KR20210010162A - Primer and TaqMan probe set for detection of Minute virus of Mice and Method for detection of Minute virus of Mice Using the Same - Google Patents

Abstract

The present invention relates to a primer set and a TaqMan probe for detecting a mouse micro-virus, and a method for detecting a mouse micro-virus using the same. More specifically, the present invention relates to a primer and probe set for detecting a mouse micro-virus and comprising a normal direction primer capable of amplifying a mouse micro-virus, a reverse direction primer, and a TaqMan probe; and a mouse micro-virus detection kit and a mouse micro-virus detection method using the same. It has been confirmed that when a primer and probe set for detecting a mouse micro-virus is used, MVM including MVMi, MVMp, MVMm, and MVMc can be specifically detected, and it has been confirmed that virus detection sensitivity is superior to that of a commercially available mouse micro-virus detection kit. Thus, the primer and probe set can be useful for a kit and a method for detecting a mouse micro-virus.

Description

Primer and TaqMan probe set for detection of Minute virus of Mice and Method for detection of Minute virus of Mice Using the Same}

The present invention relates to a set of primers and TaqMan probes for detecting micro-mouse virus, and a method for detecting micro-mouse virus using the same, and more specifically, detection of micro-mouse virus consisting of a forward primer, reverse primer, and TaqMan probe capable of amplifying micro-mouse virus It relates to a primer and probe set for, a kit for detecting micro-mouse virus and a method for detecting micro-mouse virus using the same.

Rodent-derived cell lines, Chinese hamster ovary (CHO) cells and Baby hamster kidney (BHK) cells, are mainly used for the production of cell culture-derived biologics such as antibody drugs and gene recombinant protein therapeutics. These cells can be easily infected with the micro-mouse virus (Minute Virus of Mice; MVM), and cases of contamination of these cell lines in the production process of biopharmaceuticals have been reported.

The use of contaminated biological reagents such as animal serum components, the use of contaminated excipients in the formulation stage, the use of contaminated reagents in the process of handling cells, etc. may introduce adventitious viruses in the production process. There are also cases of contamination with adventitious viruses such as epizootic hemorrhagic disease virus (EHDV), minute virus of mice (MVM), and Reo (Reovirus type 3). Viruses such as the above are non-endogenous viruses introduced from the outside (non-endogenous virus; viruses introduced from outside during culture), of which MVM belongs to the parvovirus family and single-stranded DNA It is a non-enveloped virus with a 5 kb genome composed of.

In particular, MVM has very high resistance to physicochemical treatment such as heat, organic solvents, surfactants, acids and alkalis, and drying, so it is used as a model virus to verify the safety of the cell culture-derived biopharmaceutical production process. MVM's natural host is the house mouse ( Mus muscμLus ), and if adult rats are infected, they show asymptomatic, but if newborn rats or hamsters are infected, they may become deformed or die. MVM is involved in biological processes including cell differentiation, cell proliferation, and immunosuppression, and it has been reported that viral non-structural proteins exhibit cytotoxicity (Erickson, GA, et al ., DevBiol. Stand ., 75:173 -175, 1991).

Many test methods, such as an in vitro test method, an in vivo test method, and an antibody production test method, have been used for the detection of endogenous and adventitious viruses, but these biological tests have disadvantages that require a lot of time and cost. On the other hand, the PCR test method can amplify a small amount of DNA or RNA to detect viruses, so it is more sensitive than biological test methods that use cells or animals such as the in vitro test method, the in vivo test method, and the antibody production test method. It has a high advantage, and it can analyze a large number of samples in a short time, saving time and money compared to biological test methods. In addition, since the presence or absence of a virus is detected using a virus-specific primer, the specificity is very high (Donghyuk Lee et al . , Kor . J. Microbiol . Biotechnol . , 36:12-20, 2008).

In general PCR, which is an endpoint analysis, the product after the amplification reaction is mainly separated from an agarose gel and confirmed, and it is used as a method of quantitative analysis because it is roughly estimated, but there is a problem in real-time polymerase chain reaction. (real-time polymerase chain reaction, real-time RCR, hereinafter referred to as'real-time PCR') can quantify the amount of DNA or RNA amplified in real time by online fluorescence analysis, so a small amount of contaminated virus can be detected. Can be quantitatively analyzed.

On the other hand, TaqMan probes consist of a specific nucleotide sequence with a fluorophore at the 5'end and a quencher at the 3'end, and 5'→ 3 at the extension stage during the real-time PCR process. 'Taq DNA polymeras, which has exonuclease activity, emits light as the fluorophore and quencher drop. By measuring the amount of fluorescence emitted during this process in real time, the amount of amplified nucleic acid can be quantified.

In the present invention, as a result of earnest efforts to quantitatively detect MVM in cell lines, raw materials, manufacturing processes, and finished products during the biopharmaceutical manufacturing process using CHO cell lines, all MVMs including MVMi, MVMp, MVMm and MVMc are specifically detected. As a result of performing real-time PCR by designing a set of possible primers and a specific TaqMan probe based on this, it was confirmed that various types of MVM can be specifically detected, and the present invention was completed.

