KR20150012128A - 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
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Abstract
The present invention relates to a method for detecting mycoplasma which can be easily contaminated during the production of a cell therapeutic agent and a biological drug, a primer and a probe set used in the method, and a real-time polymerase chain reaction For example. By using the newly prepared primer and probe set for real-time polymerase chain reaction of the present invention, it is possible to detect mycoplasma which can be easily contaminated when preparing, storing and administering a cell therapeutic agent and a biological drug product quickly, accurately and reproducibly. According to the real-time PCR reaction method provided in the present invention, it is possible to provide an economical, quick and accurate detection method of mycoplasma by replacing the aseptic test method of existing cell therapy agents.
Description
The present invention relates to a method for detecting mycoplasma contaminated with a cellular therapeutic agent or a biological drug using a real-time PCR, and a kit for use in the method.
Recently, the development of cell therapy methods using cultured cells (immune cells and stem cells) and the development of therapeutic methods using biopharmaceuticals (insulin, growth hormone, interferon and antibody drug) have led to the development of immune cells, stem Rapid bacteriological detection of contamination of viruses, bacteria, mycoplasma, and fungi, which are pathogenic microorganisms of cells or animal cells, is an important step that must be preceded in order to prevent secondary infections in patients before clinical application. The direct culture method, which is a standard sterile test method well known for the detection of existing microorganisms (including bacteria, fungi, and mycoplasma), takes a long time to determine the microbial contamination and is not suitable for microorganisms that are difficult to cultivate. Particularly, it is difficult to apply to a cell therapy agent which is required to be tested within 48 hours after a long-term test period. Therefore, it is necessary to develop an alternative method that can detect the high frequency contaminated microorganisms and the pathogenic microorganisms efficiently and quickly by improving the disadvantages of the existing direct culture method. Molecular diagnostics using real-time polymerase chain reaction (PCR), one of the techniques for nucleic acid amplification, is fast and sensitive and is being used for the development of a variety of molecular diagnostic products. It is used in cell therapy or biologics And also to the detection of microorganisms.
Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.
The present inventors have developed a technique for rapidly and accurately detecting mycoplasma, a pathogenic microorganism that can be contaminated with cultured cells in the process of culturing animal cells that produce immune cells or stem cells and biologics used as cell therapy agents I tried to develop it. As a result, we have developed a primer and a probe set for real-time polymerase chain reaction (RT-PCR) which can detect various kinds of mycoplasma through a single nucleic acid amplification process. The present inventors have completed the present invention by confirming that detection of various species of mycoplasma is possible.
Accordingly, it is an object of the present invention to provide a method for detecting mycoplasma contamination during cell culture using real-time PCR.
It is another object of the present invention to provide a primer and probe set for real-time PCR for detecting mycoplasma that can be contaminated during cell culture.
It is still another object of the present invention to provide a real-time PCR reaction kit for detecting mycoplasma contamination during cell culture comprising the primer and the probe set.
The objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.
According to one aspect of the present invention, there is provided a method of detecting mycoplasma contamination upon cell culture using a real time polymerase chain reaction comprising the steps of: (a) Extracting DNA from a sample to be detected; And (b) performing a real-time PCR using the extracted DNA, primer, and probe set.
Hereinafter, the method of the present invention will be described step by step.
Step (a): extracting DNA from a sample to be detected
The detection method of the present invention can be applied to a sample 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 cultured cell. For example, a biological drug produced by a biological method such as a cell culture method, an immune cell used as a cell therapy agent, or a production process of a stem cell 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, a solution for patient administration, and a raw material and an end product used in the production process of a biopharmaceutical.
