CROSS-REFERENCE TO RELATED APPLICATION

This application is a national stage application under 35 U.S.C. § 371 of International Application No. PCT/KR2019/005898, filed internationally on May 17, 2019, which claims priority to and the benefit of Korean Patent Application No. 10-2018-0057283, filed on May 18, 2018, the disclosures of which are incorporated herein by reference in their entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 717572002800SeqList.TXT, created Nov. 13, 2020, which is 2,344 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.

BACKGROUND

1. Field of the Invention

The present invention relates to a method for detectingmycoplasma, which can sensitively determine whether the infection ofmycoplasmahas occurred by using mitochondrial DNA isolated from the cell pellet of a cell infected withmycoplasmaas an internal control sample.

2. Discussion of Related Art

Mycoplasmais known to be the simplest primitive cell that lives independently among the known organisms. Since it was discovered in 1898,mycoplasmahas been reported as a pathogen that can be found in a wide range of nature such as animals including humans, birds, sewage or the like, and it can cause inflammation mainly in mammals and birds.Mycoplasmalacks a cell wall, and the outer wall is composed only of a cell membrane, and thus it is known to have specific antibiotic susceptibility unlike general microorganisms having a cell wall.

Sincemycoplasmais capable of self-replication in artificial media, it has been reported that it inhabits a culture medium during cell culture and cells to be cultured can be infected as host cells around the nucleus. Specifically, it is known that during cell culture, a range of 5% to 35% of cell lines are mainly contaminated by 6 types ofmycoplasmasuch asMycoplasma arginini, M. fermentans, M. hyorhinis, M. oraleorAcholeplasma laidlawii. For the main causes of such contamination, it may be caused by animal tissue used in the primary culture, serum used during culture, the experimenter or the like, and contamination may spread to other cell lines due to cross-contamination of cell lines in the laboratory.

Accordingly, the development ofmycoplasmadetection methods is in progress for the purpose of confirming whether contamination bymycoplasmaor infection ofmycoplasmahas occurred in an experimental environment dealing withmycoplasmaor in a patient suspected ofmycoplasmainfection, and various commercial kits have been developed. Since the existingmycoplasmadetection methods using such commercial kits use a method of detectingmycoplasmathat is contaminated in a cell culture solution or secreted from host cells infected bymycoplasma, it can merely detect the presence or absence ofmycoplasmathat is infected in a culture medium, and ultimately, it cannot directly confirm whether the infection ofmycoplasmahas occurred inside cells that are being cultured, and thus it has a limitation that the sensitivity of detection is reduced. In addition, when conventional commercialized detection kits are used, for quantitative analysis duringmycoplasmadetection, quantitative analysis is performed based on the number of amplification cycles during a PCR. Since it is greatly affected by the background, DNA bands may appear during electrophoretic analysis after the PCR, even when the DNA ofmycoplasmathat is used as an amplification target is low in quantity or not included, and thus there is a limitation that the reliability of detection results is low. Therefore, for confirmation of the presence ofmycoplasmainfected in the cell and quantitative analysis thereof, even though it is expected that a method of directly lysing a cell and confirming whethermycoplasmais detected against DNA in the cell can improve reliability, studies related thereto have not been reported yet.

As such, the present inventors have spent efforts to develop amycoplasmadetection method that is cheaper with increased detection sensitivity by directly confirming whether the DNA ofmycoplasmainfected in a host cell is included, and as a result, the present invention was completed by confirming that sensitivity was improved during the detection ofmycoplasmaand detection experiments could also be carried out at a lower cost compared to existing commercialized kits, through a PCR amplification method comprising lysing a host cell infected withmycoplasmaand using the same as a PCR template and simultaneously using mitochondrial DNA of the host cell as an internal control sample.

RELATED ART DOCUMENTS

Non-Patent Documents

(Non-Patent Document 1) Uphoff CC, Drexler HG. (2002) Comparative PCR analysis for detection ofmycoplasmainfections in continuous cell lines.In Vitro Cell Dev Biol Animl.38(2): 79-85.(Non-Patent Document 2) Uphoff CC, Drexler HG. (2004) DetectingMycoplasmacontamination in cell cultures by polymerase chain reaction.Methods Mol Med.88: 319-326.

SUMMARY OF THE INVENTION

Accordingly, an object of the present invention is to provide a method for detectingmycoplasma, in which sensitivity is improved compared to existingmycoplasmadetection methods, and the degree of infection can be quantitatively confirmed through establishing an internal control sample.

In order to achieve the above object, the present invention provides a method for detectingmycoplasma, comprising the following steps i) to iii):

i) extracting DNA from a host cell isolated from a subject suspected of being infected withmycoplasma;

ii) performing polymerase chain reaction (PCR) using the DNA extracted in step i) as a template; and

iii) confirming a DNA band by electrophoresis of the PCR product performed in step ii).

In one preferred exemplary embodiment of the present invention, the host cell of step i) may be a cell derived from human.

In one preferred exemplary embodiment of the present invention, the PCR of step ii) may amplify a target sequence using a primer set for amplifyingmycoplasmaDNA; and a primer set for amplifying mitochondrial DNA of a host cell.

