trachomatis and the like, tetracycline and macrolide are more effective than new quinolone, which is the first-line drug for the treatment of urethritis, and the selection of macrolide antibacterial drugs is particularly recommended. Therefore, mycoplasma genus and ureaplasma genus, in particular, M. pneumoniae, are also used for selecting an appropriate treatment strategy and determining the effect . There is a need for a test method that allows early detection and identification of genitalium bacteria.
  • Separation culture method is a gold standard for bacterial detection and identification, but mycoplasma culture requires a culture medium with complicated nutritional conditions and addition of carbon dioxide as a culture condition for cell growth. In many laboratory facilities, it is difficult to isolate mycoplasma cultures. Therefore, it remains impossible to truly diagnose the presence of these important pathogenic bacteria. Moreover, the growth is relatively slow and reaches only a low cell density compared to the majority of bacteria, so it is not very rapid for use in clinical diagnosis. Further, in serological tests, M.M. pneumoniae and M. pneumoniae . There was a problem in terms of specificity, such as cross-reactions with genitalium .
  • the DNA fluorescent staining method takes several days to several weeks to obtain the result, and it is remarkably lacking in speed for use in clinical diagnosis.
  • Classification based on biochemical properties is unsuitable for clinical diagnosis because more than 100 bacteria can be divided into several groups because of the few constituent enzymes of Mycoplasma bacteria.
  • nucleic acid amplification tests such as PCR, which is a molecular biological technique, have been adopted by many laboratories, test companies, and reagent companies for detecting microbial infections.
  • Nucleic acid amplification test is also used for detection of Mycoplasma and Ureaplasma, and detection by PCR method using species-specific primers has been carried out. As a result, many detections from clinical materials are reported. became.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • LAMP strand displacement amplification
  • TMA transcription-mediated amplification
  • Detection and identification of Mycoplasma and Ureaplasma bacteria using the PCR method include detection and identification of PCR amplification products by plate hybridization (Patent Document 1), decoding the base sequence of PCR products, and system analysis
  • Patent Document 3 A method for detection and identification by JP
  • Patent Document 3 A method for detection, identification and quantification by Real-time PCR targeting genitalium
  • the method for detecting and identifying Mycoplasma and Ureaplasma bacteria using the method described above is a method that requires many assay steps and requires a long assay time, or is difficult to automate. However, it cannot meet the demands of laboratories, particularly laboratories, which have a low processing capacity and need to process a large amount of specimens. As a result, there has been a situation where the purpose of promptly providing a material for selecting an appropriate treatment policy and a material for determining the effect of a therapeutic agent cannot be achieved. Further, in non-gonococcal urethritis, M. genitalium , M. et al . hominis , U .; ureallyticum and U.I.
  • the present invention has been made in view of the above problems, and has an object to detect and identify Mycoplasma and Ureaplasma bacteria with rapid, simple and high accuracy.
  • a gene amplification reaction method by PCR hereinafter sometimes referred to as a PCR method
  • an invader method an invasive cleavage structure cleavage assay method
  • the present invention relates to the following [1] to [25].
  • a method for detecting and identifying Mycoplasma and Ureaplasma bacteria comprising the following steps; (A) A step of performing PCR using a primer set set for a region common to nucleic acids contained in the bacteria group in 16s rRNA of Mycoplasma and Ureaplasma bacteria (b) The Mycoplasma and Urea A step of detecting and identifying a predetermined bacterium contained in a plasma genus bacterial group using an invader reaction.
  • the predetermined bacterium is M. pneumoniae. genitalium , M. et al . hominis , U .; ureallyticum , and U.S.A.
  • the invader reaction uses an oligonucleotide probe set including a sequence specific to the nucleic acid contained in each of the selected bacteria.
  • the predetermined bacterium is M. pneumoniae. genitalium , M. et al . hominis , U .; ureallyticum , and U.S.A. at least one selected from parvum ,
  • An oligonucleotide probe having an invader reaction that is a sequence common to the nucleic acids contained in each of the selected bacteria, and a sequence specific to the nucleic acids contained in the Mycoplasma and Ureaplasma groups The method according to [1], wherein the set is used.
  • [4] The method according to any one of [1] to [3], wherein the primer set is capable of amplifying a region consisting of positions 516 to 1049 of the base sequence represented by SEQ ID NO: 1.
  • the PCR primer set is a primer set consisting of the nucleotide sequences represented by SEQ ID NO: 5 and SEQ ID NO: 6.
  • the oligonucleotide probe sets, M. [1], [2] is an oligonucleotide probe set designed for detection and identification of genitalium, using the 320th base or its complementary base in the base sequence represented by SEQ ID NO: 37 as a target nucleic acid.
  • the oligonucleotide probe sets, M. [1], [2] is an oligonucleotide probe set designed for detection and identification of hominis and designed using the 100th base or its complementary base in the base sequence represented by SEQ ID NO: 38 as a target nucleic acid. , [4], The method in any one of [5].
  • [8] The oligonucleotide probe set described in US Pat.
  • the oligonucleotide probe set is used for detection and identification of bacteria belonging to the genus Mycoplasma and Ureaplasma, and the 264th base in the base sequence represented by SEQ ID NO: 37 or its complementary base is designed as a target nucleic acid.
  • the oligonucleotide probe set containing an allele probe represented by SEQ ID NO: 7 and an invader probe represented by SEQ ID NO: 8 is an oligonucleotide probe set according to [6], wherein the oligonucleotide probe comprising a sequence specific to the nucleic acid contained in genitalium Method.
  • the oligonucleotide probe comprising an allele probe represented by SEQ ID NO: 10 and an invader probe represented by SEQ ID NO: 11 is an oligonucleotide probe set according to [7], wherein the oligonucleotide probe comprising a sequence specific to the nucleic acid contained in hominis Method.
  • U.S. The oligonucleotide probe comprising an allele probe represented by SEQ ID NO: 13 and an invader probe represented by SEQ ID NO: 14 according to [8], wherein the oligonucleotide probe comprising a sequence specific to the nucleic acid contained in urealyticum is an oligonucleotide probe set Method.
  • the oligonucleotide probe comprising a sequence specific to the nucleic acid contained in parvum is an oligonucleotide probe set comprising the allele probe represented by SEQ ID NO: 16 and the invader probe represented by SEQ ID NO: 14 according to [9].
  • Method. [15] An oligonucleotide probe comprising a sequence common to the nucleic acid contained in each of the selected bacteria, and comprising a sequence specific to the nucleic acid contained in the Mycoplasma and Ureaplasma bacteria groups The method according to [10], which is an oligonucleotide probe set comprising an allele probe represented by SEQ ID NO: 18 or 19 and an invader probe represented by SEQ ID NO: 20 or 21.
  • [16] The method according to any one of [1] to [15], wherein a plurality of oligonucleotide probe sets used for the invader reaction are used in the same reaction vessel.
  • [17] The method according to any one of [1] to [16], wherein an internal standard substance and an oligonucleotide probe set used for an invader reaction to the internal standard substance are simultaneously present in the reaction vessel.
  • [18] The oligonucleotide probe set according to [17], wherein the oligonucleotide probe set is designed using the 329th base in the base sequence represented by SEQ ID NO: 36 or a complementary base thereof as a target nucleic acid.
  • the oligonucleotide probe set including a sequence specific to the internal standard substance is an oligonucleotide probe set including an allele probe represented by SEQ ID NO: 23 and an invader probe represented by SEQ ID NO: 24.
