TW202045913A - Method for rapid identification of bacteria by portable Raman system which has advantages of requiring no bacterial culture of patient specimens and quickly obtaining identification results in 0-15 minutes - Google Patents

Abstract

A method for rapid identification of bacteria using a portable Raman system mainly includes directly using secondary pure water to wash a sample taken from a patient without bacterial culturing, or without further concentrating to prepare the sample. In conjunction with a hydrophobic polymer film in a surface-enhanced Raman spectroscopic chip and metal particles therein to obtain a Raman spectrum to be analyzed in 0-15 minutes. Then, a peak identification method is adopted to perform full-range HQI similarity sorting and identification to both the Raman spectrum to be analyzed and pre-stored spectra, or optionally perform principal component analysis to obtain a distribution of drop points, so that single or multiple mixed bacterial species, drug-resistant strains and bacterial concentration are identified. The present invention does not require bacterial culturing of patient specimens, can quickly obtain identification results in 0-15 minutes, and is more close to the actual medical needs of a hospital.

Description

Method for rapid identification of bacteria using portable Raman system

The present invention relates to an identification method, in particular to a method that can quickly identify bacterial types, drug-resistant strains and bacterial concentrations within 15 minutes without bacterial culture.

Refer to Figure 1. The existing methods of bacterial testing mainly rely on the cultivation of bacteria and the operation of biochemical tests. For example, the Ministry of Health and Welfare announced on December 23, 102, Ministry of Health and Welfare, No. 1021951187 to amend the inspection method of food microorganisms "Salmon The test procedure of "testing for bacilli" is to add the enrichment solution to the sample and then selectively carry out enrichment culture and separation, and then conduct biochemical experiments, or use commercially available biochemical test kits to identify, or It is a serological test to determine the strain and its type. The disadvantage is that the entire inspection process is not only complicated in content, but also because it takes a long time to carry out the enrichment culture, it is not only time-consuming and laborious, but also unable to meet immediate needs.

Previously, there was also a publication number TW200932913 of the Invention Specification published by the Intellectual Property Bureau of the Republic of China on August 1, 2009, "Methods for identifying microorganisms or detecting changes in their morphology using surface enhanced Raman scattering (SERS)", It discloses the method and equipment for using surface enhanced Raman scattering (SERS) to generate a graph and the enrichment culture of microorganisms to identify microbial species, and to detect changes in the type of microorganisms caused by antibiotics, antibacterial agents, or pathogenic factors. Identify the effects of antibiotics, antibacterial agents, or pathogenic factors on bacterial cells.

To explore this invention, the active carrier used for surface-enhanced Raman scattering (SERS) is a solid film embedded or covered by nano-particles. The nano-particles are selected from the group consisting of the following substances: gold, silver , Copper, platinum, silver/gold, platinum/gold, copper/gold core-shell, and alloy particles. In the embodiment, silver/anodic alumina (AAO) carrier (silver-filled porous anodic alumina Rice tube (silver-filled porous anodic aluminum oxide nanochannels)). In addition, the invention must use a "fixed liquid" in order to prevent microorganisms from moving and keep them alive in a watery environment. The fixing liquid is selected from the group consisting of agarose gel and glycerin. group. While the bacteria sample preparation method is, for E. coli (Escherichia coli) JM109, Escherichia coli JM109 (mutant), Enterococcus faecalis (Enterococcus faecalis) 7-18uE, enterococci (Enterococcus faecium) 13631, Staphylococcus aureus (Staphylococcus aureus 13649, Staphylococcus aureus 13615, Streptococcus agalactiae 13640, Enterococcus faecalis 13641 C2, Klebsiella pneumoniae 13641 C1, Proteus mirabilis 13621 C1 , Enterobacter. cloacae ( Enterobacter. cloacae ) 13457 C2, E. coli 13594 C1, E. coli 13650, and E. coli 13634 and other 14 laboratory strains, the bacteria were cultured in 5 ml of LB broth for about 6 hours, until the OD600 value (That is, the absorbance of the bacteria at an optical wavelength of 600 nm, and the absorbance of the bacteria is used to measure the concentration of bacteria in the bacterial culture solution) when it is about 0.5, then wash 5 times with phosphate buffered saline (PBS, Phosphate buffered saline) Afterwards, it was resuspended in 0.1 ml of phosphate buffered saline, about 3 to 5 microliters of bacterial suspension was placed on the silver/anodized aluminum carrier, and 0.5% agarose gel was used to fix the bacterial sample to Inhibit the movement of bacteria.