The method for extracting DNA from the mycoplasma bacteria to be detected can be various methods known in the art. For example, 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, 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 kind of mycoplasma selected from the group consisting of the following mycoplasma: Mycoplasma hyorhinis, Mycoplasma species Mycoplasma yeastii, Mycoplasma equirhinis, Mycoplasma orale , Mycoplasma mycoides , Mycoplasma falconis , Mycoplasma arginini , Mycoplasma spp . Mycoplasma agalactiae , Mycoplasma felifaucium , Mycoplasma salivarium , Mycoplasma synoviae , Mycoplasma felis , Mycoplasma felis , Mycoplasma spp ., Mycoplasma spp ., Mycoplasma spp ., Mycoplasma spp . Mycoplas ma fermentans), (Mycoplasma gateae) a mycoplasma alkaryl lesson's (Mycoplasma alkalescens), Mycoplasma Galina erecting (Mycoplasma gallinaceum), Mycoplasma hoe varnish (Mycoplasma hominis), Mycoplasma ad Larry (Mycoplasma adleri), Mycoplasma Gatti, 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. Mycoplasma liposomes , Mycoplasma liposomes , Mycoplasma liposomes , Mycoplasma liposus , Mycoplasma liposus , Mycoplasma liposus , Mycoplasma liposus , Mycoplasma liposus , Mycoplasma liposus , Mycoplasma liposus , Mycoplasma lipofaciens , Mycoplasma hyosynoviae , Mycoplasma canadense , Mycoplasma molare , Mycoplasma pulmonis , Mycoplasma canis , Mycoplasma canis , Mycoplasma neurolyticum , Mycoplasma hyopneumoniae , Mycoplasma bovirhinis , Mycoplasma putrefaciens , Mycoplasma cottewi i ), Mycoplasma buteonis , Mycoplasma simbae , Acholeplasma laidlawii , Mycoplasma caviae , Mycoplasma testudinis , Mycoplasma spp . Acholeplasma oculi , Mycoplasma collis , Mycoplasma timone , Acholeplasma granularum , Spiroplasma citri , Spiroplasma inositol , 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 a real-time PCR using the extracted DNA, primer and probe set
The term "amplification reaction" as used herein refers to a reaction to amplify a nucleic acid molecule. Various amplification reactions have been reported in the art and include polymerase chain reaction (PCR) (US Patent Nos. 4,683,195, 4,683,202 and 4,800,159), reverse transcription-real-time polymerase Chain Reaction (RT-PCR) (Miller, HI (WO 89/06700) and Davey, C. et al. (EP 329,822), Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (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 (U.S. Patent No. 6,410,276), consensus sequence primer polymerase chain reaction (U.S. Patent No. 4,437,975), arbitrarily primed Polymerase Chain Reaction (AP-PCR) (U.S. Patent Nos. 5,413,909 and 5,861,245), nucleotide sequence (NASBA) (U.S. Patent 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 may be used are described in U.S. Patent Nos. 5,242,794, 5,494,810, 4,988,617 and U.S. Patent No. 09 / 854,317.
In the present invention, the nucleic acid amplification reaction is performed according to real-time polymerase chain reaction (RT-PCR).
The term "primer" in the context of the present invention refers to a nucleic acid that is complementary to the 5 ' end sequence and the 3 ' end sequence of the target nucleic acid region amplified in the amplification reaction of the nucleic acid, , Four different nucleoside triphosphates, and a polymerase) under the control of the oligonucleotide of the single-strand that can serve as a starting point for the polymerase reaction of the template-directed nucleic acid. The suitable length of the primer is typically 15-30 nucleotides, although it varies depending on various factors such as temperature and use of the primer. Short primer molecules generally require lower temperatures to form a sufficiently stable hybridization complex with the template.
The primer used in the present invention is hybridized or annealed at one site of the template to form a double-stranded structure. Nucleic acid hybridization conditions suitable for forming such a double-stranded structure can be found in Nucleic Acid Hybridization < RTI ID = 0.0 > (" , A Practical Approach, IRL Press, Washington, DC (1985).
Since the primer set of the present invention was designed to target a gene region coding for species conserved 16S ribosomal RNA (rRNA) in the genomic DNA sequence in mycoplasma bacteria, which can be contaminated with high frequency during the cell culture process, Type microorganism can be specifically detected.