  • Method. [20] The method according to any one of [1] to [19], wherein the step of carrying out the PCR and the step of carrying out detection and identification using the invader reaction are carried out simultaneously. [21] The method according to any one of [1] to [19], wherein a step of performing detection and identification using the invader reaction is performed subsequent to the step of performing the PCR.
  • Mycoplasma and ureaplasma bacteria comprising a primer set of SEQ ID NOS: 5 and 6 and an oligonucleotide probe set selected from at least one of the following (a) to (e): Nucleic acid probe assay reagent kit for detection and identification: (A) M.M. detection of genitalium , an oligonucleotide probe set comprising an allele probe represented by SEQ ID NO: 7 and an invader probe represented by SEQ ID NO: 8 for identification; (B) M.M.
  • an oligonucleotide probe set comprising an allele probe represented by SEQ ID NO: 10 and an invader probe represented by SEQ ID NO: 11 for identification;
  • C U.I. detection of ureallyticum , an oligonucleotide probe set comprising an allele probe represented by SEQ ID NO: 13 and an invader probe represented by SEQ ID NO: 14 for identification;
  • D U.
  • an oligonucleotide probe set comprising an allele probe represented by SEQ ID NO: 16 and an invader probe represented by SEQ ID NO: 14 for identification;
  • An oligonucleotide probe set comprising an allele probe represented by SEQ ID NO: 18 or 19 and an invader probe represented by SEQ ID NO: 20 or 21 for detection and identification of Mycoplasma and Ureaplasma bacteria groups.
  • Mycoplasma and Ureaplasma bacteria comprising a primer set of SEQ ID NOs: 5 and 6, and an oligonucleotide probe set designed with a sequence common to all Mycoplasma and Ureaplasma bacteria as a target site Nucleic acid probe assay reagent kit for detection and identification.
  • the assay can be performed in a short time with few work steps, and since it can be automated, it is possible to process a large amount of specimens with high throughput. Further, not only the operation process becomes simple, but also the container is not opened after mixing the reagents, so that contamination due to carryover or the like can be avoided.
  • all Mycoplasma and Ureaplasma bacteria that have been reported to be isolated from nongonococcal urethritis and clinical specimens can be detected with the same sensitivity, and the species of the bacteria can be identified with high specificity.
  • mycoplasma and ureaplasma bacteria that have been reported to be isolated from humans using clinical specimens are equivalent. Can be identified and identified for species that are implicated in non-gonococcal urethritis, helping to accumulate accurate information about non-gonococcal urethritis-causing fungi It is possible to quickly provide a therapeutic policy selection material and a therapeutic drug effect determination material.
  • M.M. genitalium M. et al . hominis , U .; ureallyticum , U.S.A. It is the result of aligning the sequences obtained from GenBank for four species of parvum according to the colstalW algorithm for a part of the 16srRNA region. It is the figure which described the positional information on the primer set and the various oligonucleotide probe which were designed in Example 1 based on the alignment result of FIG. It is the figure which described the positional information on the primer set and various oligonucleotide probe which were designed in the comparative example 1 based on the alignment result of FIG.
  • An embodiment of the detection and identification of Mycoplasma and Ureaplasma bacteria of the present invention is: (A) in 16s rRNA of Mycoplasma and Ureaplasma bacteria, performing PCR using a primer set set for a region common to nucleic acids contained in the bacteria group; and (b) the Mycoplasma genus and The method includes a step of detecting and identifying a predetermined bacterium contained in a group of ureaplasma bacteria using an invader reaction.
  • all Mycoplasma and Ureaplasma bacteria are to be detected and identified.
  • Mycoplasma and 2 species of Ureaplasma that have been reported to be detected from humans.
  • M. has been suggested to cause urinary tract infections . genitalium , M. et al . hominis , U .; ureallyticum , U.S.A. It is preferable to detect and identify parvum .
  • Biological samples used in embodiments of the present invention include samples that may contain one or more of the bacteria to be tested and can be obtained from humans, non-human animals, plants, or food.
  • Said biological sample can comprise a host organism, eg cells, tissues and / or DNA from a human patient, the purpose of which is to test for the presence of bacteria in that host, the host itself It is not intended to test DNA.
  • nucleic acid DNA, RNA, gene expression, code, template, promoter, primer, oligonucleotide probe, PCR, sequence, etc.
  • molecular biology genetics, genetic engineering, etc. It has the same meaning as a widely used term.
  • the nucleic acid sample is a sample containing nucleic acid and is not particularly limited as long as it is the biological sample.
  • genomic DNA or RNA obtained from all cells in the sample using blood or tissue as a sample is applicable.
  • the extraction of the nucleic acid can be performed by a known method such as a phenol / chloroform method.
  • a commercially available reagent kit for nucleic acid extraction can also be used.
  • the target site is a nucleic acid sequence in which a first oligonucleotide probe (3 ′ terminal sequence of an allele probe) and a second oligonucleotide probe (invader probe) are hybridized in a target nucleic acid sequence present in a nucleic acid sample. It is used for detection and identification of Mycoplasma and Ureaplasma bacteria.
  • the target nucleic acid refers to a base at a site where the nucleic acid sequence of the target site and the following two oligonucleotide probes among various oligonucleotide probes used in the invader method form a triple structure.
  • the base of the target nucleic acid is complementary to the base of the allele probe at the same position, and is non-complementary to the base of the invader probe at the same position.
  • Hybridization means that various oligonucleotide probes and the target nucleic acid sequences of the various oligonucleotide probes bind complementarily to all or part of the sequences.
  • Target sites may include base substitutions, insertions, deletions. Further, it may indicate a strand having a sequence encoding genetic information (hereinafter sometimes referred to as a sense strand) or a strand having a sequence complementary to the sense strand (hereinafter referred to as an antisense strand). May be shown).
  • a reaction system that simultaneously detects and identifies multiple species in the same region, many species are detected by setting target sites on the antisense strand for some species, In addition to being able to be targeted for identification, non-specific reactions can also be suppressed. At that time, the type of target site to be set on the antisense strand can be appropriately set by those skilled in the art. It also includes nucleic acid sequence changes in sequences that are not directly related to genetic information.
  • a 16s rRNA region can be used. It is preferable because detection and identification of target bacteria can be performed with high sensitivity and high accuracy.
  • primers are set for a region common to nucleic acids contained in the bacterial group in 16s rRNAs of all mycoplasma and ureaplasma bacteria.
  • Those skilled in the art can design and synthesize the primer from sequence information by a conventional method, but it is preferable to determine the primer in consideration of the detection and identification steps in the invader reaction described later.
  • a primer which can amplify a region consisting of 516th to 1049th in the base sequence represented by SEQ ID NO: 1 and preferably comprises a base sequence continuous from the 516th base to the 3 ′ end side, or A primer set comprising a primer containing a continuous base sequence capable of hybridizing to the complementary sequence from the 1049th base to the 5 ′ end side can be used.
  • the number of consecutive bases can be appropriately changed and used depending on the relationship with the oligonucleotide probe described later.
  • Particularly preferred is a combination of base sequences represented by SEQ ID NOs: 5 and 6.
  • substitution, deletion, insertion and addition of about 1 to 5 bases can be performed as long as high-sensitivity and high-precision detection of Mycoplasma and Ureaplasma bacteria is not hindered.
  • the concentration of the two kinds of primers used in PCR is not particularly limited as long as it is a concentration ratio that can obtain a double-stranded nucleic acid as a PCR product, but is preferably used at an equal concentration.