An object of the present invention is to provide a composition for detecting micro-mouse virus comprising a primer set capable of amplifying micro-mouse virus micro-mouse virus and a TaqMan probe.

Another object of the present invention is to provide a kit for detecting micro-mouse virus comprising the composition for detecting micro-mouse virus.

Another object of the present invention is to provide a method for detecting micro-mouse virus using the composition for detecting micro-mouse virus.

To achieve the above object,

The present invention detects a micro mouse virus (Minute Virus of Mice; MVM) including a forward primer represented by the nucleotide sequence of SEQ ID NO: 1, a reverse primer represented by the nucleotide sequence of SEQ ID NO: 2, and a probe represented by SEQ ID NO: 9 Primer and probe sets are provided.

According to a preferred embodiment of the present invention, the primer and probe set may detect one or more micro-mouse viruses selected from the group consisting of MVMi, MVMp, MVMm and MVMc.

According to another preferred embodiment of the present invention, the probe may be labeled with a fluorophore at the 5′ end and a quencher at the 3′ end.

According to another preferred embodiment of the present invention, the fluorophore is FAM (carboxyfluorescein), JOE (2,7-dimethoxy-4,5-dichloro-6-carboxyfluoroscein), TAMRA (5-Carboxytetramethylrhodamine), Cy3 (indocarbocyanine). -3), Cy5 (indocarbocyanine-5), Rox (6-Carboxy-X-rhodamine) may be selected from the group consisting of.

According to another preferred embodiment of the present invention, the matting agent is BHQ-1 (Black Hole Quencher 1), BHQ-2 (Black Hole Quencher 2), BHQ-3 (Black Hole Quencher 3), TAMRA (5- Carboxytetramethylrhodamine), DDQ1 (deep dark Quencher 1), DDQ2 (deep dark Quencher 2) may be selected from the group consisting of.

In addition, the present invention provides a kit for detecting micro-mouse virus comprising a set of primers and probes for detecting the micro-mouse virus.

According to a preferred embodiment of the present invention, the kit may further include a DNA polymerase, dNTP (dATP, dCTP, dGTP and dTTP) mixture, a buffer solution, and distilled water.

In addition, the present invention provides a method for detecting micro-mouse virus comprising the step of detecting a micro-mouse virus gene of an isolated biological sample by using the set of primers and probes for detecting micro-mouse virus.

It was confirmed that using the primer and probe set for detecting micro-mouse virus according to the present invention can specifically detect all MVMs including MVMi, MVMp, MVMm and MVMc, and for detecting micro-mouse viruses that are commercially available. Since it has been confirmed that the virus detection sensitivity is superior to that of the kit, it can be usefully used in kits and methods for detecting micro-mouse virus.

1 is data confirming the sensitivity of a real-time PCR kit to micro-mouse virus.
2 is data confirming the specificity of a real-time PCR kit according to each cell line when the primer and probe set for detecting the micro-mouse virus of the present invention is used.
3 is a real-time PCR result (a) using a primer and probe set for detecting micro-mouse virus of the present invention and a real-time PCR result (b) using a commercial kit (ViralSEQ ^® Mouse Minute Virus (MMV) Real-Time PCR kit) Is the data showing.

Hereinafter, the present invention will be described in detail.

In one aspect, the present invention includes a forward primer represented by the nucleotide sequence of SEQ ID NO: 1, a reverse primer represented by the nucleotide sequence of SEQ ID NO: 2, and a probe represented by SEQ ID NO: 9 (Minute Virus of Mice; MVM) to a set of primers and probes for detection.

In the present invention, the primer and probe set is characterized by detecting one or more micro-mouse viruses selected from the group consisting of MVMi, MVMp, MVMm and MVMc, but is not limited thereto, and various strains of micro-mouse viruses are used. Can be detected.

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

The "probe" of the present invention has a nucleotide sequence of SEQ ID NO: 9 capable of complementarily binding to MVM, and a fluorophore is labeled at the 5'end, and a quencher is labeled at the 3'end. do.

The fluorophore is FAM (carboxyfluorescein), JOE (2,7-dimethoxy-4,5-dichloro-6-carboxyfluoroscein), TAMRA (5-Carboxytetramethylrhodamine), Cy3 (indocarbocyanine-3), Cy5 (indocarbocyanine-5), Rox (6-Carboxy-X-rhodamine) may be selected from the group consisting of.

The matting agent is BHQ-1 (Black Hole Quencher 1), BHQ-2 (Black Hole Quencher 2), BHQ-3 (Black Hole Quencher 3), TAMRA (5-Carboxytetramethylrhodamine), DDQ1 (deep dark quencher 1), DDQ2 (deep dark Quencher 2) may be selected from the group consisting of.

In a specific embodiment of the present invention, various primer sets and TaqMan probes were produced based on the matching genes by aligning the base sequences of MVMs including MVMi, MVMp, MVMm and MVMc among the MVM genome sequences (Table 1 And Table 2), as shown in Table 3, only a primer set consisting of a forward primer represented by the nucleotide sequence of SEQ ID NO: 1 and a reverse primer represented by the nucleotide sequence of SEQ ID NO: 2, specifically detects MVM of various variants I confirmed that.