The term "probe" in the context of the present invention is a single-stranded nucleic acid molecule and includes sequences complementary to the target nucleotide sequence. The probe of the present invention can be modified to the extent that the hybridization specificity is not impaired. For example, a reporter fluorescent material or a quencher can be tagged to the end of the oligonucleotide that is the probe.
In the method using the real-time PCR reaction of the present invention, the probe uses a probe capable of complementarily binding to a partial sequence within the base sequence amplified by the primer set.
According to a preferred embodiment of the present invention, the primer and the probe set used in the real-time PCR reaction of the present invention include primers and probes of SEQ ID NOS: 1 to 25, respectively.
According to another preferred embodiment of the present invention, the primer and probe set used in the real-time PCR reaction of the present invention additionally include a set of primers for internal control of SEQ ID NO: 26 and SEQ ID NO: 27.
According to another preferred embodiment of the present invention, a reporter-fluorescent substance is tagged at the 5'-end of the probe used in the real-time PCR reaction method of the present invention, and a fluorescent-inhibiting substance quencher is tagged.
In the present invention, the reporter fluorescent substance and the fluorescence inhibition substance are not limited to specific substances. For example, the reporter fluorescent substance may be 6-FAM, JOY, TET, 6-JOE, HEX, Cy3, Cy5, VIC, EDD, And BHQ-1, BHQ-2, BHQ-3, a dichroic dark fluorescence inhibiting substance or ROX can be used as the fluorescence inhibitor.
The probe of the present invention does not emit fluorescence at the 5'-terminal reporter fluorescent substance due to the action of the fluorescence inhibiting substance present at the 3'-terminal. However, the 5 '→ 3' exonuclease activity of the Taq DNA polymerase in the extension step, which is the next step of the nucleic acid amplification reaction, causes the probe hybridized to the template to be degraded and the 5'- Separated from the probe to release fluorescence inhibition by the fluorescence inhibiting substance, thereby emitting fluorescence.
The appropriate length of the probe is designed to have a hybridization temperature of about 20 to 35 nucleotides, which is about 5-10 DEG C higher than the hybridization temperature of the primer, depending on various factors such as primer hybridization temperature and length, Can be increased.
In the nucleic acid amplification reaction of the present invention, the DNA extracted from the sample in the step (a) is used as the template DNA of the reaction.
A variety of DNA polymerases can be used in the amplification reaction, including, for example, the Klenow fragment of E. coli DNA polymerase I, the thermostable DNA polymerase and the bacteriophage T7 DNA polymerase. Preferably, the polymerase is a thermostable DNA polymerase obtainable from a variety of bacterial species, including Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, and Pyrococcus furiosus (Pfu) . The DNA polymerase that can be used in the present invention can be DNA-free polymerase from which foreign DNA has been removed by a suitable treatment. In addition, a hot-start- start) polymerase can be used. The hot-start polymerase is an enzyme having a polymerase activity at a high temperature such as 95 ° C, and inhibits the reaction at a low temperature between the primer and the polymerase to prevent primer dimer formation, thereby improving the specificity of the amplification of the 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 joiner and UDG (Uracill DNA Glycosylase). The dNTP mixture of the amplification reaction of the present invention comprises dTTP and dUTP, together with Uracil DNA Glycosylase (UDG). The UDG recognizes and cuts the Uracil contained in the template strand of the previous amplification product DNA, and the truncated DNA template strand no longer acts as a template strand, so that the amplification reaction does not occur. The present invention uses dUTP and UDG to block the generation of amplification products due to carry-over contamination of previous amplification products, thereby improving the accuracy of the test by eliminating the false positive results due to the contamination of the laboratory amplification products .
