KR20110032602A - Method for detecting bacteria or fungi contaminated in therapeutic cells by using pcr - Google Patents

Abstract

PURPOSE: A method for nucleic acid amplification is provided to quickly and accurately detect microorganism. CONSTITUTION: A method for detecting microorganism which is easily contaminated during cell therapeutic agent comprises: a step of isolating DNA from a sample; a step of performing nucleic acid amplification using the DNA and primer pair; and a step of detecting the amplified nucleic acid product. The primer pair comprises a forward primer of sequence number 1 and a reverse primer of sequence number 2.

Description

Method for Detecting Bacteria or Fungi Contaminating Cell Therapy Products Using Nucleic Acid Amplification Method {Method for Detecting Bacteria or Fungi Contaminated in Therapeutic Cells by Using PCR}

The present invention relates to a primer pair of a nucleic acid amplification method that can detect bacteria and fungi that can be easily contaminated when preparing a cell therapy, a method and a detection kit for detecting bacteria and fungi using the same.

Currently, cell therapy agents are derived from autologous, allogeneic or heterologous cells or tissues and are used for the treatment and prevention of various diseases through in vitro manipulation. Cell therapy using biological cells, such as treatment of intractable diseases such as dementia, brain injury, spinal nerve damage, arthritis, heart disease, diabetes, immune cells, such as dendritic cells, natural killer cells ( Natural killer cells, cytotoxic T cells and cytokine-induced killer cells (cytokine-induced killer cells) in the field of cancer treatment, etc. are being intensively developed, and clinical trials are being conducted in Korea. In addition, several products, such as arthritis treatment using chondrocytes and anticancer immune cell therapy, have already obtained the approval of the KFDA, and there are already more than 10 products applying for the license.

Cell therapy products are in the spotlight as a new concept of customized drugs to replace the functions of various body organs and cells, but technical, institutional and ethical constraints at some stages of development will need to be addressed for practical use. In particular, the verification of purity, efficacy and genetic stability of therapeutic cells, as well as ensuring microbiological safety in manufacturing and manipulation processes are required first (Microbial Limit Tests, USP 24, USPC, Inc., Rockville, MD, 2000, p. 1814; Sterility Tests, USP 24, USPC, Inc., Rockville, MD, 2000, p. 1818).

Cell therapies are capable of contaminating and propagating various microorganisms during in vitro manipulation with substrates such as biomaterials, biochemicals and synthetics. Therefore, the safety evaluation of various items should be conducted throughout the production and administration of cell therapy products, and in particular, microbiological safety is essential for securing the right to health of patients. Tests that can be used to detect microorganisms such as viruses, bacteria, fungi, and parasites that may be contaminated with cell therapies may include direct culture, fluorescent antibody staining, enzyme immunoreactivity, hemagglutination, and electron microscopy. Direct culture, which has disadvantages in sensitivity, economics, time, sample volume and reproducibility, and which is currently a standard sterility assay, is not suitable for sterility testing of cell therapies (Direct estimate fo active bacteria: CTC use and limitation. J Microbiol Methods. 52: 19-28. 2003; PNA for rapid microbiology.J Microbiol Methods 48: 1-17. 2002; Khuu HM, Stock F, McGann M, Carter CS, Atkins JW, Murray PR, Read EJ. of automated culture systems with a CFR / USP-compliant method for sterility testing of cell-therapy products.Cytotherapy. 2004 6 (3): 183-95; Cell therapy using human embryonic stem cells.Transpl.Imunmun. 2004 12: 203- 209: Adult neural stem ce lls: attempting to solve the identity crisis.Dev Neurosci. 26: 93-100. 2004).

Recently, FISH (Fluorescent In Situ Hybridization), PNA (Peptide Nucleic Acid) -FISH, DNA fluorescence staining method (Acridine Orange, 4,6-diamino-2-phenylindole, Propidium iodide, etc.), redox test (5-cyano-) 2,3-ditoyl tetrazolium chloride) has been developed, but it requires a large amount of bacteria and requires additional expensive equipment (fluorescence microscope, absorber) and has disadvantages in terms of sensitivity and economy.

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 tried to develop a sterility test method that can quickly and accurately determine the contamination of various microbial groups that can be contaminated throughout the manufacturing and administration of cell therapy. As a result, a nucleic acid amplification primer was developed to detect a group of microorganisms that could be contaminated with high frequency during the preparation of cell therapy products.