Accordingly, in the present invention, the forward primer represented by the nucleotide sequence of SEQ ID NO: 1, the reverse primer represented by the nucleotide sequence of SEQ ID NO: 2, and the probe represented by SEQ ID NO: 9 were finally selected for detection of micro-mouse virus.

In another aspect, the present invention relates to a kit for detecting micro-mouse virus comprising a set of primers and probes for detecting the micro-mouse virus.

In the present invention, the kit may further include a DNA polymerase, dNTP (dATP, dCTP, dGTP and dTTP) mixture, a buffer solution, and distilled water.

The kit may include at least one container including a compartmentalized carrier means for containing a sample, a container including a primer set and probe for MVM detection, a buffer for PCR reaction, and a container containing DNA polymerase, and the primer set It may include the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2, a functional combination, and a fragment thereof, and the probe may include the nucleotide sequence of SEQ ID NO: 9, a functional combination, and a fragment thereof. The carrier means is suitable for containing one or more containers, such as bottles and tubes, each container comprising the independent components used in the method of the present invention, and those of ordinary skill in the art can select the necessary formulation in the container. Can be easily distributed.

In addition, the kit of the present invention may further include a user's guide describing the optimum reaction performance conditions. The guide is a handout explaining how to use the kit, e.g., how to prepare the buffer, and the reaction conditions presented. The guide may include a brochure in the form of a brochure or flyer, a label affixed to the kit, and a description on the surface of the package containing the kit. In addition, the guide may include information disclosed or provided through an electronic medium such as the Internet.

In another aspect, the present invention relates to a method for detecting micro-mouse virus comprising the step of detecting a micro-mouse virus gene of an isolated biological sample by using the set of primers and probes for detecting micro-mouse virus.

Specifically, the micro mouse virus detection method

(a) extracting DNA from the sample;

(b) Using the extracted DNA as a template, real-time polymerase chaining using a forward primer represented by the nucleotide sequence of SEQ ID NO: 1, a reverse primer represented by the nucleotide sequence of SEQ ID NO: 2, and a probe represented by SEQ ID NO: 9 It can be carried out by a method comprising; performing a reaction.

The "sample" is, for example, cultured cells, proteins, nucleic acids, or biological pharmaceuticals containing the same, the brain, eye, heart, intestine of an animal or human. , Kidney, liver, lung, muscle, spleen, or tissue obtained from testis, blood, plasma, serum ( It may be a body fluid such as serum), urine, saliva, sweat, semen, or mucus, but is not limited thereto.

The method of extracting the DNA (genomic DNA) may be performed through various methods known in the art (Sambrook et al ., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press. 2001), and is limited to any one method. It doesn't work.

The step (b) is performed by preparing a real-time polymerase chain reaction solution containing the primer and probe set of the present invention, and the real-time polymerase chain reaction solution is a PCR template using the DNA extracted from the sample in step (a). Included as DNA. The polymerase chain reaction solution is a DNA polymerase (e.g., Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis or a thermally stable DNA polymerase obtained from Pyrococcus furiosus (Pfu)), dNTP mixture (dATP, dCTP, dGTP, dTTP), a PCR buffer, and a DNA polymerase cofactor may be included.

In the present invention, the real-time polymerase chain reaction solution of step (b) may further include MgCl ₂ , and according to a preferred embodiment of the present invention, the final concentration of the real-time polymerase chain reaction solution is 1 mM to 10 mM. It may include MgCl ₂ as possible, but is not limited thereto.

In the present invention, the polymerase chain reaction or the real-time polymerase chain reaction is performed by repeating a cycle including a denaturation step, an annealing step and/or an elongation step several times. The temperature and time conditions in the denaturation, bonding, and elongation steps may be selected by a person skilled in the art, but are not limited thereto, but according to a preferred embodiment of the present invention, the bonding temperature may be selected as 60°C.

In another specific embodiment of the present invention, real-time PCR optimization conditions were established in order to more effectively detect micro-mouse virus using the selected primer and probe set.

As shown in Example 2 and Tables 4 to 7 of the present invention, optimal conditions were established for PCR master mix, PCR mixture composition, annealing temperature, and denaturation time, and primers of the present invention And a real-time PCR kit for detecting micro-mouse virus using a probe set was prepared.

In another specific embodiment of the present invention, as a result of confirming the remodeling and detection limit of the real-time PCR kit for detecting micro-mouse virus, as shown in Table 8, the coefficient of variation (CV%) was confirmed to be 2.98 or less, It was confirmed that the detection limit was 1×10 ⁰ copies/µl. That is, it was confirmed that the real-time PCR kit for detecting micro-mouse virus of the present invention has excellent reproducibility and detection limit.