When performing the gene amplification reaction, it is preferable to provide the reaction vessel with the necessary amount of components necessary for the reaction. The appropriate amount of the components required for the amplification reaction means an amount such that the amplification reaction is not substantially restricted to the concentration of the component. It is desirable to provide the reaction mixture with a sufficient degree of amplification to achieve the joins such as Mg 2+ , dATP, dCTP, dGTP dTTP, and dUTP. All enzymes used in the amplification reaction may be active under the same reaction conditions. In fact, buffers make all enzymes close to optimal reaction conditions. Therefore, the amplification process of the present invention can be carried out in a single reaction without changing the conditions such as the addition of reactants.
In the present invention, annealing or hybridization is carried out under stringent conditions that allow specific binding between the nucleotide sequence of the target template DNA and the primer and probe sequence. The stringent conditions for annealing are sequence-dependent and vary with environmental variables.
Step (c): Detecting and analyzing the amplified nucleic acid product
The present invention further includes detecting and analyzing the amplified nucleic acid product. The amplification product of the amplified 16S rRNA coding polynucleotide sequence using the primer-probe set of the present invention is analyzed by a suitable method. For example, real-time PCR amplification can be verified in real-time by using each program provided in the instrument (ABI7500 or CFX96) to check whether the target detection gene is amplified. The detection of the real-time nucleic acid amplification reaction was carried out by detecting the fluorescence value according to the emission of the fluorescence substance from the probe labeled with the fluorescence substance capable of complementarily binding to the 16S rRNA gene as the detection target gene and the quencher (Quencher) Check for plasma infections. In the case of mycoplasma negative samples in which the fluorescence value was not measured, it was confirmed that real-time nucleic acid amplification reaction was confirmed by confirming amplification of the psbA gene of the added spinach ( Spinacia oleracea ) to confirm the real-time nucleic acid amplification reaction , and a probe complementary to the psbA gene is labeled with a Cy5 fluorescent substance that is not FAM fluorescent light and is distinguished from FAM which is a fluorescent substance of a mycoplasma positive specimen.
According to another aspect of the present invention, there is provided a primer and a probe set for a real-time PCR reaction for detecting a mycoplasma capable of contamination during cell culture, comprising: a primer comprising the primers and probes of SEQ ID NO: 1 to SEQ ID NO: 25; Provide a set of probes.
According to a preferred embodiment of the present invention, the primer and the probe set further comprise 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 an internal control primer set which can confirm whether a normal real-time PCR reaction has occurred.
According to a preferred embodiment of the present invention, the mycoplasma which can be contaminated in the cell culture is one or more of mycoplasma selected from the group consisting of 64 kinds of mycoplasma consisting of the following mycoplasma: Mycoplasma hyorhinis, Mycoplasma yeastii, Mycoplasma equirhinis, Mycoplasma orale , Mycoplasma mycoides , Mycoplasma falconis , Mycoplasma falconis , Mycoplasma equirhinis , Mycoplasma spp . Mycoplasma arninii , Mycoplasma agalactiae , Mycoplasma felifaucium , Mycoplasma salivarium , Mycoplasma synoviae , Mycoplasma felis , Mycoplasma spp . , 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 Bovy genitallium ( Myc oplasma bovigenitalium), Mycoplasma 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 Mycoplasma liposus , californicum , Mycoplasma lipofaciens , Mycoplasma hyosynoviae , Mycoplasma canadense , Mycoplasma molare , Mycoplasma pulmonis , mycoplasma Kenneth (mycoplasma canis), mycoplasma neuro utility glutamicum (mycoplasma neurolyticum), for mycoplasma Hi ohnyu monitor (mycoplasma hyopneumoniae), mycoplasma beam builder Hi varnish (mycoplasma bovirhinis), mycoplasma Fu tray Pacific Enschede (mycoplasma putrefaciens) , Mycopl 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 testus , Mycoplasma testudinis , Acholeplasma oculi , Mycoplasma collis , Mycoplasma timone , Acholeplasma granularum , Spiroplasma citri ), Spiroplasma insolitum , Spiroplasma kunkelii , Ureaplasma parvum , Spiroplasma melliferum , Spiroplasma phoeniceum , Spiroplasma mummerium , (Spiroplasma mirum), mycoplasma Four trans- (Mycoplasma penetrans) and Mycoplasma Cri three Tully (Mycoplasma cricetuli).