Accordingly, an object of the present invention is to provide a nucleic acid amplification primer pair for use in detecting a contaminating microbial group of a cell therapeutic agent.

Another object of the present invention is to provide a kit for detecting a contaminating microorganism of a cell therapeutic agent comprising the primer pair.

Still another object of the present invention is to provide a method for detecting a cell therapy contaminating microorganism through a nucleic acid amplification method using the primer pair.

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, the present invention provides a method for detecting one or more microorganisms selected from the following microbial group using a nucleic acid amplification method, comprising: (a) extracting DNA from a sample; (b) performing nucleic acid amplification using DNA and primer pairs of the extracted sample; And (c) detecting the amplified nucleic acid product, wherein the primer pair comprises: i) a primer pair consisting of an forward primer having a first sequence listing and a reverse primer having a second listing sequence; Ii) a primer pair consisting of a forward primer having SEQ ID NO: 3 and a reverse primer having SEQ ID NO: 4; Or iii) a combination of said primer pairs, said microbial population comprises the following microorganisms: Staphylococcus epidermidis , Salmonella typhimurium , Pseudomonas aeruginosa , Pseudomonas fluorescens , Enterobacter aerogenes , Bacillus subtilis , Bacillus cereus, Bacillus cereus , E. coli, Escherichia coli Bacterium glutamicum , Corynebacterium ammoniagenes , Staphylococcus aureus , Staphylococcus capitis , Streptococcus pneumococcal pyogenes Streptococcus pneumoniae , Streptococcus mytis Streptococcus mitis ), Corynebacterium diptheriae , Corynebacterium accolens , Bacteroides vulgatus , Propionibacterium acnes , Propionibacterium acnes Callis ( Entrococcus faecalis), Mycobacterium Oh Away (Mycobacterium avium), M. bovis (Mycobacterium bovis ), Mycobacterium fortuitum , Mycobacterium gordonae , Mycobacterium kansasii , Mycobacterium szulgai , Mycobacterium szulgai Mycobacterium terrae , Asperigillus niger , Candida albicans , Saccharomyces cerevisiae , Malasazia furfur .

Hereinafter, the method of the present invention will be described in detail by dividing step by step.

Step (a): extracting DNA from a sample

The detection method of the present invention can be applied to a sample that is expected to be contaminated with bacteria or fungi. Samples of the present invention include, for example, cultured cells, proteins or nucleic acids, or biological and cellular therapeutics comprising the same.

Separation of DNA from cells of bacteria or fungi to be detected may be accomplished through various methods known in the art, 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, genomic DNA of bacterial or fungal cells can be obtained by phenol-chloroform extraction method (Neumann B, et al., Rapid isolation of genomic DNA from Gram-negative bacteria., Trends Genet. 8: 332-333 (1992).

Step (b): performing a nucleic acid amplification reaction using DNA and primer pair of the extracted sample

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, including polymerase chain reaction (PCR) (US Pat. Nos. 4,683,195, 4,683,202, and 4,800,159), reverse transcription-polymerase chain reactions; 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), Riga 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 patent) No. 6,410,276), consensus sequence priming polymerase chain reaction ed 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 based 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 thermophilicity 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.

According to a preferred embodiment of the present invention, the amplification reaction is carried out according to a polymerase chain reaction (PCR). The polymerase chain reaction (PCR) is a method for selectively amplifying specific DNA fragments, which are described in Miller, H. I. (WO 89/06700) and Davey, C. et al. (EP 329,822), which is incorporated herein by reference.

Polymerase chain reaction (PCR) is the most well known method of nucleic acid amplification and many modifications and applications have been developed. For example, touchdown PCR, hot start PCR, nested PCR, and booster PCR have been developed by modifying traditional PCR procedures to enhance the specificity or sensitivity of PCR. In addition, real-time PCR, differential display PCR (DD-PCR), rapid amplification of cDNA ends (RACE), multiplex PCR, inverse polymerase chain reaction (inverse polymerase) chain reactions (IPCR), vectorette PCR, thermal asymmetric interlaced PCR (TAIL-PCR), and multiplex PCR have been developed for specific applications. For more information on PCR, see McPherson, M.J., and Moller, S.G. PCR. BIOS Scientific Publishers, Springer-Verlag New York Berlin Heidelberg, N.Y. (2000), the teachings of which are incorporated herein by reference.

