KR20050117281A - Method for detecting methicillin-resistant staphylococcus aureus by multiplex pcr - Google Patents

Abstract

The present invention relates to a method for detecting methicillin-resistant Staphylococcus aureus using multiplex PCR. More specifically, the present invention is a methicillin-resistant Staphylococcus aureus using a multiplex PCR method, characterized in that the nucleic acid sample to be analyzed by PCR amplification with a primer capable of amplifying the fem A gene, mec A gene and nuc A gene It relates to a detection method of.

The method according to the present invention has the effect of detecting methicillin-resistant Staphylococcus aureus with high sensitivity by selecting three kinds of genes specific for methicillin-resistant Staphylococcus aureus and performing multiplex PCR amplification. Since the intensity of the band generated after electrophoresis of the amplified product is uniformly performed, the error due to unbalance of the band can be minimized as compared with the conventional method of simultaneously amplifying several kinds of genes.

Description

Method for detecting methicillin-resistant Staphylococcus aureus by multiplex PCR}

The present invention relates to a method for detecting methicillin-resistant Staphylococcus aureus using multiplex PCR. More specifically, the present invention is a methicillin-resistant Staphylococcus aureus using a multiplex PCR method, characterized in that the nucleic acid sample to be analyzed by PCR amplification with a primer capable of amplifying the fem A gene, mec A gene and nuc A gene It relates to a detection method of.

Staphylococcus aureus (Stapylococcus aureus) is widely distributed in the natural world as positive and Gram belonging to the micro Coca years old child (Micrococcace ae) it has a bacteria strains present in the human skin is very closely related to the real world.

However, Staphylococcus aureus is an opportunist pathogen that can cause serious infections under appropriate circumstances, causing purulent infections and lethal sepsis, secreting endotoxins, and causing food poisoning. In particular, a patient with low immunity in the clinic is infected with Staphylococcus aureus, which can cause a number of complications. Infections caused by Staphylococcus aureus are known as pneumonia, mastitis, skin infections, osteomyelitis, endocarditis and bacteremia.

Several antibiotics, such as penicillin G and β-lactam, have been developed to treat such Staphylococcus aureus infections (Barski, P., et al., Mol. Cell. Probes. 10 ( 6): 471-475, 1996; Chamers, Henry F. Clin.Microbiol . Rev. 10 (4): 781-791, 1997; Hussain, Z. et al., J. Clin.Microbiol . 38 (6): 2051-2054, 2000). However, the emergence of strains resistant to such antibiotics has led to the development of new antibiotics. Accordingly, a new antibiotic called meticillin has been developed and used since the early 1960s as a major treatment for the treatment of Staphylococcus aureus infections.

However, among the staphylococcus aureus isolates from clinical trials, resistant strains with methicillin have also been lost, and resistant strains having a very high or high level of resistance have begun to separate. Namely, methicillin-resistant Staphylococcus aureus ; Since it was first discovered, MRSA began to be reported around the world, and the frequency of MRSA segregation among all Staphylococcus aureus is increasing (Weller TMA, J. Hosp. Infect ., 44 (3): 160-). 172, 2000). In addition, the MRSA is widely known to be resistant to various kinds of antibiotics such as β-lactam-based antibiotics, aminoglycosides, and macrolides in addition to methicillin. As described above, MRSA is not only resistant to various types of antibiotics but also affected by various growth conditions and has a very complicated genetic background. Therefore, it is difficult to detect MRSA by general antimicrobial resistance test.

In addition, it has been reported that MRSA, which was mainly detected only in the clinic, can be separated from foods (Jones, TF et al., Emer. Infect. Dis . 8 (1): 82-84, 2002). And the risk of infection of MRSA to consumers is increasing. Therefore, urgent control and epidemiological investigation of MRSA is required.

Recently, various methods for detecting the MRSA have been developed. Among them, a disk diffusion method for determining whether the strain is resistant to antibiotics by attaching the disk containing the antimicrobial agent to the surface of the solid medium on which the strain is smeared and then culturing for a predetermined time and measuring the growth inhibition band of the strain is widely used. It is becoming. However, this method has a problem such that it takes a long time and difficult conditions such as culture temperature and incubation time depending on the strain may lead to an incorrect determination.

In addition, one method for detecting MRSA is a slide latex aggregation method for measuring the presence of penicillin binding protein in a strain. The slide latex aggregation method is a method for confirming the presence or absence of methicillin resistance of Staphylococcus aureus by detecting PBP2a, a cell wall synthesis protein having low affinity with antibiotic penicillin. The antibiotic resistance mechanism of MRSA is innate by Staphylococcus aureus obtained by acquiring the mec A gene in the chromosome, and is expressed by PBP2a produced by the gene. On the other hand, the slide latex agglomeration method has a disadvantage that it is difficult to accurately detect the MRSA when there is a low ratio of MRSA in the sample or a very low generation of PBP2a under the slide latex agglomeration test conditions. Therefore, even if it is determined as negative in the slide latex agglutination method, the presence of mec A gene must be duplicated through a test that depends on other genotypes including multiplex PCR method.

