TWI416105B - Peptide biomarker for discrimninatng community-associated methicillin-resistant staphylococcus aureus and hospital associated methicillin-resistant staphylococcus aureus - Google Patents

Abstract

Rapid identification of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is essential for appropriate clinical therapy and timely intervention for cross-infection control. By screening methicillin-sensitive Staphylococcus aureus (MSSA), CA-MRSA, hospital-associated methicillin-resistant Staphylococcus aureus (HA-MRSA), heterogeneous Vancomycin-intermediate Staphylococcus aureus (h-VISA) and Vancomycin-intermediate Staphylococcus aureus (VISA) strains with MALDI-TOF, the peptide peak, GIIKX1IKX2LIEX3FTGK (wherein X1 is F or V; when X1 is F, X2 is G and X3 is K; and when X1 is V, X2 is S and X3 is Q), was found to be able to discriminate between HA-MRSA and CA-MRSA strains, and thus it can be a biomarker to identify CA-MRSA in MRSA strains.

Description

Used to distinguish community-type methicillin-resistant Staphylococcus aureus and hospital-type anti-methoxy Peptide biomarker for phenylpenicillin Staphylococcus aureus

The present invention relates to a peptide which can be used for identifying community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA), in particular for identifying the community-type anti-methoxybenzene with the peptide. Biomarker of penicillin S. aureus and its application.

Staphylococcus (Staphylococcus) is Micrococcus Branch (Micrococcaceae) is a genus, covering about thirty staphylococcal strains of Staphylococcus consists mainly Staphylococcus epidermidis (Staphylococcus epidermidis), Staphylococcus saprophyticus (Staphylococcus saprophyticus), and Staphylococcus Staphylococcus aureus , of which Staphylococcus aureus is the most common staphylococci. Staphylococci are Gram-positive bacteria, which are spherical and ubiquitous in nature. Most Staphylococcus can grow on the respiratory mucosa or on the surface of the skin. When there is a wound on the upper respiratory mucosa or on the surface of the skin, Staphylococcus can enter the tissue and cause infection. Different Staphylococcus strains can produce various toxins to cause various diseases. For example, diseases commonly caused by Staphylococcus aureus include cellulitis, pneumonia, and food poisoning.

Staphylococcus aureus , which is resistant to methicillin, has been found to be known as Methicillin-resistant Staphylococcus aureus (MRSA), which is resistant to all penicillins including methicillin. See Lowy FD. Staphylococcus aureus infections. The New England journal of medicine. 1998; 339(8): 520-32; and Fridkin SK et al, Methicillin-resistant Staphylococcus aureus disease in three communities. The New England journal of medicine 2005; 352(14): 1436-44, the entire disclosure of which is hereby incorporated by reference in its entirety, the entire disclosure of the entire disclosure of the disclosure of the disclosure of the patent application.

The methicillin-resistant Staphylococcus aureus can be divided into two types according to its SCCmec (staphylococcal cassette chromosome mec) genotype: the first one is a first type, a second type, or a third type SCCmec gene. Hospital-associated methicillin-resistant Staphylococcus aureus (HA-MRSA), or hospital-infected methicillin-resistant Staphylococcus aureus ; the second is with a fourth Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA), or community-infected anti-methicillin-resistant Staphylococcus aureus .

Hospital-type methicillin-resistant Staphylococcus aureus refers to strains that are traditionally transmitted in hospitals, such as methicillin-resistant Staphylococcus aureus infected through surgical wounds. As for the community-type methicillin-resistant Staphylococcus aureus, it is a new strain that has emerged in recent years. It is a strain that causes infection outside the hospital and is usually spread among densely populated places, such as communities or sports fields. For genotypic classification of methicillin-resistant Staphylococcus aureus , see Grundmann H et al., Tiemersma E. Emergence and resurgence of meticillin-resistant Staphylococcus aureus as a public-health threat. Lancet. 2006; 368 (9538): 874 -85; and Enright MC et al, The evolutionary history of methicillin-resistant Staphylococcus aureus (MRSA). Proceedings of the National Academy of Sciences of the United States of America. 2002; 99 (11): 7687-92, such documents The full text is hereby incorporated by reference.