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 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).

Since the primer of the present invention targets a 16S rDNA region of a species conserved bacterium or an 18S rDNA region of a fungus in a bacterial or fungi genome sequence, it is possible to specifically detect various kinds of bacteria and fungi. Can be. More specifically, the primer of the present invention investigates the homology of the nucleotide sequence of 16S or 18S rDNA conserved in a total of 31 species of microorganisms that may be contaminated with high frequency during the manufacture of cell therapy, based on the findings Primer pairs designed to detect the microorganisms.

The primer pair included in the nucleic acid amplification reaction of the present invention comprises (i) a primer pair consisting of a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2 of a sequence specific for bacterial 16S rDNA (ii) fungi (fungi) a primer pair consisting of a forward primer of SEQ ID NO: 3 and a reverse primer of SEQ ID NO: 4, and iii) a combination of the primer pairs specific for 18S rDNA.

Since the primer pair of the present invention can specifically bind to the conserved polynucleotide sequence of the microorganisms listed above, it can be used to detect not only each of the microorganisms but also two or more microorganisms simultaneously.

Among the primer pairs of the present invention, a primer pair consisting of a forward primer having SEQ ID NO: 1 and a reverse primer having SEQ ID NO: 2 is used to detect the following bacteria: Staphylococcus epidermidis ( Staphylococcus epidermidis), Salmonella typhimurium (Salmonella typhimurium), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Pseudomonas fluoro lesson's (Pseudomonas fluorescens), Enterobacter Aro to Ness (Enterobacter aerogenes), Bacillus subtilis (Bacillus subtilis), Bacillus cereus ( Bacillus cereus ), Escherichia coli , Corynebacterium glutamicum , Corynebacterium ammoniagenes , Staphylococcus aureus , Staphylococcus captis capitis), pyogenic streptococci (Streptococcus pyogenes), lung Streptococcus (Streptococcus pneumoniae), Streptococcus US tooth (Streptococcus mitis), Corynebacterium in deep Terry (Corynebacterium diptheriae), Corynebacterium Acre lances (Corynebacterium accolens), bacteriophage Death No tooth (Bacteroides vulgatus), propynyl sludge Bacterium Acnes , Proporibacterium acnes , Entrococcus faecalis , Mycobacterium avium , Mycobacterium bovis , Mycobacterium fortuitum , Mycobacterium gordonae , Mycobacterium kansasii , Mycobacterium szulgai , Mycobacterium terrae .

Among the primer pairs of the present invention, a primer pair consisting of a forward primer having SEQ ID NO: 3 and a reverse primer having SEQ ID NO: 4 is used to detect the following fungi: Aspergillus niger ( Asperigillus niger ), Candida alpicans albicans ), Saccharomyces cerevisiae , Malasazia furfur .

In the present invention, the DNA extracted from the sample in the step (a) in the nucleic acid amplification reaction 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 polymerases and bacteriophage T7 DNA polymerases.

Preferably, the polymerase is a thermostable DNA polymerase obtainable from various bacterial species, which include Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, and Pyrococcus furiosus (Pfu). Include.

In the present invention, the amplification reaction solution may include a dNTP mixture (dATP, dCTP, dGTP, dTTP), a PCR buffer and a DNA polymerase cofactor.

When carrying out a gene amplification reaction, it is desirable to provide an excess amount of components necessary for the reaction to the reaction vessel. The excess 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. So that the degree of amplification to a desired joinja, dATP, dCTP, dGTP and dTTP, such as Mg ^{2 +} can be achieved to provide the reaction mixture is desired. 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 and the primer sequence of the target template DNA. Stringent conditions for annealing are sequence-dependent and vary depending on the surrounding environmental variables.

According to a specific embodiment of the present invention, the polymerase amplification reaction in the present invention is (i) a denaturation process for 20 seconds at 95 ℃ (ii) annealing process for 30 seconds at 57 ℃ and ( Iii) repeating 40 cycles of 1 cycle of elongation at 72 ° C. for 90 seconds.

Step (c): detecting the amplified nucleic acid product

The product of the rDNA polynucleotide sequence amplified using the primer pair of the present invention is analyzed by a suitable method. For example, amplified rDNA products are analyzed by gel electrophoresis of the above-described amplification reaction products and observing and analyzing the resulting bands.