Recently, a method of identifying MRSA by amplifying a polymerase chain reaction (PCR) -specific gene has been attracting attention. Compared to conventional biochemical methods or methods such as pulsed-field gel electrophoresis (PFGE) and ribotyping, the method can separate MRSA to a high level while saving time and labor (Olive, DM). et al., J. Clin.Microbiol., 37: 1661-1669, 1999). In particular, a multiplex PCR method has been used that can reduce the time and cost of the test more efficiently by simultaneously searching for several types of genes, compared to a PCR method that searches only a single gene.

On the other hand, the multiplex PCR method for simultaneously detecting multiple types of target genes has a problem that it is difficult to standardize the PCR conditions for each step compared to the PCR method for detecting a single gene, and the sensitivity for each target gene is less There is a problem.

Therefore, the present inventors selected three genes specific for methicillin-resistant Staphylococcus aureus as a target gene while studying a method for detecting methicillin-resistant Staphylococcus aureus using a multiplex PCR method and PCR the target gene. The present invention was completed by confirming that methicillin-resistant Staphylococcus aureus can be detected with high sensitivity by a multiplex PCR method characterized by amplification.

It is therefore an object of the present invention to provide a method for detecting methicillin-resistant Staphylococcus aureus using multiplex PCR.

In order to achieve the above object, the present invention provides a method for detecting methicillin-resistant Staphylococcus aureus using multiplex PCR.

Hereinafter, the present invention will be described in detail.

The method for detecting methicillin-resistant Staphylococcus aureus of the present invention is characterized by using mec A gene, fem A gene and nuc A gene as target genes.

The mec A gene is a major causative agent of Staphylococcus aureus resistant to penicillin antibiotics including methicillin. That is, the mec A gene produces penicillin binding proteins (PBP2a), which is known to inhibit the action of antibiotics (Ryffel C., et al., Antimicrob. Agents. Chemother ., 36 (1): 25-31, 1992; Weller TMA, J. Hosp. Infect. , 44 (3): 160-172, 2000). In contrast, methicillin-sensitive S. aureus, which is not resistant to methicillin; MSSA) is not present in the mec A gene. Thus, by detecting the presence of the mec A gene in the Staphylococcus aureus, it is possible to distinguish whether the Staphylococcus aureus is MRSA or MSSA.

In addition, the present inventors have found that fem A genes specific for Staphylococcus aureus (Hussain Z. et al., J. Clin. Microbiol. , 38) together with the mec A gene that are directly involved in methicillin-resistant Staphylococcus aureus 6): 2051-2054, 2000; Vannuffel, P. et al., J. Clin.Microbiol., 33 (11): 2864-2867, 1995; Weller, TMA, J. Hosp.Infect., 44 (3) : 160-172, 2000) and nuc A gene (Brakstad, OG et al., J. Clin. Microbiol ., 30 (7): 1654-1660, 1992; Ryffel C., et al., Gene , 94 (1 ): 137-138, 1990) was used as the target gene.

The fem A gene is present in all staphylococcus aureus and is known to play an essential role in staphylococcus aureus resistance to methicillin together with the mec A gene. The fem A gene is a gene required to form a cross bridge of penta glycine which is essential for the production of PBP2a to express methicillin resistance in Staphylococcus aureus (Hussain Z. et al., J. Clin Microbiol, 38 (6): ... 2051-2054, 2000; Weller TMA, J. Hosp Infect, 44 (3):... 160-172, 2000; Vannuffel, P. et al, J. Clin Microbiol , 33 (11): 2864-2867, 1995).

In addition, the nuc A gene is present in all Staphylococcus aureus and is known to produce thermostable nucleases.

Therefore, the inventors of the present invention have a mec A gene that is directly involved in producing PBP2a, a fem A gene that is essential for forming pentaglycine cross-linking required for the production of PBP2a, and all staphylococcus aureus, which produce thermostable nucleases. By using all of the nuc A genes as target genes for the detection of methicillin-resistant Staphylococcus aureus, methicillin-resistant Staphylococcus aureus is more sensitive than when using one or two genes described above as target genes. This can be detected.

In one embodiment of the present invention using a DNA sample extracted from MRSA standard strains fem A gene, mec A gene and nuc A gene at the same time PCR amplification and electrophoresis of the amplification product. As a result, all three types of genes were detected in the MRSA standard strain. On the other hand, the band intensity of the amplified fem A gene was relatively weak compared to the mec A gene or nuc A gene (see Fig. 1).