Community-type methicillin-resistant Staphylococcus aureus strains are obtained by mixing with other strains present in the community to obtain toxic genes, such as the Panton-Valentine-Leukocidin (PVL) gene, which can cause toxins White blood cell lysis, apoptosis, and tissue necrosis are more likely to cause invasive diseases such as skin soft tissue infection, sepsis, and necrotic pneumonia. Compared with the hospital-type methicillin-resistant Staphylococcus aureus, community-type methicillin-resistant Staphylococcus aureus has a strong destructive effect on wounds and is not easy to cure. Therefore, patients who suffer from community-type methicillin-resistant Staphylococcus aureus infection, especially need special care and medication, can start the patient as soon as possible.

In clinical testing, multiplex polymerase chain reaction is mainly used to distinguish hospital-type methicillin-resistant Staphylococcus aureus and community-type methicillin-resistant Staphylococcus aureus according to SCCmec genotype. However, since the coagulase-negative staphylococci and other methicillin-sensitive S. aureus may also have the SCCmec gene, the accuracy of this test method is low. In addition, the multiplex PCR experiment procedure is complicated and the test time is long, so there are many limitations in practical applications (see Kobayashi N et al., Detection of mecA, femA, and femB genes in clinical strains of staphylococci using polymerase chain reaction. Epidemiol Infect. 1994; 113(2): 259-66, the entire disclosure of which is incorporated herein by reference.

In view of the above limitations and shortcomings of the conventional identification method, there is still a need to develop a rapid and accurate test method to help medical personnel quickly identify the type of methicillin-resistant Staphylococcus aureus, and then take A convenient treatment.

The present invention is directed to the above needs, and the inventors of the present invention have found a peptide biomarker which can be used for identifying community-type methicillin-resistant Staphylococcus aureus, which is beneficial to methicillin-resistant golden yellow grapes. Treatment of patients with cocci infection.

It is an object of the present invention to provide a peptide which is composed of the following amino acid sequence: GIIKX ₁ IKX ₂ LIEX ₃ FTGK (SEQ ID NO: 1), wherein X ₁ is F or V, and when X ₁ is In the case of F, X ₂ is G and X ₃ is K. When X ₁ is V, X ₂ is S and X ₃ is Q.

Another object of the present invention is to provide a biomarker for identifying a community-type methicillin-resistant Staphylococcus aureus which is a peptide consisting of the amino acid sequence shown by SEQ ID NO: 1.

It is still another object of the present invention to provide a method for identifying a community-type methicillin-resistant Staphylococcus aureus comprising: (a) providing a sample; and (b) detecting whether the sample contains a SEQ ID NO : The peptide consisting of the amino acid sequence shown in 1.

The detailed description of the present invention and the preferred embodiments thereof will be described in the following description.

The invention will be described in detail below with reference to the specific embodiments of the present invention. The invention may be practiced in various different forms without departing from the spirit and scope of the invention. The person stated. In addition, the terms "a", "an" and "the"

As described above, in view of the limitations and disadvantages of the conventional method for identifying methicillin-resistant Staphylococcus aureus, a rapid and highly accurate test method is required clinically to quickly identify methicillin-resistant golden yellow grapes. The type of cocci. Accordingly, the present invention provides a peptide consisting of the following amino acid sequence: GIIKX ₁ IKX ₂ LIEX ₃ FTGK (SEQ ID NO: 1), wherein X ₁ is F or V, and when X ₁ is F X ₂ is G and X ₃ is K. When X ₁ is V, X ₂ is S and X ₃ is Q. The inventor of the present study found that the community-type methicillin-resistant Staphylococcus aureus can express the peptide, so for a sample, it can be detected by a method such as mass spectrometry to detect whether the peptide is present in the test. In the body, it can be known whether the methicillin-resistant Staphylococcus aureus contained in the sample is a community-type anti-methicillin-resistant Staphylococcus aureus.

