JP2006271370A - Method and kit for detecting Staphylococcus aureus and methicillin resistant bacteria - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a product capable of readily and rapidly diagnosing MSSA, MRSA and MR-CNS which become problems in hospitals and institutions for the aged and in the cities. <P>SOLUTION: MSSA, MRSA and MR-CNS can rapidly and simply be discriminated by adapting femA primer for detection of Staphylococcus aureus and mecA primer for detecting methicillin resistance to an LAMP method. The primer of femA for detecting Staphylococcus aureus uses a specific sequence. The mecA primer for detecting methicillin resistance uses a specific sequence. MSSA can be detected as femA (+) and mecA(-) and MRSA can be detected as femA(+) and mecA(+) and MR-CNS can be detected as femA(-) and mecA(+). <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention not only quickly distinguishes and distinguishes methicillin-resistant Staphylococcus aureus and methicillin-sensitive Staphylococcus aureus, which are problematic not only from hospital infections but also from geriatric medical centers, nursing homes, nursing homes to community infections, as well as methicillin. The present invention relates to a primer capable of rapidly determining even resistant coagulase-negative staphylococci and a kit containing the same.

  Methicillin-resistant Staphylococcus aureus (MRSA) contaminates not only large hospitals but also small and medium-sized hospitals, and has a great adverse effect not only on infected patients but also on medical costs. Moreover, recently, geriatric hospitals, nursing homes and nursing homes have been contaminated, and the pollution has spread to the city. MRSA infection treatments are limited and currently there are only three drugs: vancomycin, teicoplanin, and arbekacin. In general, when infectious diseases are suspected, the first-line drugs are administered, such as β-lactams, but these antibacterial drugs have no effect on MRSA. For this reason, cases in which MRSA-infected persons become serious are recognized. In order to avoid these risks, it is considered essential to quickly and accurately detect and identify MRSA from test specimens.

  MRSA was identified as Staphylococcus aureus by a separate culture method, and then MRSA was judged from the results of antimicrobial susceptibility testing. On the other hand, it was easily determined in MRSA selection medium (medium containing antibacterial drug in S. aureus selection medium). However, culture is essential regardless of which method is used, and depending on the culture conditions, there was an uncertainty that MRSA was judged to be sensitive. This uncertainty is due in part to the fact that MRSA resistance expression is complexly controlled by regulatory genes (mecI, mecR1, blaI, blaR1). There is uncertainty.

To solve this problem, detection of the mecA gene, the main body of MRSA resistance, can be considered. The mecA gene detection method has already been put into practical use by using the polymarase chain reaction (PCR) method (Patent Literature 1, Non-Patent Literature 1), but an expensive thermal cycler is required to perform PCR. Since the reagent used for the detection is high, it has not been widely used.
JP-A-11-56371 (Ubukata, K., et al. J. Clin. Microbiol. 30, 1728-1733, 1992)

  The object of the present invention is to provide a method and kit for quickly and easily discriminating methicillin-resistant Staphylococcus aureus (MRSA), methicillin-sensitive Staphylococcus aureus (MSSA), and methicillin-resistant coagulase-negative staphylococci (MR-CNS) at low cost. It is to be.

  In order to achieve the above object, the present inventors have developed a loop-Mediated Isothermal Amplification (LAMP) method (T. Notomi et al. Nucleic Acid Research, 2000, developed by Eiken Chemical) as an inexpensive and simple isothermal amplification method. vol28, No.12, e63) is one of the solutions to the above problem that requires expensive equipment.

  However, the compatibility of the primers used in the LAMP method is the biggest problem, and the primer design method using software recommended by Eiken Chemical did not provide suitable primers. Therefore, we designed various primers on our own and tried to design primers suitable for mecA detection. At the same time, in order to identify MRSA, it was necessary to identify mecA-bearing S. aureus. As a result, femA was judged appropriate. femA is a gene possessed by staphylococci (Unal S et al. J Clin Microbiol. 1992 Jul; 30 (7): 1685-91.). Attempts were made to design primers suitable for the region and the present invention was reached.