The present inventors performed the PCR amplification step of the target gene according to the present invention divided into two steps. That is, the fem A gene whose band was detected relatively weak was partially amplified first, followed by the amplification of the remaining mec A gene and nuc A gene. As a result, the intensity between bands generated after electrophoresis was constant, indicating that distinction between different gene products was apparent (see FIG. 1). From the results of the experiment, it was confirmed that the presence or absence of the target gene in the DNA sample can be more easily identified with high sensitivity when the amplification is divided into two stages compared to the case of amplifying three kinds of genes simultaneously.

Therefore, preferably, the MRSA detection method according to the present invention is performed by dividing the PCR amplification step of the mec A gene, fem A gene and nuc A gene selected above into two stages. That is, the MRSA detection method of the present invention preferentially amplifies the nucleic acid sample with a primer capable of amplifying the fem A gene during PCR amplification of the nucleic acid sample to be analyzed, and then adds the mec A gene and nuc A gene to the amplification mixture. PCR amplification by adding a primer that can be amplified.

More specifically, the MRSA detection method of the present invention

(a) PCR amplifying the nucleic acid sample to be analyzed with a primer capable of amplifying the fem A gene;

(b) adding a primer capable of amplifying the mec A gene and the nuc A gene to the PCR amplification mixture of step (a) and PCR amplifying; And

(c) electrophoresis of the PCR amplification product of step (b).

The nucleic acid sample to be analyzed in step (a) may be a DNA or RNA sample extracted from the strain to be identified. In the case of the RNA sample, cDNA may be prepared by reverse transcription by a method known in the art and used for PCR amplification.

The method for extracting the nucleic acid sample from the strain is not particularly limited, and methods known in the art may be used. For example, phenol treatment, proteolytic enzyme addition and heat treatment methods can be used. In addition, commercially available kits for DNA or RNA extraction can also be used.

The primer that can amplify the fem A gene in step (a) is not particularly limited, and any primer that can specifically amplify the gene may be included. The primer can be easily prepared by a known method based on the fem A gene sequence of known Staphylococcus aureus. In one embodiment of the present invention, a primer pair having a nucleotide sequence represented by SEQ ID NO: 1 and SEQ ID NO: 2 was used as a primer capable of amplifying the fem A gene.

The (b) step, by some amplifying the nucleic acid sample in step (a) as primers to amplify the fem A gene was added to amplify the mec A gene and the nuc A gene in the resulting amplification mixture is a primer to mec A and nuc A genes are amplified and at the same time, the partially amplified fem A gene in step (a) is fully amplified.

A primer capable of amplifying the mec A gene and the nuc A gene in step (b) is not particularly limited, and any primers capable of specifically amplifying each gene may be included. The primer is a mec A gene sequence of known Staphylococcus aureus. Or it can be easily manufactured by a well-known method based on the nuc A gene sequence. In an embodiment of the present invention as a primer capable of amplifying the mec A gene is a primer capable of amplifying the nuc A gene, was used as a primer pair having a base sequence shown in SEQ ID NO: 3 and SEQ ID NO: 4 Was used a primer pair having a nucleotide sequence represented by SEQ ID NO: 5 and SEQ ID NO: 6.

Step (c) is a step of determining the presence of fem A, mec A and nuc A gene in the nucleic acid sample of step (a) by electrophoresis of the product amplified in step (b). That is, the presence of the genes can be determined by electrophoresis of the PCR amplification products and comparing the generated bands with molecular weight markers. If all fem A, mec A and nuc A genes are detected in the amplification product, it can be determined as methicillin-resistant Staphylococcus aureus, and methicillin if fem A and nuc A genes are detected or only nuc A genes are detected. -Can be judged as susceptible Staphylococcus aureus.

Furthermore, the present inventors can easily determine the presence of the target gene of the present invention in the nucleic acid sample to be analyzed, so that the methicillin-resistant Staphylococcus aureus is detected with high sensitivity, reaction time, reaction temperature, and primers during PCR amplification. PCR reaction conditions such as the concentration of were optimized.

Therefore, most preferably, the MRSA detection method of the present invention (a) adding a primer capable of amplifying the fem A gene to a nucleic acid sample extracted from the strain to be identified, and then denatured at 94 ° C. for 1 minute, 94 10 cycles consisting of a step of denaturing at 30 ° C., annealing at 55 ° C. for 30 seconds, and stretching at 72 ° C. for 1 minute, and maintaining the temperature at 72 ° C. for 5 minutes;

(b) adding a primer capable of amplifying the mec A gene and nuc A gene to the PCR amplification mixture of step (a) and then denaturing at 94 ° C. for 30 seconds, annealing at 55 ° C. for 30 seconds, and 72 ° C. 20 cycles consisting of a step of stretching for 1 minute at and holding at 72 ° C. for 5 minutes; And

(c) electrophoresis of the PCR amplification product of step (b).