In another specific embodiment of the present invention, when PCR is performed under the conditions established above using the real-time PCR kit for detecting micro-mouse virus of the present invention, in order to confirm whether only micro-mouse virus can be selectively and accurately detected. Specificity for CHO-K1, CHO-DG44, MDCK, Vero cell lines, which are generally known as biological/biotechnological drug production cell lines, and other human and animal cell lines, A9, FRhk-4, C8166, MA104, MPK, EBTr cell lines Experiments and robustness experiments were performed. As a result, as shown in FIG. 2 and Table 9, it was confirmed that micro-mouse virus was not detected in all of the 10 cell lines, and as shown in Table 10, the primer and probe set of the present invention had robustness. Confirmed that there is. That is, it was confirmed that the primer and probe set of the present invention can be applied to the MVM negative test in the production of a drug derived from actual cell culture.

In another specific embodiment of the present invention, in order to confirm the completeness of the real-time PCR kit for detecting micro-mouse virus of the present invention, a real-time PCR kit for detecting micro-mouse virus (ViralSEQ ^® Mouse Minute Virus (MMV) sold commercially) Real-Time PCR Detection Kit) and comparative experiments were performed. As a result, as shown in Fig. 3 and Table 11, it was confirmed that the kit of the present invention has a better detection effect and sensitivity than a commercial kit.

Hereinafter, the present invention will be described in more detail through examples.

These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples.

Primer/probe design and selection for detection of micro-mouse virus

1-1: Primer and probe design

Primer 3 software based on matching genes by aligning MVM sequences registered in the NCBI database to design primers that can amplify all MVMs including four variants of MVMi, MVMp, MVMm and MVMc The primer set and TaqMan probe were designed using the program.

The probe sequence was designed to be labeled with a fluorophore, FAM (carboxyfluorescein), at the 5′ end of the probe sequence, and BHQ-1 (Black Hole Quencher 1), a quencher at the 3′ end. The designed probe is MVMi (GenBank: M12032.1), MVMp (GenBank: V01115.1), MVMm (GenBank: DQ196317.1) and MVMc (GenBank: U34256.1) four variants of MVM DNA sequence alignment It is composed of a mixed base to cover all parts with mismatches. The nucleotide sequences of the designed primer sets and probes are described in Table 1 below, and the amplification positions of the MVM gene by the primers and probes are shown in Table 2 below.

Primers and probes for detection of micro-mouse virus Primer set Sequences Sequence number 1 primer set Forward ACC AAT CTA CCA TGG CAA GC One Reverse CCG CAA CAG GAG TAT TTG GT 2 2 primer set Forward CAG CAA GCA GAT TGA ACC AA 3 Reverse CAA CCA AGC ACA AAT CAT GG 4 3 primer set Forward CAT GAT TTG TGC TTG GTT GG 5 Reverse TGG CTG AGA GGC GTA CTT TT 6 4 primer set Forward AGT TTG CCA TGC TAT TTG C 7 Reverse ACT GGT TTA CTT GCT GTC C 8 MVM probe FAM-CCG AAA AGT ACG CCT CTC AG-BHQ1 9

Amplification site of primer and probe for detection of micro-mouse virus GenBank No. MVMi
(M12032.1) MVMp
(V01115.1) MVMm
(DQ196317.1) MVMc
(U34256.1) 1 primer set 1889-1908
2103-2122 1888-1907
2102-2121 1774-1793
1988-2007 1749-1768
1963-1982 2 primer set 1677-1696
1856-1875 1676-1695
1855-1874 1562-1581
1741-1760 1537-1556
1716-1735 3 primer set 1857-1876 2020-2039 1856-1875
2019-2038 1742-1761
1905-1924 1717-1736
1880-1899 4 primer set 1389-1407 1606-0624 1388-1406
1605-1623 1274-1292
1491-1509 1249-1267
1466-1482 MVM probe 2017-2036 2016-2035 1902-1921 1877-1896

1-2: Culture and quantification of MVM

A9 cells (ATCC CCL-1.4) were used for the culture and quantification of MVM (ATCC VR-1346). A9 cells were cultured in DMEM (Dulbecco's Modified Eagles Medium; Hyclone) medium to which 10% FBS (Hyclone) was added. After each virus was infected with the monolayer cells cultured in a T-150 flask, the cytopathic effect (CPE) was observed periodically. When CPE was clearly observed, the freezing and thawing processes were repeated three times to crush the pellet, and then centrifuged at 2,000 rpm for 3 minutes to obtain a supernatant. The supernatant was filtered through a 0.45 μm filter, then subdivided and stored at -70°C.

For the quantification of MVM, the titer of the infectious virus was expressed as TCID ₅₀ (50% tissue culture infectious dose). MVM was diluted 7 times using DMEM medium containing 2% FBS, and then inoculated into 24 well-plates by 0.25 mL. 0.25 mL of the cell culture medium was inoculated as a negative control, and then CPE was continuously observed under a microscope while culturing in an incubator at 35° C. and 5% CO ₂ .

1-3: Primer and probe selection

In the present invention, in order to select the optimal primer set among the primer sets prepared as shown in Table 1, a real-time PCR system device (7500 Fast Real-Time PCR System, Thermo Fisher scientific, USA) was used to perform a TaqMan probe real-time PCR method. The detection effectiveness was compared.