According to a preferred embodiment of the present invention, the amplified rDNA polynucleotide sequence products can be analyzed in more detail by the type of bacteria and fungi through RFLP (Restriction Polymorphism Length Polymorphism) analysis.

According to a specific embodiment of the present invention, the restriction enzyme Sau3AI or HhaI treatment of the amplified rDNA polynucleotide product using a primer pair consisting of a forward primer having SEQ ID NO: 1 and a reverse primer having SEQ ID NO: 2 RFLP typing analysis is then performed.

According to another specific embodiment of the present invention, the PCR product amplified rDNA polynucleotide product using a primer pair consisting of a forward primer having SEQ ID NO: 3 and a reverse primer having SEQ ID NO: 4 to the restriction enzyme SauA3AI Processing to perform RFLP typing analysis.

According to an aspect of the present invention, the present invention provides a primer pair consisting of: i) a forward primer having SEQ ID NO: 1 and a reverse primer having reverse SEQ ID NO: ii) an forward primer having SEQ ID NO: 3, and A primer pair consisting of a reverse primer having a sequence number 4 sequence or iii) A primer pair for nucleic acid amplification for detecting one or two or more microorganisms selected from the group consisting of the following microorganisms is provided. : Staphylococcus epidermidis , Salmonella typhimurium , Pseudomonas aeruginosa , Pseudomonas fluorescens , Enterobacter agenyls suberos ( Bacillus subtilis ), Bacillus cereus , E. coli ( Escherichia coli , Corynebacterium glutamicum , Corynebacterium ammoniagenes , Staphylococcus aureus , Staphylococcus capitis , Purulent Streptococcus Streptococcus pyogenes), Streptococcus pneumoniae (Streptococcus pneumoniae), Streptococcus US Tees (Streptococcus mitis), Corynebacterium deep Terry at (Corynebacterium diptheriae), Corynebacterium Ako Lawrence (Corynebacterium accolens), bacteriophage death No Bluetooth (Bacteroides vulgatus , Propionibacterium acnes , Enterococcus picas , Encococcus faecalis , Mycobacterium avium , Mycobacterium bovis , Mycobacterium fortuitium ( Mycobacterium fortuitum ), Mycobacterium gordier onae ), Mycobacterium kansasii , Mycobacterium szulgai , Mycobacterium terrae , Asperigillus niger , Candidadia pepican Candida albicans , Saccharomyces cerevisiae , Malasazia furfur .

According to another aspect of the present invention, the present invention provides a detection kit for nucleic acid amplification for detecting one or two or more microorganisms selected from the group consisting of the microorganisms, including the primer pair.

According to a specific embodiment of the present invention, the primer pair consisting of a forward primer having the first sequence listing and the reverse primer having a sequence listing the second sequence of the present invention (primary pair) and sequence pairs for bacteria detection Nucleic acid amplification method is carried out by mixing a primer pair for fungi detection consisting of a forward primer having a sequence and a reverse primer having a fourth sequence of SEQ ID NO: 4 or more microorganisms selected from the group consisting of Detect at the same time.

According to a preferred embodiment of the invention, the kit comprises (a) a dNTP mixture (dATP, dCTP, dGTP, dTTP); (b) a DNA polymerase and (c) a buffer solution.

The detection kit of the present invention may optionally contain reagents necessary for nucleic acid amplification, such as buffers, DNA polymerases (eg, Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis or Pyrococcus furiosus (Pfu). Thermally stable DNA polymerases), DNA polymerase cofactors and dNTPs.

Kits of the invention can be prepared in a number of separate packaging or compartments containing the reagent components described above.

As described in detail above, the present invention provides a nucleic acid amplification method that can detect bacteria and fungi that can be easily contaminated during cell therapy preparation, primer pairs used in the method, and a kit for detecting microorganisms including the same. It is about. By using the newly prepared nucleic acid amplification primer pair of the present invention, microorganisms that can be easily contaminated during cell therapy preparation, storage and administration can be detected quickly, accurately and reproducibly. According to the nucleic acid amplification method proposed in the present invention, it is possible to provide an economical, rapid and accurate sterility test method by replacing the sterility test method of the existing cell therapy.

