KR102051678B1 - Method for Detecting Mycoplasma Contaminated in Therapeutic Cells or Biological Medicine By Using Real-time Polymerase Chain Reaction and Kit for the Same Method - Google Patents
Method for Detecting Mycoplasma Contaminated in Therapeutic Cells or Biological Medicine By Using Real-time Polymerase Chain Reaction and Kit for the Same Method Download PDFInfo
- Publication number
- KR102051678B1 KR102051678B1 KR1020130087578A KR20130087578A KR102051678B1 KR 102051678 B1 KR102051678 B1 KR 102051678B1 KR 1020130087578 A KR1020130087578 A KR 1020130087578A KR 20130087578 A KR20130087578 A KR 20130087578A KR 102051678 B1 KR102051678 B1 KR 102051678B1
- Authority
- KR
- South Korea
- Prior art keywords
- mycoplasma
- spiroplasma
- real
- plasma
- chain reaction
- Prior art date
Links
Images
Classifications
-
- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
-
- 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
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
-
- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
-
- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Toxicology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention provides a method for detecting mycoplasma that can be easily contaminated in the manufacture of cell therapeutics and biological drugs, primers and probe sets used in the method, and real-time polymerase chain reaction for detecting mycoplasma including the same. It is related to a kit. Using the newly prepared primer and probe set for real-time polymerase chain reaction of the present invention can quickly, accurately and reproducibly detect mycoplasma that can be easily contaminated during the preparation, storage and administration of cellular and biological drugs. The real-time polymerase chain reaction method provided by the present invention has the effect of providing an economical, rapid and accurate detection method of mycoplasma by replacing the sterility test method of the existing cell therapy.
Description
The present invention relates to a method for detecting mycoplasma contaminated with a cell therapy or biological drug using a real-time polymerase chain reaction, and a kit for use in the method.
Recently, with the development of cell therapies using cultured cells (immune cells and stem cells) and therapies using biopharmaceuticals (insulin, growth hormone, interferon and antibody drugs), immune cells, stem cells cultured as biopharmaceuticals and cell therapy products have been developed. Rapid fungal detection for contamination of viruses, bacteria, mycoplasma, and fungi, which are pathogenic microorganisms of cells or animal cells, is an important process that must be preceded to prevent secondary disease infection in patients prior to clinical application.
Direct cultivation, a standard sterility test method well-known for existing microorganisms (including bacteria, fungi, and mycoplasma), takes a long time to determine microbial contamination and is not suitable for difficult microorganisms. In particular, it is difficult to apply practically to cell therapies which are usually used within 48 hours after preparation because a long test period is required.
Therefore, it is necessary to develop an alternative method that can efficiently and rapidly detect high frequency contaminating microbial groups and pathogenic microbial groups by improving the disadvantages of the existing direct culture method. Molecular diagnostics using Real-time Polymerase Chain Reaction (PCR), one of the technologies for amplifying nucleic acids, is used for the development of various molecular diagnostic products due to its rapid and sensitive sensitivity and contaminated with cell or biological drugs. It is also applied to the microorganism detection method.
Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.
The inventors of the present invention have a technique for quickly and accurately detecting mycoplasma, a pathogenic microorganism that may be contaminated with cultured cells in the culture of immune cells or stem cells and animal cells producing biopharmaceuticals. Research efforts have been made to develop.
As a result, we developed a set of primers and probes for real-time polymerase chain reaction (RT-PCR) that can detect various species of mycoplasma through a single nucleic acid amplification process. The present invention was completed by confirming that a real-time nucleic acid amplification method using a set can detect various species of mycoplasma.
Accordingly, it is an object of the present invention to provide a method for detecting contaminable mycoplasma in cell culture using real-time polymerase chain reaction.
Another object of the present invention is to provide a primer and probe set for real-time polymerase chain reaction for detecting contaminant mycoplasma in cell culture.
Still another object of the present invention is to provide a real-time polymerase chain reaction kit for detecting contaminant mycoplasma during cell culture including the primer and probe set.
The objects and advantages of the invention will become apparent from the following detailed description, claims and drawings.
delete
According to one aspect of the present invention, the present invention provides a method for detecting contaminable mycoplasma in cell culture using a real time polymerase chain reaction comprising the following steps: (a) Extracting DNA from a sample to be detected; And (b) performing a real-time polymerase chain reaction using the extracted DNA, primers and probe sets.
Hereinafter, the method of the present invention will be described in detail by dividing step by step.
Step (a): extracting DNA from a sample to be detected
delete
delete
The detection method of the present invention can be applied to a sample that is expected to be contaminated with Mycoplasma bacteria. In the present invention, the sample to be detected is preferably a biological sample, more preferably a biopharmaceutical or a cultured cell. For example, a biopharmaceutical produced by a biological method such as a cell culture method, an immune cell used as a cell therapy, or a stem cell production process or a final product thereof may be used as a sample. More specifically, the sample may be a culture solution before and after cell culture, sap for patient administration, raw materials and final products used in the production of biopharmaceuticals.
As a method for extracting DNA from the mycoplasma bacteria to be detected, various methods known in the art may be used, and specific methods thereof may be used in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001). Which is incorporated herein by reference. For example, in the present invention, genome DNA (genomic DNA) in mycoplasma cells can be extracted by applying a phenol-chloroform extraction method.