The invention also provides a biomarker useful for identifying community-type methicillin-resistant Staphylococcus aureus, which is a peptide consisting of the following amino acid sequence: GIIKX ₁ IKX ₂ LIEX ₃ FTGK (SEQ ID NO: 1 Where X ₁ is F or V, and when X ₁ is F, X ₂ is G and X ₃ is K, and when X ₁ is V, X ₂ is S and X ₃ is Q. As described above, whether the methicillin-resistant Staphylococcus aureus is a community-type anti-methicillin gold is detected by mass spectrometry or the like to detect the presence or absence of the biomarker/peptide. Staphylococcus aureus. Specifically, when (1) a peptide having the amino acid sequence of SEQ ID NO: 1 (wherein X ₁ is F, X ₂ is G and X ₃ is K), (2) having SEQ ID NO a peptide of the amino acid sequence of 1 (wherein X ₁ is V, X ₂ is S and X ₃ is Q), or (3) has the amino acid sequence of SEQ ID NO: 1 (wherein X ₁ is F, When the peptide having X ₂ is G and X ₃ is K) and the peptide having the amino acid sequence of SEQ ID NO: 1 (where X ₁ is V, X ₂ is S and X ₃ is Q), it can be confirmed The test system contains community-type methicillin-resistant Staphylococcus aureus.

The peptide/biomarker of the present invention is a phenol-soluble modulin alpha 1 (PSMα1, amino acid sequence: MGIIAGIIKVIKSLIEQFTGK (SEQ ID NO: 2)) and phenol A proteolytically therapeutic peptide fragment produced by enzymatic hydrolysis of a phenol-soluble modulin alpha 2 (PSMα2, amino acid sequence: MGIIAGIIKFIKGLIEKFTGK (SEQ ID NO: 3)). The phenol soluble regulatory peptide is known to be one of the toxic proteins of staphylococci, which can destroy human neutrophils.

The invention also provides a method for identifying a community-type methicillin-resistant Staphylococcus aureus comprising: (a) providing a sample; and (b) detecting whether the sample contains a SEQ ID NO: 1 A peptide consisting of the amino acid sequence shown.

In step (a) of the method of the present invention, the sample may be, but is not limited to, a biological sample such as body fluid or tissue; wherein the body fluid may be, for example, blood, serum, saliva, digestive juice, tears, sweat, or urine. Etc., and the tissue can be, for example, wound tissue, epithelial tissue, or muscle tissue.

In step (b) of the method of the present invention, based on the general knowledge and the teachings of the present specification, a suitable amino acid detection method may be selected as needed to detect whether the sample to be tested contains a SEQ. ID NO: The peptide consisting of the amino acid sequence shown in 1. For example, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis, liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) analysis, liquid chromatography serialization Mass spectrometry (LC-MS/MS) analysis, gas chromatography mass spectrometry (GS/MS) analysis, high performance liquid chromatography (HPLC) method, and ultra high performance liquid chromatography (UPLC) method, etc., to carry out the step (b) ) detection. In one embodiment of the invention, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is used for detection analysis.

In the step (b), when the detection result indicates that the detection system contains the peptide consisting of the amino acid sequence shown by SEQ ID NO: 1 (ie, the biomarker of the present invention), it can be determined that the detection system contains Community-type methicillin-resistant Staphylococcus aureus. Because community-type methicillin-resistant Staphylococcus aureus is more destructive to wounds in patients, it is less likely to be cured. Therefore, other antibiotics should be considered or used in combination with special care to enable patients to be treated. Healed as soon as possible.

As shown in the appended examples, the method of the present invention provides a high degree of assay accuracy by determining the presence of a particular peptide. In addition, since the method of the present invention does not require a complicated and time-consuming multiplex PCR technique, it can be known whether the sample contains the community-type methicillin-resistant Staphylococcus aureus, and therefore has a procedure compared to the conventional detection method. Relatively simple, time-saving and fast.

The invention is further illustrated by the following specific embodiments. The embodiments are provided by way of illustration only and are not intended to limit the scope of the invention.