That is, the present invention is as follows.
1. 4 oligonucleotides of SEQ ID NO: 1, 2, 3 and 4 or 4 oligonucleotides of SEQ ID NOs: 13, 14, 15 and 16 (wherein any thymine (t) is substituted with uracil (u)) A method for detecting methicillin-resistant bacteria by the Loop-Mediated Isothermal Amplification (LAMP) method using a gene amplification reaction primer.
2. LAMP characterized by using four oligonucleotides of SEQ ID NOs: 5, 6, 7 and 8 (wherein any thymine (t) may be substituted with uracil (u)) as a gene amplification reaction primer Method for detecting Staphylococcus aureus
3. 4 kinds of oligonucleotides of SEQ ID NO: 1, 2, 3 and 4 or 4 kinds of oligonucleotides of SEQ ID NOs: 13, 14, 15 and 16 each containing 4 kinds of oligonucleotides A kit for detecting methicillin-resistant bacteria.
4). A kit for detecting Staphylococcus aureus, comprising the four oligonucleotides according to item 2.
5. 4 oligonucleotides of SEQ ID NO: 1, 2, 3 and 4 or 4 oligonucleotides of SEQ ID NO: 13, 14, 15 and 16 and 4 oligonucleotides of SEQ ID NO: 5, 6, 7 and 8 (provided that A detection kit for Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA) or methicillin-resistant coagulase-negative staphylococci (MR-CNS) containing any thymine (t may be replaced with uracil (u)).

  The present inventor tried to create primers that can amplify the mecA and femA genes reliably by the LAMP method, and found a primer that specifically reacts with S. aureus with femA. Furthermore, in mecA, the primer which reacts not only with MRSA but with MR-CNS was found. The present invention provides a femA detection primer containing a Staphylococcus aureus-specific region that can be used in the LAMP method and a mecA detection primer that can be used in the LAMP method, and these are used alone or in combination. Thus, MSSA, MRSA, and MR-CNS can be detected quickly and easily at low cost. If both S. aureus-specific femA and mecA involved in methicillin resistance are detected, MRSA can be confirmed, and if only S. aureus-specific femA is detected, it can be confirmed as susceptible S. aureus. Moreover, MR-CNS can be diagnosed if only mecA is used.

The present invention is described in detail below.
The LAMP method used in the present invention uses four types of primers set for six regions of a target gene and a strand displacement type DNA polymerase, and uses the strand displacement activity of this DNA polymerase to maintain a constant temperature. The detection reaction can be performed in one step. The amplification mechanism consists of an initial reaction until the starting structure of the amplification cycle is completed and an amplification cycle in which amplification products are continuously generated.

  The femA primers for detecting S. aureus used in the present invention are 4 types of SEQ ID NOs: 5 to 8, and the mecA primer for detecting methicillin resistance is 4 types of SEQ ID NOs: 1 to 4 or SEQ ID NOs: 13 to 16 There are four types. These primers may be artificially synthesized products or synthesized using an automatic DNA synthesizer. The MRSA detection method of the present invention is carried out according to the usual LAMP method, except that four types of primers for femA and mecA are used. That is, the amplified gene is detected by performing the LAMP method by isothermal amplification using four types of femA primers for detecting Staphylococcus aureus and four types of mecA primers for detecting methicillin resistance separately.

  As a specimen in the detection method of the present invention, various clinical test materials, blood, pus juice, sputum, cerebrospinal fluid, skin, nasal cavity, pharynx, feces, urine, or the like, or a bacterial liquid or bacterial colony obtained therefrom may be used. it can. The nucleic acid extraction method from the specimen can be performed by heat treatment, enzyme treatment, physical destruction, etc., and the obtained nucleic acid extract is used after being treated by a general nucleic acid purification method such as phenol treatment or ethanol treatment. You can also

The nucleic acid amplification method basically follows the LAMP method developed by Eiken Chemical. That is, a nucleic acid amplification reaction using four types of primers is performed. In the case of this primer, the reaction is preferably carried out at an isothermal temperature of 63 ° C. for 10 minutes to 1 hour.
Judgment of DNA amplification uses the commonly used electrophoresis method, hybridization method with a labeled probe, and the property that pyrophosphate and magnesium produced during DNA amplification bind to become insoluble (magnesium pyrophosphate). However, in order to more easily determine DNA amplification, a nucleic acid amplification reaction such as PCR or LAMP is performed using a primer with a distinguishable label, and labeled amplified DNA. The method is advantageous in that it is captured and identified on a solid phase, but any method is applicable to the present detection method.