According to a preferred embodiment of the present invention, the mycoplasma detectable in the present invention is at least one mycoplasma selected from the group consisting of the following mycoplasma: Mycoplasma hyorhinis, Mycoplasma isti ( Mycoplasma yeastii, Mycoplasma equirhinis, Mycoplasma orale , Mycoplasma mycoides , Mycoplasma falconis , Mycoplasma falconis , Mycoplasma arginini arginini Mycoplasma agalactiae , Mycoplasma felifaucium , Mycoplasma salivarium , Mycoplasma synoviae , Mycoplasma feliplasma felis mycoplasma Mycoplas ma fermentans ), Mycoplasma alkalescens , Mycoplasma gallinaceum , Mycoplasma hominis , Mycoplasma adleri , Mycoplasma adleri , Mycoplasma gatieae Mycoplasma art utility disk (Mycoplasma arthritidis), Mycoplasma Albi (Mycoplasma alvi), mycoplasma-based piece (Mycoplasma gypis), Maiko plasma pneumoniae (Mycoplasma pneumoniae), Mycoplasma not seriseu (Mycoplasma anseris), Mycoplasma indie Enschede (mycoplasma indiense), mycoplasma pirum (mycoplasma pirum), mycoplasma brother-less (mycoplasma auris), as mycoplasma Jenny de Solarium (mycoplasma lagogenitalium), mycoplasma's peoma topil room (mycoplasma spermatophilum), mycoplasma Bobby Jenny de Solarium (Mycoplasma bovigenitalium), M. Raj town Leoni cap TV (Mycoplasma leonicaptivi), Mycoplasma portion Calle (Mycoplasma buccale), Mycoplasma Leo parin Kish (Mycoplasma leopharyngis), Mycoplasma Jenny de Solarium (Mycoplasma genitalium), Mycoplasma potassium pony glutamicum (Mycoplasma californicum), M. Plasma lipofaciens , Mycoplasma hyosynoviae , Mycoplasma canadense , Mycoplasma molare , Mycoplasma pulmonis , Mycoplasma pulmonis Mycoplasma canis , Mycoplasma neurolyticum , Mycoplasma hyopneumoniae , Mycoplasma bovirhinis , Mycoplasma putrefaciens coat, Mycoplasma putrefaciens coat Stomach ( Mycoplasma cottewi i), Mycoplasma portion Theo varnish (Mycoplasma buteonis), Mycoplasma simbe (Mycoplasma simbae), O Collet plasma Lai Raise weaving (Acholeplasma laidlawii), Mycoplasma cabinets (Mycoplasma caviae), mycoplasma test tudi varnish (Mycoplasma testudinis a) , Acholeplasma oculi , Mycoplasma collis , Mycoplasma timone , Acholeplasma granularum , Spiroplasma citri , Spiroplasma insole ritum (Spiroplasma insolitum), spiro plasma Kuhn kelriyi (Spiroplasma kunkelii), Ureaplasma urealyticum Parr boom (Ureaplasma parvum), spiro plasma mellitic peorum (Spiroplasma melliferum), spiro plasma Finney erecting (Spiroplasma phoeniceum), spiro plasma deferring (Spiroplasma mirum), Mycoplasma penetrans ) And Mycoplasma cricetuli.
Step (b): performing real time polymerase chain reaction using the extracted DNA, primer and probe set
As used herein, the term “amplification reaction” means a reaction that amplifies a nucleic acid molecule. Various amplification reactions have been reported in the art, which include polymerase chain reaction (PCR) (US Pat. Nos. 4,683,195, 4,683,202, and 4,800,159), reverse transcription-Real-time Polymerase Chain Reaction; RT-PCR) (Sambrook et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001)), Miller, HI (WO 89/06700) and Davey, C. et al. (EP 329,822) Methods, ligase chain reaction (LCR) (17, 18), Gap-LCR (WO 90/01069), repair chain reaction (EP 439,182), transcription-mediated amplification TMA) (19) (WO 88/10315), self sustained sequence replication (20) (WO 90/06995), selective amplification of target polynucleotide sequences (US Pat. No. 6,410,276), consensus sequence priming polymerase chain reaction (consensus se) quence primed Polymerase Chain Reaction (CP-PCR) (US Pat. No. 4,437,975), arbitrarily primed Polymerase Chain Reaction (AP-PCR) (US Pat. Nos. 5,413,909 and 5,861,245), nucleic acid base sequences Nucleic acid sequence based amplification (NASBA) (US Pat. Nos. 5,130,238, 5,409,818, 5,554,517, and 6,063,603), strand displacement amplification (21, 22) and ring-mediated constant temperature Loop-mediated isothermal amplification; LAMP) 23, but is not limited thereto. Other amplification methods that can be used are described in US Pat. Nos. 5,242,794, 5,494,810, 4,988,617 and US Pat. No. 09 / 854,317.
Nucleic acid amplification reaction in the present invention is carried out according to the real-time polymerase chain reaction (RT-PCR).
As used herein, the term "primer" is complementary to the 5 'and 3' terminal sequences of the target nucleic acid site to be amplified during the amplification reaction of the nucleic acid, respectively, and is suitable conditions in a suitable buffer at a suitable temperature (i.e. , Single-stranded oligonucleotides that can act as the starting point for the polymerase reaction of the template-directed nucleic acid under four different nucleoside triphosphates and polymerases. Suitable lengths of primers are typically 15-30 nucleotides, although varying depending on various factors, such as temperature and the use of the primer. Short primer molecules generally require lower temperatures to form hybridization complexes that are sufficiently stable with the template.
Primers used in the present invention are hybridized or annealed to one site of the template to form a double chain structure. Suitable nucleic acid hybridization conditions for forming such a double-chain structure include Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) and Haymes, BD, et al., Nucleic Acid Hybridization , A Practical Approach, IRL Press, Washington, DC (1985).
The primer set of the present invention was designed to target gene regions encoding species conserved 16S ribosomal RNA (rRNA) in genome DNA sequences in mycoplasma bacteria that could be contaminated with high frequency during cell culture. Kinds of mycoplasma bacteria can be detected specifically.
The term "probe" in the context of the present invention is a single-chain nucleic acid molecule, and includes a sequence complementary to the target nucleotide sequence. Probes of the invention can be modified within a range in which hybridization specificity is not impaired. For example, a reporter phosphor or a quencher may be tagged at the end of the oligonucleotide as a probe.
In the method using the real-time polymerase chain reaction of the present invention, the probe uses a probe capable of complementarily binding to some sequences inside the nucleotide sequence amplified by the primer set.