First, DNA extraction of MVM was isolated using the Prep-sure™ Genomic DNA Extraction Kit (BioPS, Korea) according to the manufacturer's method. Then, 2 μl of extracted MVM DNA, 10 μl of 2× Realtime PCR Master Mix (TOYOBO, Japan), 1 μl of 10 pmol forward primer, 1 μl of 10 pmol reverse primer, 0.5 μl of 10 pmol TaqMan probe, nuclease-free distilled water (Nuclease-Free Water) 5.5 µl was added and the final volume was adjusted to 20 µl, and real-time PCR was performed. The nucleic acid amplification reaction was carried out for 40 cycles under conditions of pre-incubation at 95°C for 2 minutes, then denaturation at 95°C for 30 seconds and annealing at 60°C for 30 seconds.

Validation of detection of each primer set Primer set selection (Ct value) TCID ₅₀ /mL 1 x 10 ⁴ 1 x 10 ³ 1 x 10 ² 1 x 10 ¹ 1 x 10 ⁰ Negative control 1 primer set 24.94 24.64 29.33 29.30 32.60 32.12 37.11 35.26 35.51 N/A N/A N/A 2 primer set 25.88 26.48 29.82 30.76 33.54 33.25 36.57 32.81 37.06 37.73 36.67 36.43 3 primer set 24.23 24.28 28.39 28.52 32.28 31.75 34.74 34.91 35.74 35.35 35.07 36.73 4 primer set 24.64 24.84 28.58 28.83 31.43 32.71 35.42 35.37 35.98 35.33 N/A 36.79 Values indicate threshold cycle (Ct) value
N/A, Not Applicable; real-time PCR signals were not detected

As shown in Table 3, as a result of comparing the Ct value of each primer set according to the MVM concentration (Titer, TCID ₅₀ ), all primer sets were detectable up to 10 ¹ TCID ₅₀ /ml, except for 1 primer set. Ct values were detected in the negative control. Based on this result, 1 primer set was selected.

Establishment of real-time PCR optimal conditions

2-1: PCR master mix selection

In order to more effectively detect micro-mouse virus using the selected primer and probe set, it was attempted to establish optimal conditions for real-time PCR.

First, in order to select the PCR master mix, the detection effectiveness of 5 commercially available PCR master mixes was compared. The five PCR master mixes are as follows, and a mixture was prepared for each of the five PCR master mixes.

1) 2 × Realtime PCR Master Mix (TOYOBO, Japan),

2) 2 × Premix Ex Taq™ (Probe qPCR, TAKARA, Japan),

3) 2 × AccuPower ^® DualStar™ qPCR PreMix (BIONEER, Korea),

4) 2 × Multi-Star Real-Time PCR Master Mix (For Probe, BIOFACT, Korea),

5) TOPreal™ qPCR 2 × PreMIX (Enzynomics, Korea).

2 µl of extracted MVM DNA, 10 µl of 2 × PCR master mix, 1 µl of 10 pmol forward and reverse primer mix, 0.5 µl of 10 pmol TaqMan probe, 6.5 µl of Nuclease-Free Water were added, and the final Real-time PCR was performed by adjusting the volume to 20 μl. Nucleic acid amplification reaction was performed under the conditions of pre-incubation at 50°C for 4 minutes and 95°C for 10 minutes, then denaturation at 95°C for 30 seconds, and annealing at 60°C for 30 seconds. Cycle was performed.

Validation of detection according to PCR master mix type PCR master mix selection (Ct value) TCID ₅₀ /mL 1 x 10 ³ 1 x 10 ² 1 x 10 ¹ 1 x 10 ⁰ Negative control Realtime PCR Master Mix 26.17 26.19 29.82 29.84 32.88 32.90 37.81 35.92 N/A N/A Premix Ex Taq™ 27.47 27.84 30.87 31.08 33.86 33.51 38.42 38.05 N/A N/A AccuPower ^® DualStar™ qPCR PreMixr set 24.28 23.06 27.75 28.36 30.16 31.74 33.72 33.94 35.07 36.73 Multi-Star Real-Time PCR Master Mix 25.46 25.23 28.60 28.53 32.63 32.52 36.54 37.56 N/A N/A TOPreal™ qPCR 2 × PreMIX 34.83 35.01 39.19 38.93 N/A N/A N/A N/A N/A N/A Values indicate threshold cycle (Ct) value
N/A, Not Applicable; real-time PCR signals were not detected

As shown in Table 4, as a result of comparing the Ct value of each PCR master mix according to the MVM concentration (Titer, TCID ₅₀ ), when compared with the foreign PCR master mix, Multi-Star Real-Time in the domestic PCR master mix PCR Master Mix (For Probe, BIOFACT, Korea) showed equal or better results, and the other two products showed Ct values or low sensitivity in the negative control.

Based on the above results, the PCR master mix was selected as domestic, which is easy to secure, and the Multi-Star Real-Time PCR Master Mix was selected.

2-2: PCR Mixture composition selection

PCR Mixture was prepared with three compositions, and the effectiveness of each composition was compared.

The composition of each of the three is as follows, and the remaining additives are added equally to 2 µl of MVM DNA and 10 µl of 2 × PCR master mix, and the final volume is determined using Nuclease-Free Water. Adjusted to 20 μl.