In step (a), the primers capable of amplifying the fem A gene are preferably added at a concentration of 0.6 μM, and in step (b), the primers capable of amplifying the mec A gene and the nuc A gene are 0.4 μM and 0.2, respectively. It is preferred to add at a concentration of μM.

In one embodiment of the present invention, the DNA isolated from the strain isolated from clinical, strain isolated from gimbap, MRSA standard strain and MSSA standard strain as a sample to perform a multiplex PCR divided into two steps according to the method of the present invention fem The presence of A, mec A and nuc A genes was measured. As a result, the mec A gene was detected in 44 strains of clinically isolated strains, and the 44 strains were MRSA. In the case of strains isolated from gimbap , fem A and nuc A genes were detected, but the mec A gene was not detected. In the MRSA standard strain, fem A gene, nuc A gene and mec A gene were all detected, and in the MSSA standard strain, only fem A gene and nuc A gene were detected (see FIG. 2).

Furthermore, the inventors of the present invention, to compare the separation ability and sensitivity of the multiplex PCR method according to the present invention using a conventionally known method, staphylococcus aureus isolated from clinical, staphylococcus aureus isolated from gimbap, MRSA standard strain and The phenotype of each gene was examined in MSSA standard strain.

In the comparative example of the present invention, the phenotype of each strain was examined by measuring the presence of thermostable nuclease produced by the nuc A gene. As a result, it was confirmed that the thermostable nuclease exists in all strains. Therefore, the strains were all assumed to have nuc A gene (see Comparative Example 1).

In another comparative example, the phenotype of each strain was examined by measuring the presence of penicillin binding protein (PBP2a) produced by the mec A gene using a slide-latex aggregation method. As a result, 42 of the 55 Staphylococcus aureus isolates were clinically identified as MRSA strains producing PBP2a, and all 50 Staphylococcus aureus isolates from gimbap were determined to be MSSA strains that did not produce PBP2a. In addition, MRSA standard strain Staphylococcus aureus ATCC 43300 was positive, and MSSA standard strain Staphylococcus aureus ATCC 19095 was negative (see Comparative Example 2). Therefore, from the above experimental results, it was estimated that all the strains measured to produce PBP2a had mec A gene.

In another comparative example of the present invention, antibiotic susceptibility tests were performed using strains isolated from clinical trials and strains separated from gimbap. As a result, 44 strains isolated from clinical trials were determined to be antibiotic resistant strains, and all isolates from gimbap were determined to be antibiotic sensitive strains (see Comparative Example 3).

Comparing the experimental results of the comparative example and the experimental results according to the method of the present invention, all strains in Example 1-4) nuc consistent with the results of confirming that all the strains in Comparative Example 1 to generate a heat stable nuclease It was confirmed that it has the A gene.

In addition, the results of Example 1-4) according to the method of the present invention and the slide-latex aggregation method of Comparative Example 2 and the antibiotic susceptibility test of Comparative Example 3 showed similar results, but the slides were isolated from clinically isolated strains. Latex coagulation revealed that the mec A gene was present in two strains that did not contain PBP2a. Antibiotic susceptibility testing revealed the presence of the mec A gene in one strain that was determined to be antibiotic susceptible. The difference in these results may be due to the presence of the mec A gene in the strain, which was not expressed due to any cause or the amount of PBP2a produced was very low.

Therefore, the MRSA detection method according to the present invention can detect methicillin-resistant Staphylococcus aureus with high sensitivity, and also has an advantage of minimizing errors caused by unbalanced band intensities generated in conventional multiplex PCR. have.

<Example 1>

Detection of Methicillin-Resistant Staphylococcus Aureus According to the Multiplex PCR Method of the Present Invention

1-1) Preparation of DNA Sample

55 Staphylococcus aureus isolated from clinical trials, 50 Staphylococcus aureus isolated from gimbap, Staphylococcus aureus ATCC 43300 and MRSA standard strain for detecting methicillin-resistant Staphylococcus aureus according to the multiplex PCR method of the present invention; DNA was extracted from the MSSA standard strain Staphylococcus aureus ATCC 19095.

Staphylococcus aureus isolated from the clinical trial was used to receive 55 Staphylococcus aureus isolated from patients hospitalized by the hospital, Professor Kim Jong-bae, Department of Clinical Pathology, Yonsei University Health Sciences. Staphylococcus aureus isolated from food was used by receiving 50 strains isolated from gimbap distributed in five cities in Korea by the Korea Food and Drug Administration. Staphylococcus aureus ATCC 43300 was used as the MRSA standard strain, and Staphylococcus ATCC 19095 was used as the MSSA standard strain.