  • the invader reaction is a method for detecting and identifying a target site utilizing the property of an enzyme called flap endonuclease (chrybase). That is, when a flap endonuclease forms a triple structure (hereinafter sometimes referred to as a cleavage structure) in which three bases of a target nucleic acid, an allele probe, and an invader probe are aligned at the position of the target nucleic acid, 'Recognize the part where the sequence on the terminal side is a flap, and cut the flap part. Allele probes are designed to contain the same sequence as the target site and a sequence complementary to the FRET probe. The flap portion released from the allele probe is then hybridized with a FRET probe having a complementary sequence.
  • a flap endonuclease that is, when a flap endonuclease forms a triple structure (hereinafter sometimes referred to as a cleavage structure) in which three bases of a target nucleic acid, an allele
  • the base located at the 5 ′ end of the flap portion enters and enters the complementary binding site of the FRET probe itself to form a cleavage structure.
  • the flap endonuclease again recognizes this cleavage structure and cleaves the reporter base with the fluorescent dye.
  • the cleaved fluorescent dye is not affected by the quencher and exhibits fluorescence.
  • the allele probe does not have the same sequence as the target site, for example, when it is a sequence derived from another bacterium, a cleavage structure is not formed. Therefore, since the cleavage by the flap endonuclease and the release of the sequence of the flap portion do not occur, the fluorescence bound to the reporter base is not detected.
  • Detection is by measuring these fluorescences. For example, the fluorescence intensity detected by the fluorescence detector gradually increases from the start of the reaction and eventually reaches a plateau. For this reason, conditions can also be set from the fluorescence intensity when a specific time has elapsed from the start of the reaction, the time until the fluorescence intensity reaches a plateau, the fluorescence intensity at that time, and the like.
  • the invader reaction shows higher target specificity than the method using hybridization alone because the fluorescence intensity is amplified by repeating the reaction with the excess signal probe or FRET probe present in the reaction reagent.
  • a plurality of universal FRET probes can be used, it has an advantage that it is more cost effective than the melting curve method or TaqMan method that requires a fluorescent probe for each target site. According to such a method using an invader reaction, it is possible to prevent contamination that may occur due to opening the reaction vessel lid after PCR, and a highly sensitive analysis can be performed easily and quickly.
  • the invader reaction can be performed simultaneously with the nucleic acid amplification reaction by PCR.
  • This method is generally called an invader method.
  • the invader method is performed by dividing the PCR process. Only the nucleic acid amplification by PCR is performed in the first few cycles, and detection and identification by the nucleic acid amplification reaction and the invader reaction are simultaneously performed in the second PCR cycle. Since the invader method performs nucleic acid amplification by PCR before the invader reaction, the sensitivity is increased by performing nucleic acid amplification, and detection can be performed more quickly than when detection is performed directly from a nucleic acid sample. In addition, since background increases when many templates are brought in, it is preferable to set reaction conditions by adjusting the amount of nucleic acid brought into the reaction system.
  • the invader plus method has the advantage that amplification and subsequent detection can be performed in one tube.
  • Each method is properly used according to the usage scene.
  • the invader method can be used for SNP analysis, and the invader plus method can be used for detection of microorganisms.
  • the real-time invader method that measures the increase in fluorescence caused by the invader reaction in real time, the amount of nucleic acid in the nucleic acid sample can be accurately quantified.
  • oligonucleotide probes FRET probe and Invader probe
  • the nucleic acid amplification reaction by PCR is performed under conditions that do not serve as primers.
  • Taq polymerase is inactivated at a high temperature, and then the invader reaction is performed by maintaining the optimum temperature for the invader reaction for a certain time.
  • the oligonucleotide probe used in the invader reaction means that it has a base sequence capable of forming a stable hydrogen bond with a target site under hybridization conditions. It means a polymer having units or nucleobase subunits, and includes DNA and / or RNA or analogs thereof. A perfect match between the nucleic acids of the various oligonucleotide probes is not required.
  • various oligonucleotide probes labeled with a labeling compound can be used.
  • various oligonucleotide probes will be described by way of example in which the target site is set to the sense strand.
  • the allele probe is composed of a first oligonucleotide probe and a third oligonucleotide probe, and is a hybridization region capable of forming a complementary strand by hybridization with a region containing a base from the target nucleic acid to the 5 ′ end of the target site.
  • First oligonucleotide probe and a flap region (third oligonucleotide probe) that has a sequence unrelated to the sequence of the target site and does not hybridize with the target site. It is arranged on the 5 ′ end side of the oligonucleotide probe.
  • the base corresponding to the target nucleic acid in the first oligonucleotide probe is located on the most 5 'side of the first oligonucleotide probe and has a sequence specific to the target nucleic acid.
  • specific means that it hybridizes with the nucleic acid sequence of the target site of the target bacterium and does not substantially hybridize with the nucleic acid sequence of other bacteria.
  • an oligonucleotide probe is considered to hybridize with other bacteria, if the bacteria are rarely detected in clinical specimens to be examined, or usually If a non-specific reaction due to a cross-reaction with a rarely detected bacterium is detected, there will be no problem in the actual detection and identification of the target bacterium, so the use of such an oligonucleotide probe is allowed. .
  • the first oligonucleotide probe which is the 5 'terminal sequence of the allele probe, is labeled with a labeling compound without using the FRET probe described later, and is designed to emit fluorescence when cleaved with a flap endonuclease. Thus, it can be used for detection and identification of target bacteria.
  • An invader probe (hereinafter sometimes referred to as a second oligonucleotide probe) is an unlabeled oligo that hybridizes with a region containing a base from the target nucleic acid to the 3 ′ end of the target site to form a complementary strand.
  • the base located at the 3 ′ end of the invader probe and corresponding to the target nucleic acid may be any base, and may be designed not to hybridize. Therefore, when a nucleic acid sample containing a target nucleic acid is hybridized with an allele probe or an invader probe, one base of the invader probe is interrupted at the position of the target nucleic acid where the target site and the first oligonucleotide probe are hybridized.
  • the flap endonuclease is a flap region (third oligo) which is a sequence on the 5 ′ end side of an allele probe when three bases of a target nucleic acid, an allele probe and an invader probe are aligned to form a cleavage structure.
  • the third oligonucleotide probe is released. Since this third oligonucleotide probe has a sequence complementary to a predetermined region of the FRET probe described later, it hybridizes with the FRET probe.
  • the detection is performed using a FRET probe (hereinafter sometimes referred to as “fourth oligonucleotide probe”) (hereinafter referred to as “fourth oligonucleotide probe”).
  • the FRET probe is designed so that its 5 ′ terminal sequence self-associates to form a loop structure, and is composed of three regions. That is, the region from the 5 ′ end of the FRET probe to the loop structure (region 1), the region forming the stem loop (region 2), and the region from the stem loop to the hybridization of the third oligonucleotide probe (region 3) is there.
  • Region 1 faces the region 2 to form a loop structure and is designed to be a complementary sequence. Therefore, region 1 forms a complementary strand with region 2 itself.
  • a flap that is, a region (region 3) that has a sequence complementary to the third oligonucleotide probe and hybridizes with the third oligonucleotide probe. Since the FRET probe has a sequence completely unrelated to the target sequence, it is possible to select and set the sequence according to the purpose of detection and identification regardless of the type of the target sequence, and to set a common sequence. it can. It is also possible to appropriately set an optimal combination of the sequence of the first oligonucleotide probe and the invader probe designed specifically for the target site and the flap portion of the allele probe, that is, the sequence of the third oligonucleotide probe.
  • a reporter is labeled on the base at the 5 'end, and a quencher is bound downstream thereof. Therefore, in this state, the quencher absorbs the fluorescence, and the fluorescence detector cannot detect the fluorescence.