According to a preferred embodiment of the present invention, the primers and probe sets used in the real-time polymerase chain reaction of the present invention include the primers and probes of SEQ ID NO: 1 to SEQ ID NO: 25.
According to another preferred embodiment of the present invention, the primer and probe set used in the real-time polymerase chain reaction of the present invention further comprises a primer set for the internal control of SEQ ID NO: 26 and SEQ ID NO: 27.
According to another preferred embodiment of the present invention, the reporter-fluorescent material is tagged at the 5'-end of the probe used in the real-time polymerase chain reaction method of the present invention, and the fluorescent inhibitor (at the 3'-end) ( quencher) is tagged.
In the present invention, the reporter fluorescent material and the fluorescent inhibitor are not limited to specific materials. For example, the reporter fluorescent material is 6-FAM, JOY, TET, 6-JOE, HEX, Cy3, Cy5, VIC, EDD, TAMRA. Fluorescence inhibitors may be used, and BHQ-1, BHQ-2, BHQ-3, Dipsil dark fluorescent inhibitors or ROX may be used.
In the probe of the present invention, the 5'-terminal reporter fluorescent substance does not emit fluorescence by the action of the fluorescent inhibitor present at the 3'-terminal. However, by the 5 '→ 3'exonuclease activity of Taq DNA polymerase in the extension step, which is the next step of the nucleic acid amplification reaction, the probe hybridized to the template is decomposed, and the 5'-end fluorescent material is decomposed. It is separated from the probe to release the fluorescence by fluorescence inhibition by the fluorescence inhibitor.
Suitable lengths of probes vary depending on various factors, such as primer hybridization temperature and length, but are typically 20-35 nucleotides designed to have a hybridization temperature that is about 5-10 ° C. above the hybridization temperature of the primer, thereby real-time polymerase reaction. Can increase the specificity of.
In the nucleic acid amplification reaction of the present invention, the DNA extracted from the sample in step (a) is used as template DNA of the reaction.
Various DNA polymerases can be used in the amplification reaction, including, for example, Klenow fragments of E. coli DNA polymerase I, thermostable DNA polymerase and bacteriophage T7 DNA polymerase. Preferably, the polymerase is a thermostable DNA polymerase obtained from a variety of bacterial species, which include Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, and Pyrococcus furiosus (Pfu). Include. In addition, the DNA polymerase usable in the present invention may use a DNA-free polymerase from which foreign DNA has been removed by a suitable treatment, and also has a hot-start function with a hot-start function. start) polymerase can be used. Hot-start polymerase is an enzyme having polymerase activity at high temperature such as 95 ° C., which enhances the specificity of the polymerase amplification reaction by inhibiting primer dimer formation by inhibiting the reaction at low temperature between primer and polymerase. .
In the present invention, the amplification reaction solution may include a mixture of dATP, dCTP, dGTP, dTTP, and dUTP as a dNTP mixture, a PCR buffer, a DNA polymerase cofactor, and UDG (Uracill DNA Glycosylase). The dNTP mixture of the amplification reaction of the present invention includes dTTP and dUTP, together with UDG (Uracil DNA Glycosylase). The UDG recognizes and cuts the uracils contained in the previous amplification product DNA template strands, and the cut DNA template strands no longer serve as template strands, and thus no amplification reaction occurs. The present invention blocks the generation of amplified products due to carry-over contamination of previous amplification products by using dUTP and UDG, thereby improving the accuracy of the test by excluding false positive results due to laboratory contamination of the previous amplification products. .
When carrying out a gene amplification reaction, it is desirable to provide the reaction vessel with an appropriate amount of components necessary for the reaction. An appropriate amount of components necessary for the amplification reaction means an amount such that the amplification reaction is not substantially limited to the concentration of the components. It is preferred to provide cofactors such as Mg 2+ , dATP, dCTP, dGTP dTTP, and dUTP to the reaction mixture to such an extent that sufficient amplification can be achieved. All enzymes used in the amplification reaction may be active under the same reaction conditions. In fact, the buffer ensures that all enzymes are close to optimal reaction conditions. Thus, the amplification process of the present invention can be carried out in a single reactant without changing conditions such as addition of reactants.
Annealing or hybridization in the present invention is carried out under stringent conditions allowing specific binding between the nucleotide sequence of the target template DNA and the primer and probe sequences. Stringent conditions for annealing are sequence-dependent and vary depending on the surrounding environmental variables.
Step (c): detecting and analyzing the amplified nucleic acid product
The present invention further includes the step of detecting and analyzing the amplified nucleic acid product. Amplification products of 16S rRNA coding polynucleotide sequences amplified using the primer-probe sets of the invention are analyzed in a suitable manner. For example, real-time PCR amplification can be confirmed in real time whether the target gene is amplified using each program provided by the user equipment (ABI7500 or CFX96). The real-time nucleic acid amplification reaction was confirmed by detecting the fluorescence value due to the luminescence caused by the cleavage of the fluorescent material from the probe labeled with the fluorescent material and the fluorescent inhibitor capable of complementarily binding to the 16S rRNA gene, the detection target gene Check for plasma infection. In the case of mycoplasma negative samples where the fluorescence value is not measured, the real-time nucleic acid amplification reaction was clearly confirmed by confirming the amplification of the psbA gene of spina ( Spinacia oleracea ) added to confirm the real-time nucleic acid amplification reaction. , Probes complementary to the psbA gene are labeled with Cy5 fluorescent material, not FAM fluorescence, and are detected separately from FAM, a fluorescent material of mycoplasma positive samples.
According to another aspect of the present invention, the present invention provides a primer and probe set for real-time polymerase chain reaction for detecting contaminable mycoplasma in cell culture, primers comprising primers and probes of SEQ ID NO: 1 to SEQ ID NO: 25 and Provide a probe set.
According to a preferred embodiment of the invention, the primer and probe set further comprises a primer set of SEQ ID NO: 26 and SEQ ID NO: 27. The primer set of SEQ ID NO: 26 and SEQ ID NO: 27 is a primer set for the internal control that can determine whether normal real-time polymerase chain reaction.