The standard strain and the separated strain were inoculated in 5 ml LB (luria bertani) broth, respectively, and incubated at 37 ° C. for 24 hours. The culture was harvested by centrifuging the culture medium at 15,000rpm for 5 minutes, and then washed twice with 1 ml of 1M NaCl and centrifuged under the same conditions to harvest the cells. A suspension was prepared by adding 500 μl of 1 × TE (10 mM Tris-HCl, 1 mM EDTA) to the harvested cells. 1 mg / ml of 10 μl lysostaphin (Sigma, U.S.A.) and 10 μl of 30 μl RNase A (Qiagen, U.S.A.) were added to the cell suspension and left at 37 ° C. for 20 minutes. At this time, 10% of 60µl SDS (Sigma, U.S.A.) was added when it was not digested with lysostaphin. 60 μl proteinase K (proteinase K, Sigma, U.S.A) was added to the suspension and reacted at 37 ° C. for 1 hour. Then, the same amount of phenol was added to the decomposed cell suspension, mixed, and centrifuged at 15,000 rpm for 5 minutes to obtain an aqueous solution layer. The same procedure was repeated once again, followed by mixing the same amount of chloroform, followed by mixing under the same conditions. Centrifugation gave the supernatant. To precipitate DNA, 2.5 times the volume of supernatant was added to ethanol (absolute ethanol), which was allowed to stand at -70 ° C for 60 minutes, and then centrifuged at 15,000 rpm for 5 minutes. The precipitate obtained by centrifugation was dried and 50 μl of 1 × TE (10 mM Tris-HCl, 1 mM EDTA) was added to dissolve DNA.

1-2) Multiplex PCR with amplification of mec A gene, fem A gene and nuc A gene

Multiplex PCR was performed using DNA extracted from Staphylococcus aureus ATCC 43300, the MRSA standard strain of Example 1-1, as a sample.

PCR reactions include 1 μl DNA sample, 5 μl 10 × reaction buffer (100 mM KCl, 1 mM DTT, 0.1 mM EDTA, 50% glycerol, 0.5% Tween20, 0.5% Noniche-P40 and 20 mM Tris-HCl, pH 8.0 ), A mixture of ₂ mM MgCl ₂ , 0.2 mM dNTPs (deoxyribonucleoside-5'-triphosphate, Bioneer, Korea) and 2 units of Taq DNA polymerase (Bioneer, Korea) was used. Here, primers for detecting fem A, mec A and nuc A genes were added to 0.6 μM, 0.4 μM and 0.2 μM, respectively, and the volume of the entire reaction solution was 50 μl using sterile distilled water.

Primers used to detect the three genes (Bioneer, Korea) are shown in Table 1.

Primers Used for Multiplex PCR gene primer Sequence SEQ ID NO: fem A fem A1  5'-CTT ACT TAC TGG CTG TAC CTG-3 ' One fem A2  5'-ATG TCG CTT GTT ATG TGC-3 ' 2 mec A  RSM2647  5'-AAA ATC GAT GGT AAA GGT TGG C-3 ' 3  RSM2648  5'-AGT TCT GCA GTA CCG GAT TTG C-3 ' 4 nuc A nuc A1  5'-GCG ATT GAT GGT GAT ACG GTT-3 ' 5 nuc A2  5'-AGC CAA GCC TTG ACG AAC TAA AGC-3 ' 6

PCR amplification was repeated 30 times in a cycle consisting of a total denaturation step at 92 ° C. for 3 minutes, a denaturation step at 92 ° C. for 1 minute, an annealing step at 56 ° C. for 1 minute, and an extension step for 1 minute at 72 ° C., at 72 ° C. as a post-extension step. After maintaining for 3 minutes, the PCR reaction was terminated. The product obtained through the multiplex PCR was subjected to electrophoresis on a 2% agarose gel.

As a result, it was confirmed that fem A, mec A and nuc A gene bands were all detected in the MRSA standard strain. On the other hand, when the three types of genes were simultaneously amplified and electrophoresed, the band intensity of the amplified fem A gene was relatively weak compared to the mec A gene or nuc A gene (see FIG. 1).

1-3) Multiplex PCR amplified by dividing mec A gene, fem A gene and nuc A gene in two stages

The multiplex PCR was performed in the same manner as in Example 1-2), but the PCR amplification step was performed in two steps.