In addition, the primer set for amplifyingmycoplasmaDNA may comprise a forward primer comprising a nucleotide sequence of SEQ ID NO: 1; a forward primer comprising a nucleotide sequence of SEQ ID NO: 2; a forward primer comprising a nucleotide sequence of SEQ ID NO: 3; a forward primer comprising a nucleotide sequence of SEQ ID NO: 4; a forward primer comprising a nucleotide sequence of SEQ ID NO: 5; a forward primer comprising a nucleotide sequence of SEQ ID NO: 6; a reverse primer comprising a nucleotide sequence of SEQ ID NO: 7; a reverse primer comprising a nucleotide sequence of SEQ ID NO: 8; and a reverse primer comprising a nucleotide sequence of SEQ ID NO: 9.

In addition, the primer set for amplifying mitochondrial DNA of a host cell may comprise a forward primer comprising a nucleotide sequence of SEQ ID NO: 10; and a reverse primer comprising a nucleotide sequence of SEQ ID NO: 11.

In addition, the present invention provides a method for providing information for diagnosingmycoplasmainfection, comprising the following steps i) to iii):

i) extracting DNA from a host cell isolated from a subject suspected of being infected withmycoplasma;

ii) performing polymerase chain reaction (PCR) using the DNA extracted in step i) as a template; and

iii) determining that the subject is infected withmycoplasma, if the amplification ofmycoplasmaDNA is confirmed in the PCR of step ii).

In one preferred exemplary embodiment of the present invention, the PCR of step ii) may be performed using a forward primer comprising a nucleotide sequence of SEQ ID NO: 1; a forward primer comprising a nucleotide sequence of SEQ ID NO: 2; a forward primer comprising a nucleotide sequence of SEQ ID NO: 3; a forward primer comprising a nucleotide sequence of SEQ ID NO: 4; a forward primer comprising a nucleotide sequence of SEQ ID NO: 5; a forward primer comprising a nucleotide sequence of SEQ ID NO: 6; a reverse primer comprising a nucleotide sequence of SEQ ID NO: 7; a reverse primer comprising a nucleotide sequence of SEQ ID NO: 8; a reverse primer comprising a nucleotide sequence of SEQ ID NO: 9; a forward primer comprising a nucleotide sequence of SEQ ID NO: 10; and a reverse primer comprising a nucleotide sequence of SEQ ID NO: 11.

In one preferred exemplary embodiment of the present invention, the confirmation of the amplification ofmycoplasmaDNA in step iii) may confirm that DNA having a size of 500 bp has been amplified.

In addition, the present invention provides a kit for detectingmycoplasma, comprising the following a) to c):

a) a cell lysis buffer;

b) a primer set for amplifyingmycoplasmaDNA; and

c) a primer set for amplifying mitochondrial DNA of a host cell.

In one preferred exemplary embodiment of the present invention, the b) primer set for amplifyingmycoplasmaDNA may comprise a forward primer comprising a nucleotide sequence of SEQ ID NO: 1; a forward primer comprising a nucleotide sequence of SEQ ID NO: 2; a forward primer comprising a nucleotide sequence of SEQ ID NO: 3; a forward primer comprising a nucleotide sequence of SEQ ID NO: 4; a forward primer comprising a nucleotide sequence of SEQ ID NO: 5; a forward primer comprising a nucleotide sequence of SEQ ID NO: 6; a reverse primer comprising a nucleotide sequence of SEQ ID NO: 7; a reverse primer comprising a nucleotide sequence of SEQ ID NO: 8; and a reverse primer comprising a nucleotide sequence of SEQ ID NO: 9.

In one preferred exemplary embodiment of the present invention, the c) primer set for amplifying mitochondrial DNA of a host cell may comprise a forward primer comprising a nucleotide sequence of SEQ ID NO: 10; and a reverse primer comprising a nucleotide sequence of SEQ ID NO: 11.

Further, the present invention provides a composition for detectingmycoplasmainfection, comprising:

a forward primer comprising a nucleotide sequence of SEQ ID NO: 1;

a forward primer comprising a nucleotide sequence of SEQ ID NO: 2;

a forward primer comprising a nucleotide sequence of SEQ ID NO: 3;

a forward primer comprising a nucleotide sequence of SEQ ID NO: 4;

a forward primer comprising a nucleotide sequence of SEQ ID NO: 5;

a forward primer comprising a nucleotide sequence of SEQ ID NO: 6;

a reverse primer comprising a nucleotide sequence of SEQ ID NO: 7;

a reverse primer comprising a nucleotide sequence of SEQ ID NO: 8; and

a reverse primer comprising a nucleotide sequence of SEQ ID NO: 9.

The present invention also provides a use of a primer set in detection or infection diagnosis ofmycoplasma, the primer set comprising:

a forward primer comprising a nucleotide sequence of SEQ ID NO: 1;

a forward primer comprising a nucleotide sequence of SEQ ID NO: 2;

a forward primer comprising a nucleotide sequence of SEQ ID NO: 3;

a forward primer comprising a nucleotide sequence of SEQ ID NO: 4;

a forward primer comprising a nucleotide sequence of SEQ ID NO: 5;

a forward primer comprising a nucleotide sequence of SEQ ID NO: 6;

a reverse primer comprising a nucleotide sequence of SEQ ID NO: 7;

a reverse primer comprising a nucleotide sequence of SEQ ID NO: 8; and

a reverse primer comprising a nucleotide sequence of SEQ ID NO: 9.

