KR101491904B1 - Primer set, TaqMan probe, and real-time PCR method for the detection of mycoplasma - Google Patents

Abstract

The present invention relates to a method for confirming the presence or absence of Mycoplasma contamination using an optimized TaqMan real-time PCR technique. More specifically, the present invention relates to a method for confirming the presence or absence of Mycoplasma contamination by using a primer set and / And a TaqMan real-time PCR technique using a probe represented by the nucleotide sequence of SEQ ID NO: 9. Since the optimized TaqMan real-time PCR technique according to the present invention has a useful effect capable of specifically detecting a wide range of mycoplasma, it is possible to quickly and accurately detect the presence or absence of mycoplasma contamination prior to the production of biologics, There is an effect that can be usefully used by law.

Description

(Primer set, TaqMan probe, and real-time PCR method for the detection of mycoplasma) using primer set, TaqMan probe for confirming presence or absence of mycoplasma contamination,

The present invention relates to a method for identifying the presence or absence of mycoplasma contamination using an optimized TaqMan real-time PCR method.

Mycoplasma belongs to the Mollicutes river, which has no cell wall. It is the smallest microorganism capable of self-propagation in the artificial medium, widely distributed in people, animals, insects and plants. And is a microorganism capable of causing various diseases in these. To date, more than 180 species of eight genera are known to exist. (Chang Myung Woong, 1994, AIDS and Mycoplasma, Life Science, 4: 64-69). 20 types of mycoplasma and Acholeplasma genus are the major sources of cell culture, and cell lines of 5 to 35% or more are found in six species of M. arginini , M. fermentans , M. hyorhinis , M. et al . orale , Acholeplasma laidlawii ). The source of contamination is caused by animal tissues used in primary culture, serum used in culture, or by the experimenter, and contamination may spread to other cell lines due to cross-contamination of the cell lines in the laboratory (Jeon Woo Jin, et al., 2005, Application of PCR technique for mycoplasma detection in live animal virus vaccines, The Korean Journal of Microbiology, 41: 269-274). Unlike bacterium with cell walls, mycoplasma is easy to change its shape because it has no cell wall. It has a diameter of 0.2 ~ 2 μm and can pass through 0.22 ~ 0.45 μm membrane filter used for cell culture medium filtration. (Hay, RJ, et al., 1989, Mycoplasma infection of cultured cells, Nature, 339: 487-488).

Prior to the development of biopharmaceuticals, the presence of mycoplasmas should be verified through the negative test of 7 weeks of mycoplasma as specified in the European Pharmacopoeia. Of the methods for confirming the presence of mycoplasma contamination, the direct culture method is not only time consuming, but also has a disadvantage that it can be influenced by many factors such as the composition of the separation medium, the characteristics of the sample and the culture conditions. Recently, DNA fluorochrome staining, enzyme immunoassay, fluorescence staining, biochemistry, and DNA probe techniques have been reported as methods for more rapid and accurate detection of mycoplasma contamination in host cells and seed viruses 1993, Detection of Mycoplasmas Contamination in Cultured Cell Lines, Korean Journal of Microbiology, 28: 209-221 and Spaepen, M., et al., 1992, Detection of Bacterial and Mycoplasma DNA Polymerase Chain Reaction, FEMS Microdiol. Lett., 99: 89-94). However, these techniques also have limitations in detecting all types of mycoplasma contamination that can be contaminated in the interpretation of results and cell culture. Currently, the PCR method for specific rRNA of mycoplasma has been reported as the most rapid and easy-to-use contamination detection method, and some developed kits have been commercialized (Hopert, A., et al., 1993, Specificity and Sensitivity 16: 91-100., Tang, J., et al., 2000, A polymerase chain (PCR) in comparison with other methods for the detection of mycoplasma contamination in cell lines, J. Immunol. 1993, Rapid and sensitive PCR method for identification of Mycoplasma species in tissue culture (Jung et al., 1993) , p. 257-260).

However, in addition to the usual SYBR Green real-tim PCR technique, it is necessary to develop a new method that can detect the presence of mycoplasma more rapidly and accurately.

Therefore, the present inventors constructed a new primer set such as a universal primer and designed a specific TaqMan probe based on the primer set. As a result, TaqMan real-time PCR technique was performed. As a result, various types of mycoplasma And the TaqMan real-time PCR test method of the present invention was completed.

Accordingly, an object of the present invention is to provide a method for confirming the presence or absence of contamination of Mycoplasma.

It is still another object of the present invention to provide a kit for confirming whether Mycoplasma is contaminated.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

According to an aspect of the present invention, there is provided a method of detecting a DNA fragment, comprising: (a) extracting a genomic DNA from a sample; (b) preparing a Real-Time Polymerase Chain Reaction solution containing the extracted genomic DNA, a primer set, and a probe composed of the nucleotide sequence of SEQ ID NO: 9 step; And (c) performing a real-time polymerase chain reaction (PCR) on the Mycoplasma.

In one embodiment of the present invention, the real-time PCR reaction solution of step (b) further comprises magnesium chloride (MgCl ₂ ).

In another embodiment of the present invention, the primer set includes a forward primer consisting of the nucleotide sequence of SEQ ID NO: 1, and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 2 .

In another embodiment of the present invention, the probe may include a sequence complementary to a mycoplasma labeled with a fluorescent reporter dye at the 5 'end and a quencher at the 3' end, .

In another embodiment of the present invention, the fluorescent coloring agent is selected from the group consisting of carboxyfluorescein (FAM), 2,7-dimethoxy-4,5-dichloro-6-carboxyfluoroscein (JOE), 5-carboxytetramethylrhodamine (TAMRA), indocarbocyanine- ), indocarbocyanine-5 (Cy5), and 6-Carboxy-X-rhodamine (Rox).

In another embodiment of the present invention, the quencher is selected from the group consisting of Black Hole Quencher 1 (BHQ-1), Black Hole Quencher 2 (BHQ-2), Black Hole Quencher 3 (BHQ-3), 5-Carboxytetramethylrhodamine , deep dark Quencher 1 (DDQ 1) and deep dark Quencher 2 (DDQ 2).

In another embodiment of the present invention, the mycoplasma is selected from the group consisting of A. laidlawii , M. gallisepticum , M. fermentans , climb will be Hi varnish (M. hyorhinis), mycoplasma oleyl (M. orale), mycoplasma pneumoniae selected from the group consisting of (M. pneumoniae), and (M. synoviae) a mycoplasma sinobi.

The present invention also provides a primer set comprising the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2, a probe consisting of the nucleotide sequence of SEQ ID NO: 9, or a probe for detecting the presence or absence of mycoplasma contamination Provide a kit.

The TaqMan real-time PCR method using universal primers has a wide range of mycoplasma detection capability, allowing rapid and accurate detection of mycoplasma contamination prior to biopharmaceutical production. It is effective.