As far as the technical content disclosed in the TW200932913 case is concerned, the bacteria used in the test are all self-cultivating in the laboratory. The bacteria cultivated in the laboratory have been adjusted and changed, and are not the same as those in the patient’s specimens obtained clinically in the hospital. , It will harm the human body and cause disease, even bacteria that are resistant to some antibiotics. In addition, in the process of detection and determination, the process of enrichment culture is very time-consuming. For example, it takes about 6 hours in LB broth. After that, it must be tested for 30 to 60 minutes to confirm the OD600 value, and the number of bacteria will be obtained. The test can be performed after reaching a certain amount. If the number of bacteria is insufficient than the expected amount, the cultivation time must be extended and the OD600 value must be reconfirmed. In this way, the process of excessively long and repeated bacteria enrichment culture and confirmation of the OD600 value will not only take time For patients, this period of time is that the body is being attacked and attacked by bacteria, and it also increases the bacterial infection in the hospital and exposes more patients and medical staff to the risk of infection.

Moreover, the culture medium used in the process of enrichment culture is rich in salts, so the obtained Raman scattering spectrum has too much noise, which makes it impossible to accurately identify and analyze the bacteria; the agarose gel used to fix the bacteria gel) It takes time to wait for coagulation. If the coagulation is not complete, it will cause the bacteria to be unable to be fixed and subsequent detection cannot be performed. In addition, the actual use of the technical content disclosed in the TW200932913 case must be restricted to the use of the Jobin Yvon Raman microscope (Model LabRAM HR800). The current asking price is about NT$10~18 million. Yuan, a high-end Raman instrument, and because of its large model and inconvenient carrying, it is not widely used in general medical institutions. At the same time, the operation of the Yobin Ivan Raman microscope is difficult to adjust due to the small focus distance. To the most suitable focal length confocal surface for observation, it is not only time-consuming in operation, but also requires a lot of money to train related operating manpower.

According to statistics, nearly 7 million of the more than 300 million people in the United States visit a doctor for urinary tract infections each year. A further query of the relevant information of the General Hospital of the Tri-Services shows that in the urology clinic, there is an average of about one out of every three to four people. Is suffering from urinary tract infection. However, in observing the clinically infected patients in hospitals, it is often not just a single strain of bacteria. At the same time, there are also cases of infection with more than two kinds of bacteria. This situation will make it more difficult for the hospital to control bacterial infections.

In addition, according to the research report on the establishment of a reference laboratory for drug-resistant bacteria in the 95-year self-research plan of the Executive Yuan Department of Health, it can be seen that in terms of bacterial resistance testing, whether it is traditional medium drug sensitivity test, pulse electrophoresis, polymerization Enzyme chain reaction or other molecular diagnostic methods are time-consuming and labor-intensive and cannot meet immediate needs. However, the treatment of medically resistant bacteria and the nosocomial infections caused by them is urgent.

In view of the above, the existing bacteria types, concentration testing methods, and bacterial resistance testing still need to be improved to quickly and accurately detect single or multiple mixed bacteria types, bacterial concentration and drug resistance, so as to carry out follow-up medical treatment. And other applications.

Therefore, the purpose of the present invention is to provide a method for rapid identification of bacteria using a portable Raman system that does not require bacterial culture and can be quickly detected within 0-15 minutes.

Therefore, the present invention uses a portable Raman system to quickly identify bacteria in urinary tract inflammation, including one step (a), one step (b), one step (c), one step (d), and one step (e) ).