  • Use of dyes such as 6-FAM, TET, HEX, Cy3, VIC, TAMRA, ROX, LC Red, Cy5, BHQ-1, BHQ-2, BHQ-3, Yakima Yellow, Redmond Red, etc.
  • usable dyes are not limited to these, and those skilled in the art can appropriately select and use a dye having a wavelength that can be detected by an instrument actually used for measurement. .
  • FAM has an excitation wavelength of 494 nm and a detection wavelength of 518 nm
  • VIC has an excitation wavelength of 538 nm and a detection wavelength of 552 nm
  • ROX has an excitation wavelength of 587 nm and a detection wavelength of 607 nm.
  • Yakima Yellow has an excitation wavelength of 531 nm and a detection wavelength of 550 nm, which is almost equivalent to VIC, it can be detected and identified using a filter set for VIC.
  • Redmond Red has an excitation wavelength of 579 nm and a detection wavelength of 595 nm, which is substantially equivalent to ROX, and therefore can be detected and identified using a filter set for ROX. In this way, it is possible to construct a detection and identification reaction by combining a plurality of dyes.
  • the third oligonucleotide probe hybridizes in the region 3 of the FRET probe, it hybridizes in such a way that the base of the third oligonucleotide probe enters between the complementary strands forming a loop on the 5 ′ end side of the FRET probe.
  • the third oligonucleotide probe becomes an invader probe to form a cleavage structure (hereinafter sometimes referred to as a second cleavage structure).
  • a second cleavage structure cleavage structure
  • the FRET probe utilizes the property of forming a stem loop
  • factors affecting the properties of the FRET probe as a probe include the length of the self-complementary region, the length of the overlap region, Watson-Crick base Stability of hairpins or stem-loop structures as predicted by the presence or absence of pairs and specially stable loop sequences is envisaged, but those skilled in the art will also consider the design of the sequences that form the stem loops.
  • these fourth oligonucleotide probes can be appropriately designed.
  • the fluorescence intensity emitted from the FRET probe is measured, detected, and identified, but a determination criterion can also be set in order to quickly report the test result.
  • a determination criterion can also be set in order to quickly report the test result.
  • the amount of fluorescence when the plasmid is 0 copies / reaction is used as the background .
  • genitalium is 6,000
  • M. hominis is 6,000
  • U.I. parvum 1,700
  • ureallyticum is 800
  • Mycoplasma and Ureaplasma genus hereinafter abbreviated as ALL Myco
  • ALL Myco Myco
  • the internal standard is set to 1,700 as the cut-off value. Can do.
  • a site having a species-specific sequence in a region used for detection and identification of all Mycoplasma and Ureaplasma bacteria can be used.
  • the target site is set with respect to a continuous base sequence before and after the sequence portion specific to the bacterium to be detected and identified as a target nucleic acid .
  • 320 th base of the base sequence represented by SEQ ID NO: 37 in the case of genitalium M. 100 th base of the base sequence represented by SEQ ID NO: 38 in the case of hominis, U. 312 th base of the base sequence represented by SEQ ID NO: 39 in the case of urealyticum, U.
  • the target site can be set using the 329th base of the base sequence represented by SEQ ID NO: 36 as the target nucleic acid.
  • the number of consecutive bases in each oligonucleotide probe that hybridizes to the target site can be adjusted as appropriate, taking into account the calculation of adjacent bases and the calculation of the free energy of a given structure in addition to the Tm value. It is not limited to the number of bases.
  • the invader reaction uses a reaction in which a nucleic acid sequence to be detected and identified at a target nucleic acid position, an allele probe and an invader probe form a triple structure. Therefore, before the allele probe hybridizes to the target site, the invader probe must enter and hybridize. Therefore, the invader probe can be designed and used so as to form a triple structure by making the Tm value of the invader probe smaller than that of the allele probe.
  • oligonucleotide probes can usually be designed by selecting species-specific sequences based on the sequence information. However, those skilled in the art should appropriately design and use them according to known methods. Is possible. As described above, in the invader reaction, since the invader probe enters between the target sequence and the allele probe to form a cleavage structure, the invader probe has a smaller Tm value than the allele probe. It is possible to adjust appropriately based on the presence or absence of a higher order structure of the corresponding sequence part, the GC content, etc. For example, it is possible to design by consigning to Hologic and to select a sequence by considering an optimal combination with primers and probes. Moreover, those skilled in the art can appropriately manufacture various designed oligonucleotide probes according to known methods. For example, production may be outsourced to a company such as Hologic.
  • a plurality of oligonucleotide probes are designed based on the general index as described above, and a plurality of Mycoplasma and Ureaplasma bacteria are simultaneously detected and identified using these oligonucleotide probes.
  • the conditions of the invader reaction suitable for the reaction can be set as appropriate, but when the invader reaction conditions are to be set to specific conditions, each oligonucleotide probe exhibits the same reactivity under the conditions, It is also possible to improve the oligonucleotide probe sequence side so that each oligonucleotide probe can react with high specificity with the target site of the bacteria to be detected and identified.
  • the oligonucleotide probe sequence is partially modified, or a plurality of oligonucleotide probes having similar sequences. And can be determined as appropriate by selecting one that exhibits the most appropriate reaction under specific conditions.
  • species-specific oligonucleotide probes can be designed to identify each species for Mycoplasma or Ureaplasma bacteria. A plurality of species can be detected and identified simultaneously by labeling different dyes on the oligonucleotide probes designed for each species.
  • the newly reported bacterium is phylogenetic close to either the Mycoplasma or Ureaplasma genus, i.e. the organisms are close to each other in an evolutionary sense, and thus far away. It is only necessary that the entire nucleic acid sequence has higher homology than the organism, and it is not necessary that the bacteria are closely related to the four types of bacteria and have high homology.
  • the oligonucleotide probe set includes an allele probe and an invader probe, and can detect and identify target Mycoplasma and Ureaplasma bacteria without using a FRET probe.
  • the end of the allele probe should be labeled and designed to emit fluorescence when cleaved by a flap endonuclease.
  • a FRET probe can also be included as needed.
  • the allele probe having the base sequence in which the sequence of the first oligonucleotide probe is located after the third oligonucleotide probe sequence per target site to be detected and identified, the second oligo There is an invader probe consisting of a nucleotide probe sequence and a FRET probe consisting of a fourth oligonucleotide probe sequence.
  • Oligonucleotide probe sets can be used by appropriately selecting what oligonucleotide probe sets are combined depending on the purpose and situation of detection and identification. For example, at least one or more sets of detection and identification can be performed using only a species-specific oligonucleotide probe set to be identified. These may be used in one set of oligonucleotide probe sets or in combination of two or more sets of oligonucleotide probes.
  • M.M. Detection of genitalium in the case of identification, the oligonucleotide probe set containing invader probe represented by the allele probe and SEQ ID NO: 8 of SEQ ID NO: 7, M. Detection of hominis, in the case of identification, the oligonucleotide probe set containing invader probe represented by the allele probe and SEQ ID NO: 11 of SEQ ID NO: 10, U. Detection of urealyticum, in the case of identification, the oligonucleotide probe set containing invader probe represented by the allele probe and SEQ ID NO: 14 of SEQ ID NO: 13, U.
  • an oligonucleotide probe set comprising an allele probe represented by SEQ ID NO: 16 and an invader probe represented by SEQ ID NO: 14, the target species is detected with high sensitivity and high accuracy. It is preferable because it can be identified.
  • allele probes and invader probes including a first oligonucleotide probe specific to the target site are designed, and each species specific It can also be used simultaneously with the designed oligonucleotide probe.