That is, the PCR amplification mixture was prepared in the same manner as 1-2) using DNA extracted from Staphylococcus aureus ATCC 43300, which is the MRSA standard strain of Example 1-1, and only the fem A gene was partially first. In order to amplify, only the primers capable of amplifying the fem A gene (SEQ ID NO: 1 and SEQ ID NO: 2) were added, followed by a pre-denaturation step at 94 ° C for 1 minute, and a denaturation step at 94 ° C for 30 seconds. 10 cycles consisting of an annealing step at 55 ° C. for 30 seconds, an extension step at 72 ° C. for 1 minute (PTC-100, MJ research, USA), and maintained at 72 ° C. for 5 minutes Later, as a second step, two pairs of primers (SEQ ID NO: 3 to SEQ ID NO: 6) capable of amplifying the mec A and nuc A genes were added to the fem A amplification mixture, followed by a denaturation step at 94 ° C. for 30 seconds. Annealing at 55 ° C. for 30 seconds, 72 ° C. Standing for one minute elongation cycle (cycle) consisting of (extension) step after after repeating 20 times a total PCR reaction was terminated by maintaining height (post-extention) step for 5 minutes. The product obtained through the multiplex PCR was subjected to electrophoresis on a 2% agarose gel.

As a result, all fem A gene, mec A gene and nuc A gene were detected in the MRSA standard strain, and the band intensities of all three types of genes were consistently observed (FIG. 1).

1-4) Detection of MRSA Using Two-Step Multiplex PCR

DNA extracted from 55 Staphylococcus aureus, 50 Staphylococcus aureus isolated from Gimbap, Staphylococcus aureus ATCC 43300, MRSA standard strain, and Staphylococcus aureus ATCC 19095, MSSA standard strain Two-step multiplex PCR was performed.

Two-step multiplex PCR was performed in the same manner as in Example 1-3).

As a result, there were some strains in which fem A, nuc A and mec A genes were all detected, and some strains in which only fem A and nuc A genes were detected (FIG. 2). More specifically, fem A and nuc A genes were detected in all 55 strains isolated from clinical trials. Therefore, it was confirmed that the strain isolated in the clinical was Staphylococcus aureus. In addition, 44 strains among the 55 isolates were determined to have mec A gene, and the 44 strains were found to be MRSA, and the remaining 11 strains did not have mec A gene. It turned out to be MSSA. In all 50 isolates isolated from gimbap, fem A and nuc A genes were detected, but no mec A gene was present. All the isolates from gimbap were determined to be MSSA.

In addition, fem A gene, nuc A gene, and mec A gene were all detected in the DNA sample extracted from the MRSA standard strain Staphylococcus aureus (ATCC 43300) and confirmed as MRSA, and Staphylococcus aureus (ATCC 19095), the MSSA standard strain, was detected. The fem A gene and nuc A gene were detected in the DNA sample extracted from the mec A gene but not detected as MSSA strain.

Comparative Example 1

Investigation of Staphylococcus Aureus Production of Thermostable Nucleases

We investigated the generation of thermostable nucleases produced by the nuc A gene in Staphylococcus aureus. For this purpose, Staphylococcus aureus isolated from the clinical of Example 1-1), Staphylococcus aureus isolated from food, MRSA standard strain and MSSA standard strain were used.

Toludine blue DNA agar medium was prepared to measure the heat stable nuclease produced by the nuc A gene in Staphylococcus aureus. DNA used in the medium was used as a spum nuclei type 2 (Sigma, USA). The toludine blue DNA agar was coated to about 3 ml on the slide glass before hardening, and about 10 wells having a diameter of about 2 mm per slide glass were made. BHI (brain heart infusion, Difco, USA) broth inoculated with the staphylococcus aureus cultured 200μL each in a test tube and heated for 15 minutes in boiling water and then cooled to room temperature. 10 μl of the culture solution cooled to room temperature was taken and placed in the well of the toludine blue DNA agar prepared above. The wells were cultured at 35 ° C. for 4 hours in a chamber, and then positively determined for strains that changed from blue to pink at least 1 mm around the wells.

As a result of the experiment, it was confirmed that 1 to 5 mm was changed from blue, which is the original medium color, to pink around the well of toludine blue DNA agar in all strains.

From the results of the experiment it was confirmed that all kinds of Staphylococcus aureus produces thermostable nuclease. Therefore, it could be assumed that it contains nuc A gene which produces thermostable nuclease.

Comparative Example 2

Detection of Penicillin-binding Protein in Staphylococcus Aureus Using Slide-Latex Aggregation

Strain isolated from the clinic of Example 1-1) using slide-latex agglutination, strain isolated from gimbap, MRSA standard strain (yellow staphylococcus ATCC 43300) and MSSA standard strain (yellow staphylococcus) A penicillin binding protein (PBP2a) produced by the mec A gene was detected in cocci ATCC 19095).