[Examples]

[Strain collection and identification]

16 strains of vancomycin were collected and identified by different clinical patients to reduce Staphylococcus aureus (VISA), 8 heterogeneous vancomycin-sensitive strains of Staphylococcus aureus (hVISA), and 126 strains of methicillin-sensitive gold Methillillin-susceptible Staphylococcus aureus (MSSA), 213 strains of methicillin-resistant Staphylococcus aureus (MRSA); and 99 strains of methicillin-resistant Staphylococcus aureus from the "SMART study" in 2003 ( See, Ho CM et al., Prevalence and accessory gene regulator (agr) analysis of vancomycin-intermediate Staphylococcus aureus among methicillin-resistant isolates in Taiwan--SMART program, 2003. Eur J Clin Microbiol Infect Dis. 2010;29(4): 383-9; and Wang WY et al, Molecular and phenotypic characteristics of methicillin-resistant and vancomycin-intermediate staphylococcus aureus isolates from patients with septic arthritis. J Clin Microbiol. 2009;47(11): 3617-23, the full text of these documents And here for reference).

(1) Sensitivity test of antibiotic reagents

Strain using the above-described system BD Phoenix ^TM Automated Microbiology System (available from Becton Dickinson) identified as Staphylococcus aureus, and the strain sensitivity to such antibiotic agents according to the development of clinical assay and Laboratory Standards Institute standards (see Clinical and Laboratory Standards Institute Performance standards for antimicrobial susceptibility testing. Wayne, PA. CLSI document M100-S20. CLSI (Twentieth informational supplement), which is incorporated herein by reference in its entirety.

(2) SCCmec genotype identification

Multiplex PCR was performed using the genomic DNA of MRSA strain as a template, using 14 primers: 2 for the mecA gene, 4 for the 4 class mec gene complex (class A to D), and 8 for Box of chromosome recombinase gene (ccr) complexes (class 1 to 5 ccr). The reaction temperature of the multiplex PCR was as follows: first, the template DNA was separated by heating at 94 ° C for 2 minutes; then, heating was repeated at 94 ° C for 2 minutes (denaturation separation), heating at 60 ° C for 1 minute (adhesion primer), and heating at 72 ° C for 2 minutes (extension) The (extension) step was a total of 30 cycles; it was then heated at 72 ° C for 2 minutes for final extension. Next, the results of multiplex PCR were analyzed by gel electrophoresis (100 volts, 30 minutes). The reference standard strains of SCCmec type I, type II, type III, type IV, and type 5 were NCTC10442. , N315, 85/2082, Iva, and WIS. Since the MRSA strain of the first type of SCCmec is currently not readily available in clinical samples due to evolutionary relationships, only one standard MRSA strain of SCCmec type I (i.e., NCTC 10442) was analyzed in this example.

The analysis showed that there were 185 strains of MRSA with the genotype of SCCmec type 2 or type 3, which were identified as HA-MRSA; there were 127 strains of MRSA with the genotype of SCCmec type 4 or type 5, It was identified as CA-MRSA. For analysis and identification of MRSA strains, see Kondo Y et al., Combination of multiplex PCRs for staphylococcal cassette chromosome mec type assignment: rapid identification system for mec, ccr, and major differences in junkyard regions. Antimicrobial agents and chemotherapy. 2007; 51(1): 264-74; Grundmann H et al., Emergence and resurgence of meticillin-resistant Staphylococcus aureus as a public-health threat. Lancet. 2006;368(9538): 874-85; and Chambers HF et al., Waves And resistance: Staphylococcus aureus in the antibiotic era. Nat Rev Microbiol. 2009; 7(9): 629-41, the entire disclosure of which is hereby incorporated by reference.

[Example 1] Matrix-assisted laser desorption ionization time-of-flight mass spectrometry

(1) Preparation of samples

The colonies were added to 500 μl of 70 vol% ethanol (purchased from Merck), and after the suspension was centrifuged at 12000 g for 3 minutes, the strain was washed and the supernatant was removed. 50 microliters of 70% by volume or 2.5% by volume of formic acid (liquid chromatography-mass spectrometry (LC-MS) grade, purchased from Sigma Aldrich, Missouri, USA) was added to the strain as an extraction solution and shaken. Next, 50 μl of acetonitrile (LC-MS grade, purchased from JT-Baker, Mallinckrodt Baker) was added and shaken, and then the sample was centrifuged at 12000 g for 3 minutes. The supernatant was taken for mass spectrometry analysis.