Hereinafter, the present invention will be described by way of examples.
Reference example 1
Test strains were (1) Staphylococcus aureus FDA 209P, (2) MRSA (clinical isolate), (3) Staphylococcus epidermidis (clinical isolate), (4) MRSE (clinical isolate), (5) Micrococcus luteus ATCC9341, 6) Streptococcus pyogenes (clinical isolate), (7) Streptococcus pneumoniae (clinical isolate), (8) Streptococcus agalactiae (clinical isolate) were used. Staphylococcus genus was cultured on Muller Hinton agar medium, and Streptococcus genus was a colony cultured on blood agar medium. The cell DNA was simply extracted with saturated phenol / chloroform after cell destruction by hot water treatment. For the purpose of evaluating the primers used in this LAMP method, femA and mecA were also confirmed by conventional PCR. As primers for PCR, femA is SEQ ID NO: 9 and 10, mecA is SEQ ID NO: 11 and 12 (PCR products 519 bp), and amplification reaction was performed with AmpliTaq Gold (Applied Biosystems).

PCR reaction solution is 0.2 μL of extracted DNA (sterilized distilled water is added as a control), 0.2 μL of each primer of 100 pmol / μL, 0.4 μL of 5.0 U / μL AmpliTaq Gold (Applied Biosystems), 2 mM dTNPmix (GeneAmp) 1.0 μL, 25 mM MgCl ₂ 3.0 μL, 10 × PCR buffer (Applied Biosystems) 5.0 μL, and distilled water 40.0 μL were added. Cycling time and temperature setting for femA, after heating at 95 ° C for 12 minutes, (94 ° C, 30sec. → 52 ° C, 1min. → 70 ° C, 1min.) X 40 cycles, treated at 70 ° C for 10 minutes did. For mecA, after heating at 95 ° C. for 12 minutes, (94 ° C., 30 sec. → 52 ° C., 1 min. → 70 ° C., 1 min.) × 40 cycles and treatment at 70 ° C. for 10 minutes. As a thermal cycler, GeneAmp PCR System 9700 (Applied Biosystems) was used. The reaction result was confirmed by electrophoresis. In other words, after the reaction was completed, 8.0 μL of the reaction solution was taken, 3.0 μL of bromophenol blue was added and mixed, and the mixture was applied to a 2% agarose gel made with Tris (hydroxymethyl) aminomethane-acetic acid-Disodium EDTA (pH 8.0) buffer. After loading each sample with 8 μL and 2 μL of DNA ladder, electrophoresis was performed in Tris (hydroxymethyl) aminomethane-acetic acid-Disodium EDTA (pH 8.0) buffer with a voltage of 100 V for 35 minutes. After completion of electrophoresis, the agarose gel was immersed in 1.0 μg / mL ethidium bromide for 20 minutes and then immersed in purified water for 10 minutes. The band was confirmed with a UV lamp. The results summarized in Table 1 are shown in FIG. 1 and the electrophoresis results are shown. femA was detected only in S. aureus (1) and (2), and mecA was detected in methicillin-resistant staphylococci (2), (4), and (5).

Example 1
Test bacteria, DNA extraction method, and test bacteria are in accordance with Reference Example 1. The femA primers for the LAMP method are SEQ ID NO: 5 (f-1), SEQ ID NO: 6 (f-2), SEQ ID NO: 7 (f-3), and SEQ ID NO: 8 (f-4).
In addition, SEQ ID NO: 1 (m-1), SEQ ID NO: 2 (m-2), SEQ ID NO: 3 (m-3), and SEQ ID NO: 4 (m-4) were used as primers for mecA for detecting methicillin resistance. .
The LAMP reaction solution was 1.0 μL of the extracted DNA (sterilized distilled water was added as a control), 100 pmol / μL of each primer, f-1, f-2 and m-1, m-2 were 0.05 μL, f- For 3, f-4, m-3, and m-4, 0.4 μL and 8.0 U / μL Bst DNA Polymerase was added in an amount of 1 μL, and a nucleic acid amplification reaction was performed. In addition to the above primers and enzyme, the reaction solution is 20 mM Tris HCl (pH 8.8), 10 mM KCl, 8 mM MgSO ₄ , 10 mM (NH ₄ ) ₂ SO ₄ , 0.1% Tween 20, 0.8 M Betaine, 1.4 mM each of dNTPs It is contained in 25 μL of the reaction solution. The reaction solution was heated at 63 ° C. for 60 minutes and then treated at 82 ° C. for 2 minutes to inactivate the enzyme. The reaction results were confirmed by electrophoresis according to Reference Example 1. The results summarized in Table 2 are shown in FIGS. 2-1 and 2-2. femA was detected by S. aureus (1) Staphyloccocus aureus FDA 209P and (2) MRSA92-1191, but it was not detected by bacteria other than S. aureus, especially the closely related CNS, and its high specificity Proved. MecA responsible for methicillin resistance was detected in all methicillin-resistant staphylococci (2) MRSA92-1191, (4) MRSE-001, (5) MRSE-002, but sensitive to β-lactam drugs that do not possess mecA Was not detected in any bacteria. This proves the high specificity of the mecA primer.