In the step (a), at least one surface-enhanced Raman spectroscopy chip is placed in at least one of the plurality of spaced substrate grooves of the portable Raman system, and the at least one surface-enhanced Raman spectroscopy chip has a substrate, A hydrophobic polymer film formed on the top surface of the substrate, and a majority of metal particles distributed in the hydrophobic polymer film and having a size range of nanometers, wherein the hydrophobic polymer film is selected from the following Formed by at least one of the polymer materials forming the group: polyimide, sol-gel, maleic acid, polyvinylidene fluoride, polyethylene, polypropylene, polyamide, polystyrene, polycarbonate , And polymethyl methacrylate, wherein the sol-gel is composed of methyltrimethoxysilane, tetramethoxysilane and octadecyltrimethoxysilane with a weight composition ratio of 10:2:1. The particles are formed of at least one selected from the following group: gold, silver, graphene, and copper.

In this step (b), a sample taken from a human body is directly selected from washing and diluting with pure water twice, and condensing without bacterial culture, and processed into at least one sample to be tested with different bacterial concentrations.

The step (c) immediately places the at least one sample to be tested on the at least one surface-enhanced Raman spectroscopy chip so that the at least one sample to be tested directly contacts the hydrophobic polymer film and is affected by the metal particles .

In the step (d), the light source of the portable Raman spectrometer system is used to irradiate the at least one sample to be tested placed on the at least one surface enhanced Raman spectroscopy chip to obtain at least one Raman spectrum to be analyzed.

This step (e) uses the peak identification method to compare the at least one Raman spectrum to be analyzed and most of the pre-stored and corresponding to different types of bacteria. When the comparison similarity exceeds the preset value, it is determined that the corresponding The sample to be tested for the Raman spectrum to be analyzed contains bacteria corresponding to the spectrum.

In particular, the present invention is applied portable Raman system for rapid identification of bacteria, wherein the step (e) in the x-axis is also distinguished 600-750cm ^-1, 950-1100cm ^-1, 1400-2000cm ^-1 three areas The segments are sorted by similarity to determine the type of bacteria contained in the sample to be tested.

In particular, the method for rapidly identifying bacteria using the portable Raman system of the present invention further includes a step (f), using the to-be-analyzed Raman spectrum to perform the principal component analysis of the whole section to obtain the drop point distribution to identify at least one bacterial species, At least one of strain resistance and bacterial concentration.

In particular, the present invention uses a portable Raman system to quickly identify bacteria, wherein the specimens taken from the human body in step (b) include, but are not limited to, urine, ascites, blood, plasma, serum, sputum, and cerebrospinal fluid. With fluid, pleural fluid, or tissue fluid.

In particular, the present invention uses a portable Raman system to quickly identify bacteria, wherein in step (c), an amount of 3-10 μl is placed on the at least one surface enhanced Raman spectroscopy wafer.

In particular, the present invention uses a portable Raman system to quickly identify bacteria. In step (d), laser light with a wavelength of 785 nm is used as a light source to illuminate the sample to be tested to obtain the Raman spectrum to be analyzed.

The effect of the present invention is to provide a method that can directly obtain the spectrum to be analyzed by adding a Raman spectroscopy chip to the surface of the patient's bacterial specimen, and then use the peak identification method to sort the similarity and identify the drop point distribution of the principal component analysis of the whole section , To identify single or multiple mixed strains, drug-resistant strains and bacterial concentration, to improve the existing identification process of bacterial species, because the need for bacterial specimen culture or other factors lead to time-consuming, excessive noise, and unable to quickly , Accurate identification of single or multiple mixed bacteria, bacterial concentration and drug resistance analysis.

Before the present invention is described in detail, it should be noted that in the following description, similar elements are represented by the same numbers.

2 and 3, an embodiment of the method for rapidly identifying bacteria using a portable Raman system of the present invention includes a step (a) 201, a step (b) 202, a step (c) 203, and a step ( d) 204, a step (e) 205, and a step (f) 206, which are used to quickly and accurately perform single or multiple mixed bacterial species identification, bacterial concentration and drug resistance analysis.