Figure 1 shows the nucleotide sequence alignment for the common primer construction common to mycoplasma from the NCBI database.
Figure 2 shows the nucleotide sequence alignment for the common probe fabrication common to mycoplasma from the NCBI database.
Figure 3 shows the results of a general PCR using a common primer set (A), (B) M16 primer set, (C) MGSO primer set, (D) M. genus primer set at various annealing temperatures, Lane 2, 59 ° C .; Lane 3, 58 ° C .; Lane 4, 57 ° C .; Lane 5, 56 ° C. The results of detection of Mycoplasma fermentans (Lane M, 100 bp DNA ladder; Lane 6, 55 ° C; Lane 7, 54 ° C; Lane 8, 53 ° C).
Figure 4 shows the results of Mycoplasma spp. By performing TD-PCR using (A) a common primer set, (B) M16 primer set, (C) MGSO primer set, and (D) M. genus primer set. It shows the results of detection (Lane M, 100 bp DNA ladder ; Lane 1, M. fermentans; Lane 2, M. gallisepticum; Lane 3, M. pneumoniae; Lane 4, M. hyorhinis; Lane 5, A. laidlawii; Lane 6, M. orale , Lane 7, M. synoviae , Lane 8, negative control).
Figure 5 shows the results of an experiment on the sensitivity of real-time PCR to Mycoplasma fermentans .
FIG. 6 shows a standard curve by regression analysis of the crossing point value for the initial concentration.
Figure 7 shows the results of a comparison between genomic DNA extraction kit according to the manufacturer (□ Exgene ™ Blood SV mini kit (GeneALL, Korea); QIAamp® DNA mini kit (QIAGEN, German); ○ Genomic DNA prep kit , Korea); I-genomic BYF DNA Extraction min kit (INTRON, Korea);
Figure 8 shows the results of a comparison of genomic DNA extraction kits according to the manufacturer (□ PrepSEQ ™ Nucleic Acid Extraction Kit (Applied Biosystems, USA) □ Exgene ™ Blood SV mini Kit (GeneALL, Korea);
Figure 9 shows the results of an experiment for the specificity of the real-time PCR technique according to the present invention (□ Mycoplasma fermentation ; CHO-K1; ○ CHO-DG44; ● negative control group).
Figure 10 shows the results of an experiment for the specificity of the real-time PCR technique according to the present invention (□ Mycoplasma fermentation ; ■ A9; ○ FRhk-4; ● C8166; Vero; MA104; ◇ MPK; ◆ EBtr; ▷ negative control group).
FIG. 11 shows a state in which cap colors are different from each other to distinguish components of box 1 (FIG. 11A) and box 2 (FIG. 11B) while forming a formulation of MycoSure ™ according to the present invention This is the picture shown.
12 shows a workflow of a Mycoplasma detection kit (MycoSure ™) according to the present invention.
13 to 15 show results of detection of mycoplasma of samples A, B and C using a conventional mycoplasma detection kit (MycoSEQ).
16 to 18 show the results of detection of mycoplasma of Samples A, B and C using a Mycoplasma detection kit (MycoSure (TM)) according to the present invention.

The present invention relates to an optimized real-time PCR method using a primer set and a TaqMan probe, more specifically, real-time PCR is performed using the primer set and a TaqMan probe, (Mycoplasma) contamination can be detected quickly and accurately.

The present inventors performed real-time PCR using a TaqMan probe as a method for quickly and accurately detecting the presence or absence of mycoplasma contamination.

That is, according to one embodiment of the present invention, universal primers having high specificity and sensitivity, and various primers capable of detecting mycoplasma genes were prepared on their own. Based on the products made using common primers, (See Example 1) to optimize TaqMan real-time PCR conditions (see Example 3).

According to another embodiment of the present invention, the optimized TaqMan real-time PCR method has revealed the specificity of the primer set and the probe, and as a result, it has been shown that only mycoplasma nucleic acid can be selectively detected (see Example 7 ) And the linearity and reproducibility of the optimized TaqMan real-time PCR method showed that the coefficient of determination (r ² ) between the crossing point values (y) for log CFU (CFU / mL; x) The regression coefficient between the log CFU and the crossing point value is high at 0.99 or more and the coefficient of variation (CV) is constant without change, and the variation according to the variable is insignificant, indicating that the method of the present invention is highly reliable ). In addition, the robustness of the optimized TaqMan real-time PCR method was tested and found to be unaffected by general variables (see Example 9).

From the above results, the present inventors have found that TaqMan real-time PCR can be used for mycoplasma detection by performing optimized TaqMan real-time PCR using a common primer consisting of the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2 and a probe there was.

Therefore, the present invention can provide a method for confirming whether Mycoplasma is contaminated, comprising the steps of:

(a) extracting genomic DNA from a sample;

(b) preparing a Real-Time Polymerase Chain Reaction solution containing the extracted genomic DNA, a primer set, and a probe composed of the nucleotide sequence of SEQ ID NO: 9 step; And

(c) performing a real-time PCR reaction.

The term " sample " is used herein to refer to a sample, for example a cultured cell, a protein, a nucleic acid, or a biologic comprising it, an animal or human brain, eye, heart, tissue, blood, plasma, blood, etc. obtained from the kidney, kidney, liver, lung, muscle, spleen, or testis, But is not limited to, a body fluid such as serum, urine, saliva, sweat, semen, or mucus.

Methods for extracting genomic DNA can be accomplished through a variety of methods known in the art (Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001)), But is not limited to.

The primer set of step (b) may be an forward primer consisting of the nucleotide sequence of SEQ ID NO: 1, and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 2.

The term " primer " means an oligonucleotide complementary to a 5 ' end sequence and a 3 ' end sequence adjacent to a target nucleic acid used in a polymerase chain reaction and the term " forward primer " The term " reverse primer " means a primer that binds to the 3 'and 5' ends of a certain region of a gene amplified by a polymerase chain reaction, respectively.

Since the primers of the present invention are based on the species conserved 16S ribosomal RNA gene in the mycoplasma genomic sequence, it is possible to specifically detect various species of mycoplasma.

A "probe" of the present invention has a nucleotide sequence of SEQ ID NO: 9 capable of complementarily binding to mycoplasma, wherein a fluorescent reporter dye is labeled at the 5 'end and a quencher ).

The fluorescent colorant may be selected from the group consisting of carboxyfluorescein (FAM), 2,7-dimethoxy-4,5-dichloro-6-carboxyfluoroscein (JOE), 5-carboxytetramethylrhodamine (TAMRA), indocarbocyanine- 6-Carboxy-X-rhodamine (Rox) may be used. According to one embodiment of the present invention, carboxyfluorescein (FAM) may be preferably used.

Further, the quencher may be selected from the group consisting of Black Hole Quencher 1, Black Hole Quencher 2, Black Hole Quencher 3, 5-Carboxytetramethylrhodamine (TAMRA), deep dark Quencher 1 (DDQ 1) , Or deep dark quencher 2 (DDQ2). According to one embodiment of the present invention, Black Hole Quencher 1 (BHQ-1) may be preferably used, but is not limited thereto.

In addition, the real-time PCR reaction solution of the present invention includes the genomic DNA extracted from the sample in step (a) as a template DNA of the PCR. The polymerase chain reaction solution may be a DNA polymerase such as a thermostable DNA polymerase obtained from Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis or Pyrococcus furiosus (Pfu) (dATP, dCTP, dGTP, dTTP), a PCR buffer, and a DNA polymerase joiner.

In the present invention, the real-time PCR reaction solution of step (b) may further include magnesium chloride (MgCl ₂ ). According to a preferred embodiment of the present invention, the final concentration of the real-time PCR reaction solution is 1 mM to 10 mM, but may be such that the containing MgCl _2, but is not limited to this.

In the present invention, a polymerase chain reaction or a real-time PCR is performed by repeating cycles including denaturation, annealing and / or elongation. The temperature and time conditions in the denaturation, binding, and elongation steps may be selected by those skilled in the art, but the present invention is not limited thereto. According to a preferred embodiment of the present invention, the binding temperature can be selected to be 60 캜.