According to a preferred embodiment of the present invention, the mycoplasma contaminated during cell culture is at least one mycoplasma selected from the group of 64 mycoplasma consisting of the following mycoplasma: Mycoplasma hyorhinis, Mycoplasma Mycoplasma yeastii, Mycoplasma equirhinis, Mycoplasma orale , Mycoplasma mycoides , Mycoplasma falconis , Mycoplasma argconis (mycoplasma arginini), mycoplasma Agar lock thienyl (mycoplasma agalactiae), mycoplasma pellets Pau sieom (mycoplasma felifaucium), mycoplasma raised barium (mycoplasma salivarium), for mycoplasma sinobi (mycoplasma synoviae), mycoplasma Felice (mycoplasma felis) , my A plasma peomen Tansu (Mycoplasma fermentans), Mycoplasma alkaryl lesson's (Mycoplasma alkalescens), Mycoplasma Galina erecting (Mycoplasma gallinaceum), Mycoplasma hoe varnish (Mycoplasma hominis), Mycoplasma ad Larry (Mycoplasma adleri), Mycoplasma Gatti ( Mycoplasma gateae), Mycoplasma art utility disk (Mycoplasma arthritidis), Mycoplasma Albi (Mycoplasma alvi), mycoplasma-based piece (Mycoplasma gypis), Maiko plasma pneumoniae (Mycoplasma pneumoniae), Mycoplasma not seriseu (Mycoplasma anseris), mycoplasma indicator Enschede (mycoplasma indiense), mycoplasma pirum (mycoplasma pirum), mycoplasma brother-less (mycoplasma auris), Jenny as mycoplasma deionized Solarium (mycoplasma lagogenitalium), mycoplasma's peoma topil room (mycoplasma spermatophilum), mycoplasma Bobigenitarium ( Myc oplasma bovigenitalium , Mycoplasma leonicaptivi , Mycoplasma buccale , Mycoplasma leopharyngis , Mycoplasma genitalium , Mycoplasma mycoplasma mycoplasma californicum), Mycoplasma lipoprotein Pacifico Enschede (Mycoplasma lipofaciens), the mycoplasma Rio sinobi (Mycoplasma hyosynoviae), Mycoplasma Cana dense (Mycoplasma canadense), Mycoplasma Mora les (Mycoplasma molare), Mycoplasma pulmo Nice (Mycoplasma pulmonis), Mycoplasma canis , Mycoplasma neurolyticum , Mycoplasma hyopneumoniae , Mycoplasma bovirhinis , Mycoplasma putrepasiens ( Mycoplasma putrefacien ) , MyCouple Lightning Court above (Mycoplasma cottewii), Mycoplasma portion Theo Nice (Mycoplasma buteonis), Mycoplasma simbe (Mycoplasma simbae), Oh Collet plasma Rai Raise weaving (Acholeplasma laidlawii), a mycoplasma-Kebbi (Mycoplasma caviae), mycoplasma testing tudi Mycoplasma testudinis , Acholeplasma oculi , Mycoplasma collis , Mycoplasma timone , Acholeplasma granularum , Spiroplasma citri ), spiro plasma led ritum (Spiroplasma insolitum), spiro plasma Kuhn kelriyi (Spiroplasma kunkelii), Ureaplasma urealyticum Parr boom (Ureaplasma parvum), spiro plasma mellitic peorum (Spiroplasma melliferum), spiro plasma Finney erecting (Spiroplasma phoeniceum), spiro plasma deferring (Spiroplasma mirum), mycoplasma Four trans- (Mycoplasma penetrans) and Mycoplasma Cri three Tully (Mycoplasma cricetuli).
According to another aspect of the present invention, the present invention provides a kit for real-time polymerase chain reaction for detecting contaminable mycoplasma in cell culture comprising the primer and probe set described above as an active ingredient.
According to a preferred embodiment of the invention, the kit optionally comprises reagents necessary for real-time PCR amplification, such as buffers, DNA polymerases [eg, Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermally stable DNA polymerases obtained from Thermococcus literalis or Pyrococcus furiosus (Pfu)], DNA polymerase cofactors, dNTP mixtures consisting of dATP, dCTP, dGTP, dTTP and dUTP and Ultra DNA DNA Gylcosylase (UDG).
The present invention relates to a method for detecting mycoplasma that can be easily contaminated in the manufacture of cell therapies and biological drugs, a set of primers and probes used in the method, and a kit for real-time mycoplasma detection comprising the set. . Using the newly-produced real-time nucleic acid amplification primers and probe sets of the present invention, it is possible to detect contaminated mycoplasma rapidly, accurately and reproducibly during the manufacture, storage and administration of cellular and biological drugs. According to the nucleic acid amplification method provided by the present invention, it is possible to provide an economical, rapid and accurate detection method of mycoplasma by replacing the sterility test method of the existing cell therapy agent.
Figure 1a is a result showing the detection results of mycoplasma species using a standard sample. Standard specimens include Acholeplasma laidlawii , Acholeplasma oculi , Mycoplasma arthritidis , Mycoplasma fermentans , and Mycoplasma genie purchased from ATCC. Thallium ( Mycoplasma genitalium ), Mycoplasma hominis , Mycoplasma hyopneumoniae , Mycoplasma hyorhinis, Mycoplasma hyosynoviae , Mycoplasma hyosynoviae Eurail ( Mycoplasma orale ), Mycoplasma pirum , Mycoplasma pulmonis , Mycoplasma spermatophilum , Mycoplasma arginini , Spiroplasma citri , Mycoplasma pneumoniae and Mycoplasma penetrans strains were used.