In the step (a) 201, at least one surface-enhanced Raman spectroscopy chip 3 is placed on at least one of a plurality of regularly spaced substrate grooves 511 of a substrate 51 of the portable Raman system 5; the at least one surface The enhanced Raman spectroscopy chip 3 has a substrate 31, a hydrophobic polymer film 32 on a top surface 311 of the substrate 31, and a majority of metals distributed on the hydrophobic polymer film 32 and having a size range of nanometers. Particles 33, wherein the hydrophobic polymer film 32 has a rough surface 321 and is selected from the group consisting of polyimide, sol-gel, maleic acid, polyvinylidene fluoride , Polyethylene, polypropylene, polyamide, polystyrene, polycarbonate, and polymethyl methacrylate, where the sol-gel is composed of methyl trimethoxysilane with a weight composition ratio of 10: 2: 1 , Tetramethoxysilane and octadecyltrimethoxysilane; the metal particles 33 range in size from 60 to 120 nanometers, and are selected from gold, silver, graphene, copper, or a combination of the foregoing, Therefore, when performing light excitation to generate scattering, a spectrum that can be easily distinguished, compared and identified can be obtained sensitively and without noise.

This step (b) 202 can be performed before step (a) 201 or simultaneously. This step (b) 202 can directly take samples from the patient, such as urine, ascites blood, plasma, serum, sputum, Cerebrospinal fluid, pleural fluid, or tissue fluid does not need to be cultured directly as the sample to be tested 900 for immediate testing, or, the sample is washed and diluted with secondary pure water (ddH ₂ O), or concentrated (15 minutes) into the sample 900 to be tested; among them, the process of washing and diluting or concentrating the sample 900 with twice pure water is to pack the sample from the patient in a test tube and cool and centrifuge at 700 rpm for 5 minutes After that, the supernatant was obtained, and then the supernatant was taken out after cooling and centrifugation at 13000 rpm for 5 minutes. The second pure water was added to the original test tube and the supernatant was taken out after cooling and centrifuging at 13000 rpm for 5 minutes. Add secondary pure water to the test tube and dilute or concentrate to obtain the sample 900 to be tested.

The prepared sample 900 does not need to be cultured by bacteria, and can be immediately placed in the substrate groove 511 of the surface enhanced Raman spectroscopy wafer 3 for this step (c) 203, this step (d) 204, and this step within 0-15 minutes. (e) 205, and this step (f) 206 perform rapid analysis and determination.

In this step (c) 203, the sample 900 to be tested is placed in the substrate groove 511 of the surface enhanced Raman spectroscopy wafer 3, so that the sample 900 directly contacts the rough surface 321 of the hydrophobic polymer film 32, and the sample to be tested 900 is affected by the metal particles 33 in the hydrophobic polymer film 32 so as to obtain a spectrum that is easy to distinguish, compare and appraise sensitively and without noise during subsequent light excitation to generate scattering. Preferably, the amount is 3~10μl. In this example, the amount of 3μl is placed on the surface enhanced Raman spectroscopy wafer 3, and, for example, the concentration of the test sample 900 is placed on a surface enhanced Raman spectroscopy wafer Attention must be paid to the problems of confusion and cross-contamination in the substrate groove 511 of 3

In step (d) 204, one of the light sources 52 of the portable Raman spectrometer system 5, such as 785nm laser light, is used to irradiate the surface-enhanced Raman spectroscopy wafer 3 on which the sample 900 is placed for excitation The corresponding Raman scattered light is generated to obtain at least one Raman spectrum to be analyzed; step (e) 205 uses the peak identification method to compare the similarity of the whole section to the at least one Raman spectrum to be analyzed and the majority of the pre-stored and correspondingly different The spectrogram of the bacterial species, and when the comparison similarity exceeds the preset value, it is determined that the test sample corresponding to the Raman spectrum to be analyzed contains the bacteria corresponding to the spectrogram; the step (f) 206 uses the Raman to be analyzed Principal component analysis of the whole section of the spectrum is performed to obtain a drop point distribution to identify at least one of at least one bacterial species, bacterial resistance, and bacterial concentration.

Refer to Figure 4, Figure 4 is the implementation of the above step (d) 204, and testing a number of samples containing different concentrations of Escherichia coli (Escherichia coli, E. coli) to be analyzed and obtained each to-be-analyzed Raman spectrum, where The x-axis is Raman Shift (Raman Shift) representation.