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: Bacteria and Fungi Strains Used in Experiments of the Invention

Tables 1 and 2 below show microorganisms that are contaminated with high frequency during the preparation of cell therapies, and indicate bacterial and fungal strains used in the experiments of the present invention.

Strain ATCC number Where to buy Staphylococcus epidermidis 12228 KCTC 1917 Salmonella typhimurium 13311 KCTC 2514 Pseudomonas aeruginosa 9027 KCTC 2513 Pseudomonas fluorescens 49642 KCTC 12028 Enterobacter aerogenes 13048 KCTC 2190 Bacillus subtilis 6633 KCTC 1021 Bacillus cereus 14579 KCTC 3624 Escherichia coli - MG1655 lab stock strain Corynebacterium glutamicum 13032 KCTC 1445 Corynebacterium ammoniagenes 6871 KCTC 1018 Staphylococcus aureus 6538 KCTC 3881 Staphylococcus capitis 35661 KCCM 41466 Streptococcus pyogenes 19615 KCTC 3208 Streptococcus pneumoniae 33400 KCTC 5412 Streptococcus mitis 49456 KCTC 13047 Corynebacterium diptheriae 11913 KCTC 3075 Corynebacterium accolens 49725 KCTC 3431 Bacteroides vulgatus 8482 ATCC 8482 Propionibacterium acnes 6919 KCTC 3314 Entrococcus faecalis 19433 KCTC 3206 Mycobacterium avium BAA-968 ATCC BAA-968D Mycobacterium bovis (BCG) ATCC BAA-968D ATCC 19015D Mycobacterium fortuitum 11440 - Mycobacterium gordonae 35760 ATCC 35760D Mycobacterium kansasii 12478 - Mycobacterium szulgai 29717 - Mycobacterium terrae 15755 -

Strain ATCC number Where to buy Aspergillus niger 16404 KCTC 6317 Candida albicans 10231 KCTC 7965 Saccharomyces cerevisiae - W303 lab stock strain Malassezia furfur 14521 KCTC 7545

In addition, Figures 1a and 1b is a photograph showing the form of the bacterial and fungal strains used in this experiment.

Example 2 Design of Primer for Bacterial or Fungal Detection

Bacteria or fungi shown in Example 1 are microorganisms that are contaminated with high frequency during the preparation of cell therapy products. Primer pairs were designed to specifically detect multiple bacteria or fungi simultaneously.

16S rDNA sequences of prokaryote such as bacteria are very well preserved. Therefore, 16S rDNA sequences of the bacteria were selected as amplification targets. Comparison of 16S rDNA Gene Sequencing of 27 Bacteria Using Clustal W (http://www.ccbb.re.kr/clustalW.php), a multiple sequence alignment program developed by Julie D. Thompson et al. In 1994 The nucleotide sequence of 27 kinds of bacteria was selected from the analysis results. In this case, if the base sequences of 27 bacteria are a common part but some bases are not 100% identical, some bases which are not matched to be combined with the target DNA sequencing of various bacteria are mixed base (C / Designed by replacing T-> Y, T / A-> W). Primers designed by the above method are shown in Table 3 below.

Specificity designation order Length (mer) Tm (℃) Omnidirectional MB-F1 Sequence Listing First Sequence GCYTA AYACA TGCAA GT 17 46 Reverse MB-R2 Sequence Listing Second Sequence TGAYT TGACG TCATC CCCAC CTTCC 25 76

18S rDNA sequences of the fungi were selected as amplification targets in the same manner as described in the above bacteria, and similarities of 18S rDNA gene sequences of the fungi of the species were compared using a multi-sequence alignment program Clustal W. From the analysis results, a common sequence portion of four fungi was selected. At this time, if the four fungal nucleotide sequences are a common part, but some of the bases are not 100% identical, some bases which are not matched to be combined with the target DNA nucleotide sequences of the various fungi are mixed bases (mixed base C / T). -> Y, T / A-> W). Primers designed by the above method are shown in Table 4 below.

Specificity designation order Length (mer) Tm (℃) Omnidirectional YE-F Sequence Listing Third Sequence TTCAT TCAAA TWTCT GCCCT ATCAA C 26 70 Reverse YE-R2 Sequence Listing IV ACTTT GATTT CTCGT AAGGT GCCG 24 70

Example  3: each of the present invention Primer pair  Bacterial Detection Specificity Test

Detection specificity for bacteria was measured using the pair of primers for detecting bacteria prepared by the method described in Example 2 MB-F1 (SEQ ID NO: 1) and MB-R2 (SEQ ID NO: 2). First, the specificity of detection for Staphylococcus epidermidis , one of the highly contaminated bacteria during cell therapy, was examined.