According to another aspect of the present invention, there is provided a kit for real-time PCR for detecting mycopheresis capable of contamination upon cell culture comprising the above-described primer and probe set as an active ingredient.
According to a preferred embodiment of the invention, the kit optionally comprises a reagent, such as a buffer, a DNA polymerase (e.g., Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermostable DNA polymerase obtained from Thermococcus literalis or Pyrococcus furiosus (Pfu), DNA polymerase joinder, dNTP mixture consisting of dATP, dCTP, dGTP, dTTP and dUTP and UDG (Uracil DNA Gylcosylase).
The present invention relates to a method for detecting mycoplasma which can be easily contaminated during the production of a cell therapeutic agent and a biological drug, a primer and a probe set used in the method, and a kit for real-time mycoplasma detection comprising the set . Using the newly prepared real-time nucleic acid amplification primer and probe set of the present invention, it is possible to rapidly, accurately and reproducibly detect contaminated mycoplasma when preparing, storing and administering a cell therapeutic agent and a biological drug. According to the nucleic acid amplification method provided in the present invention, it is possible to provide an economical, quick and accurate method for detecting mycoplasma by replacing the sterility test method of existing cell therapy agents.
1A shows results of detection of mycoplasma species using standard samples. As a standard sample, there can be mentioned , for example, Acholeplasma laidlawii , Acholeplasma oculi , Mycoplasma arthritidis , Mycoplasma fermentans , deionized Solarium (mycoplasma genitalium), mycoplasma hoe varnish (mycoplasma hominis), mycoplasma Hi ohnyu monitor the (mycoplasma hyopneumoniae), mycoplasma Hi climb Hi varnish (mycoplasma hyorhinis), for mycoplasma Rio sinobi (mycoplasma hyosynoviae), mycoplasma Mycoplasma orale , Mycoplasma pneumonia , Mycoplasma pulmonis , Mycoplasma spermatophilum , Mycoplasma arginini , Spiroplasma citri , Mycoplasma pneumoniae , Mycoplasma spp . Mycoplasma pneumoniae and Mycoplasma penetrans strains were used.
FIG. 1B shows the results of detection of 16S rRNA detection sites of 19 target species which can not be purchased or sold, using synthetic template strands obtained by artificial synthesis. The 19 strains of the synthetic template strands were Mycoplasma equirhinis, Mycoplasma agalactiae , Mycoplasma felifaucium , Mycoplasma felis , Mycoplasma felis , Mycoplasma gallinaceum , Mycoplasma gateae , Mycoplasma gypis , Mycoplasma lagogenitalium , Mycoplasma buccale , Mycoplasma leopardinus , Mycoplasma gallinaceum , Mycoplasma leopharyngis , Mycoplasma californicum , Mycoplasma lipofaciens , Mycoplasma cricetuli, Mycoplasma neurolyticum , Mycoplasma cocteoides ( Mycoplasma spp. ), Mycoplasma spp . Mycoplasma cottewii), M. platforms A lightning unit Theo varnish (Mycoplasma buteonis), Mycoplasma Collins (Mycoplasma collis), Mycoplasma timone (Mycoplasma timone), Mycoplasma mikoyi deuseu (Mycoplasma mycoides).
FIG. 2 is a result of checking the specificity of mycoplasma detection using the mycoplasma detection kit of the present invention.
FIGS. 3A to 3D are the results of checking the sensitivity of mycoplasma detection using the mycoplasma detection kit of the present invention. FIG.
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .
Example
Example 1: Mycoplasma strains used in the experiment
Table 1 and Table 2 below show mycoplasma species used in this experiment as mycoplasma contaminated with a high frequency in the manufacture of cell therapeutic agents and biological drugs. Table 1 shows 17 kinds of mycoplasma strains and 25 kinds of microorganisms and specimens as negative specimens purchased from ATCC, a foreign standard sample sales company. In the case of strains which can not be purchased or sold, the target gene, Mycoplasma 16S rRNA gene region was synthesized and used as a template strand for detection and validation (see Table 2).
Example 2: Design of primers and probes for mycoplasma detection
A primer-probe set capable of specifically detecting mycoplasma contaminated at a high frequency in the preparation of the cell therapy agent and the biological drug shown in Example 1 was designed. The 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 / 98 / NT. Nucl. Acids. Symp. Ser. 41: 95-98.). The sites containing the common nucleotide sequence with the highest similarity in the target species were selected as primer sites. In the case where bases in some base sequence regions do not coincide with each other in the selected target base sequence region, each target primer is added in order to increase the detection specificity, and finally 17 kinds of forward primers and 7 kinds of reverse primers a reverse primer and a probe capable of exhibiting one kind of fluorescence were selected. In addition, a 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 ) and was designed to confirm that the real-time polymerase chain reaction in the mycoplasma negative sample reacted correctly. Table 3 below shows the sequences of primers and probes for mycoplasma detection, and Table 4 lists the detectable mycoplasma species according to each primer combination.
Example 3: Detection of mycoplasma using the primer and probe set of the present invention
3-1. Preparation of sample
The detection specificity of mycoplasma was examined using the primer-probe set for mycoplasma detection prepared in Example 2 above. First, as negative specimens for the specificity test, human genome DNA, seven fungi, Malassezia furfur , Candida albicans , Aspergillus niger , My process Spanish sheath (Paecilomyces sp.), Candida Tropical faecalis (Candida tropicalis), the My process serenity busy as Saccharomyces (Saccharomyces cerevisiae), Candida glabrata (Candida glabrata), Bacillus cereus (Bacillus cereus of 17 kinds of bacteria ), Bacillus subtilis (Bacillus subtilis), watermelon Tero rise beolga tooth (Bacteroides vulgatus), Corynebacterium ammoniagenes jeneseu (Corynebacterium ammoniagenes), Enterobacter aero to Ness (Enterobacter aerogenes), Enterococcus faecalis (Enterococcus faecalis) , Propionibacterium acnes , Pseudomonas aeruginosa , Staphylococcus auretat ( St < RTI ID = 0.0 > aphylococcus arlettae , Staphylococcus capitis , Staphylococcus equorum , Stenotrophomonas maltophilia , Streptococcus alactolyticus , Streptococcus gallina , Staphylococcus aureus , Streptococcus gallinaceus , Streptococcus intermedius , Streptococcus mitis , and Streptococcus pneumoniae were used. Positive specimens were 17 kinds of mycoplasma species, such as Acholeplasma laidlawii , Mycoplasma arthritidis , Mycoplasma fermentans , Mycoplasma genitalia, Mycoplasma genitalium , Mycoplasma hominis , Mycoplasma hyorhinis , Mycoplasma orale , Mycoplasma pirini , Mycoplasma arginini , Spiroplasma citri , Mycoplasma pneumoniae , Acholeplasma oculi , Mycoplasma arculini , Mycoplasma pneumoniae , Mycoplasma hyopneumoniae , Mycoplasma hyosynoviae , Mycoplasma pulmonis , Mycoplasma spermatophilum and Mycoplasma penetrans strains were tested for their genomes DNA (genomic DNA) was extracted and used. Mycoplasma equilhinis, Mycoplasma agalactiae , Mycoplasma felifaucium , Mycoplasma felis , Mycoplasma gallinaceum , Mycoplasma gallinaceum , Mycoplasma spp . Mycoplasma griseus , Mycoplasma gypis , Mycoplasma lagogenitalium , Mycoplasma buccale , Mycoplasma leopharyngis , Mycoplasma gypis , Mycoplasma spp . Mycoplasma californicum , Mycoplasma lipofaciens , Mycoplasma cricetuli, Mycoplasma neurolyticum , Mycoplasma cottewii , Mycoplasma cortewii , Mycoplasma spp . 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). Genomic DNA, and extraction of plasmid DNA synthesis, using the Solgent TM Genomic DNA Prep Kit (Cat.No. SGD41-C100, Solgent, Korea) was performed according to the contents of the instructions included in the kit. The extracted gDNA was measured with a UV spectrometer (range 10-100 pg) and purity was measured after A260 / A280 ratio (≥ 1.8).