Referring to FIG. 5, FIG. 6, FIG. 5 is a Raman spectrum analysis of the x-axis is divided into three segments ^{600-750 cm -1, 950-1100 cm -1,} 1400-2000 cm -1 for distinguishing section 4 to be obtained FIG. Perform similarity ranking and identification, and obtain the results of similarity ranking and identification. In more detail, the peak identification method in the peak identification system developed in JAVA language can compare the spectrum library for the peak of the bacterial specimen of the test patient to generate Hit Quality Index (hereinafter referred to as HQI), which indicates the degree of similarity between the Raman spectrum to be analyzed and the pre-stored spectra with respect to a specific peak. The higher the similarity, the greater the HQI. Figure 5 shows that the HQI of the sample to be tested containing Escherichia coli (E. coli) is 0.67, 0.45, and 0.36 after the peak identification method is used to strengthen the differentiation of the detailed segments, which is obviously different from the HQI of the pre-stored bacteria library standard. The similarity can be recognized immediately, that is, it is proved that the present invention can confirm the bacterial species to identify single or multiple mixed bacterial species. Referring to Figure 6, the HQI obtained by distinguishing the analytical Raman spectrum from the pre-stored bacterial spectrum by the detailed section is 0.89, 0.65, 0.45, and it is proved that the peak identification method of the present invention is divided into three sections to strengthen the distinction of the detailed section to be analyzed. Mann spectroscopy performs similarity ranking and principal component analysis, which can identify single or multiple mixed bacterial species, and can improve the accuracy of the results of the bacterial species to be confirmed.

Referring to Fig. 7, Fig. 8, and Fig. 9, the above embodiments of the present invention are implemented, and the sample to be tested containing Escherichia coli (E. coli) and subjected to the second pure water cleaning treatment is used for the peak identification method to test The results of the comparison of the whole range of the sample wave peaks, the HQI obtained are 0.70, 0.81, and 0.67, which confirms that the sample to be tested obtained by the "secondary pure water cleaning" process of the present invention can still be accurately compared with the wave peak identification method. Accurate identification and analysis of bacterial species, and it can be seen from FIG. 9 that the HQI of the sample to be tested after the second pure water cleaning treatment in the embodiment of the present invention is increased from 0.70 to 0.79, which proves that bacterial species identification and analysis can be performed more accurately. analysis.

Referring to Figure 10, Figure 11, Figure 12, the present invention contains drug-resistant Escherichia coli (Escherichia coli ESBL, E. coli ESBL), Proteus mirabilis (P. mirabilis), Escherichia coli (Escherichia coli, E. coli) sample is concentrated into the sample to be tested, and the Raman spectrum to be analyzed is obtained, and the principal component analysis of the whole spectrum spectrum shows that the bacterial spectrum signal is significantly enhanced.

Refer to Figure 13 and Figure 14, and further focus on the samples from patients with urinary tract inflammation that contain Escherichia coli (E. coli) before being concentrated, and the HQI is 0.66, 0.69, 0.61; after the concentration of the embodiment of the present invention Later, the HQI is 0.86, 0.72, 0.49, which can verify that the concentration process of the present invention increases the analytical identification to distinguish bacterial species.

The following further describes the identification of specimens containing two kinds of mixed bacteria according to the present invention.

Referring to Figure 15, samples from patients with urinary tract inflammation, including two kinds of mixed bacteria Citrobacter freundii (C. freundii) and Proteus mirabilis (P. mirabilis), were obtained after obtaining the Raman samples for analysis. After spectroscopy, import several full-range Raman spectrum data into the drop point distribution software, set the variables and analysis conditions for each factor, then perform full-range analysis, and then select the type of statistical graph generated based on the factor analysis result to get the drop point The distribution and observation show that several data points of the specimens that also include Citrobacter freundii have a tendency to gather in a specific area. Therefore, this specific area can be circled, and when the test is unknown and waiting When confirming the Raman spectrum data of the whole section of the specimen of the bacterial species and obtaining its drop point, it can be determined in the specimen of the bacterial species to be confirmed according to whether the position of the drop point approaches or falls within this specific area The possibility that the bacteria contained is Citrobacter freundii is higher.