  • the sequence of the third oligonucleotide probe that is cleaved from the allele probe and released, the sequence of the FRET probe to which the third oligonucleotide probe binds, and the fluorescently labeled compound are the sequence of the oligonucleotide probe and the fluorescence that are designed specifically for the species, respectively. Designed differently from the labeled compound.
  • a first oligonucleotide probe and an invader probe are designed that have a sequence specific to genitalium as a target site. Further, a third oligonucleotide probe that is cleaved from the allele probe and released, and a FRET probe that is complementary-bonded to the third oligonucleotide probe and labeled with FAM at the base on the 5 ′ end side are prepared. On the other hand, a first oligonucleotide probe and an invader probe are designed using a sequence common to all Mycoplasma and Ureaplasma bacteria as a target site.
  • a third oligonucleotide probe that is released when the first oligonucleotide probe is cleaved and a FRET probe that is complementary-bonded to the third oligonucleotide probe and labeled with a VIC on the 5 ′ terminal side are prepared. All these reagents are mixed and an invader reaction is performed. At this time, an invader method in which PCR and an invader reaction are simultaneously performed may be performed, or an invader plus method in which an invader reaction is performed after the amplification reaction by the PCR reaction may be performed. The invader plus method is preferable because highly sensitive analysis can be performed easily and rapidly.
  • Detection is performed by measuring the fluorescence intensity of FAM and VIC with a fluorescence detector. That is, as a result of detection, if VIC fluorescence can be detected in addition to strong FAM fluorescence, it means that the nucleic acid contained in the sample is a bacterium belonging to the genus Mycoplasma or Ureaplasma.
  • the sequence is M.M. It means having a genitalium specific sequences, M. can be identified as genitalium .
  • fluorescence VIC of FAM are, M. It can be presumed that they are bacteria of the genus Mycoplasma or Ureaplasma other than genitalium .
  • an oligonucleotide probe set for detecting and identifying the internal standard substance may be used in combination.
  • an internal standard substance can be added to the reaction solution.
  • the internal standard is amplified by the primer set used, but does not react with any of the species-specific oligonucleotide probes used in the invader reaction process, and the oligonucleotide probe designed for detection and identification of the internal standard That have a sequence that reacts only with
  • PCR conditions can be changed or primers can be added. However, it is preferable to add the primer so as not to greatly affect the reaction without increasing the number of primer sets.
  • M.M An artificially modified sequence of the genitalium sequence (X77334) can be used.
  • a reagent kit comprising a nucleic acid probe assay composition according to an embodiment of the present invention is configured to detect and identify Mycoplasma and Ureaplasma bacteria using PCR and an invader reaction according to an embodiment of the present invention.
  • a primer set (SEQ ID NOs: 5 and 6) designed in a 16s rRNA region common to all mycoplasma and ureaplasma bacteria of interest, and a sequence common to all mycoplasma and ureaplasma bacteria of interest
  • Oligonucleotide probe set designed as a target site and / or oligonucleotide probe set designed as a target site with at least one species-specific sequence selected from all Mycoplasma and Ureaplasma bacteria can be included.
  • Oligonucleotide probe sets can be used by appropriately selecting what kind of oligonucleotide probe sets are combined according to the purpose and situation of detection and identification. It is possible to select a combination according to the identification method.
  • the oligonucleotide probe set includes an allele probe and an invader probe, and may include a FRET probe as necessary. In the case where the FRET probe is not used, the allele probe end may be labeled and designed to emit fluorescence when cleaved by a flap endonuclease.
  • at least one or more sets of detection and identification target species-specific oligonucleotide probe sets can be included.
  • oligonucleotide probe sets may contain one set of oligonucleotide probe sets or two or more sets of oligonucleotide probe sets.
  • the oligonucleotide probe set include an oligonucleotide probe set selected from at least one of the following (a) to (e).
  • (A) M.M. detection of genitalium an oligonucleotide probe set comprising an allele probe represented by SEQ ID NO: 7 and an invader probe represented by SEQ ID NO: 8 for identification
  • B M.M. detection of hominis
  • an oligonucleotide probe set comprising an allele probe represented by SEQ ID NO: 10 for identification and an invader probe represented by SEQ ID NO: 11,
  • C U.I.
  • oligonucleotide probe set comprising an allele probe represented by SEQ ID NO: 13 and an invader probe represented by SEQ ID NO: 14 for detection and identification of urealyticum
  • D U. detection of parvum , an oligonucleotide probe set comprising an allele probe represented by SEQ ID NO: 16 and an invader probe represented by SEQ ID NO: 14 for identification
  • E An oligonucleotide probe set comprising an allele probe represented by SEQ ID NO: 18 or 19 and an invader probe represented by SEQ ID NO: 20 or 21 for detection and identification of Mycoplasma and Ureaplasma bacteria groups.
  • the oligonucleotide probe set constituting the reagent kit can be appropriately selected and used in accordance with the purpose of the test and the target bacterial species.
  • an internal standard substance and an oligonucleotide probe set for detection and identification of the internal standard substance can also be included.
  • the internal standard can be appropriately selected and used.
  • An oligonucleotide probe set including an allele probe represented by SEQ ID NO: 23 and an invader probe represented by SEQ ID NO: 24 can be used by using a modified part of the genitalium sequence.
  • oligonucleotide probe set for detection and identification of oligonucleotide probe set can also be included.
  • a set of oligonucleotide probes comprising an allele probe represented by SEQ ID NO: 13 and an invader probe represented by SEQ ID NO: 14 for detection and identification of urealyticum ;
  • D U. detection of parvum , an oligonucleotide probe set comprising an allele probe represented by SEQ ID NO: 16 and an invader probe represented by SEQ ID NO: 14 for identification;
  • E an oligonucleotide probe set comprising an allele probe represented by SEQ ID NO: 18 or 19 and an invader probe represented by SEQ ID NO: 20 or 21 for detection and identification of Mycoplasma spp.
  • Ureaplasma spp The bacterial oligonucleotide probe set of interest can be selected and included from any of the above.
  • A M.M. detection of genitalium , an oligonucleotide probe set comprising an allele probe represented by SEQ ID NO: 7 and an invader probe represented by SEQ ID NO: 8 for identification;
  • B M.M. detection of hominis , an oligonucleotide probe set comprising an allele probe represented by SEQ ID NO: 10 for identification and an invader probe represented by SEQ ID NO: 11,
  • C U.I. a set of oligonucleotide probes comprising an allele probe represented by SEQ ID NO: 13 and an invader probe represented by SEQ ID NO: 14 for detection and identification of urealyticum ;
  • D U.
  • an oligonucleotide probe set comprising an allele probe represented by SEQ ID NO: 16 and an invader probe represented by SEQ ID NO: 14 for identification;
  • an oligonucleotide probe set comprising an allele probe represented by SEQ ID NO: 18 or 19 and an invader probe represented by SEQ ID NO: 20 or 21 for detection and identification of Mycoplasma spp.
  • Ureaplasma spp Ureaplasma spp.
  • an internal standard substance and an oligonucleotide probe set for detection and identification of the internal standard substance can also be included.
  • Example 1 Design of primers and oligonucleotide probes
  • M.M. genitalium X77334; SEQ ID NO: 1
  • M. et al . hominis M96660; SEQ ID NO: 2
  • U. urealyticum AF073450; SEQ ID NO: 3
  • GenBank National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov/genbank/
  • Each obtained sequence was aligned using the software GENEX (manufactured by Genetics) according to the algorithm of ClustalW, which is a multiple alignment program. The alignment result is shown in FIG.