3-2. Preparation of Positive Control Molds
The template used as a positive control was amplified from Mycoplasma Species gene 16S ribosomal RNA and one species of Spinacia oleracea psbA gene, and the amplified product was cloned into T vector and used as a positive control template.
3-3. Real-time polymerase chain reaction
The reaction mixture of RT-PCR (Real-time Polymerase Chain Reaction) was prepared with the following composition 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 And the volume of the total reaction solution was adjusted to 25 μl. The RT-PCR reaction was carried out under the following conditions. The UDG reaction was performed at 3O < 0 > C at 50 < 0 > C, the initial RT-PCR activation was 15 cycles of 1 / 15min at 95 [deg.] C, denaturation at 95 [deg.] C for 20 seconds, annealing at 56 [deg.] C for 30 seconds, And an extension of 40 seconds in 45 cycles. Real-time detection of RT-PCR-amplified products was detected using a program provided by the equipment manufacturer. When Life Technologies ABI 7500 was used, the detection target 16S rRNA amplification was confirmed by detecting the FAM fluorescence value, and the fluorescence value detected within the RT-PCR cycle number of 40 (ct) And the fluorescence positivity value detected from the cycle number 41 was confirmed by rechecking to confirm whether or not it was positive. In addition, to confirm the real - time nucleic acid amplification reaction, the fluorescence value of Cy5 luminescence from the attached psbA gene was measured and the accuracy of the real - time nucleic acid amplification reaction was confirmed to confirm the negative of the mycoplasma negative sample. If the fluorescence value of Cy5 emitted from the psbA gene is lower than the reference value or emits at a cycle number of 40 (ct value) or more, the RT-PCR reaction is not accurately performed, and this case is re-confirmed. The composition of 2X Multiplex PCR Smart mix (with UDG) in the reaction solution used in the present invention is shown in Table 5 below.
Genomic DNA extracted from the above 17 kinds of mycoplasma and 19 kinds of synthetic plasmid DNAs were subjected to real-time PCR reaction, and the results are shown in FIGS. 1A and 1B. From the results of FIGS. 1A and 1B, it was confirmed that various types of microplasma bacteria can be detected simultaneously by using the RT-PCR method using the primer-probe set of the present invention.
Example 4: Detection of mycoplasma using the 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 specimens include human genomic DNA, seven fungi such as Malassezia furfur , Candida albicans , Aspergillus niger , system (Paecilomyces sp.), Candida Tropical faecalis (Candida tropicalis), the My process serenity busy as Saccharomyces (Saccharomyces cerevisiae), Candida glabrata (Candida glabrata), Bacillus cereus (Bacillus cereus) of the 17 kinds of bacteria, Bacillus But are not limited to, Bacillus subtilis , Bacteroides vulgatus , Corynebacterium ammoniagenes , Enterobacter aerogenes , Enterococcus faecalis , Propionibacterium acnes , Pseudomonas aeruginosa , Staphylococcus ( Staphylococcus aureus), Staphylococcus arctetae , Staphylococcus capitis , Staphylococcus equorum , Stenotrophomonas maltophilia , Streptococcus alactolyticus , Streptococcus gallinaceae , Streptococcus gallinaceus , Streptococcus intermedius , Streptococcus mitis , Streptococcus pneumoniae were used. Mycoplasma collis was used as a positive specimen. The results of specificity tests using positive and negative samples are shown in Fig. From the results of FIG. 2, it was confirmed that only mycoplasma collis was detected in the real-time PCR reaction using the primer-probe set of the present invention, and no negative specimen was detected.