1) 1 μl of 10 pmol TaqMan probe in 2 μl of 10 pmol forward primer and reverse primer mix,

2) 1.5 µl of 10 pmol TaqMan probe to 1.5 µl of 10 pmol forward primer and reverse primer mix,

3) 0.5 μl of 10 pmol TaqMan probe to 2.5 μl of 10 pmol forward primer and reverse primer mix.

Nucleic acid amplification reaction was performed for 40 cycles under conditions of pre-incubation at 95°C for 10 minutes, then denaturation at 95°C for 30 seconds and annealing at 60°C for 30 seconds.

Confirmation of detection effectiveness according to PCR Mixture composition PCR Mixture condition selection (Ct value) TCID ₅₀ /mL 1 x 10 ³ 1 x 10 ² 1 x 10 ¹ 1 x 10 ⁰ Negative control 2 µl of Primer mix
1 μl of probe 24.45 24.63 28.41 28.51 31.63 31.42 35.56 34.89 N/A N/A 1.5 µl of Primer mix
Probe 1.5 µl 24.86 24.94 28.37 28.76 31.58 31.64 34.95 35.26 N/A N/A Primer mix 2.5 µL
0.5 μl of probe 25.14 24.71 28.67 28.19 31.96 31.36 35.83 35.89 N/A N/A Values indicate threshold cycle (Ct) value
N/A, Not Applicable; real-time PCR signals were not detected

As shown in Table 5, as a result of comparing the Ct value of each PCR Mixture composition according to the MVM concentration (Titer, TCID ₅₀ ), the Ct values according to the three PCR Mixture compositions were confirmed very similarly, and the second composition Phosphorus 10 pmol forward primer and reverse primer mix 1.5 µl and 10 pmol TaqMan probe 1.5 µl were selected.

2-3: Annealing temperature setting

The effectiveness of each binding temperature was compared based on the primer set and PCR Mixture composition selected according to the results of Example 1, Examples 2-1 and 2-2. The bonding temperature was set to 56°C, 58°C, and 60°C for comparative experiments. Nucleic acid amplification reaction was performed under conditions of pre-incubation at 95°C for 10 minutes, then denaturation at 95°C for 30 seconds, and annealing at 56°C, 58°C and 60°C for 30 seconds, respectively. 40 cycles were performed.

Validation of detection according to bonding temperature Annealing temperature selection (Ct value) TCID ₅₀ /mL 1 x 10 ³ 1 x 10 ² 1 x 10 ¹ 1 x 10 ⁰ Negative control Annealing temperature 56℃ 24.01 24.01 28.08 28.08 31.64 31.58 35.49 35.41 N/A N/A Annealing temperature 58℃ 24.55 24.34 28.14 28.07 31.29 31.62 34.78 34.78 N/A N/A Annealing temperature 60℃ 24.86 24.94 28.37 28.76 31.58 31.64 34.95 35.26 N/A N/A Values indicate threshold cycle (Ct) value
N/A, Not Applicable; real-time PCR signals were not detected

As shown in Table 6, as a result of comparing the Ct values for each bonding temperature according to the MVM concentration (Titer, TCID ₅₀ ), the Ct values according to the three bonding temperatures were very similar, and the bonding temperature was selected as 60°C. I did.

2-4: Set the reaction time of the denaturation step

Based on the primer set and PCR Mixture composition selected according to the results of Example 1, Examples 2-1 and 2-2, the effectiveness of the denaturation step by reaction time was compared. The denaturation time was set to 10 seconds, 20 seconds, and 30 seconds to perform comparative experiments. Nucleic acid amplification reaction was performed under conditions of pre-incubation at 95°C for 10 minutes, followed by denaturation at 95°C for 10 seconds, 20 seconds, 30 seconds, and annealing at 60°C for 30 seconds. 40 cycles were performed.

Confirmation of detection effectiveness according to reaction time of denaturation step Denaturation time selection (Ct value) TCID ₅₀ /mL 1 x 10 ³ 1 x 10 ² 1 x 10 ¹ 1 x 10 ⁰ Negative control Denaturation time
10 sec 25.71 25.41 29.16 29.34 32.12 31.95 35.00 N/A N/A N/A Denaturation time
20 sec 24.95 25.28 28.60 28.32 32.12 32.17 35.43 35.12 N/A N/A Denaturation time
30 sec 24.86 24.94 28.37 28.76 31.58 31.64 34.95 35.26 N/A N/A Values indicate threshold cycle (Ct) value
N/A, Not Applicable; real-time PCR signals were not detected

As shown in Table 7 and FIG. 1, as a result of comparing the Ct values for each bonding temperature according to the MVM concentration (Titer, TCID ₅₀ ), the Ct values according to the three denaturation times were very similar, and the denaturation time was 30 It was selected as seconds.

As shown in Tables 4 to 7 above, optimal conditions were established for the PCR master mix, PCR mixture composition, annealing temperature, and denaturation time, and the primer and probe set of the present invention were used. A kit for detecting mouse virus was prepared.

Reproducibility and detection limit experiment using MVM detection kit

In the present invention, the detection limit indicating the reproducibility of the MVM detection kit and the minimum amount of detectable nucleic acid was confirmed.