  • Primers and oligonucleotide probes were designed with reference to the alignment data. Primers were designed by selecting sequence sites common to these species. Oligonucleotide probes were designed by selecting species-specific sequences. M.M. Genitalium designed the oligonucleotide probe to hybridize to the antisense strand, all others designed the oligonucleotide probe to hybridize to the sense strand.
  • the primer sequences used for the PCR reaction are as follows.
  • Myco_Urea_F 5′-CGCGGGTAATACATGGTTGCAACGTTTATC-3 ′ (SEQ ID NO: 5)
  • Rv1 5′-GCACCACCTGTCACTCTGTTAACCTC-3 ′ (SEQ ID NO: 6)
  • the oligonucleotide probe sequences used for each type are as follows. M.M.
  • the genitalium- specific oligonucleotide probe is as follows. In the allele probe, the flap sequence, that is, the sequence that is cleaved by the flap endonuclease to become the third oligonucleotide probe is shown in lower case.
  • the fourth oligonucleotide probe designed for detection and identification of genitalium was purchased from Hologic Co., which had its 5 ′ end labeled with FAM.
  • the hominis specific probe is as follows. In the allele probe, the flap sequence, that is, the sequence that is cleaved by the flap endonuclease to become the third oligonucleotide probe is shown in lower case.
  • Allele probe Mh2_63_P1_arm7 5′-tccgcgcgtccCGCCTCGCTTTGG-3 ′ (SEQ ID NO: 10)
  • Second oligonucleotide probe (invader probe) Mh2_inv2: 5′-GAGTTAAATCCCGGGGCTCAACCCA-3 ′ (SEQ ID NO: 11)
  • Third oligonucleotide probe 5′-tccgcgcgtcc-3 ′ (SEQ ID NO: 12) M.M.
  • a fourth oligonucleotide probe designed for detection and identification of hominis was purchased from Hologic Co., Ltd. with its 5 ′ end labeled with FAM.
  • the urealyticum- specific oligonucleotide probe is as follows. In the allele probe, the flap sequence, that is, the sequence that is cleaved by the flap endonuclease to become the third oligonucleotide probe is shown in lower case.
  • the fourth oligonucleotide probe designed for detection and identification of urealyticum was purchased from Hologic, with its 5 'end labeled with Redmond Red.
  • the parvum specific oligonucleotide probe is as follows. In the allele probe, the flap sequence, that is, the sequence that is cleaved by the flap endonuclease to become the third oligonucleotide probe is shown in lower case. Allele probe Up_63_P1_arm1: 5′-cgcgccgaggCCGAATGGGTCGGT-3 ′ (SEQ ID NO: 16) Second oligonucleotide probe (invader probe) is, U. A common sequence (SEQ ID NO: 14) with urealyticum was designed and used. Third oligonucleotide probe: 5′-cgcgccgagg-3 ′ (SEQ ID NO: 17) U. The fourth oligonucleotide probe designed for parvum detection and identification was purchased from Hologic, whose 5 ′ end was labeled with Yakima Yellow.
  • oligonucleotide probes common to all Mycoplasma and Ureaplasma bacteria were designed as follows.
  • the flap sequence that is, the sequence that is cleaved by the flap endonuclease to become the third oligonucleotide probe is shown in lower case.
  • Ureaplasma spp U.urealyticum, U.parvum detection of the fourth oligonucleotide probes for the identification is designed to be common sequences, the 5 'terminus U.
  • the oligonucleotide probe for detection of urealyticum is Yakima, U.
  • the oligonucleotide probe for detection of parvum was labeled with Redmond.
  • Yakima has an excitation wavelength of 531 nm and a detection wavelength of 550 nm, which is almost equivalent to VIC, and therefore can be detected and identified using a filter set for VIC.
  • Redmond since Redmond has an excitation wavelength of 579 nm and a detection wavelength of 595 nm, which is substantially equivalent to ROX, it can be detected and identified using a filter set for ROX.
  • oligonucleotide probes specific to the internal standard were designed.
  • the flap sequence that is, the sequence that is cleaved by the flap endonuclease to become the third oligonucleotide probe is shown in lower case.
  • Allele probe Myco_IC_P_arm1 5′-cgcgccgaggCGACAGCTAGTATCTACG-3 ′ (SEQ ID NO: 23)
  • the fourth oligonucleotide probe designed for internal standard substance detection and identification was purchased from Hologic Co., Ltd. with its 5 ′ end labeled with Yakima Yellow.
  • the base located at the 3 ′ end of all the third oligonucleotide probes is synthesized so as to bind to the base located at the 5 ′ end of the first oligonucleotide probe by a phosphodiester bond, and one is not mixed when mixing the reagents.
  • One oligonucleotide probe is present in the reaction solution.
  • Table 1 shows a list of primers and each oligonucleotide probe.
  • the third oligonucleotide probe is shown in lower case in the sequence described as an invader probe.
  • IC indicates an internal standard substance.
  • strain Strains of Mycoplasma and Ureaplasma spp. Bacteria were purchased from ATCC (American Type Culture Collection) . genitalium , M. et al . hominis , U .; ureallyticum , U.S.A. Parvum mycoplasma and ureaplasma strains were used. In addition, 41 types of bacteria and yeasts other than Mycoplasma and Ureaplasma bacteria used in the cross test were all purchased from ATCC and used. Table 2 shows a list of strains used.
  • DNA used as a positive control for detection and as a template for PCR reaction was prepared. M. to be detected and identified . genitalium , M. et al . hominis , U .; ureallyticum , U.S.A. DNA extraction was performed using an automated purification reagent (QIAsymphony SP, manufactured by QIAGEN) as 4 strains of parvum . Using the extracted DNA as a template, the primer set consisting of SEQ ID NO: 5 and SEQ ID NO: 6 designed in Example 1 was used for amplification under normal PCR reaction conditions. M.M.
  • genitalium contains bases 516 to 1037
  • hominis contains the 512th to 1019th bases
  • ureallyticum contains bases 492-1013
  • U. parvum amplified the 496th to 1017th bases.
  • the obtained PCR amplification product was inserted into a plasmid for gene recombination (pT7 Blue T-vector, manufactured by Novagen) and cloned according to the attached protocol. This was used as a positive target for detection and a template for PCR reaction.
  • An internal standard substance used for confirming the presence or absence of inhibition during the PCR reaction was prepared.
  • the M.M. Using a plasmid inserted with DNA having the genitalium sequence as a template, The genitalium sequence portion was amplified by a PCR reaction according to a conventional method. After purifying the obtained PCR product, a plasmid for gene recombination (pT7 Blue T-vector, manufactured by Novagen) obtained by artificially modifying a part of the base sequence (corresponding to positions 769 to 930 of X77334) The product was inserted and cloned in accordance with the attached protocol, and used as an internal standard (SEQ ID NO: 36).
  • reagent kit 1 M.
  • a reagent set comprising an oligonucleotide probe set specific to genitalium , an oligonucleotide probe set common to all Mycoplasma and Ureaplasma bacteria (hereinafter referred to as ALL Myco), and an oligonucleotide probe set designed for an internal standard As kit 2, M.M. hominis , U .; ureallyticum , U.S.A. Prepared as a reaction solution for detecting a specific oligonucleotide probe set for each of parvum .
  • Example 3 Examination of PCR condition cycle number
  • the conditions under which each bacterium of parvum can be detected and identified were examined.
  • the plasmid prepared in Example 2 was used so as to be 10 6 to 10 1 copies per reaction.