4-2. Detection sensitivity
The detection sensitivity of mycoplasma was measured using a mycoplasma detection kit for real-time PCR in the present invention. Examples of the detection sensitivity include, but not limited to, Acholeplasma laidlawii , Acholeplasma oculi , Mycoplasma arthritidis , Mycoplasma fermentans , Jenny de Solarium (mycoplasma genitalium), mycoplasma hoe varnish (mycoplasma hominis), mycoplasma Hi ohnyu monitor the (mycoplasma hyopneumoniae), mycoplasma Hi climb Hi varnish (mycoplasma hyorhinis), for mycoplasma Rio sinobi (mycoplasma hyosynoviae), M. Mycoplasma orale , Mycoplasma pneumonia , Mycoplasma pulmonis , Mycoplasma spermatophilum , Mycoplasma arginini , Spiroplasma citri , Mycoplasma pneumoniae , Mycoplasma spp . Mycoplasma pneumoniae and Mycoplasma penetrans were used. Also, 19 strains of Mycoplasma equirhinis, Mycoplasma agalactiae , Mycoplasma pellifolia, Mycoplasma felifaucium , Mycoplasma felis , Mycoplasma gallinaceum , Mycoplasma gateae , Mycoplasma gypis , Mycoplasma lagogenitalium , Mycoplasma spp ., Mycoplasma spp ., Mycoplasma spp. ), 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 the sensitivity test was performed cottewii), using a synthetic template DNA for mycoplasma portion Theo varnish (mycoplasma buteonis), mycoplasma Collins (mycoplasma collis), mycoplasma timone (mycoplasma timone), mycoplasma mikoyi deuseu (mycoplasma mycoides) . The results of the inspection 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 of about 1-10 3 copies according to the mycoplasma species.
4-3. Identification of mycoplasma detectable species
The detection possibility of mycoplasma species that can be detected using the real-time mycoplasma detection kit prepared in the present invention was examined. The detection of the detectable species was performed by collecting the 16S rRNA gene of 64 mycoplasma species selected as a detection target from the National Center for Biotechnology Information (NCBI) and then using the BioEdit Sequence Alignment Editor program 28 of mycoplasma strains showing at least 80% similarity to selected primer sequences SEQ ID Nos. 1 to 25 were selected. The results are shown in Table 6, And the matching rate of the base sequence of the detection site binding to each primer was 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, Mycoplasma testudinis , and Spiroplasma phoeniceum , but not all of them.
While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.
≪ 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> <223> PCR primer <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> <223> PCR primer <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> <223> PCR primer <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> <223> PCR primer <400> 22 tgttaacctc cattatatct ctataa 26 <210> 23 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 23 tagcctcggc tatatctcta tag 23 <210> 24 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 24 taacctccac tgtatttcta cag 23 <210> 25 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> PCR probe <400> 25 tgtgccagca gccgcggtaa tacat 25 <210> 26 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 26 cgaatacacc agctacacct aa 22 <210> 27 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 27 tacaatggtg gtccttatga act 23
Claims (7)
(a) extracting DNA from a sample to be detected; And
(b) performing a real-time PCR using the extracted DNA, primer and probe set.
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| 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 |
| CN115976238A (en) * | 2022-11-15 | 2023-04-18 | 无锡生基医药科技有限公司 | qPCR (quantitative polymerase chain reaction) rapid detection kit containing 15 mycoplasma, and use method and application thereof |
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| 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 |
| CN115976238A (en) * | 2022-11-15 | 2023-04-18 | 无锡生基医药科技有限公司 | qPCR (quantitative polymerase chain reaction) rapid detection kit containing 15 mycoplasma, and use method and application thereof |
| 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 |
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