The slide-latex agglomeration was performed by purchasing a latex agglutination kit (Oxoid, U.K.), and all procedures related to the slide-latex agglomeration were performed based on the guidelines proposed by Oxoid. First, Mueller Hinton agar (Difco, USA) was smeared with the standard strains and isolates of the Staphylococcus aureus, and 1 μg of oxacillin (oxacillin) contained in the most smeared areas in each medium. The disc was placed and incubated at 37 ° C. for 24 hours. Colonies grown around the disc were taken about 1 loop and mixed in 200 μl extraction buffer 1 and diluted in boiling water for 3 minutes. After cooling to room temperature, 50 μl extraction buffer 2 was mixed, centrifuged at 3,000 rpm, and 50 μl of the supernatant was taken. A control latex and a test latex solution were added dropwise to the supernatant and then mixed well using a sterile rod included in the kit. Thereafter, the strain in which aggregation was observed was determined as an MRSA strain producing PBP2a, and the strain in which no aggregation reaction occurred was determined as an MSSA strain that did not produce PBP2a.

As a result, 42 strains among 55 Staphylococcus aureus isolates were clinically identified as MRSA strains producing PBP2a, and the remaining 13 strains were determined as MSSA strains. All 50 Staphylococcus aureus isolates from gimbap were determined to be MSSA strains that do not produce PBP2a. In addition, MRSA standard strain Staphylococcus aureus ATCC 43300 was positive, and MSSA standard strain Staphylococcus aureus ATCC 19095 was negative. Therefore, the strain determined to produce PBP2a above could be assumed to have the mec A gene.

Comparative Example 3

Antibiotic Susceptibility Testing of Staphylococcus Aureus

The susceptibility test for the antibiotic oxacillin (oxacillin) was performed using the strain isolated from the clinical of Example 1-1) and the strain isolated from food.

Antibiotic susceptibility testing was performed according to Kirby-Bauer's disk diffusion methods. The isolate was inoculated in muller hinton broth (MHB) and incubated at 37 ° C. for 24 hours. The culture solution was diluted with an ELISA reader (Molecular Devies Co., CF., USA) at 600 nm to measure absorbance and dilute to adjust the OD value to 0.5. The culture medium with the OD value adjusted was evenly spread on a muller hinton plate (MHP) with a sterile swab. After the plate was dried for 5 minutes, the plate was dried, and then the sterilized tweezers was fixed on the plate for keeping the antibiotic susceptibility test disk at a distance of 20 mm or more. Fixed plate medium was incubated for 24 hours in a 37 ℃ incubator and the diameter of growth resistant ring was measured. The results according to the growth refrigeration diameter are shown by National Committe for Susceptibility was read according to Clinical Laboratory Standardization).

As a result, out of 55 isolates from clinical trials, 43 strains showed antibiotic resistance, and all isolates from gimbap were susceptible to antibiotics.

<Comparative Example 4>

Comparison of the results according to the method of the multiplex PCR method of the present invention with the method of the comparative example

The separation ability and sensitivity of the multiplex PCR method of the present invention were compared with the measurement results of Comparative Examples 1 to 3.

Comparing the experimental results of Example 1-4) with the results of investigating whether thermally stable nucleases were produced in Comparative Example 1, all the strains in Example 1-4) were found to have nuc A genes. All strains 1 also showed that they produce heat stable nucleases (Table 2).

Comparing the experimental results of Example 1-4) with the results of the latex agglomeration method of Comparative Example 2, the results of the method of the present invention and the latex agglomeration method were found to be the same in the case of the strain isolated from gimbap, In the case of strains, the two strains showed inconsistent results between the method of the present invention and the latex aggregation method. That is, two strains determined to not produce PBP2a in Comparative Example 2 were determined to have the mec A gene in the method of the present invention (Table 2). The difference in these results may be due to the presence of the mec A gene in the two strains isolated from the clinic but was not expressed due to any cause or the production of PBP2a was very low.

In addition, when comparing the experimental results of Example 1-4) and the antibiotic susceptibility test results of Comparative Example 3, it was found that the same for the strain isolated from gimbap, but there was a difference in the strain isolated from clinical. That is, one strain that was not determined to have antibiotic resistance in Comparative Example 3 was determined to have the mec A gene in the method of the present invention (Table 2). The difference in the experimental results may be due to the presence of the mec A gene in the clinically isolated strain, such as when the difference was observed in the latex aggregation method.