Staphylococcus epidermidis (Staphylococcus epidermidis ) was centrifuged at 250 g for 3 minutes to take a supernatant, and then placed in a new microtube, heated at 95 ° C. for 5 minutes, and centrifuged to extract genomic DNA. The extracted DNA was stored at -20 ° C until used as a template for PCR reaction.

The PCR reaction was carried out with 25 μl of the total amount of the reactants. In order to reduce the error between the PCR reaction samples, all the reactants except the DNA extract to be used as a template were mixed to make a master mix. 2X Taq premix 2 (SolGent, STD02-M50h) was used to easily set up PCR conditions to optimize the detection of bacteria and fungi.

In addition to the sample to be analyzed, PCR was performed under the same conditions as the analytical sample, using distilled water, human genomic DNA or mouse genomic DNA, or Saccharomyces cerevisiae genomic DNA as a template.

10 μl of each reaction solution terminated with PCR was loaded on a 2.5% NuSieve: Seckem agarose gel and electrophoresed at 150 V. The electrophoretic agarose gel was stained with EtBr, and then irradiated with a UV trans-illuminator to confirm the presence or absence of bands and sizes of PCR products.

The detection specificity test results are shown in FIG. 2A. As can be seen from the results of Figure 2a, the primer detection pair MB-F1 (SEQ ID NO: 1) and MB-R2 (SEQ ID NO: 2) of the present invention is Staphylococcus epidermidis Specific detectability).

Subsequently, the detection specificity of a total of 27 bacteria which are high frequency contaminants of the cell therapy agent of the primer pair was examined. Isolation of bacterial genomic DNA and performance of PCR reactions were performed in the same manner as described above. The test results are shown in Figure 2b. As can be seen from the results of Figure 2b, the bacterial detection primer pair of the present invention showed a specific detection capacity for 27 bacteria.

Example  4: each of the present invention Primer pair  Fungal detection specificity test

The detection specificity of the fungi of PCR using the fungal detection primer pairs YE-F (SEQ ID NO: 3) and YE-R2 (SEQ ID NO: 4) prepared by the method described in Example 2 was measured. .

The extraction of genomic DNA, the method of performing PCR and the detection of the PCR product were performed by the same method as described in Example 3 above. At this time, PCR was performed under the same conditions as the analytical sample, using distilled water, human genomic DNA or mouse genomic DNA, or Staphylococcus epidermidis genomic DNA as a template in addition to the sample to be analyzed. The test results are shown in Figure 2c.

As can be seen from the results shown in Figure 2c, the primer pair for fungal detection of the present invention is Aspergillus niger ( Aspergigillus niger ), Candida albicans ( Cedida albicans ), Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) and Malasazia furfur fungi.

Example  5: PCR Amplified products RFLP  ( restriction fragment length polymorphism ) analysis

5-1. Against germs PCR Amplification RFLP  analysis

The PCR amplification products for the bacteria obtained in Example 3 were purified and subjected to RFLP analysis by restriction enzyme Sau3AI treatment or HhaI treatment. The analysis results are shown in FIGS. 3A and 3B. RFLP analysis of the PCR amplification products of bacteria can confirm the specific types of bacteria detected.

5-2. Against fungi PCR Amplification RFLP  analysis

The PCR amplification product for the fungi obtained in Example 4 was purified and subjected to RFLP analysis by restriction enzyme Sau3AI treatment. The analysis results are shown in Figure 3c. RFLP analysis of the PCR amplification products of fungi can be used to identify the specific species of fungi detected.

Example  6: of the present invention PCR  Detection sensitivity test of each strain by the method

6-1. Of the present invention against bacteria PCR  Sensitivity test of the method

For each bacterial strain of the PCR method of the present invention using a pair of bacteria detection primer pair MB-F1 (SEQ ID NO: 1) and MB-R2 (SEQ ID NO: 2) prepared by the method described in Example 2 Detection sensitivity was measured. After measuring the concentration of genomic DNA of each strain 10-fold dilution series was converted to the copy number, the PCR reaction was carried out according to the PCR method of the present invention using genomic DNA as a template from about 100,000 copies to 0.1 copy. Detection sensitivity measurement results for each bacterial strain of the PCR method of the present invention is shown in Figures 4a and 4b. From the results shown in Figures 4a and 4b it was confirmed that the method of the present invention has a very high detection sensitivity as a method capable of detecting bacterial copy number at the level of 1-1000.