(2) Mass spectrometry

0.5 μl of the extract was placed on a matrix-assisted laser-desorption ionization (MALDI) sample tray (purchased from Bruker Daltonic) with 0.5 μl of substrate solution (1 mg of α-cyano-4-hydroxyl) Cinnamic acid (CHCA, available from Bruker Daltonics) was dissolved in 500 microliters (μl) of water, 500 microliters (μl) of 100% acetonitrile, and 1 microliter (μl) of 100% trifluoroacetic acid) to cover the sample.

Samples of all S. aureus strains were then analyzed by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) using an Ultraflex III mass spectrometer equipped with a smart laser, available from Bruker Daltonics. Mass spectra of each strain were obtained after 600 laser shots. In linear mode, mass spectrometry detects masses ranging from 1000 to 10,000 mass-to-charge ratios; in reflector mode, mass spectrometry detects masses ranging from 1000 to 4000 mass-to-charge ratios.

Figure 1 is a mass spectrum of a sample treated with 70% by volume of formic acid. Figure 1A shows the linear mode, which has better sensitivity to high-quality areas and is therefore suitable for macromolecule detection, such as protein detection. The results showed that the three standard strains (MSSA: ATCC29213; MRSA: N315; and VISA: Mu50, the minimum inhibitory concentration of vancomycin was 8 mg / liter), the maximum intensity signal appeared in the mass-to-charge ratio of 3000 Da (Dalton) ) to 10,000 Da. In order to further analyze the differences between the various samples, mass spectrometry is performed in the reflection mode (this mode has better resolution for lower mass regions, so it is suitable for detection of small molecules, such as peptide detection). 1B, since the results showed that the signal value of the sample treated with 70% by volume of formic acid was weak, the sample treated with 2.5% by volume of formic acid was further analyzed, and the results are shown in Fig. 2.

Fig. 2 and Fig. 3 are mass spectra of samples treated with 2.5% by volume of formic acid, wherein Fig. 2A is a linear mode analysis and Fig. 2B is a reflection mode analysis. The results showed that the mass spectrum of the sample treated with 2.5% by volume of formic acid and analyzed in the reflection mode had different ion signals in the MSSA, MRSA and VISA strains and the intensity of the mass spectrum peak signal was large.

(3) Analysis of results

All MALDI-TOF mass spectra were analyzed and compared to identify mass spectrometric signals that could be used to identify community-type methicillin-resistant Staphylococcus aureus. Figure 3 shows the reflection pattern analysis of MSSA strain and SCCmec type 1 to type 5 MRSA strains. The results show that SCCmec type I to type III MRSA strains have similar mass spectrometry signals, and MSSA strain, SCCmec fourth The type and the MRSA strain of the fifth type have similar mass spectrum signals.

Mass spectrometry signals of all CA-MRSA (127 strains) and HA-MRSA (185 strains) were analyzed using ClinProTools software (purchased from Bruker Daltonics), and mass spectrometry signals with P values less than 0.01 were screened as available to distinguish The possible markers of CA-MRSA and HA-MRSA showed that the mass spectrum of most MSSA strains and CA-MRSA strains showed 1774 Da and 1792 Da signals (the mass value was rounded to the whole number). As shown in Table 1, among the 126 MSSA strains, the mass spectrum of 117 strains also had signals of 1774 Da and 1792 Da; among the 127 CA-MRSA strains, 121 strains had 1774 Da and 1792 Da simultaneously. However, among the 185 strains of HA-MRSA, only 15 strains have signals of 1774 Da and 1792 Da; and among 8 strains of hVISA and 16 strains of VISA, only 1 strain of VISA has 1774 Da and 1792. Signal of Da. The above results showed that among all the methicillin-resistant Staphylococcus aureus strains (HA-MRSA and CA-MRSA), the mass spectral peak signals of 1774 Da and 1792 Da were used to identify the community-type anti-methicillin golden yellow. Staphylococcus biomarkers have a sensitivity of up to 95.2% (121/127) and a specificity of 91.8% (170/185).