Further, the present invention provides a primer set consisting of the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2, and a probe consisting of the nucleotide sequence of SEQ ID NO:

The present invention also provides a kit for confirming the presence or absence of contamination of mycoplasma comprising the primer set and the probe.

The " kit " may include one or more containers containing a container containing partitioned carrier means for containing a sample, a container containing a set of primers for detecting mycoplasma and a probe, a buffer for PCR reaction and a container containing a DNA polymerase, The primer set may include the nucleotide sequence, the functional combination, and the fragment thereof shown in SEQ ID NO: 1 and SEQ ID NO: 2, and the probe may include the nucleotide sequence, the functional combination, and the fragment thereof shown in SEQ ID NO: The carrier means is suitable for containing one or more containers such as bottles, tubes, and each container comprises the independent components used in the method of the present invention, Easy to distribute.

The mycoplasma that can be detected by the methods or kits of the present invention is selected from the group consisting of A. laidlawii , M. gallisepticum , M. fermentans , < RTI ID = 0.0 > M. & lt ; / RTI > Hi varnish (M. hyorhinis), mycoplasma oleyl (M. orale), mycoplasma pneumoniae (M. pneumoniae), and may be selected from the group consisting of a mycoplasma sinobi (M. synoviae), but limited to, It is not.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

Maiko Plasma  For detection primer  And Of the probe  design

<1-1> Used strain  And culture

The strains used in this example are shown in Table 1 below, and seven kinds of mycoplasma standard strains were cultured in the medium described in the American Type Culture Collection (ATCC). That is to say, there can be mentioned, for example, M. fermentans , M. hyorhinis , M. orale , M. gallisepticum , Achilleplasma laidlawii ) was cultured in ATCC # 243 medium, and M. pneumoniae was cultured in ATCC # 988 Spiroplasma medium sp-4 medium for mycoplasma inoculation test. M. synoviae was cultured in ATCC # 486 Modified Chalquist's anigen medium. The culture conditions were as shown in Table 1 below. In order to confirm the concentration of the culture, the culture was diluted to 10-fold with a new medium, 100 L of each culture was inoculated into the agar medium, The average was calculated by counting the properties in units of colony forming unit (CFU).

Species Source Condition Media A. laidlawii ATCC ^a 23206 Plates: 37 ° C, 5% CO ₂
Broth: Aerobic # 243 media ^b M. gallisepticum ATCC ^a 19610 Plates: 37 ° C, 5% CO ₂
Broth: Aerobic # 243 media ^b M. fermentans ATCC ^a 19989 37 ° C, Anaerobic # 243 media ^b M. hyorhinis ATCC ^a 17981 Plates: 37 ° C, 5% CO ₂
Broth: Aerobic # 243 media ^b M. Orale ATCC ^a 23714 37 ° C, Anaerobic # 243 media ^b M. pneumoniae ATCC ^a 15531 Plates: 37 ° C, 5% CO ₂
Broth: Aerobic # 988 media ^b M. synoviae ATCC ^a 25204 Plates: 37 ° C, 5% CO ₂
Broth: Aerobic # 486 media ^{b a} ATCC, American Type Culture Collection.
^b ATCC medium.

<1-2> primer  And Probe  design

The oligonucleotide primer sequence used to amplify seven mycoplasma genes is based on the 16S ribosomal RNA gene of mycoplasma registered in the NCBI database and the 16S ribosomal RNA gene known to have little gene mutation using Primer 3 Software A universal primer was designed as shown in Fig. 1, and a primer for mycoplasma detection was designed. The designed common primers were 7 kinds of mycoplasma (Accore Plasma Ride Lowe, Maiko Plasmagliacepticum, Maiko Plasmapermans, Maiko Plasma Hiornis, Maiko Plasma Oreil, Maiko Plasma New Moonia, Maiko Plasma Sinobie ) DNA sequence and a homology with a high degree of minimum mismatches in alignment. 2, the TaqMan probe was designed to have a Tm (Melting temperature) value higher than that of the primer by about 8 to 10 ° C, and at the 5 'end, a guanidine guanidine) did not come and the same nucleotide sequence did not occur more than 4 times. A fluorescent reporter dye, carboxyfluorescein (FAM), was designed at the 5 'end of the probe sequence, and Black Hole Quencher 1 (BHQ-1) was used as a quencher at the 3' end. The designed probes were composed of mixed bases in order to include all mismatches in the alignment of the seven kinds of mycoplasma DNA sequences, and the nucleotide sequences of the primer sets and probes designed in the following Table 2 were described.

Primer set Sequences SEQ ID NO: Common primer Forward GGC GAA TGG GTG AGT AAC ACG One Reverse CGG ATA ACG CTT GCG ACC TAT G 2 M-16 primer Forward TTT TGG TGG ATG CCT TGG 3 Reverse TTT CCC CAT TCG GAA ATC 4 MGSO primer Forward GGG AGC AAA CAG GAT TAG ATA CCC T 5 Reverse TGC ACC ATC TGT CAC TCT GTT AAC CTC 6 M. genus primers Forward ACT CCT ACG GGA GGC AGC AGTA 7 Reverse TGC ACC ATC TGT CAC TCT GTT AAC CTC 8 Mycoplasma probe * FAM- YGR CWA ACT ATG TGC CAG CAG YCG CG -BHQ1 9 * IUB Code with mixed base
Y: C, TR: A, GW: A, T

Maiko Plasma  dielectric DNA  ( Genomic DNA ) Extraction

Genomic DNA extraction of mycoplasma was carried out using the Exgene ™ Blood SV mini Kit (GeneALL, Korea) according to manufacturer's instructions.

PCR  Establishment of optimization conditions through execution

<3-1> General PCR  Reaction conditions

In order to compare the detection effectiveness of the primer set designed in Example 1 above, the detection effectiveness of the primer set designed by the conventional PCR (conventional PCR) and the TD-PCR (touch down PCR) method using a Kyratec (Australia) Super Cycler Gradient Cycler device Were compared. To control the annealing temperature of general PCR, 2 μL of genomic DNA, 10 μL of 2 × GoTaq ^® Green Master Mix (Promega, USA) and 10 μL of forward primer of 0.5 pmol were added to Mycoplasma fermentans using Mycoplasma fermentans μL, and 10 pmol reverse primer, add 1.6 μL of MgCl ₂ (25 mM) and 5.4 μL of Nuclease-Free Water, and adjust the final volume to 20 μL and the final MgCl ₂ concentration to 4 mM. The nucleic acid amplification reaction was pre-incubated at 94 ° C. for 90 seconds and then denaturation at 94 ° C. for 30 seconds and annealing at 53 ° C., 54 ° C., 55 ° C., 56 ° C. and 57 ° C. 40 cycles were performed under conditions of 58 ° C, 59 ° C and 60 ° C for 30 seconds and an extension time of 72 ° C for 40 seconds. After the final cycle, the cells were reacted at 72 ° C for 4 minutes. After PCR, the PCR product was confirmed by electrophoresis using 2% agarose (Sigma Co., USA) gel at 100 V for 30 minutes and staining with ethidium bromide (EtBr).