FIG. 1B is a result of detection using synthetic template strands obtained by artificially synthesizing the 16S rRNA detection sites of 19 kinds of detection target species that cannot be purchased or sold. The 19 strains of the synthetic template strand were Mycoplasma equirhinis, Mycoplasma agalactiae , Mycoplasma felifaucium , Mycoplasma felis , Mycoplasma felis , Mycoplasma felis Mycoplasma gallinaceum , Mycoplasma gateae , Mycoplasma gypis , Mycoplasma lagogenitalium , Mycoplasma buccale , Mycoplasma leoparin ( Mycoplasma leopharyngis ), Mycoplasma californicum , Mycoplasma lipofaciens , Mycoplasma cricetuli, Mycoplasma neurolyticus , Mycoplasma neurolyticum Mycoplasma cottewii), M. platforms A lightning unit Theo varnish (Mycoplasma buteonis), Mycoplasma Collins (Mycoplasma collis), Mycoplasma timone (Mycoplasma timone), Mycoplasma mikoyi deuseu (Mycoplasma mycoides).
2 is a result of testing mycoplasma detection specificity using the mycoplasma detection kit of the present invention.
3A to 3D are results of testing mycoplasma detection sensitivity using the mycoplasma detection kit of the present invention.
Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .
delete
Example 1 Mycoplasma Strains Used in Experiments
Table 1 and Table 2 show the mycoplasma strains used in this experiment as mycoplasmas contaminated with high frequency during cell therapy and biological drug preparation. Table 1 shows 17 mycoplasma strains and 25 microorganisms and samples as positive samples purchased from ATCC, an overseas standard sample sales company. For strains that cannot be purchased or sold, the Mycoplasma 16S rRNA gene region, which is a detection target gene, was synthesized and used as a template strand for detection verification (see Table 2).
Example 2 Design of Primer and Probe for Mycoplasma Detection
A primer-probe set capable of simultaneously detecting mycoplasmas contaminated with a high frequency in the preparation of the cellular and biological drugs shown in Example 1 was designed. BioEdit Sequence Alignment Editor program (Ver7.1.11, Hall, TA 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95 /) for 16S rRNA genes of 64 mycoplasma bacteria selected for detection. 98 / NT, Nucl.Acids.Symp.Ser. 41: 95-98. The site containing the most similarity of the common nucleotide sequence in the target species was selected as the primer position. In addition, if the bases of some of the nucleotide sequences do not match in the selected target nucleotide sequence, each target primer is added to increase the detection specificity. Finally, 17 forward primers and 7 reverse directions are added. (reverse) primers and probes capable of exhibiting one fluorescence were selected. In addition, PCRC primer was added to confirm the positive reaction of the polymerase chain reaction. The PCRC primer was designed to amplify the psbA gene of spinach ( Spinacia oleracea ) was designed to confirm that the real-time polymerase chain reaction in the mycoplasma negative sample correctly reacted. Table 3 below shows the sequences of mycoplasma detection primers and probes, and Table 4 shows the detectable mycoplasma species according to each primer combination.
Example 3 Detection of Mycoplasma Using the Primer and Probe Set of the Invention
3-1. Preparation of Sample
The detection specificity of mycoplasma was examined using the primer-probe set for detecting mycoplasma prepared in Example 2. First, as a negative sample for specificity test, human genome DNA, seven fungi Malassezia furfur , Candida albicans , Aspergillus niger , and pasillo Paecilomyces sp. , Candida tropicalis , Saccharomyces cerevisiae , Candida glabrata , 17 bacteria Bacillus cereus ) , Bacillus subtilis , Bacteroides vulgatus , Corynebacterium ammoniagenes , Enterobacter aerogenes , Enterococcus faecalis , PROFIBUS sludge tumefaciens arc Ness (Propionibacterium acnes), Pseudomonas brother Reggie labor in (Pseudomonas aeruginosa), Staphylococcus are Queretaro (St aphylococcus arlettae , Staphylococcus capitis , Staphylococcus equorum , Stenotrophomonas maltophilia , Streptococcus alactolyticus , Streptococcus alactolyticus , Streptococcus gallinaceus , Streptococcus intermedius , Streptococcus mitis , Streptococcus pneumoniae were used. Positive specimens were 17 mycoplasma, Acholeplasma laidlawii , Mycoplasma arthritidis , Mycoplasma fermentans and Mycoplasma genitalium purchased from ATCC. ( Mycoplasma genitalium ), Mycoplasma hominis , Mycoplasma hyorhinis , Mycoplasma orale , Mycoplasma pirum , Mycoplasma arginini , Spiroplasma citri , Mycoplasma pneumoniae , Acoleplasma oculi , Mycoplasma hiuli Mycoplasma hyopneumoniae , Mycoplasma hyosynoviae , Mycoplasma pulmonis , Mycoplasma spermatophilum , Mycoplasma penetrans trans DNA (genomic DNA) extraction was used. In addition, Mycoplasma equirhinis, Mycoplasma agalactiae , Mycoplasma felifaucium , Mycoplasma felis , Mycoplasma gallinaco , Mycoplasma gallinaceum galceceum ( Mycoplasma equirhinis ) Mycoplasma gateae , Mycoplasma gypis , Mycoplasma lagogenitalium , Mycoplasma buccale , Mycoplasma leopharyngis Cali, Mycoplasma Mycoplasma californicum , Mycoplasma lipofaciens , Mycoplasma cricetuli, Mycoplasma neurolyticum , Mycoplasma cottewma , Mycoplasma cottewma vu Nice ( Mycoplasma bute onis), Mycoplasma Collins (Mycoplasma collis), Mycoplasma timone (Mycoplasma timone), Mycoplasma mikoyi deuseu (was used for plasmid DNA containing the 16S rRNA gene region synthesized for mycoplasma of 19 species of Mycoplasma mycoides). Extraction of genome DNA and synthetic plasmid DNA was performed using the Solgent ™ Genomic DNA Prep Kit (Cat. No. SGD41-C100, Solgent, Korea) in accordance with the instructions contained in the kit. The extracted gDNA was measured using a UV spectrometer to measure the concentration (range of 10-100 pg), and the purity was measured after measuring the A260 / A280 ratio (≥ 1.8).