Multiplex PCR Results According to the Invention Phenotype Genotype Isolated strain Number of isolates Toludine Blue DNA Agar Staining Latex Agglomeration Antibiotic sensitivity test Multiplex PCR Thermostable nuclease  PBP2a Oxacillin nuc A mec A fem A + - + - S ³⁾ R ⁴⁾ + - + - + - WCH ¹⁾ 55 55 42 13 12 43 55 44 11 55 KB ²⁾ 50 50 50 50 50 50 50 gun 105 105 42 63 62 43 105 44 61 105

1) WCH: Clinically Isolated Strains

2) KB: Strain Isolated from Gimbap

3) S: shows sensitivity to oxacillin

4) R: shows resistance to oxacillin

5) +: positive,-: negative

The method according to the present invention has the effect of detecting methicillin-resistant Staphylococcus aureus with high sensitivity by selecting three kinds of genes specific for methicillin-resistant Staphylococcus aureus and performing multiplex PCR amplification. Since the intensity of the band generated after electrophoresis of the amplified product is uniformly performed, the error due to unbalance of the band can be minimized as compared with the conventional method of simultaneously amplifying several kinds of genes.

1 shows electrophoresis results of PCR amplification products obtained by performing PCR amplification and PCR amplification in two steps according to the method for detecting methicillin-resistant Staphylococcus aureus of the present invention. (M: molecular weight marker, when lane 1: PCR amplification was performed simultaneously, lane 2: PCR amplification was performed in two steps).

Figure 2 shows the detection results of MRSA by the multiplex method performed by PCR amplification in two steps according to the detection method of methicillin-resistant staphylococcus of the present invention (M: molecular weight marker, lane 1: MRSA standard Strain, lane 2: MSSA standard strain, lane 3: clinically isolated MRSA strain, lane 4: clinically isolated MSSA strain, lane 5: kimbap isolated MSBA strain).

<110> KIM, Keun-Sung <120> Method for detecting methicillin-resistant Staphylococcus aureus by multiplex PCR <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Sense primer femA1 <400> 1 cttacttact ggctgtacct g 21 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Antisens primer femA2 <400> 2 atgtcgcttg ttatgtgc 18 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Sense primer RSM2647 <400> 3 aaaatcgatg gtaaaggttg gc 22 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Antisense primer RSM2648 <400> 4 agttctgcag taccggattt gc 22 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Sense primer nucA1 <400> 5 gcgattgatg gtgatacggt t 21 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Antisens primer nucA2 <400> 6 agccaagcct tgacgaacta aagc 24

Claims (7)

Method for detecting methicillin-resistant Staphylococcus aureus characterized in that the nucleic acid sample to be analyzed by PCR amplification with a primer capable of amplifying the fem A gene, mec A gene and nuc A gene. The method of claim 1, (a) PCR amplifying the nucleic acid sample to be analyzed with a primer capable of amplifying the fem A gene; (b) adding a primer capable of amplifying the mec A gene and the nuc A gene to the PCR amplification mixture of step (a) and PCR amplifying; And (C) method of detecting methicillin-resistant Staphylococcus aureus comprising the step of electrophoresis of the PCR amplification product of step (b). The method of claim 2, (a) adding a primer capable of amplifying the fem A gene to the nucleic acid sample to be analyzed, followed by full denaturation at 94 ° C. for 1 minute, denaturation at 94 ° C. for 30 seconds, annealing at 55 ° C. for 30 seconds, PCR amplification by repeating the cycle consisting of stretching for 1 minute at 72 ° C 10 times and maintaining the temperature at 72 ° C for 5 minutes; (b) adding a primer capable of amplifying the mec A gene and nuc A gene to the PCR amplification mixture of step (a) and then denaturing at 94 ° C. for 30 seconds, annealing at 55 ° C. for 30 seconds, and 72 ° C. PCR amplification by repeating the cycle consisting of stretching for 1 minute at 20 times and then maintained for 5 minutes at 72 ℃ as a post-extension step; And (C) method of detecting methicillin-resistant Staphylococcus aureus comprising the step of electrophoresis of the PCR amplification product of step (b). The methicillin-resistant yellow staphylococcus according to any one of claims 1 to 3, wherein the primers capable of amplifying the fem A gene of step (a) are represented by SEQ ID NO: 1 and SEQ ID NO: 2. Methods of detecting cocci. The methicillin-resistant yellow staphylococcus according to any one of claims 1 to 3, wherein the primers capable of amplifying the mec A gene of step (b) are represented by SEQ ID NO: 3 and SEQ ID NO: 4. Methods of detecting cocci. The methicillin-resistant yellow staphylococcus according to any one of claims 1 to 3, wherein the primers capable of amplifying the nuc A gene of step (b) are represented by SEQ ID NO: 5 and SEQ ID NO: 6. Methods of detecting cocci. The method according to any one of claims 1 to 3, wherein a primer capable of amplifying the fem A gene of step (a) is added at a concentration of 0.6 μM, and the mec A gene and nuc A gene of step (b) are amplified. A method for detecting methicillin-resistant Staphylococcus aureus, which comprises adding primers at a concentration of 0.4 µM and 0.2 µM, respectively.