As a result, as shown in FIG. 3, when DNA was used as a template, DNA bands were observed at 400-500 bp for a universal primer, and 100-200 bp for a M16 primer DNA bands between bp were identified. The DNA band was confirmed between 300-400 bp for the MGSO primer and the DNA band was found between 600-700 bp for the M. genus primer. In addition, the reactivity was observed at 60 ° C to 53 ° C, and it was observed that the DNA band became more dense at lower temperatures.

<3-2> Touch Down PCR  Reaction conditions

According to the results of the above Example <3-1>, TD-PCR was performed with the binding temperature set at 56 ° C, and the detection effectiveness of the respective primers against seven mycoplasma species was compared. Add 1.6 μL of MgCl ₂ (25 mM) and 5.4 μL of Nuclease-Free Water to a mixture of 2 μL of Genomic DNA, 10 μL of 2 × GoTaq ^® Green Master Mix (Promega, USA), 0.5 μL of 10 pmol forward primer and 0.5 μL of 10 pmol reverse primer To a final volume of 20 μL and a final MgCl ₂ concentration of 4 mM. The nucleic acid amplification reaction was pre-incubated at 94 ° C for 90 seconds, followed by 40 cycles of denaturation at 94 ° C for 30 seconds, annealing at 66-56 ° C for 30 seconds, extension at 72 ° C for 40 seconds, Lt; 0 &gt; C for 4 minutes. The binding temperature was initiated at 66 ° C, followed by 20 cycles of 1 ° C down to 56 ° C for 2 cycles, and 20 cycles at 56 ° C. After PCR, 2% agarose (Sigma Co., USA) gel was used for electrophoresis at 100 V for 30 minutes and then stained with EtBr to confirm PCR reaction products.

As a result, as shown in Fig. 4, a common primer was used to amplify the plasmid DNA of the present invention using the following primers: Acoreplasma ridrowii, Maikoplasma galliicepticum, Maikoplasmapermentans, Maikoplasmahihorinis, Maikoplasmaorailii, , Mycoplasma synovia, and negative control, specific bands were observed between 400-500 bp for all mycoplasma except negative control. On the other hand, when the M16 primer was used, a specific band was observed between 100-200 bp against Maiko Plasmapermans, Maiko Plasmagriscepticum, and Accore Plasma Ridrow. When MGSO primers were used, it was possible to observe Maiko Plasmapermans, Maiko Plasma hyoriensis, Accore Plasma Raid Loi and Maiko Plasmasinobia, and the size of the amplified product was found to be Mycoplasma permentans , A specific band between 200-300 bp for mycoplasma hyorienis, and a specific band between 100-200 bp for mycoplasma synovia, while a specific band between 200-300 bp for acoreplasma hioronis Non - specific bands were observed between 400 and 500 bp. When M. genus primers were used, mycoplasma pertensans, mycoplasma galli cephictumum, acoreplasma lidrowii, and mycoplasma oleyl could be observed. The size of the amplified product was found to be in the range of 600-700 bp for mycoplasma pertensin, acoreplasma lidolite and mycoplasma oryla, depending on the species of mycobacteria, while mycoplasma gallicacepticum was found to be between 500-600 bp And a non-specific band between 700-800 bp.

From the above, it can be seen that detection specific amplification products of all seven kinds of microplasma in the common primer can be confirmed, and thus it can be used for broad mycoplasma detection.

<3-3> primer  Set and Of the probe  For Sensitivity Measurements real - time PCR  optimization

Real-time PCR master mix (TOYOBO, Japan) was performed on the basis of the PCR conditions established by general PCR using Mycoplasma fermentans measured by CFU in order to measure the sensitivity of the primer set and probe Real-time PCR (qPCR) conditions were established using real-time PCR. The real-time PCR conditions were optimized by varying the binding temperature and the MgCl ₂ concentration, and the mycoplasma pertensin was sequentially added to 1 × 10 ⁴ CFU / mL, 1 × 10 ² CFU / mL, 1 × 10 ⁰ CFU / mL The binding temperature was 50 ℃, 52 ℃, 54 ℃, 56 ℃, 58 ℃ and 60 ℃, respectively. MgCl ₂ concentration was 3 mM, 4 mM, 5 mM and 6 mM respectively Experiments were performed. For PCR, 10 μL of real-time PCR master mix (TOYOBO, Japan), 1.5 μL of forward primer of 10 pmol common primer, and 10 pmol of common primer of 10 pmol common primer were used for PCR. To the mixture of 1.5 μL of reverse primer, 0.5 μL of 10 pmol specific probe, and 5 μL of template DNA, 0.8 μL of MgCl ₂ (25 mM) and 0.7 μL of Nuclease-Free purified water were added to the final volume of 20 μL and the final MgCl ₂ concentration Was adjusted to 4 mM. Nucleic acid amplification was pre-incubated at 94 ° C for 90 seconds, followed by 45 cycles of denaturation at 94 ° C for 30 seconds and annealing at 60 ° C for 40 seconds.

CFU / mL Annealing temperature 50 ℃ 52 ℃ 54 ℃ 56 ℃ 58 ℃ 60 ° C 1 x 10 ⁴ 19.82 * 20.55 20.42 20.24 20.53 19.87 20.09 20.25 20.42 19.93 20.39 20.67 1 x 10 ² 28.51 28.38 28.39 27.99 27.78 27.61 27.69 27.78 27.45 27.57 27.72 27.51 1 x 10 ⁰ 35.66 37.10 34.63 35.79 35.09 34.64 35.79 35.59 34.82 34.18 33.91 33.97 Buffer control N / A N / A N / A N / A N / A N / A N / A N / A N / A N / A N / A N / A * Values indicating crossing point (Cp) value
N / A, Not Applicable; real-time PCR signals were not detected

CFU / mL MgCl ₂ concentration 3 mM 4 mM 5 mM 6 mM 1 x 10 ⁴ 18.95 * 19.48 19.68 19.51 19.65 19.41 20.05 19.79 1 x 10 ² 26.73 26.46 27.34 27.06 28.48 27.11 27.44 27.57 1 x 10 ⁰ N / A 29.82 30.22 30.46 31.75 31.04 N / A N / A Buffer control N / A 29.75 N / A N / A 31.96 31.88 N / A N / A * Values indicating crossing point (Cp) value
N / A, Not Applicable; real-time PCR signals were not detected

As a result of comparing the reactivity of the bonding temperature, as shown in Table 3, it was found that the influence of any parameter was insignificant at all temperatures and the optimum bonding temperature was selected at 60 캜.

In addition, as shown in Table 4, MgCl ₂ concentration was experimentally optimized. As shown in Table 4, the MgCl ₂ concentration was hardly affected at all concentrations, and the optimum MgCl ₂ concentration was selected to be 4 mM.

Wherein the fit in the optimized conditions CFU is 1 × 10 ⁷ CFU / mL of Mycoplasma FER men Tansu sequentially ^{1 × 10 6 CFU / mL,} 1 × 10 5 CFU / mL, 1 × 10 4 CFU / mL, 1 × The PCR product was diluted to 10 ³ CFU / mL, 1 × 10 ² CFU / mL, 1 × 10 ¹ CFU / mL, 1 × 10 ⁰ CFU / mL and 1 × 10 ^-1 CFU / A curve was created. The standard curve was generated by converting the crossing point value detected by Real-Time PCR to CFU / mL according to the concentration of genomic DNA. The crossing point represents the number of cycles in which the PCR cycle enters the exponential phase.