Referring to Figure 16, Figure 17, Figure 18, based on the above, Figure 16 about Proteus mirabilis (P. mirabilis), Figure 17 about Escherichia coli (Escherichia coli, E. coli), and Figure 18 about Proteus mirabilis (P. mirabilis) can also be obtained respectively. Pseudomonas aeruginosa (Pseudomonas aeruginosa, P. aeruginosa) is located, and a specific area is delineated to determine what bacteria are contained in the specimen to be confirmed.

Referring to Figure 19, it will also include Citrobacter freundii (Citrobacter freundii, C. freundii), Proteus mirabilis (P. mirabilis), Escherichia coli (E. coli), Pseudomonas aeruginosa , P. aeruginosa) of the whole section of the specimen to be analyzed Raman spectroscopy data to obtain the drop point distribution, according to the aforementioned method to delineate the specific range of each bacterial aggregation in the same coordinate plane, can be used to identify the mixture to be confirmed The mixed bacterial species contained in the sample of bacteria.

Refer to Figure 20, taking patient A (labeled as patient A in the figure) as an example. After obtaining the Raman spectrum to be analyzed from the specimen, the drop point labeled as patient A can be analyzed, because patient A and French lemon Acid bacillus (Citrobacter freundii, C. freundii), Proteus mirabilis (P. mirabilis) are closer, and are similar to Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa) Obvious distinction, so it can be determined that the specimen taken from patient A includes two kinds of mixed bacteria, and the bacterial species are Citrobacter freundii (C. freundii) and Proteus mirabilis (P. mirabilis).

Refer to Figure 21, Figure 22, Figure 23, Figure 24, Figure 25, Figure 26, Figure 21 is about specimens including Escherichia coli (E. coli), Klebsiella pneumoniae (Klebsiella pneumoniae, K. pneumoniae) ) Two kinds of mixed bacteria, Figure 22 is about the specimens including Proteus mirabilis (P. mirabilis) and Pseudomonas aeruginosa (P. aeruginosa) two kinds of mixed bacteria, Figure 23 is about the specimens including Freund Citrobacter freundii (Citrobacter freundii, C. freundii), Klebsiella pneumoniae (Klebsiella pneumoniae, K. pneumoniae) two kinds of mixed bacteria, Figure 24 is about specimens including Escherichia coli (E. coli), gram Klebsiella pneumoniae (Klebsiella pneumoniae, K. pneumoniae) two kinds of mixed bacteria, Figure 25 is about the specimens including Citrobacter freundii (Citrobacter freundii, C. freundii), Klebsiella pneumoniae (Klebsiella pneumoniae, K. . pneumoniae) two kinds of mixed bacteria, Figure 26 is about the specimens including Klebsiella pneumoniae (Klebsiella pneumoniae, K. pneumoniae), drug-resistant Klebsiella pneumoniae (Klebsiella pneumoniae ESBL, K. pneumoniae ESBL) two species The mixed bacteria is used to identify the two mixed bacterial species of the specimen; in particular, because the drop points fall outside the delineated range, it can be further determined that the bacterial species of the specimen to be tested are different, so as to verify that the present invention can indeed Distinguish the mixed strains.

Referring to Figure 27, the embodiment of the present invention is used to identify three kinds of mixed bacteria, which will be taken from patients with urinary tract inflammation including Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa) and coagulation After coagulase-negative staphylococci (CoNS) specimens are analyzed in the whole segment, the drop point distribution as shown in Figure 27 is obtained. After delineating the range of various bacterial species, it can be used to identify Escherichia coli, Pseudomonas aeruginosa and coagulation Enzyme-negative staphylococcal strains to be tested; and the drop point of the tested sample is different from Acinetobacter baumannii/calcoaceticus complex (ABC), Enterobacter aerogenes, E. Aerogenes) and Serratia marcensess (S. marcensess, S. marcensess) strains to be tested are clearly distinguishable, and it is verified that the present invention can indeed distinguish mixed bacterial strains.