3-2. Preparation of Positive Control Molds
The template to be used as a positive control group was used to amplify the Mycoplasma Species gene 16S ribosomal RNA and psbA gene of one species of Spinacia oleracea , and cloned the amplification product into a T vector and used it as a positive control template.
3-3. Real Time Polymerase Chain Reaction
The reaction solution of Real-time Polymerase Chain Reaction (RT-PCR) was prepared in the following component ratios: 12.5 μl of 2X Multiplex PCR Smart mix (with UDG), 2 μl of primer-probe set, and Nuclease free Water and DNA template samples. The total reaction volume was adjusted to 25 μl by addition. RT-PCR reaction was performed under the following conditions. UDG reaction at 1/3 cycle at 50 ° C, initial RT-PCR activation at 1/1 cycle at 95 ° C, 20 seconds of denaturation at 95 ° C, annealing at 56 ° C and 30 ° C, and 72 ° C A cycle consisting of an extension of 40 seconds at 40 seconds was performed at 45 cycles. The real-time detection of the product amplified RT-PCR was detected using a program provided by the equipment sales company. In the case of using the ABI 7500 of Life Technology, the detection target 16S rRNA amplification was confirmed by detecting the FAM fluorescence value, and the mycoplasma positive negative value was detected through the fluorescence value detected within 40 (ct value) of the RT-PCR cycle. The positive fluorescence value detected from the cycle number 41 was reconfirmed through reexamination to confirm whether it was positive or negative. Further, in order to confirm the real-time nucleic acid amplification reaction, the fluorescent value of Cy5 emitting from the attached psbA gene was measured to confirm the accuracy of the real-time nucleic acid amplification reaction to confirm the negativeness of the mycoplasma negative sample. If the fluorescence value of Cy5 luminescent from the psbA gene was below the reference value or the luminescence was over 40 cycles (ct value), the RT-PCR reaction was not performed correctly. The composition of the 2X Multiplex PCR Smart mix (with UDG) in the reaction solution used in the present invention is shown in Table 5 below.
Real-time PCR was performed on the genome DNA and 19 synthetic plasmid DNAs extracted from the 17 mycoplasmas, and the results are shown in FIGS. 1A and 1B. It is confirmed from the results of FIGS. 1A and 1B that the RT-PCR method using the primer-probe set of the present invention can accurately detect various kinds of microplasma bacteria at the same time.
Example 4 Detection of Mycoplasma Using Mycoplasma Detection Kit of the Present Invention
4-1. Detection specificity
The detection specificity of mycoplasma was examined using the mycoplasma detection kit produced in the present invention. In the specificity test, negative samples were human genome DNA, seven fungi Malassezia furfur , Candida albicans , Aspergillus niger , and pacillomyces sp. Paecilomyces sp. , Candida tropicalis , Saccharomyces cerevisiae , Candida glabrata , 17 bacteria Bacillus cereus , Bacillus Bacillus subtilis , Bacteroides vulgatus , Corynebacterium ammoniagenes , Enterobacter aerogenes , Enterococcus faecalis Propionibacterium acnes , Pseudomonas aeruginosa , Staphylococcus arlettae ), Staphylococcus capitis , Staphylococcus equorum , Stenotrophomonas maltophilia , Streptococcus alactolyticus , Streptococcus gallina Streptococcus gallinaceus , Streptococcus intermedius , Streptococcus mitis , Streptococcus pneumoniae were used. In addition, mycoplasma collis was used as a positive sample. The results of the specificity test using the positive sample and the negative sample are shown in FIG. 2. From the results of FIG. 2, it was confirmed that only Mycoplasma collis bacteria, which were the detection target species, were detected in the real-time polymerase chain reaction using the primer-probe set of the present invention.
4-2. Sensitivity
The detection sensitivity of mycoplasma was measured using the mycoplasma detection kit for real-time polymerase chain reaction prepared in the present invention. In the measurement of detection sensitivity, specimens such as Acholeplasma laidlawii , Acholeplasma oculi , Mycoplasma arthritidis , Mycoplasma fermentans and Mycoplasma Zenitarium ( Mycoplasma genitalium ), Mycoplasma hominis , Mycoplasma hyopneumoniae , Mycoplasma hyorhinis, Mycoplasma hyosinobier ( Mycoplasma hyosynoviae ) Plasma Orail ( Mycoplasma orale ), Mycoplasma pirum , Mycoplasma pulmonis , Mycoplasma spermatophilum , Mycoplasma arginini , Spiroplasma citri , Mycoplasma pneumoniae , Mycoplasma penetrans were used, and 19 strains, Mycoplasma equirhinis, Mycoplasma agalactiae and Mycoplasma felipau, were used. Mycoplasma felifaucium , Mycoplasma felis , Mycoplasma gallinaceum , Mycoplasma gateae , Mycoplasma gypis , Mycoplasma gypis lagitalium plasmatalagium ), Mycoplasma Bukkale ( Myc oplasma buccale), Mycoplasma Leo parin Kish (Mycoplasma leopharyngis), Mycoplasma Carly Pony Qom (Mycoplasma californicum), Mycoplasma lipoprotein Pacifico Enschede (Mycoplasma lipofaciens), Mycoplasma Neuro utility Qom (Mycoplasma neurolyticum), Mycoplasma courts above (Mycoplasma cottewii ), Mycoplasma buteonis , Mycoplasma collis , Mycoplasma timone , Mycoplasma mycoides were tested for sensitivity using synthetic template DNA. . The test results are shown in FIGS. 3A to 3D. From the results of FIGS. 3A to 3D, it was confirmed that the mycoplasma detection kit of the present invention had detection sensitivity up to about 1-10 3 copies depending on the mycoplasma species.