  • the method of detecting and identifying by the invader reaction simultaneously with the amplification by the PCR reaction is the PCR amplification using a thermal cycler (PRISM 7900HT, manufactured by Applied Biosystems) in which the reagent mixed as described above is heated to 95 ° C. Reaction conditions are (95 ° C., 10 seconds) + (95 ° C., 30 seconds + 70 ° C., 1 minute) ⁇ 2 + (95 ° C., 15 seconds + 70 ° C., 1 minute) ⁇ 10 + (95 ° C., 15 seconds + 63 ° C., 1 minute) ) X40.
  • PRISM 7900HT manufactured by Applied Biosystems
  • the Invader Plus method which detects and identifies by the Invader method after the PCR reaction, uses a thermal cycler (PRISM7900HT, manufactured by Applied Biosystems) in which the reagent mixed as described above is heated to 95 ° C.
  • the reaction conditions were (95 ° C., 10 seconds) + (95 ° C., 15 seconds + 70 ° C., 1 minute) ⁇ 30, followed by heating at 99 ° C. for 10 minutes to inactivate Taq polymerase, (63 ° C., 30 Second) x 60, fluorescence data was acquired by the invader method. Comparison results between the invader method and the invader plus method are shown in FIG. 4 (invader method) and FIG. 5 (invader plus method).
  • the PCR reaction cycle number was amplified as 20, 27, 30, and 40 cycles, followed by heating at 99 ° C. for 10 minutes to inactivate Taq polymerase, and then the invader reaction was performed at 63 ° C. And 60 cycles of fluorescence data were acquired (fluorescence data was acquired every 30 seconds at 63 ° C. for 30 minutes), and the determination results are shown in FIG. 5 (PCR: 30 cycles), FIG. 6 (PCR: 40 cycles), and FIG. (PCR: 20 cycles), shown in Table 3.
  • detection by PCR can be detected from a nucleic acid sample having a low copy number by increasing the number of PCR cycles.
  • the amount of PCR amplification product and the intensity of fluorescence over time regardless of whether the copy number contained in the nucleic acid sample is high or low, It is possible to detect an increasing situation.
  • the amount of the PCR amplification product becomes excessive as the number of PCR reaction cycles increases, so that the background fluorescence intensity increases and it becomes difficult to detect in a high copy number nucleic acid sample. It was found that some species could be excluded from detection despite having a sufficient amount of nucleic acid, which could cause misjudgment (FIG. 6).
  • Example 4 Detection and identification of nucleic acid derived from Mycoplasma and Ureaplasma bacteria
  • the plasmid prepared in Example 2 was used so as to be 10 6 to 10 1 copies per reaction.
  • PCR amplification reaction conditions were 95 ° C., 10 seconds + (95 ° C., 15 seconds + 70 ° C., 1 minute) ) ⁇ 34 cycles + 99 ° C., 10 minutes + (63 ° C., 30 seconds) ⁇ 60 cycles
  • the invader reaction was determined by obtaining fluorescence data of 60 cycles at 63 ° C. (acquiring fluorescence data every 30 seconds at 63 ° C. for 30 minutes).
  • the fluorescence detection amount of the specific oligonucleotide probe was measured to determine the detection criterion.
  • the amount of fluorescence when the plasmid was 0 copies / reaction was used as the background .
  • genitalium is 6,000
  • M. hominis is 6,000
  • U.I. parvum is 1,700 .
  • ureallyticum was set to 800, 800 for Mycoplasma and Ureaplasma bacteria (hereinafter abbreviated as ALL Myco), and 1,700 for the internal standard substance.
  • Table 4 shows the amount of fluorescence of each detected oligonucleotide probe. It was confirmed that the amount of fluorescence detected for any of the various oligonucleotide probes exceeded the judgment value up to 10 copies per reaction.
  • Example 5 Confirmation of reproducibility
  • Detection sensitivity and reproducibility were confirmed using a reagent kit containing primers and oligonucleotide probes designed in the present invention.
  • M.M. genitalium M. et al . hominis , U .; ureallyticum , U.S.A.
  • Three lots were prepared so that the concentration of the plasmid DNA incorporating the PCR amplification product of parvum was 10 2 , 10 1 , and 0 copies per reaction. These three lots were measured 6 times as confirmation of simultaneous reproducibility, and further, confirmation measurement of day difference reproducibility by changing the day was performed 3 times.
  • the concentration at which the detection rate was 100% was defined as the minimum detection limit.
  • the detection and identification methods were the same as in Example 4. As a result, in the 18 measurements carried out, the number of copies with a detection rate of 100% was M.P. genitalium , M. et al . hominis , U .; urealyticum, ALL Myco is 50 copies / test, U. parvum , the internal standard was 100 copies / test and sufficiently satisfied the detection sensitivity required in the laboratory.
  • Example 6 Cross-reactivity test
  • Table 6 shows the results of cross-reactivity studies with 41 bacteria other than Mycoplasma and Ureaplasma that have been reported to be infected with the urinary tract. Shown in Each oligonucleotide probe designed specifically for the species was confirmed to react only with the target bacterium, and a non-specific reaction that reacted with the non-target bacterium was not detected. In addition, when the ALLMyco probe designed with a sequence common to all Mycoplasma and Ureaplasma bacteria as a target site was used, detection of Proteus mirabilis and Vibrio parahaemolyticus was confirmed. It was not detected from yeast. Even when a species other than the target species has a highly homologous sequence, it seems that there is substantially no problem for a species that has almost no separation from clinical specimens.
  • Example 7 Detection from clinical specimen
  • the control method a comparison was made using a method disclosed in JP-A-2004-24206 (hereinafter referred to as the control method). Nucleic acid extraction from the sample was carried out by the same detection and identification method as in Example 3, except that a nucleic acid sample was prepared using QIAsymphony Virum / Bacteria Midi Kit (manufactured by QIAGEN).
  • M.M. In the case of genitalium , M. cerevisiae was also detected from samples not detected by the control method . It was confirmed that genitalium can be detected and identified. Similarly it was compared for the other species results, M. hominis and U.I. detected in not detected analyte by also control method for urealyticum, identification was confirmed to be possible. U. As for parvum , specimens in which the results were inconsistent between the control method and the method of the present invention were observed.
  • the nucleic acid sample contained in the sample has a low copy number close to the minimum detection limit, which is the detection lower limit, and infection of multiple bacteria is suspected It turned out to be a complex infection.
  • the forward primer has the same sequence as that designed in Example 1 (SEQ ID NO: 5), and the reverse primer is designed so that the primer used in the example is shifted 12 bases downstream.
  • the sequence of the reverse primer used is shown below.
  • the oligonucleotide probe sequences used for each type are as follows.
  • the flap sequence that is, the sequence that is cleaved by the flap endonuclease to become the third oligonucleotide probe is shown in lower case.
  • M.M. A genitalium- specific oligonucleotide probe was designed for the sense strand.
  • the part used as the 3rd oligonucleotide probe of a flap part is not changed.
  • Third oligonucleotide probe 5′-tccgcgcgtcc-3 ′ SEQ ID NO: 29
  • the fourth oligonucleotide probe was prepared by the M.I. The same fourth oligonucleotide probe for detection and identification of genitalium was used.
  • the urealyticum- specific oligonucleotide probe two types of allele probes were used.
  • the third oligonucleotide probe to be the sequence of the flap portion was changed, and the sequence of the fourth oligonucleotide probe was changed accordingly.
  • the flap sequence that is, the sequence that is cleaved by the flap endonuclease to become the third oligonucleotide probe is shown in lower case.
  • Uu — 63_P1 — 2 arm3: 5′-acggacgcgggagTCGAACGAGGTCTGTT-3 ′ (SEQ ID NO: 31)
  • the second oligonucleotide probe (invader probe) was the same as that designed in Example 1 (SEQ ID NO: 14).