MVM plasmid DNA was prepared and quantified in copies/µl and used as a positive control, and MVM plasmid DNA was used as 1×10 ² copies/µl, 1×10 ¹ copies/µl, and 1×10 ⁰ copies/µl, respectively. It was carried out by serial dilution. At least three serially diluted panels were independently prepared and repeated 24 times for each dilution multiple to obtain results, and statistical analysis was performed on the obtained results.

Check reproducibility and detection limits of MVM real-time PCR kit Run Copies/µl Limit of detection 1 × 10 ² 1 × 10 ¹ 1 × 10 ⁰ One 31.79 31.47 31.33 34.10 31.30 34.61 36.51 36.28 35.33 10 ⁰ copies/µl 2 32.32 32.20 31.99 33.55 35.21 35.92 38.46 38.64 38.95 10 ⁰ copies/µl 3 30.43 30.21 30.09 33.50 33.74 33.79 36.99 37.21 37.18 10 ⁰ copies/µl 4 30.38 29.90 30.21 33.50 33.46 33.58 37.82 37.28 37.36 10 ⁰ copies/µl 5 30.50 30.18 30.05 33.62 33.78 33.60 37.07 37.25 37.52 10 ⁰ copies/µl 6 30.43 30.15 30.13 33.37 33.74 33.94 37.57 37.34 37.60 10 ⁰ copies/µl 7 30.38 29.98 29.80 33.56 33.73 33.50 37.08 36.99 36.20 10 ⁰ copies/µl 8 29.38 29.14 29.07 32.93 32.59 32.50 36.11 36.15 35.59 10 ⁰ copies/µl SD 0.91 0.87 0.89 AVE 30.48 3.63 37.10 CV(%) 2.98 2.60 2.40

As a result, as shown in Table 8, it was confirmed that the coefficient of variation (CV%) was 2.98 or less, and the detection limit was 1×10 ⁰ copies/µl. From the above, it was confirmed that the optimized real-time PCR method of the present invention has excellent reproducibility and detection limits.

MVM detection kit specificity test

In the present invention, a specificity experiment was performed to determine whether only MVM nucleic acids can be selectively and accurately detected even under conditions in which other foreign substances are present. Specificity tests were performed on the CHO-K1, CHO-DG44, MDCK, and Vero cell lines, which are biotechnology drug production cell lines, and the A9, FRhk-4, C8166, MA104, MPK, and EBTr cell lines, which are human and animal cell lines. I did. At this time, DNA was extracted from 1×10 ⁶ cells/ml of all cell lines, and real-time PCR was performed.

Confirmation of specificity of real-time PCR kit for MVM detection Species Cell/mL Specificity 10 ⁶ CHO-K1 N/A N/A N/A Y CHO-DG44 N/A N/A N/A Y MDCK N/A N/A N/A Y Vero N/A N/A N/A Y A9 N/A N/A N/A Y FRhK-4 N/A N/A N/A Y C8166 N/A N/A N/A Y MA104 N/A N/A N/A Y MPK N/A N/A N/A Y EBTr N/A N/A N/A Y Positive control
(MVM plasmid DNA) 27.84 27.83 27.67 Y Negative control N/A N/A N/A Y N/A, Not Applicable; real-time PCR signals were not detected.

As a result, as shown in Table 9 and FIG. 2, it was found that all of the total 10 cell lines did not respond to the kit of the present invention. From the above, it was confirmed that the primer set and probe of the present invention and the MVM detection method using the same can be applied to an actual process for negative MVM testing in human cell lines, CHO cell lines and CHO cell cultures.

Robustness test of MVM detection kit

In order to find out the robustness of the MVM detection kit of the present invention, 3×10 ⁰ copies/µl, which is a concentration equivalent to 3 times the detection limit of 1×10 ⁰ copies/μL, for 20 randomly selected negative samples. After addition, real-time PCR was performed using a kit for MVM detection.

Confirmation of the robustness of the MVM detection kit Run Ct value 3×10 ⁰ copies/µl Negative Control One 33.60 NA 2 32.75 NA 3 33.11 NA 4 33.59 NA 5 33.58 NA 6 33.24 NA 7 32.49 NA 8 33.06 NA 9 33.72 NA 10 33.45 NA 11 33.94 NA 12 34.25 NA 13 33.39 NA 14 33.34 NA 15 32.71 NA 16 32.36 NA 17 33.49 NA 18 33.62 NA 19 33.72 NA 20 34.00 NA SD of Ct 0.50 Mean of Ct 33.37 CV (%) 1.50

As a result of the robustness experiment, as shown in Table 10, it was confirmed that the MVM plasmid DNA was detected in all samples used in the test, and thus the robustness was present.

Comparison of the kit for MVM detection of the present invention and the kit for commercial MVM detection

In order to find out the completeness of the kit for MVM detection of the present invention, a comparative experiment was performed with the ViralSEQ ^® Mouse Minute Virus (MMV) Real-Time PCR Detection Kit sold on the market.