Refer to Fig. 28, Fig. 29, Fig. 30, Fig. 31, Fig. 32, Fig. 33, Fig. 34, Fig. 35, Fig. 36, Fig. 37, Fig. 38, the implementation of the present invention is used to identify specimens that additionally include three mixed bacteria, wherein , Figure 28 is about a sample of three mixed bacteria including Escherichia coli (E. coli), Entercoccus faecalis (E. faecalis) and coagulase-negative staphylococci (CoNS), Figure 29 It is about a sample of three mixed bacteria including Escherichia coli (E. coli), Klebsiella pneumoniae (K. pneumoniae) and coagulase-negative staphylococci (CoNS). 30 is about a sample that includes Klebsiella pneumoniae (Klebsiella pneumoniae, K. pneumoniae), Entercoccus faecalis (E. faecalis) and coagulase-negative staphylococci (CoNS). Figure 31 is a sample of three mixed bacteria including Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa) and coagulase-negative staphylococci (CoNS), Figure 32 It is about a sample of three mixed bacteria including Escherichia coli (E. coli), Klebsiella pneumoniae (Klebsiella pneumoniae, K. pneumoniae) and Proteus mirabilis (P. mirabilis), Figure 33 is Regarding the specimens including three mixed bacteria of Escherichia coli (E. coli), Proteus mirabilis (P. mirabilis) and coagulase-negative staphylococci (CoNS), Figure 34 is about Klebsiella pneumoniae (Klebsiella pneumoniae, K. pneumoniae), enterobacter cloacae (Enterobacter cloacae, E. cloacae), and Pseubacterium aeruginosa (Pseu domonas aeruginosa, P. aeruginosa) three mixed bacteria specimens, Figure 35 is about including Klebsiella pneumoniae (Klebsiella pneumoniae, K. pneumoniae), Pseudomonas aeruginosa (P. aeruginosa) and vancomycin resistance Enterococcus (Vancomycin resistant enterococci, VRE) three mixed bacteria specimens, Figure 36 is about Escherichia coli (E. coli), Entercoccus faecalis (Entercoccus faecalis, E. faecalis) and coagulase-negative staphylococci (coagulase -negative staphylococci, CoNS) Three kinds of mixed bacteria specimens, the drop point distribution obtained by full-segment analysis is used to identify the test specimens including the three mixed bacteria species, and the bacterial species of the test specimens are different Bacterial strains, verifying that the present invention can indeed distinguish mixed bacterial strains.

Referring to Fig. 37, Fig. 38, and Fig. 39, the implementation of the present invention to identify drug-resistant strains will be described below. Samples taken from patients including Methicillin-resistant Staphylococcus aureus (MRSA) bacterial specimens and non-multidrug resistant Staphylococcus aureus (MSSA) specimens were purified twice Water is diluted 1 times, 3 times, 5 times, and 10 times into multiple test samples (including the test samples of multi-drug resistant Staphylococcus aureus, respectively marked as MRSA-1X, MRSA-3X, MRSA- -5X, MRSA-10X; samples to be tested including non-multidrug resistant Staphylococcus aureus are labeled as MSSA--1X, MSSA--3X, MSSA--5X, MSSA--10X), and the obtained samples are to be analyzed Mann spectrograms are shown in Figure 37 and Figure 38. Principal component analysis is performed with the full range spectrum to obtain the drop point distribution shown in Figure 39. The test specimens based on different drug susceptibility are clearly divided into two blocks. Therefore, it is verified that the present invention can indeed distinguish drug resistance of strains.

Refer to Figure 40, Figure 41, and Figure 42, and the following samples are taken from patients including drug-resistant Escherichia coli (Escherichia coli ESBL, E. coli ESBL) and Escherichia coli (E. coli) using secondary pure samples. After dilution with water, the principal component analysis was performed with the full-segment spectrum to obtain the drop point distribution, verifying that the present invention can quickly and reliably distinguish the drug resistance of strains based on different drug susceptibility strains.

Refer to Figure 43, Figure 44, Figure 45, the following are taken from patients including drug-resistant Klebsiella pneumoniae ESBL (K. pneumoniae ESBL), Klebsiella pneumoniae ESBL (Klebsiella pneumoniae ESBL, K. pneumoniae After the specimens of) were diluted with pure water twice, the principal component analysis was performed on the full-segment spectrum to obtain the drop point distribution, verifying that the present invention can quickly and reliably distinguish the drug resistance of strains based on different drug susceptibility strains.