4-3. Identification of Mycoplasma Detectable Species
Using the real-time mycoplasma detection kit produced in the present invention, the detectability of detectable mycoplasma species was investigated. The detection of the detectable species was collected from NCBI (National Center for Biotechnology Information) of 64 mycoplasma bacteria selected as detection targets and then sequenced using multiple sequence alignments using the BioEdit Sequence Alignment Editor program. The similarity of the gene sequences was compared to confirm the detectability, and 28 mycoplasma strains showing at least 80% or more similarity with the selected primers SEQ ID NO: 1 to SEQ ID NO: 25 were selected. The matching rate of the nucleotide sequence of the detection site which can be combined and combined with each primer is indicated. Probes for matching rate, the detection target 64 species Mycoplasma species of mycoplasma Jenny de Solarium (Mycoplasma genitalium), Mycoplasma pirum (Mycoplasma pirum), (Mycoplasma pneumoniae ), Mycoplasma Albi (Mycoplasma alvi) for Mycoplasma pneumoniae, Not marked as 100% identical in all except Mycoplasma testudinis and Spiroplasma phoeniceum .
Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.
<110> Solgent Co., Ltd. <120> Method for Detecting Mycoplasma Contaminated in Therapeutic Cells or Biological Medicine By Using Real-time Polymerase Chain Reaction and Kit for the Same Method <130> PN130288 <160> 27 <170> KopatentIn 2.0 <210> 1 <211> 25 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 1 ggaagaaaaa atagaatagg aaatg 25 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 2 ggaagaaaaa actagatagg aaatg 25 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 3 aagaaaagct tagggaggaa atg 23 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 4 gaagaaaaag tagcttagga aatg 24 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 5 gaagaaaaag tagttgagga aatg 24 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 6 agaacacctg gttgaggaaa tg 22 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 7 gaagaacatt tgcaatagga aatg 24 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 8 gaagaatgac tttagcaggt aatg 24 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 9 aagaatggct agcagaggaa atg 23 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 10 gaagaaacgc taaaatagga aatg 24 <210> 11 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 11 taaaaattga cggtaccata tgaat 25 <210> 12 <211> 25 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 12 ggaataaaaa acagtgtagg aaatg 25 <210> 13 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 13 ggaagaaaaa atattataag aaaagat 27 <210> 14 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 14 ggaagaacag taagtatagg aaatg 25 <210> 15 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 15 gaaagaaaaa atagggtagg aaatg 25 <210> 16 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 16 agaacacctg gtagaggaaa tg 22 <210> 17 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 17 acgaattgta agaagaggaa atg 23 <210> 18 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 18 taacctccac tatatctcta tag 23 <210> 19 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 19 taacctccat tatgtttcca taa 23 <210> 20 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 20 tagcctccga acttatttct aag 23 <210> 21 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 21 taacctccac tatgtctcca tag 23 <210> 22 <211> 26 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 22 tgttaacctc cattatatct ctataa 26 <210> 23 <211> 23 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 23 tagcctcggc tatatctcta tag 23 <210> 24 <211> 23 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 24 taacctccac tgtatttcta cag 23 <210> 25 <211> 25 <212> DNA <213> Artificial Sequence <220> PCR probe <400> 25 tgtgccagca gccgcggtaa tacat 25 <210> 26 <211> 22 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 26 cgaatacacc agctacacct aa 22 <210> 27 <211> 23 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 27 tacaatggtg gtccttatga act 23
Claims (7)
(a) extracting DNA from a sample to be detected; And
(b) performing a real-time polymerase chain reaction using the extracted DNA and a primer and probe set comprising primers and probes of SEQ ID NO: 1 to SEQ ID NO: 25.
Mycoplasma hyorhinis, Mycoplasma yeastii, Mycoplasma equirhinis, Mycoplasma orale , Mycoplasma mycoides , Mycoplasma mycoides , Mycoplasma mycoides Mycoplasma falconis , Mycoplasma arginini , Mycoplasma agalactiae , Mycoplasma felifaucium , Mycoplasma salivarium , Mycoplasma salivarium (mycoplasma synoviae), mycoplasma Felice (mycoplasma felis), mycoplasma peomen Tansu (mycoplasma fermentans), mycoplasma alkaryl lesson's (mycoplasma alkalescens), mycoplasma Galina erecting (mycoplasma gallinaceum), mycoplasma hoe varnish (mycoplasma hominis), Mycoplasma Adnan Larry (Mycoplasma adleri), the mycoplasma Gatti (Mycoplasma gateae), Mycoplasma art utility disk (Mycoplasma arthritidis), Mycoplasma Albi (Mycoplasma alvi), mycoplasma-based piece (Mycoplasma gypis), the Mycoplasma pneumoniae (Mycoplasma pneumoniae), mycoplasma not seriseu (mycoplasma anseris), mycoplasma indicator Enschede (mycoplasma indiense), mycoplasma pirum (mycoplasma pirum), mycoplasma brother-less (mycoplasma auris), Jenny as mycoplasma deionized Solarium (mycoplasma lagogenitalium), M. Plasma Spermatophilum ( Mycoplasma spermatophilum ), Mycoplasma bovigenitalium , Mycoplasma leonicaptivi , Mycoplasma buccale , Mycoplasma leoparingius , Mycoplasma Mycopl Zenitium asma genitalium , Mycoplasma californicum , Mycoplasma lipofaciens , Mycoplasma hyosynoviae , Mycoplasma canadense mora mycoarea mycoarea ), Mycoplasma pulmonis , Mycoplasma canis , Mycoplasma neurolyticum , Mycoplasma hyopneumoniae , Mycoplasma bovirhinis , Mycoplasma putrefaciens , Mycoplasma cottewii , Mycoplasma buteonis , Mycoplasma simbae , Acoleplasma laidlawii , Acholeplasma laidlawii Mycoplas ma caviae), mycoplasma testing tudi Nice (Mycoplasma testudinis), Oh Collet plasma Oh Cooley (Acholeplasma oculi), Mycoplasma Corliss (Mycoplasma collis), Mycoplasma timone (Mycoplasma timone), Oh Collet plasma Gras press rarum (Acholeplasma granularum) , Spiroplasma citri , Spiroplasma insolitum , Spiroplasma kunkelii , Ureaplasma parvum , Spiroplasma meliferum, Spiroplasma melliferum ( Spiroplasma phoeniceum ), Spiroplasma mirum , Mycoplasma penetrans and Mycoplasma cricetuli.