6-2. Of the present invention against fungi PCR  Sensitivity test of the method

Each fungal strain for the PCR method of the present invention using the fungal detection primer pair YE-F (SEQ ID NO: 3) and YE-R2 (SEQ ID NO: 4) prepared by the method described in Example 2 above. Detection sensitivity was measured. After measuring the concentration of genomic DNA of each strain 10-fold dilution series was converted to the copy number, the PCR reaction was carried out according to the PCR method of the present invention using genomic DNA as a template from about 10,000 copies to 1 copy. Detection sensitivity measurement results for each fungal strain of the PCR method of the present invention is shown in Figure 4c. From the results shown in Figure 4c it was confirmed that the method of the present invention has a very high detection sensitivity as a method detectable at the level of 100-1000 fungal copy number.

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. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Figure 1a is a photograph showing the form of 20 kinds of bacteria to be detected by the detection method of the present invention. A: Bacillus cereus ), B: Bacillus subtilis subtilis ), C: Bacteroides vulgatus ), D: Enterobacter Arogenes ( Enterobacter aerogenes ), E: Escherichia coli ), F: Entrococcus faecalis , G: Propionibacterium acnes , H: Pseudomonas aeruginosa , I: Pseudomonas fluorescens , J: Streptococcus Streptococcus mitis , K: Streptococcus pneumoniae , L: Streptococcus pyogenes , M: Staphylococcus aureus , N: Staphylococcus capitis , O : Staphylococcus epidermidis (Staphylococcus in Corynebacterium deep Terry: epidermidis), P: Salmonella typhimurium (Salmonella typhimurium), Q: Corynebacterium Acre lances (Corynebacterium accolens), R: Corynebacterium ammoniagenes to Ness (Corynebacterium ammoniagenes), S ( Corynebacterium diptheriae ), T: Corynebacterium glutamicum .

Figure 1b is a photograph showing the form of four types of fungi that are detected by the detection method of the present invention. A: Asperigillus niger ), B: Saccharomyces cerevisiae ( Saccharomyces cerevisiae ), C: Candida albicans , D: Malasazia furfur ).

FIG. 2A illustrates PCR amplification of Staphylococcus epidermidis , a highly contaminated bacterium, using a primer pair consisting of a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2. The amplification product is a photo showing that confirmed on the agarose gel.

Figure 2b is a PCR amplification reaction for a total of 27 bacteria using a primer pair consisting of the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2 sequence and confirmed that the amplification product on the agarose gel It is a picture showing.

Figure 2c is a PCR amplification reaction for a total of four fungi using a primer pair consisting of the forward primer of SEQ ID NO: 3 and the reverse primer of SEQ ID NO: 4 sequence and confirmed that the amplification product on the agarose gel It is a picture showing.

Figure 3a is a diagram showing the result of RFLP analysis of the PCR amplification product obtained in Example 3 treated with restriction enzyme Sau3AI.

Figure 3b is a diagram showing the result of RFLP analysis of the PCR amplification product obtained in Example 3 treated with restriction enzyme HhaI.

Figure 3c is a diagram showing the result of RFLP analysis of the PCR amplification product obtained in Example 4 treated with restriction enzyme Sau3AI.

4A and 4B are diagrams showing the results of measuring detection sensitivity of each bacterial strain of a PCR method using a primer pair consisting of a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2 of the present invention; to be.

Figure 4c is a view showing the result of measuring the detection sensitivity of each fungal strain of the PCR method using a primer pair consisting of the forward primer of the sequence listing 3rd sequence and the reverse primer of the sequence 4th sequence of the present invention.