Figure 4A shows the HA-MRSA strain (SSCmec type 2 or type 3) and the CA-MRSA strain (SSCmec type 4) with the signal intensity of 1774 Da as the horizontal axis and the signal intensity of 1792 Da as the vertical axis. Or the data of the fifth type) is made into a 2-D cluster plot; the fourth panel is the HA-MRSA strain (SSCmec type 2 or type 3) and the CA-MRSA strain (SSCmec fourth) Two-dimensional analysis chart made of data of type or type 5), wherein the left Y axis is the number of colonies; the right Y axis is the color depth, which can display the mass spectrum signal intensity; the X axis is the mass to charge ratio (m/z) ) Separate dimensions. The darker the color of the black dot in the figure, the stronger the signal of the mass spectrum peak of the specific mass-to-charge ratio in the mass spectrum of the strain. The results of Figures 4A and 4B show that most of the CA-MRSA strains (SSCmec type 4 or type 5) have both mass spectrometry signals of 1774 Da and 1792 Da, with only a few HA-MRSA (SSCmec). The second or third type strain has such mass spectral peak signals.

4C and 4D are the receiver operating characteristic curves (ROC curves, which are a statistical representation) of the mass spectral peak signals of 1774 Da and 1792 Da, respectively, to analyze the mass spectral peak signals for identification of community type. The specificity and sensitivity of methicillin-resistant Staphylococcus aureus. Among them, the area under the ROC plot (AUC) of the 1774 Da mass spectral peak signal is 0.968, and the area under the ROC curve of the mass spectral peak signal of 1792 Da is 0.973. The closer the AUC area is to 1, the higher the identification accuracy. Show that these mass spectral peak signals can be used to accurately identify community-type methicillin-resistant Staphylococcus aureus.

[Example 2] Matrix-assisted laser desorption ionization time-of-flight mass spectrometry

The signals of 1774 Da and 1792 Da were further analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI TOF/TOF Mass Spectrometer). The results are shown in Figures 5A and 5B, 1774 Da and 1792 Da. The signals represent the amino acid sequence of GIIKX ₁ IKX ₂ LIEX ₃ FTGK (SEQ ID NO: 1). Wherein, the signal of 1774 Da represents a peptide having the sequence of SEQ ID NO: 1, and wherein X ₁ is V, X ₂ is S, and X ₃ is Q (ie, amino acid sequence of GIIKVIKSLIE QFTGK); signal of 1792 Da Represents a peptide having the sequence of SEQ ID NO: 1, wherein X ₁ is F, X ₂ is G and X ₃ is K (ie, the amino acid sequence of GIIKFIKGLIEKFTGK). After sequence alignment with the NCBInr database, it was found that the peptide represented by the 1774 Da signal is the peptide fragment (PSMα1 Δ1-5) produced by the enzymatic treatment of the phenol soluble regulatory protein α1 (PSMα1, MGIIAGIIKVIKSLIEQFTGK) of Staphylococcus. The peptide represented by the 1792 Da signal is a peptide fragment (PSMα2 Δ1-5) produced by enzymatic hydrolysis of the phenol soluble regulatory protein α2 (PSMα2, MGIIAGIIKFIKGLIEKFTGK) of Staphylococcus.

From the above experimental results, it is known that GIIKX ₁ IKX ₂ LIEX ₃ FTGK (SEQ ID NO: 1, where X ₁ is F or V, and when X ₁ is F, X ₂ is G and X ₃ is K, when X ₁ is At V, the amino acid sequence of X ₂ is S and X ₃ is Q) is mainly present in the MSSA strain and the CA-MRSA strain. In clinical diagnosis, when a patient is suspected to be infected with Staphylococcus aureus, the sensitivity of the sample to methicillin can be tested by conventional bacterial culture method. When the test result shows that the sample has methicillin resistance. Then, it can be judged to be an MRSA strain, and then, the peptide/biomarker of the present invention can be further utilized to identify whether the specimen has a CA-MRSA strain. Alternatively, the peptide/biomarker of the present invention can be used to identify whether the sample has the MSSA strain or the CA-MRSA strain, and further, the medium containing methicillin is used to identify whether the sample contains CA-MRSA.