  • the fourth oligonucleotide probe designed for detection and identification of urealyticum was purchased from Hologic, with its 5 ′ end labeled with Yakima Yellow.
  • the fourth oligonucleotide probe was common to Ureaplasma bacteria ( U. urealyticum , U. parvum ), and its 5 ′ end was labeled with Yakima for detection and identification of Ureaplasma bacteria.
  • the oligonucleotide probes common to all Mycoplasma and Ureaplasma bacteria are as follows.
  • the flap sequence that is, the sequence that is cleaved by the flap endonuclease to become the third oligonucleotide probe is shown in lower case.
  • Allele probe ALL2_63_P2-2_arm6 5′-cgcgaggccgAGATACCCTAGTAGTCCCAC-3 ′ (SEQ ID NO: 33)
  • the 5 ′ end of the second oligonucleotide probe designed for detection and identification was labeled with “(labeled compound)”.
  • Third oligonucleotide probe 5′-cgcgaggccg-3 ′ (SEQ ID NO: 35)
  • the fourth oligonucleotide probe designed for detection and identification of all Mycoplasma and Ureaplasma bacteria was purchased from Hologic Co., Ltd. with its 5 ′ end labeled with Yakima Yellow.
  • Table 7 shows a list of primers and each oligonucleotide probe.
  • IC indicates an internal standard substance.
  • a reagent kit containing the primer and oligonucleotide probe designed in Comparative Example 1 was prepared.
  • the reagent kit 1 includes M.M. Includes detection of genitalium , oligonucleotide probe for identification, internal standard detection, oligonucleotide probe for identification, detection of all Mycoplasma and Ureaplasma bacteria, and oligonucleotide probe for identification.
  • Reagent Kit 2 contains M.M.
  • hominis U .; ureallyticum , U.S.A. It contains oligonucleotide probes for detecting and identifying each of the parvum .
  • primer sets that have sequences designed according to conventional methods from sequence information may not be sufficient.
  • highly specific reactions that take into account the specificity in the detection and identification process in the invader reaction. It turns out that it is necessary to build a system. Therefore, primer sets and oligonucleotide probe sets designed by known methods may not be sufficient for use in the detection and identification of Mycoplasma and Ureaplasma bacteria and may not be used.
  • Test systems that use primers and various oligonucleotide probes are particularly described in M.M. genitalium , M. et al . hominis , U .; ureallyticum , and U.S.A.
  • reagents required for PCR amplification reaction, detection by invader method, and identification reaction are mixed at once in one container, and separation from humans has been reported using clinical samples as materials.
  • All Mycoplasma and Ureaplasma bacteria can be detected with comparable sensitivity and identified for species that are suggested to be associated with nongonococcal urethritis.
  • it helps to accumulate accurate information about non-gonococcal urethritis-causing bacteria, and can promptly provide a material for selecting an appropriate treatment policy and a material for determining the effect of a therapeutic agent.
  • this invention was demonstrated along the specific aspect, the deformation
  • SEQ ID Nos: 7, 10, 13, 16, 18, 19, 23, 24, 27, 30, 31, 33 in the sequence listing are Mg_SE_P1_arm7, Mh2_63_P1_arm7, Uu_63_P1_arm6, Up_63_P1_arm1, ALL2_SE_P1_arm4, ALL2_SE_P1_G_, Uu_63_P1_arm3, Uu_63_P1_2_arm3, ALL2_63_P2-2_arm6.
  • Each base sequence of SEQ ID Nos: 9, 12, 15, 17, 22, 25, 29, 32, and 35 is a third oligonucleotide probe sequence.
  • the base sequence of SEQ ID NO: 36 is an internal standard substance.

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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé pour la détection et l'identification rapides, faciles et précises d'une bactérie appartenant au genre Mycoplasma ou Ureaplasma. Le procédé comprend : une étape (a) pour réaliser une PCR à l'aide d'un ensemble d'amorces, ledit ensemble d'amorces étant conçu pour une région commune aux acides nucléiques contenus dans les bactéries appartenant aux genres Mycoplasma et Ureaplasma dans les ARNr 16s des bactéries susmentionnées ; et une étape (b) pour détecter et identifier une bactérie définie appartenant au genre Mycoplasma ou Ureaplasma à l'aide d'une réaction d'envahissement.
PCT/JP2013/082779 2012-12-07 2013-12-06 Procédé pour détecter et identifier des bactéries appartenant au genre mycoplasma et ureaplasma WO2014088088A1 (fr)

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JP2017209063A (ja) * 2016-05-26 2017-11-30 学校法人福岡大学 絨毛膜羊膜炎関連微生物同定ならびに検出方法、絨毛膜羊膜炎関連微生物検出用プライマーセットならびにアッセイキット、および絨毛膜羊膜炎検出方法
JP2018126089A (ja) * 2017-02-08 2018-08-16 学校法人日本大学 ウレアプラズマ属細菌検出用プライマーセット及びその使用
CN110643723A (zh) * 2019-09-26 2020-01-03 刘君 用于检测微小脲原体的引物组
JP2020089339A (ja) * 2018-12-07 2020-06-11 一般財団法人日本生物科学研究所 プライマーセット、並びにプライマーセットを用いた核酸増幅方法、細菌の同定方法、及び罹患ブタの細菌感染診断方法
CN114480663A (zh) * 2021-11-15 2022-05-13 贵州省畜牧兽医研究所 用于检测猪源差异脲原体的引物对、试剂盒及检测方法

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JP2004024206A (ja) * 2002-06-28 2004-01-29 Mitsubishi Kagaku Bio-Clinical Laboratories Inc マイコプラズマおよびウレアプラズマの検出方法
JP2007244349A (ja) * 2006-03-20 2007-09-27 Bml Inc 歯周病菌の検出方法
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JP2001299352A (ja) * 2000-04-20 2001-10-30 Mitsubishi Kagaku Bio-Clinical Laboratories Inc マイコプラズマ属およびウレアプラズマ属の細菌の検出方法並びにマイコプラズマ属およびウレアプラズマ属の菌種の同定方法
JP2004024206A (ja) * 2002-06-28 2004-01-29 Mitsubishi Kagaku Bio-Clinical Laboratories Inc マイコプラズマおよびウレアプラズマの検出方法
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2017209063A (ja) * 2016-05-26 2017-11-30 学校法人福岡大学 絨毛膜羊膜炎関連微生物同定ならびに検出方法、絨毛膜羊膜炎関連微生物検出用プライマーセットならびにアッセイキット、および絨毛膜羊膜炎検出方法
JP2018126089A (ja) * 2017-02-08 2018-08-16 学校法人日本大学 ウレアプラズマ属細菌検出用プライマーセット及びその使用
JP2020089339A (ja) * 2018-12-07 2020-06-11 一般財団法人日本生物科学研究所 プライマーセット、並びにプライマーセットを用いた核酸増幅方法、細菌の同定方法、及び罹患ブタの細菌感染診断方法
JP7267001B2 (ja) 2018-12-07 2023-05-01 一般財団法人日本生物科学研究所 プライマーセット、並びにプライマーセットを用いた核酸増幅方法、細菌の同定方法、及び罹患ブタの細菌感染診断方法
CN110643723A (zh) * 2019-09-26 2020-01-03 刘君 用于检测微小脲原体的引物组
CN114480663A (zh) * 2021-11-15 2022-05-13 贵州省畜牧兽医研究所 用于检测猪源差异脲原体的引物对、试剂盒及检测方法

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