<110> Chungbuk National University Industry Academic Cooperation Foundation          Korea Food and Drug Administration <120> Method for Detecting Bacteria or Fungi Contaminated in          Therapeutic Cells by Using PCR <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 17 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 1 gcytaayaca tgcaagt 17 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 2 tgayttgacg tcatccccac cttcc 25 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 3 ttcattcaaa twtctgccct atcaac 26 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 4 actttgattt ctcgtaaggt gccg 24  

Claims (6)

As one or more microorganism detection method selected from the following microbial group using a nucleic acid amplification method, (a) extracting DNA from a sample; (b) performing nucleic acid amplification using DNA and primer pairs of the extracted sample; And (c) detecting the amplified nucleic acid product, The primer pair is i) a primer pair consisting of a forward primer having SEQ ID NO: 1 and a reverse primer having SEQ ID NO: 2; Ii) a primer pair consisting of a forward primer having SEQ ID NO: 3 and a reverse primer having SEQ ID NO: 4; or Iii) a combination of the primer pairs, The microbial group is a detection method consisting of the following microorganisms: Staphylococcus epidermidis , Salmonella typhimurium , Pseudomonas aeruginosa , Pseudomonas fluorescens , Enterobacter agrogenes ( Enterobacter aerogenes subtilis) Bacillus subtilis , Bacillus cereus , Escherichia coli , Corynebacterium glutamicum , Corynebacterium ammoniagenes , Staphylococcus aureus , Staphylococcus capitis , Streptococcus pyogenes , Streptococcus pneumoniae , Streptococcus mitis , Corynebacterium diphtherier Corynebacterium dipthee Solarium Acre lances (Corynebacterium accolens), bacteriophage death Gatu's (Bacteroides vulgatus), propionic sludge tumefaciens arc Ness (Propionibacterium acnes), Enterococcus blood faecalis (Entrococcus faecalis), Mycobacterium Oh Away (Mycobacterium avium), M. bovis (Mycobacterium bovis), M. Mycobacterium fortuitum , Mycobacterium gordonae , Mycobacterium kansasii , Mycobacterium szulgai , Mycobacterium terraium Mycobacterium terrae ), Asperigillus niger , Candida albicans , Saccharomyces cerevisiae , Malasazia furfur . The method of claim 1, further comprising performing restriction fragment length polymorphism (RFLP) analysis by treating the nucleic acid amplified product of step (c) with restriction enzymes Sau3AI or HhaI. The method of claim 1, wherein the nucleic acid amplification reaction of step (c) is a polymerase chain reaction (PCR). As a pair of primers for nucleic acid amplification for detection of cell therapy contaminating microorganisms, i) a primer pair consisting of a forward primer having SEQ ID NO: 1 and a reverse primer having SEQ ID NO: 2; or Ii) a primer pair consisting of a forward primer having SEQ ID NO: 3 and a reverse primer having SEQ ID NO: 4, Primer pairs for nucleic acid amplification for detecting one or more microorganisms selected from the group consisting of: Staphylococcus epidermidis , Salmonella typhimurium , Pseudomonas aeruginosa , Pseudomonas fluorescens , Enterobacter aroseneus subtilis, Enterobacter aerogenes Bacillus subtilis ), Bacillus cereus , Escherichia coli ), Corynebacterium glutamicum , Corynebacterium ammoniagenes , Staphylococcus aureus , Staphylococcus capitis , Pseudomonas aeruginosa ( Coli ) Streptococcus pyogenes), Streptococcus pneumoniae (Streptococcus pneumoniae), Streptococcus US Tees (Streptococcus mitis), Corynebacterium deep Terry at (Corynebacterium diptheriae), Corynebacterium Ako Lawrence (Corynebacterium accolens), bacteriophage death No Bluetooth (Bacteroides vulgatus , Propionibacterium acnes , Enterococcus picas , Encococcus faecalis , Mycobacterium avium , Mycobacterium bovis , Mycobacterium fortuitium (Mycobacterium fortuitum), Mycobacterium choose to Donny (Mycobacterium gordonae), Matt M. Te Solarium Khan strabismus (Mycobacterium kansasii), Mycobacterium Chlef Guy (Mycobacterium szulgai), M. Te Solarium TB (Mycobacterium terrae), Aspergillus and this (Asperigillus niger), Kanda Dia al pecan's (Candida albicans ), Saccharomyces cerevisiae , Malasazia furfur . The primer pair of claim 4, wherein the nucleic acid amplification is by a polymerase chain reaction (PCR). Kit for detecting a cell therapy contaminant microorganism comprising the primer pair for nucleic acid amplification according to claim 4.

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