After adding 900 µl of MDCK cells to three tubes, 100 µl of MVM of 1×10 ³ TCID ₅₀ /ml, 1×10 ² TCID ₅₀ /ml, and 1×10 ¹ TCID ₅₀ /ml were added to each tube. Then, DNA was extracted using each kit, and real-time PCR was performed using a 7500 Fast Real-Time PCR instrument. As a positive control, a positive control included in each kit and an MVM of 1×10 ³ TCID ₅₀ /ml were used.

MVM detection kit of the present invention and commercial MVM detection kit comparison result Kit Sample name Ct value MVM Real-Time PCR Kit 1×10 ² TCID ₅₀ /ml 31.59 31.72 31.91 1×10 ¹ TCID ₅₀ /ml 34.13 35.13 34.60 1×10 ⁰ TCID ₅₀ /ml 35.25 34.82 35.97 PC (plasmid DNA) 30.21 30.56 29.97 PC (TCID ₅₀ /ml) 28.65 28.75 29.16 NC N/A N/A N/A ViralSEQ ^® Mouse Minute Virus (MMV) Real-Time PCR Detection Kit 1×10 ² TCID ₅₀ /ml 32.64 32.68 32.53 1×10 ¹ TCID ₅₀ /ml 36.49 36.70 36.69 1×10 ⁰ TCID ₅₀ /ml N/A N/A N/A PC (plasmid DNA) 28.98 28.90 28.81 PC (TCID ₅₀ /ml) 28.86 28.04 28.88 NC N/A N/A N/A N/A, Not Applicable; real-time PCR signals were not detected.
PC: Positive control, NC: Negative control.

As a result of the comparative experiment, as shown in Table 11 and FIG. 3, it was confirmed that the commercial MVM detection kit can detect up to 1 × 10 ¹ TCID ₅₀ / ㎖, whereas the MVM detection kit of the present invention is 1 × 10 ⁰ TCID. _Since it was confirmed that detection was possible up to ₅₀ /ml, it was confirmed that the kit of the present invention has a detection capability equal to or higher than that of a commercial kit.

<110> BioPS Co., Ltd. Hannam University Institute for Industry-Academia Cooperation Korea Research Institute of Chemical Technology <120> Primer and TaqMan probe set for detection of Minute virus of Mice and Method for detection of Minute virus of Mice Using the Same <130> 1065967 <160> 9 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer set 1_F <400> 1 accaatctac catggcaagc 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer set 1_R <400> 2 ccgcaacagg agtatttggt 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer set 2_F <400> 3 cagcaagcag attgaaccaa 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer set 2_R <400> 4 caaccaagca caaatcatgg 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer set 3_F <400> 5 catgatttgt gcttggttgg 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer set 3_R <400> 6 tggctgagag gcgtactttt 20 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer set 4_F <400> 7 agtttgccat gctatttgc 19 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer set 4_R <400> 8 actggtttac ttgctgtcc 19 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MVM probe <400> 9 ccgaaaagta cgcctctcag 20

Claims (8)

A primer for detecting a micro mouse virus (Minute Virus of Mice; MVM) comprising a forward primer represented by the nucleotide sequence of SEQ ID NO: 1, a reverse primer represented by the nucleotide sequence of SEQ ID NO: 2, and a probe represented by SEQ ID NO: 9, and Probe set.
The group consisting of MVMi (GenBank: M12032.1), MVMp (GenBank: V01115.1), MVMm (GenBank: DQ196317.1) and MVMc (GenBank: U34256.1) according to claim 1, wherein the primer and probe set A set of primers and probes for detecting micro-mouse virus, characterized in that detecting at least one micro-mouse virus selected from.
The set of primers and probes according to claim 1, wherein the probe is labeled with a fluorophore at the 5′ end and a quencher at the 3′ end.
The method of claim 3, wherein the fluorophore is FAM (carboxyfluorescein), JOE (2,7-dimethoxy-4,5-dichloro-6-carboxyfluoroscein), TAMRA (5-Carboxytetramethylrhodamine), Cy3 (indocarbocyanine-3), Cy5 ( Indocarbocyanine-5), Rox (6-Carboxy-X-rhodamine), characterized in that selected from the group consisting of a set of primers and probes for detecting micro-mouse virus.
The method of claim 3, wherein the matting agent is BHQ-1 (Black Hole Quencher 1), BHQ-2 (Black Hole Quencher 2), BHQ-3 (Black Hole Quencher 3), TAMRA (5-Carboxytetramethylrhodamine), DDQ1 (deep Dark Quencher 1), DDQ2 (deep dark Quencher 2), characterized in that selected from the group consisting of a set of primers and probes for detecting micro-mouse virus.
A kit for detecting micro-mouse virus comprising a set of primers and probes for detecting micro-mouse virus according to any one of claims 1 to 5.
According to claim 6, The kit is a DNA polymerase, dNTP (dATP, dCTP, dGTP and dTTP) mixture, a buffer solution (buffer solution) and distilled water, characterized in that it further comprises a micro-mouse virus detection kit.
A method for detecting micro-mouse virus comprising the step of detecting a micro-mouse virus gene of an isolated biological sample by using the primer and probe set for detecting the micro-mouse virus of any one of claims 1 to 5.

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