As a result, as shown in FIG. 5 and FIG. 6, the coefficient of determination (r ² ) between the crossing point value (y) for the log CFU (CFU / mL; x) of mycoplasma pertensans was 0.9948, The regression between the log CFU and crossing point values of Tans was high.

dielectric DNA  ( Genomic DNA ) Comparison method of extraction method

Optimized DNA extraction method The manufacturer, a filter method, performed comparative experiments on four different types of DNA prep kit. DNA prep kit used for the comparison experiments Exgene ™ Blood SV mini Kit (GeneALL , Korea), QIAamp ® DNA mini kit (QIAGEN, German), Genomic DNA prep kit (Solgent, Korea), I-genomic BYF DNA Extraction min kit ( INTRON, Korea). DNA was extracted from mycoplasma pertensin with a CFU of 1 × 10 ⁵ CFU / mL according to the manufacturer's method, and the extracted DNA stock was extracted using a StepOne Plus ™ Real-Time PCR System (Applied Biosystems, USA) And an experiment was conducted using the optimized method of Example 3 above. The selected filter type DNA prep kit and the magnetic particles type PrepSEQ ™ Nucleic Acid Extraction Kit were tested in the same manner.

Comparison of DNA extraction methods. As shown in FIG. 7, it was confirmed that the DNA extraction ability of the Exgene ™ Blood SV mini Kit (GeneALL, Korea) was the best.

As shown in FIG. 8, using the selected Exgene ™ Blood SV mini kit and the magnetic particles PrepSEQ ™ Nucleic Acid Extraction Kit, the microcuppliar microhardness with CFU of 1 × 10 ⁶ CFU / mL was prepared The DNA extraction of the PrepSEQ ™ Nucleic Acid Extraction Kit showed better DNA extraction according to the manufacturer's manual.

Using an optimized method Maiko Plasma  Reactivity experiment

To investigate the reactivity of mycoplasma using an optimized real-time PCR method, 1 × 10 ⁴ CFU / mL of A. laidlawii , M. gallisepticum ) is 1 × 10 ⁵ CFU / mL, mycoplasma FER men Tansu (M. fermentans) is 1 × 10 ⁵ CFU / mL, mycoplasma Hi Hi climb varnish (M. hyorhinis) is 1 × 10 ⁶ CFU / mL, M. plasma oleyl (M. orale) is 1 × 10 ⁴ CFU / mL, mycoplasma pneumoniae (M. pneumoniae) is (M. synoviae) to 1 × 10 ⁴ CFU / mL, mycoplasma sinobi is 1 × 10 ⁴ The PCR was carried out by diluting mycoplasma with the concentration of CFU / mL to 1 × 10 ^-1 CFU / mL. The crossing point value was converted into CFU / mL, and the crossing point represents the number of cycles in which the PCR cycle enters the logarithmic phase.

Mycoplasma
species CFU / ml Regression curve
Y = -aX + b r ² 10 ⁵ 10 ⁴ 10 ³ 10 ² 10 ¹ 10 ⁰ 10 ^-1 A. laidlawii · · 24.94 * 28.63 32.70 35.85 N / A Y = -3.680X + 36.053 0.998 M. gallisepticum · 22.94 26.28 29.58 33.32 35.72 N / A Y = -3.259X + 36.085 0.996 M. fermentans · 22.81 26.50 29.57 32.95 36.46 N / A Y = -3.374X + 36.406 0.999 M. hyorhinis 17.79 21.08 24.84 28.25 31.65 34.97 N / A Y = -3.458X + 35.076 1,000 M. Orale · · 25.46 29.12 32.82 35.50 N / A Y = -3.382X + 35.798 0.995 M. pneumoniae · · 24.88 28.40 31.94 35.04 N / A Y = -3.402X + 35.17 0.999 M. synoviae · · 26.36 29.67 32.87 36.45 N / A Y = -3.346X + 39.702 0.999 r ² : the coefficient of determination
* Values indicating crossing point (Cp) value
N / A, Not Applicable; real-time PCR signals were not detected

As a result, as shown in Table 5, reactivity was confirmed up to 1 x 10 &lt; ⁰ &gt; CFU / mL for all seven kinds of mycoplasmas.

Detection limit experiment using optimized method

Using the optimized real-time PCR method, the detection limit, which means the minimum amount of nucleic acid detectable in each mycoplasma, was examined. In this example, mycoplasma, which was cultured and quantified as CFU / mL, was used as a standard, and at least three consecutive diluted panels were independently prepared, and 24 replicate tests per dilution were performed. Were analyzed statistically. The seven kinds of mycoplasma used in the experiment were Acoreplasma ridrowii, Maikoplasma galliicepticum, Maikoplasmapermansans, Maikoplasma hyoriensis, Maikoplasma oryl, Maikoplasmus pneumoniae, Maikoplasmusinobiene, each of 1 × 10 ⁴ to ^{CFU / mL, 1 × 10 3} CFU / mL, 1 × 10 2 CFU / mL, 1 × 10 1 CFU / mL, 1 × 10 0 CFU / mL, 1 × 10 -1 CFU / mL The optimized real-time PCR method was performed by serial dilution.

Run CFU / mL ( A. laidlawii ) Limit of detection 10 ³ 10 ² 10 ¹ 10 ⁰ 10 ^-1 One + + + + + + + + + + + + - - - 10 &lt; ⁰ &gt; CFU / mL 2 + + + + + + + + + + + + - - - 10 &lt; ⁰ &gt; CFU / mL 3 + + + + + + + + + + + - - - - 10 &lt; ⁰ &gt; CFU / mL 4 + + + + + + + + + + - + - - - 10 &lt; ⁰ &gt; CFU / mL 5 + + + + + + + + + + + + + - + 10 &lt; ⁰ &gt; CFU / mL 6 + + + + + + + + + + + + - - + 10 &lt; ⁰ &gt; CFU / mL 7 + + + + + + + + + - + - - - - 10 &lt; ⁰ &gt; CFU / mL 8 + + + + + + + + + + + + - - - 10 &lt; ⁰ &gt; CFU / mL +, positive; -, negative

Run CFU / mL ( M. gallisepticum ) Limit of detection 10 ⁴ 10 ³ 10 ² 10 ¹ 10 ⁰ 10 ^-1 One + + + + + + + + + + + + + - + - - - 10 &lt; ⁰ &gt; CFU / mL 2 + + + + + + + + + + + + + + + - - - 10 &lt; ⁰ &gt; CFU / mL 3 + + + + + + + + + + + + + + - - - - 10 &lt; ⁰ &gt; CFU / mL 4 + + + + + + + + + + + + + + + - - - 10 &lt; ⁰ &gt; CFU / mL 5 + + + + + + + + + + + + + - + - - - 10 &lt; ⁰ &gt; CFU / mL 6 + + + + + + + + + + + + + + + - - - 10 &lt; ⁰ &gt; CFU / mL 7 + + + + + + + + + + + + + + + - - - 10 &lt; ⁰ &gt; CFU / mL 8 + + + + + + + + + + + + - + + - - - 10 &lt; ⁰ &gt; CFU / mL +, positive; -, negative