The above embodiments are merely illustrative of the principles and effects of the invention and are not intended to limit the invention. Modifications and variations of the above-described embodiments can be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, the scope of the invention should be defined as defined in the appended claims.

<110> China Medical University

<120> Shengyuetai Biomarker for the identification of community-type methicillin-resistant Staphylococcus aureus

<130> None

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<170> PatentIn version 35

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<213> Staphylococcus

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Figure 1A shows a matrix-assisted laser-desorption ionization time-of-flight mass spectrometry analysis of a S. aureus sample treated with a 70% formic acid extraction solution in a linear mode;

Figure 1B is a graph showing matrix-assisted laser desorption ionization time-of-flight mass spectrometry of a S. aureus sample treated with a 70% formic acid extraction solution in a reflective mode;

Figure 2A shows a matrix-assisted laser-desorption ionization time-of-flight mass spectrometry analysis of a S. aureus sample treated with a 2.5% formic acid extraction solution in a linear mode;

Figure 2B is a graph showing the matrix-assisted laser desorption ionization time-of-flight mass spectrometry of a S. aureus sample treated with a 2.5% formic acid extraction solution in a reflective mode;

Figure 3 is a graph showing the matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of methicillin-sensitive Staphylococcus aureus and methicillin-resistant Staphylococcus aureus;

Figure 4A shows a 2-D cluster diagram of the data for the mass spectrometry signal of 1774 Da and 1792 Da of methicillin-resistant Staphylococcus aureus; Figure 4B shows the methicillin-resistant gold a two-dimensional analysis of data from the mass spectrometry signal of Staphylococcus aureus;

Figure 4C shows the receiver operating characteristic curve prepared by the data of the mass spectrometry signal of 1774 Da of methicillin-resistant Staphylococcus aureus;

Figure 4D is a graph showing the receiver operating characteristic curve prepared by the data of the mass spectrum peak signal of 1792 Da of methicillin-resistant Staphylococcus aureus;

Figure 5A shows the mass spectrum of the fracture fragment of 1774 Da using matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry;

Figure 5B shows the mass spectrum of the fractured fragment of 1792 Da using matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry.

Claims (9)

A peptide consisting of the following amino acid sequence: GIIKX ₁ IKX ₂ LIEX ₃ FTGK (SEQ ID NO: 1), wherein X ₁ is F or V, and when X ₁ is F, X ₂ is G And X ₃ is K, and when X ₁ is V, X ₂ is S and X ₃ is Q. One is used to distinguish between community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) and hospital-associated methicillin-resistant Staphylococcus aureus (hospital-associated methicillin-resistant Staphylococcus aureus) . A biomarker of HA-MRSA) which is a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1. A method for distinguishing between community-type methicillin-resistant Staphylococcus aureus and hospital-type methicillin-resistant Staphylococcus aureus comprises: (a) providing a sample containing methicillin-resistant Staphylococcus aureus; (b) detecting whether the sample contains a peptide consisting of the amino acid sequence shown by SEQ ID NO: 1. The method of claim 3, wherein the test system fluid or tissue. The method of claim 4, wherein the body fluid is selected from the group consisting of blood, serum, saliva, digestive juice, tear fluid, sweat, urine, and combinations thereof. The method of claim 4, wherein the tissue is selected from the group consisting of wound tissue, epithelial tissue, muscle tissue, and combinations of the foregoing. The method of claim 3, wherein in step (b), the group is selected from the group consisting of Method for detection: matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis, liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) analysis, liquid chromatography tandem mass spectrometry ( LC-MS/MS) analysis, gas chromatography mass spectrometry (GS/MS) analysis, high performance liquid chromatography (HPLC), ultra high performance liquid chromatography (UPLC), and combinations of the foregoing. The method of claim 7, wherein in step (b), matrix-assisted laser desorption ionization time-of-flight mass spectrometry is used for detection. The method of any one of claims 3 to 8, wherein if the peptide is detected in step (b), the sample is judged to comprise a community-type methicillin-resistant Staphylococcus aureus.