Example 2
Under the conditions of Reference Example 1 and Example 1, 42 methicillin-susceptible Staphylococcus aureus (MSSA) and 47 methicillin-resistant Staphylococcus aureus were examined for reactivity by PCR and LAMP. As controls, methicillin-sensitive S. aureus FDA209P strain and methicillin-resistant Staphylococcus aureus (MRSA) 92-1191 strain were used as controls. A summary of the results is shown in Table 3.
Both PCR and LAMP obtained consistent results for all clinical isolates. That is, femA was positive in all S. aureus regardless of methicillin sensitivity. MecA was negative for all methicillin-sensitive staphylococci and positive for methicillin-resistant staphylococci. The excellent characteristics of each of the four types of primers, femA and mecA, that can be used in the newly developed LAMP method were proved from the above examples, and the present invention was completed.

Example 3
MecA was detected by the LAMP method in the same manner as in Example 1 except that SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 were used as primers for mecA for detecting methicillin resistance. As in the case of Example 1, as a result, mecA responsible for methicillin resistance was detected in all methicillin-resistant staphylococci, but not in bacteria sensitive to β-lactam drugs that do not have mecA.

Example 4
42 strains of methicillin-sensitive Staphylococcus aureus (MSSA) were used in exactly the same manner as in Example 2 except that SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 were used as primers for mecA to detect methicillin resistance. And 47 strains of methicillin-resistant Staphylococcus aureus were examined for reactivity by the LAMP method.
As a result, as in Example 2, mecA was negative for all methicillin-sensitive staphylococci and positive for methicillin-resistant staphylococci.

  By applying the femA and mecA detection primers of the present invention to the LAMP method, MSSA, MRSA, and MR-CNS can be distinguished in a short time without using expensive equipment such as a thermal cycler. MRSA continues to be a problem as a hospital-acquired infectious agent, but recently it has been isolated from the city, and a diagnosis at the level of a practitioner has been required. In addition, outbreaks have been reported in geriatric facilities, nursing homes, nurseries, kindergartens, etc., and it is easy to use for those who are not practitioner level or bacteriological specialists to prevent and respond to the spread. There is a need for a rapid discrimination kit. In order to meet these demands, the rapid diagnostic method for MSSA, MRSA, MR-CNS of the present invention was developed.

It is the electrophoresis photograph showing the result of confirmation of femA and mecA by conventional PCR. It is an electrophoresis photograph showing the result of confirmation of femA and mecA by LAMP method which is the method of the present invention.

Claims (5)

  4 oligonucleotides of SEQ ID NO: 1, 2, 3 and 4 or 4 oligonucleotides of SEQ ID NOs: 13, 14, 15 and 16 (wherein any thymine (t) is substituted with uracil (u)) A method for detecting methicillin-resistant bacteria by the Loop-Mediated Isothermal Amplification (LAMP) method using a gene amplification reaction primer.   LAMP characterized by using four oligonucleotides of SEQ ID NOs: 5, 6, 7 and 8 (wherein any thymine (t) may be substituted with uracil (u)) as a gene amplification reaction primer Method for detecting Staphylococcus aureus   A kit for detecting a methicillin-resistant bacterium comprising the four oligonucleotides of SEQ ID NOs: 1, 2, 3, and 4 according to claim 1 or the four oligonucleotides of SEQ ID NOs: 13, 14, 15, and 16. .   A kit for detecting Staphylococcus aureus, comprising the four oligonucleotides according to claim 2. 4 oligonucleotides of SEQ ID NO: 1, 2, 3 and 4 or 4 oligonucleotides of SEQ ID NO: 13, 14, 15 and 16 and 4 oligonucleotides of SEQ ID NO: 5, 6, 7 and 8 (provided that A detection kit for Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA) or methicillin-resistant coagulase-negative staphylococci (MR-CNS) containing any thymine (t may be replaced with uracil (u)).

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