Run CFU / mL ( M. fermentans ) Limit of detection 10 ⁴ 10 ³ 10 ² 10 ¹ 10 ⁰ 10 ^-1 One + + + + + + + + + + + + + + + - - - 10 &lt; ⁰ &gt; CFU / mL 2 + + + + + + + + + + + + + + + - - - 10 &lt; ⁰ &gt; CFU / mL 3 + + + + + + + + + + + + + + + - - + 10 &lt; ⁰ &gt; CFU / mL 4 + + + + + + + + + + + + + + + - - - 10 &lt; ⁰ &gt; CFU / mL 5 + + + + + + + + + + + + + + + - - - 10 &lt; ⁰ &gt; CFU / mL 6 + + + + + + + + + + + + + + + - - - 10 &lt; ⁰ &gt; CFU / mL 7 + + + + + + + + + + + + - - + - - - 10 ¹ CFU / mL 8 + + + + + + + + + + + + - - - - - - 10 ¹ CFU / mL +, positive; -, negative

Run CFU / mL ( M. hyorhinis ) Limit of detection 10 ⁴ 10 ³ 10 ² 10 ¹ 10 ⁰ 10 ^-1 One + + + + + + + + + + + + + + + - + - 10 &lt; ⁰ &gt; CFU / mL 2 + + + + + + + + + + + + - + + - - - 10 &lt; ⁰ &gt; CFU / mL 3 + + + + + + + + + + + + + + + - - - 10 &lt; ⁰ &gt; CFU / mL 4 + + + + + + + + + + + + + + - - - - 10 &lt; ⁰ &gt; CFU / mL 5 + + + + + + + + + + + + + - + - - - 10 &lt; ⁰ &gt; CFU / mL 6 + + + + + + + + + + + + + + + - - - 10 &lt; ⁰ &gt; CFU / mL 7 + + + + + + + + + + + + + + + - - - 10 &lt; ⁰ &gt; CFU / mL 8 + + + + + + + + + + + + + + + - - - 10 &lt; ⁰ &gt; CFU / mL +, positive; -, negative

Run CFU / mL ( M. orale ) Limit of detection 10 ³ 10 ² 10 ¹ 10 ⁰ 10 ^-1 One + + + + + + + + + - + + - - + 10 &lt; ⁰ &gt; CFU / mL 2 + + + + + + + + + - - + - - - 10 ¹ CFU / mL 3 + + + + + + + + + + + - - - - 10 &lt; ⁰ &gt; CFU / mL 4 + + + + + + + + + - - - - - - 10 ¹ CFU / mL 5 + + + + + + + + + + - - - - - 10 ¹ CFU / mL 6 + + + + + + + + + - - - - - - 10 ¹ CFU / mL 7 + + + + + + + + + + + - - - - 10 &lt; ⁰ &gt; CFU / mL 8 + + + + + + + + + + + - - - - 10 &lt; ⁰ &gt; CFU / mL +, positive; -, negative

Run CFU / mL ( M. pneumoniae ) Limit of detection 10 ³ 10 ² 10 ¹ 10 ⁰ 10 ^-1 One + + + + + + + + + - + - - - - 10 ¹ CFU / mL 2 + + + + + + + + + - + + - - - 10 &lt; ⁰ &gt; CFU / mL 3 + + + + + + + + + - - - - - - 10 ¹ CFU / mL 4 + + + + + + + + + + + - - - - 10 &lt; ⁰ &gt; CFU / mL 5 + + + + + + + + + + - + - - - 10 &lt; ⁰ &gt; CFU / mL 6 + + + + + + + + + + + - - - - 10 &lt; ⁰ &gt; CFU / mL 7 + + + + + + + + + + - + - + - 10 &lt; ⁰ &gt; CFU / mL 8 + + + + + + + + + + - + - - - 10 &lt; ⁰ &gt; CFU / mL +, positive; -, negative

Run CFU / mL ( M. synoviae ) Limit of detection 10 ³ 10 ² 10 ¹ 10 ⁰ 10 ^-1 One + + + + + + + + + + + + - - + 10 &lt; ⁰ &gt; CFU / mL 2 + + + + + + + + + + + + - - + 10 &lt; ⁰ &gt; CFU / mL 3 + + + + + + + + + + + + - - - 10 &lt; ⁰ &gt; CFU / mL 4 + + + + + + + + + + + + - - - 10 &lt; ⁰ &gt; CFU / mL 5 + + + + + + + + + + - + - - - 10 &lt; ⁰ &gt; CFU / mL 6 + + + + + + + + + + + + - - - 10 &lt; ⁰ &gt; CFU / mL 7 + + + + + + + + + + + + - - - 10 &lt; ⁰ &gt; CFU / mL 8 + + + + + + + + + + + + - - + 10 &lt; ⁰ &gt; CFU / mL +, positive; -, negative

Mycoplasma species Limit of detection
CFU / ml (or DNA Copies) A. laidlawii 10 CFU / ml M. gallisepticum 10 CFU / ml M. fermentans 10 CFU / ml M. hyorhinis 10 CFU / ml M. orale 10 CFU / ml M. pneumoniae 10 CFU / ml M. synoviae 1 CFU / ml

As a result, as shown in Tables 6 to 13, each of the detection limitations was found to be in the order of A. laidlawii , M. gallisepticum , M. fermentans 1 x 10 &lt; ¹ &gt; CFU / mL for M. hyorhinis , M. pneumoniae , M. orale and mycoplasma pneumoniae, And 1 x 10 &lt; ⁰ &gt; CFU / mL for plasma synovia ( M. synoviae ).

primer  Set and Of the probe  Specificity experiment

Specificity experiments were conducted to determine if only mycoplasma nucleic acids could be selectively detected even under the presence of other foreign substances. In this example, the CHO DG-44 cell line derived from human T-cell-derived C8166 cell line and chinese hamster ovary (CHO) cell line was subjected to a specificity test. At this time, the C8166 cell line and the CHO DG-44 cell line were 1 × 10 ⁶ cells / mL, 1 × 10 ⁴ cells / mL, 1 × 10 ³ cells / mL, 1 × 10 ² cells / 1 × 10 ¹ cells / mL, and real-time PCR was performed. For the analysis of the results, the detected crossing point value was converted into CFU / mL, and the crossing point represents the number of cycles in which the PCR cycle enters the logarithmic phase.

Species CFU / mL or Cell / mL Specificity 10 ⁴ 10 ³ 10 ² 10 ¹ Human keratinocyte N / A N / A N / A N / A Y Human mesenchymal stem cell N / A N / A N / A N / A Y Negative control N / A N / A N / A N / A · N / A, Not Applicable; real-time PCR signals were not detected

As a result, as shown in Table 14, it was found that mycoplasma was not detected in the C8166 cell line and the CHO DG-44 cell line.

In addition, a CHO-K1, CHO-DG44 and the other cell line A9, FRhk-4, C8166, Vero, MA104, MPK, EBtr, total nine kinds of cells to 1 × 10 ⁶ cell / mL DNA of the CHO cell-derived The optimized real-time PCR method was performed.

Species  Cell / mL Specificity 10 ⁶ CHO - K1 N / A Y CHO-DG44 N / A Y A9 N / A Y FRhK-4 N / A Y C8166 N / A Y Vero N / A Y MA104 N / A Y MPK N / A Y EBtr N / A Y Negative control N / A · N / A, Not Applicable; real-time PCR signals were not detected.

As a result, as shown in Table 15, FIG. 9, and FIG. 10, all of the nine cell lines were found not to respond to the probe.

From the above, it can be seen that the primer sets and probes of the present invention can be used for mycoplasma inoculation tests in human cell lines, CHO cell lines and CHO cell culture fluids. In addition, if the PCR is positive, it is essential to identify the contaminating strains through sequence analysis of the PCR result, so that the PCR test method of the present invention can be applied to actual processes.