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020130087578A KR102051678B1 (en) | 2013-07-24 | 2013-07-24 | Method for Detecting Mycoplasma Contaminated in Therapeutic Cells or Biological Medicine By Using Real-time Polymerase Chain Reaction and Kit for the Same Method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020130087578A KR102051678B1 (en) | 2013-07-24 | 2013-07-24 | Method for Detecting Mycoplasma Contaminated in Therapeutic Cells or Biological Medicine By Using Real-time Polymerase Chain Reaction and Kit for the Same Method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| KR20150012128A KR20150012128A (en) | 2015-02-03 |
| KR102051678B1 true KR102051678B1 (en) | 2019-12-03 |
Family
ID=52488317
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020130087578A KR102051678B1 (en) | 2013-07-24 | 2013-07-24 | Method for Detecting Mycoplasma Contaminated in Therapeutic Cells or Biological Medicine By Using Real-time Polymerase Chain Reaction and Kit for the Same Method |
Country Status (1)
| Country | Link |
|---|---|
| KR (1) | KR102051678B1 (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106319080A (en) * | 2016-10-26 | 2017-01-11 | 武汉科前生物股份有限公司 | PCR detection kit for rapidly identifying mycoplasma hyopeumoniae, mycoplasma synoviae and mycoplasma hyorhinis, and applications of PCR detection kit |
| USD881352S1 (en) * | 2018-03-28 | 2020-04-14 | Delta Faucet Company | Faucet spout |
| CN111118185A (en) * | 2020-01-19 | 2020-05-08 | 石家庄海关技术中心 | Real-time fluorescent RPA primer, probe and kit for detecting mycoplasma capricolum and subspecies pneumonia of goat and using method of real-time fluorescent RPA primer, probe and kit |
| CN115976238B (en) * | 2022-11-15 | 2024-01-30 | 无锡生基医药科技有限公司 | qPCR rapid detection kit containing 15 mycoplasma, and use method and application thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100974861B1 (en) * | 2008-01-23 | 2010-08-11 | 충북대학교 산학협력단 | PCR Primer Set for Identification of Mycoplasma from Biological Medical Preparation and Detection Kit for Mycoplasma Comprising the Same |
| KR101191305B1 (en) * | 2009-09-23 | 2012-10-16 | 대한민국 (식품의약품안전청장) | Method for Detecting Bacteria or Fungi Contaminated in Therapeutic Cells by Using PCR |
| KR20130047122A (en) * | 2011-10-31 | 2013-05-08 | (주)지노첵 | Method and kit for detecting mycoplasma contaminated in cell culture using real-time pcr |
-
2013
- 2013-07-24 KR KR1020130087578A patent/KR102051678B1/en active IP Right Grant
Non-Patent Citations (2)
| Title |
|---|
| In Vitro Cell Dev Biol Anim, 2006 Mar-Apr, 42(3-4): 63-69 (2006.04.)* |
| 국내공개특허공보 10-2013-0047122 (2013.05.08.)* |
Also Published As
| Publication number | Publication date |
|---|---|
| KR20150012128A (en) | 2015-02-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US9074260B2 (en) | Methods, compositions and kits for detection and analysis of antibiotic-resistant bacteria | |
| JP5830461B2 (en) | PCR primer set, PCR reaction solution, detection method for food poisoning bacteria | |
| EP2977465B1 (en) | Selective detection method for mycobacterium tuberculosis and nontuberculous mycobacteria and kit using same | |
| KR101865899B1 (en) | Diagnostic Kit for Simultaneously Detecting Mycobacterium complex, Non-tuberculosis Mycobacteria and Multidrug-resistant Tuberculosis | |
| EP2989212B1 (en) | Strand-invasion based dna amplification method | |
| KR102051678B1 (en) | Method for Detecting Mycoplasma Contaminated in Therapeutic Cells or Biological Medicine By Using Real-time Polymerase Chain Reaction and Kit for the Same Method | |
| KR102055447B1 (en) | Method for Detecting Fungi Contaminated in Therapeutic Cells or Biological Medicine By Using Polymerase Chain Reaction and Real-time Polymerase Chain Reaction and Kit for the Same Method | |
| US8377656B2 (en) | Compositions and methods for detecting Cryptococcus neoformans | |
| KR101865898B1 (en) | Diagnostic Kit for Simultaneously Detecting Mycobacterium Complex and Non-tuberculosis Mycobacteria | |
| KR102055006B1 (en) | Method for Detecting Mycoplasma Contaminated in Therapeutic Cells or Biological Medicine By Using Polymerase Chain Reaction and Kit for the Same Method | |
| KR101191305B1 (en) | Method for Detecting Bacteria or Fungi Contaminated in Therapeutic Cells by Using PCR | |
| KR101443716B1 (en) | Methods for Simultaneously Detecting Nontuberculous mycobacteria and Kits Using the Same | |
| KR102084217B1 (en) | Method for Detecting Bacteria Contaminated in Therapeutic Cells or Biological Medicine By Using Polymerase Chain Reaction and Real-time Polymerase Chain Reaction | |
| ES2439227T3 (en) | New methods and kits related to the detection, identification and quantification of yeasts in wine, beer and juices | |
| US20230193405A1 (en) | Compositions and methods for detection of candida auris | |
| KR101886379B1 (en) | Method for detecting rapidly yeast growing in presence of a carbonic acid and detection kit | |
| JP2005229838A (en) | Method for detecting / identifying strictly anaerobic gram-negative bacterium |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| E902 | Notification of reason for refusal | ||
| E701 | Decision to grant or registration of patent right | ||
| GRNT | Written decision to grant |