TaqMan Real - Time PCR  Method of Linearity  And Reproducibility Experiment

In order to investigate the linearity and reproducibility of the optimized real-time PCR method, seven kinds of mycoplasma species, Acoreplasma ridrowii, Maikoplasma galliicepticum, Maikoplasmapermentans, Maikoplasma hyoriensis, Mycoplasma oleyl, M. each of the plasma pneumoniae, mycoplasma sinobi ^{1 × 10 4 CFU / mL,} 1 × 10 3 CFU / mL, 1 × 10 2 CFU / mL, 1 × 10 1 CFU / mL, 1 × 10 0 CFU / mL and 1 × 10 ^-1 CFU / mL, respectively. For the analysis of the results, the detected crossing point value was converted into CFU / mL, and the crossing point represents the number of cycles in which the PCR cycle enters the logarithmic phase.

Mycoplasma
species CFU / ml Regression curve
Y = -aX + b r ² 10 ⁴ 10 ³ 10 ² 10 ¹ 10 ⁰ 10 ^-1 A. laidlawii · 20.56 * 23.61 27.05 30.17 N / A Y = -3.178X + 30.007 0.997 · 20.63 23.56 26.77 29.50 N / A · 20.28 23.59 26.90 30.27 35.06 CV (%) · 0.90 0.10 0.52 1.39 · M. gallisepticum 19.12 22.64 26.07 29.11 N / A N / A Y = -3.319X + 32.634 0.999 19.21 22.70 26.11 29.43 32.39 N / A 19.57 22.69 26.05 29.65 32.57 N / A CV (%) · 0.65 0.27 0.20 0.41 · M. hyorhinis 18.71 21.88 25.49 28.77 N / A N / A Y = -3.346X + 32.343 0.997 19.03 21.90 25.00 28.66 32.65 N / A 18.67 21.72 25.33 28.86 32.60 34.78 CV (%) 1.04 0.45 0.98 0.34 0.10 · M. fermentans 21.74 25.28 28.72 32.31 34.86 N / A Y = -3.35X + 32.074 0.996 21.78 25.17 28.61 32.22 34.90 N / A 21.92 25.45 28.98 32.51 35.31 N / A CV (%) 0.43 0.55 0.66 0.45 0.71 · M. gallisepticum 19.12 22.64 26.07 29.11 N / A N / A Y = -3.458X + 35.729 0.99 19.21 22.70 26.11 29.43 32.39 N / A 19.57 22.69 26.05 29.65 32.57 N / A CV (%) 1.23 0.14 0.11 0.92 0.39 · M. pneoumoniae · 25.43 28.47 31.99 N / A N / A Y = -3.495X + 35.656 0.998 · 25.30 28.76 32.34 35.85 N / A · 25.01 28.46 32.06 35.62 N / A CV (%) · 0.85 0.59 0.57 0.45 · M. orale · 25.44 29.19 32.29 35.41 N / A Y = -3.291X + 32.669 0.999 · 25.49 28.66 31.74 36.6 N / A · 25.33 28.77 31.87 N / A N / A CV (%) · 0.32 0.96 0.89 2.33 · r ² : the coefficient of determination
* Values indicating crossing point (Cp) value
N / A, Not Applicable; real-time PCR signals were not detected

Linearity and reproducibility As a result of experiments, it was found that seven kinds of mycoplasma species, A. laidlawii , M. gallisepticum , M. fementans , Maiko Plasmus hyoriensis Crossing point for log CFU (CFU / mL; x) for M. hyorhinis , M. orale , M. pneumoniae , M. synoviae , It can be seen that the coefficient of determination (r ² ) between the values (y) is higher than 0.99 and the regression between the log CFU and the crossing point value is high, and the coefficient of variation (CV) is constant without change.

From the above, it can be seen that the optimized real-time taqman PCR method of the present invention is excellent in linearity and reproducibility.

TaqMan Real - Time PCR  Method robustness experiment

To determine how the optimized real-time PCR assay of the present invention is affected by the method parameters, a robustness experiment was performed. Therefore, we investigated whether changes in MgCl ₂ concentration and the effects of primer makers could affect the results even with small changes. For this purpose, mycoplasma hyorhinis was serially diluted with 1 × 10 ³ CFU / mL, 1 × 10 ² CFU / mL, and 1 × 10 ¹ CFU / mL and performed in triplicate. For the analysis of the results, the detected crossing point value was converted into CFU / mL. The crossing point represents the number of cycles in which the PCR cycle enters the logarithmic phase.

CFU / ml Cp values obtained with different MgCl ₂ concentrations Below optimal
concentration Optimal concentration Above optimal
concentration Mean of Cp SD of Cp CV (%) 1 x 10 ³ 22.47 22.24 22.37 22.41 21.76 22.08 21.88 21.65 21.80 22.07 0.31 1.41 1 x 10 ² 25.87 25.60 25.68 25.55 25.59 25.71 25.24 25.40 25.30 25.55 0.20 0.79 1 x 10 ¹ 29.38 31.07 28.93 29.05 29.04 29.06 29.14 28.89 28.87 29.27 0.69 2.36 Negative Control N / A N / A N / A N / A N / A N / A N / A N / A N / A N / A, Not Applicable; real-time PCR signals were not detected

CFU / ml Cp values obtained with different primer sets Vendor Vendor B Vendor C Mean of Cp SD of Cp CV (%) 1 x 10 ³ 21.82 21.93 21.66 22.19 21.91 21.70 22.41 21.76 22.08 21.94 0.24 1.12 1 x 10 ² 25.19 25.18 25.16 25.28 25.29 25.39 25.55 25.59 25.71 25.37 0.20 0.79 1 x 10 ¹ 28.73 28.91 28.46 29.06 28.93 29.02 29.05 29.04 29.06 28.92 0.20 0.70 Negative Control N / A N / A N / A N / A N / A N / A N / A N / A N / A N / A, Not Applicable; real-time PCR signals were not detected

As a result of the hardness test, as shown in Tables 17 and 18, MgCl ₂ And the primer manufacturer's variables.

From the above, it can be seen that the optimized real-time PCR method of the present invention shows high robustness and therefore the reliability obtained from the optimized real-time PCR method is high.

Prototype of Maiko Plasma  detection Kit ( MycoSure ^TM ) Produce

<10-1> Test method Quality management  for DNA positive control ( Plasmid DNA Production

Microplasma DNA positive control (Plasmid DNA) was prepared as follows. That is, the microplasma plasmid DNA positive control contains a region of the microplasma universal primer, and the region including the base 508 to the base 698 ( Acholeplasma laidlawii , GenBank: M23932.1) was cloned into a TA vector. The microplate plasmid DNA positive control was transformed into E. coli DH5α, and E. coli was cultured. The microplate plasmid DNA positive control was isolated from the E. coli culture using the QIAfilter ™ Plasmid Midi kit. The concentration of the positive control DNA was quantified by UV spectrophotometer. Based on the number of copies.

<10-2> Maiko Plasma  detection Of kit Formulation composition

In this example, the formulation of the mycoplasma detection kit was constructed with two box types (box 1 and box 2), and the specific formulation is shown in Table 19 below.

Package Description Cap color Stroage Inside box 1: Mycoplasma Taqman Probe Real-time PCR Reagent Mycoplasma Real-time PCR Primer Mix (10x) Red -15 to -25 ° C Mycoplasma Real-time PCR TaqMan probe (10x) Blue -15 to -25 ° C Real-time PCR Master Mix (2x) Black -15 to -25 ° C Negative control White -15 to -25 ° C Inside box 2: Mycoplasma Taqman Probe Real-time PCR DNA control Mycoplasma Real-time PCR DNA Positive Control Yellow -15 to -25 ° C Mycoplasma Real-time PCR DNA Spiking Positive Control Green -15 to -25 ° C Mycoplasma Real-time PCR External Control ( M. pneumoniae ) Purple -15 to -25 ° C

The real-time PCR primer mix (10x), the Real-time PCR TaqMan probe (10x), and the Real-time PCR master mix (2x) ) And negative control, and the cap color was discriminated by different colors for distinguishing each constituent (see FIG. 11A). Box 2 was constructed to contain the mycoplasma DNA positive control prepared in the above <10-2>. In addition, the box 2 contained DNA spiking positive control (plasmid DNA) for spiking control and inhibition control, M. pneumoniae standard strains may contain external control. box 2 also distinguishes the cap color by different colors for distinguishing each constituent (see FIG. 11B).

Prototype Myco Plasma Detection Kit (MycoSure ^TM ) Detection performance

<11-1> Microplasma  Sample preparation for negative test

Based on the Good PCR Practice, samples were prepared in the order listed in Table 20 below.

Sample Preparation Negative control · 15 μL of premix solution
· 5 μL of Negative control Unknown sample
(Test sample) · 15 μL of premix solution
· 5 μL of DNA extracted from test sample Spiking control · 15 μL of premix solution
· 5 μL of DNA extracted from test sample plus DNA positive control Inhibition control · 15 μL of premix solution
· 5 μL of DNA extracted from test sample
0.5 μL of DNA positive control (10 ³ copy / L) Mycoplasma DNA positive control · 15 μL of premix solution
5 μL of DNA positive control (10 ² copy / L) Mycoplasma external control · 15 μL of premix solution
5 μL of DNA extracted from Mycoplasma species

Spiking control is a control to check whether mycoplasma DNA has been extracted normally from a test sample. A specific amount of mycoplasma DNA positive control was added to the test sample and DNA was extracted and then real-time PCR was performed.

Inhibition control is a control to check whether real-time PCR is inhibited due to the substances present in the sample. Real-time PCR was performed after adding a specific amount of mycoplasma DNA positive control after extracting DNA from the sample .

<11-2> Criteria for Microplasma Negative Test

As shown in the following Table 21, micro-plasma negative test acceptance criteria were defined.

Criteria Sample Cp value Criteria for test samples
Negative sample N / A Positive sample Cp? 38 Criteria for controls Negative sample N / A Spiking control Cp? 38 Inhibition control Cp? 38 Mycoplasma DNA positive control Cp? 38 Mycoplasma external control Cp? 38 Spiking control Cp < 2 Inhibition control Cp < 2

&Lt; 11-3 > Maiko Plasma  detection Kit and  Conventional Maiko Plasma  detection Kit  Detection performance

In order to confirm the detection performance of the mycoplasma detection kit (MycoSure ™) according to the present invention, experiments were conducted as follows.

That is, the sensitivity test of the conventional mycoplasma detection kit (MycoSEQ ™) and the mycoplasma detection kit (MycoSure ™) according to the present invention was performed on the samples A, B, and C of the biopharmaceutical production process. 22, and Figs. 13 to 18. 13 to 15 show results of detection of mycoplasma of samples A, B and C using a conventional mycoplasma detection kit (MycoSEQ ™), respectively. FIGS. 16 to 18 show results of detection of mycoplasma And Mycoplasma detection results of Samples A, B and C using a detection kit (MycoSure). At this time, the workflow of the mycoplasma detection kit according to the present invention is shown in Fig.

Test article Result judgment
Conventional Mycoplasma Detection Kit (MycoSEQ) The mycoplasma detection kit (MycoSure ™) A Negative Negative B Negative Negative C Negative Positive

As shown in Table 22, samples A, B and C were judged to be negative in the conventional mycoplasma detection kit (MycoSEQ ™), while samples MyoSure ™ in the mycoplasma detection kit (MycoSure ™) , And the sample C was determined to be positive. From the results, it can be seen that the sensitivity of the mycoplasma detection kit (MycoSure ™) according to the present invention is better than that of the conventional detection kit.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

<110> Hannam University Institute for Industry-Academia Cooperation <120> Primer set, TaqMan probe, and real-time PCR method for the          detection of mycoplasma and real-time PCR method <130> PB13-11335 <150> 10-2012-0052168 <151> 2012-05-16 <160> 9 <170> Kopatentin 2.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> universal primer forward <400> 1 ggcgaatggg tgagtaacac g 21 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> universal primer reverse <400> 2 cggataacgc ttgcgaccta tg 22 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> M-16 primer forward <400> 3 ttttggtgga tgccttgg 18 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> M-16 primer reverse <400> 4 tttccccatt cggaaatc 18 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> MGSO primer forward <400> 5 gggagcaaac aggattagat accct 25 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> MGSO primer reverse <400> 6 tgcaccatct gtcactctgt taacctc 27 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> M. genus primer forward <400> 7 actcctacgg gaggcagcag ta 22 <210> 8 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> M. genus primer reverse <400> 8 tgcaccatct gtcactctgt taacctc 27 <210> 9 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> mycoplasma probe <400> 9 ygrcwaacta tgtgccagca gycgcg 26

Claims (8)

A method for determining the presence or absence of Mycoplasma contamination comprising the steps of:
(a) extracting genomic DNA from a sample;
(b) a primer set comprising the extracted genomic DNA, an forward primer consisting of the nucleotide sequence of SEQ ID NO: 1 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 2, , And a probe comprising a nucleotide sequence of SEQ ID NO: 9; and a step of preparing a real-time polymerase chain reaction solution containing the probe; And
(c) performing a real-time PCR reaction. The method according to claim 1,
Wherein the real-time PCR reaction solution of step (b) further comprises magnesium chloride (MgCl ₂ ). delete The method according to claim 1,
Wherein the probe has a sequence complementary to a mycoplasma labeled with a fluorescent reporter dye at the 5 'end and a quencher at the 3' end. 5. The method of claim 4,
The fluorescent colorant may be selected from the group consisting of carboxyfluorescein (FAM), 2,7-dimethoxy-4,5-dichloro-6-carboxyfluoroscein (JOE), 5-carboxytetramethylrhodamine (TAMRA), indocarbocyanine- 6-Carboxy-X-rhodamine (Rox). 5. The method of claim 4,
The quencher may be selected from the group consisting of Black Hole Quencher 1 (BHQ-1), Black Hole Quencher 2 (BHQ-2), Black Hole Quencher 3 (BHQ-3), 5-Carboxytetramethylrhodamine (TAMRA) deep dark Quencher 2 (DDQ2). The method according to claim 1,
The mycoplasma may be selected from the group consisting of A. laidlawii , M. gallisepticum , M. fermentans , M. hyorhinis , Characterized in that it is selected from the group consisting of plasma ores ( M. orale ), M. pneumoniae , and M. synoviae . A primer set consisting of the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2; And a probe comprising the nucleotide sequence of SEQ ID NO: 9.

Images