Refer to Figure 46, Figure 47, Figure 48, the following are the samples taken from patients including Carbapenem-Resistant Enterobacter cloacae (CR E. cloacae) and Enterobacter cloacae (E. cloacae) respectively. After being diluted with the sub-pure water, the principal component analysis is performed with the full range spectrum to obtain the drop point distribution, verifying that the present invention can quickly and reliably distinguish the drug resistance of the strains based on the different drug susceptibility strains.

Refer to Figure 49, Figure 50, and Figure 51. The following samples are taken from patients, including drug-resistant Enterobacter aerogenes (Carbapenem-Resistant Enterobacter aerogenes, CREA) and Enterobacter aerogenes (E. aerogenes) respectively. After the second pure water dilution, the principal component analysis is performed with the full-range spectrum to obtain the drop point distribution, which verifies that the present invention can quickly and reliably distinguish the drug resistance of strains based on strains with different drug susceptibility.

Refer to Figure 52, Figure 53, Figure 54, the following are taken from patients including drug-resistant Citrobacter freundii (Carbapenem-Resistant Citrobacter freundii, CR C. freundii), Citrobacter freundii (Citrobacter freundii, C. freundii) After the samples were diluted with pure water for the second time, the principal component analysis was performed with the full-segment spectrum to obtain the drop point distribution, verifying that the present invention can quickly and indeed distinguish the drug resistance of the strains based on different drug susceptibility strains.

Refer to Figure 55, Figure 56, Figure 57, the following are the samples taken from the patient including the drug-resistant Proteus mirabilis ESBL (P. mirabilis ESBL) and Proteus mirabilis (P. mirabilis) respectively. After being diluted with the sub-pure water, the principal component analysis is performed with the full range spectrum to obtain the drop point distribution, verifying that the present invention can quickly and reliably distinguish the drug resistance of the strains based on the different drug susceptibility strains.

Refer to Figure 58, Figure 59, and Figure 60. The following samples are taken from patients, including multidrug-resistant Pseudomonas aeruginosa (MDRPA) and Pseudomonas aeruginosa (P. aeruginosa). After the pure water is diluted, the principal component analysis is performed with the full range spectrum to obtain the drop point distribution, verifying that the present invention can quickly and reliably distinguish the drug resistance of the strains based on the strains with different drug sensitivity.

Refer to Figure 61, Figure 62, Figure 63, the following are taken from patients, including multidrug-resistant Acinetobacter baumannii/calcoaceticus complex (MDR-ABC), Acinetobacter baumannii/calcoaceticus complex (ABC) After the specimens of) were diluted with pure water twice, the principal component analysis was performed on the full-segment spectrum to obtain the drop point distribution, verifying that the present invention can quickly and reliably distinguish the drug resistance of strains based on different drug susceptibility strains.

Refer to Figure 64, Figure 65, and Figure 66. The following samples were taken from patients, including Vancomycin resistant enterococci (Vancomycin resistant enterococci, VRE) and Enterococcus faecalis (Enterococcus faecalis, E. faecalis). After dilution, the principal component analysis is performed with the full-segment spectrum to obtain the drop point distribution, verifying that the present invention can quickly and reliably distinguish the drug resistance of strains based on different drug susceptibility strains.

Based on the above analysis results of the multiple bacterial species and the drop point of the drug-resistant bacterial species, it can be reliably verified that the present invention can quickly and accurately identify single or multiple mixed bacterial species, and can also quickly identify drug-resistant strains and bacterial concentrations.

Referring to Fig. 67 and Fig. 68, it is explained that the present invention is used to distinguish specimens containing different concentrations of E. coli. Dilute samples with different concentrations of Escherichia coli that are 10 times, 100 times, 1000 times, and 10000 times diluted with secondary pure water. After obtaining the Raman spectrum to be analyzed, perform principal component analysis with the full range spectrum, you can see Obviously distinguish the landing spots of the specimens with different concentrations to be tested, thereby verifying that the present invention can indeed distinguish different E. coli concentrations in the specimens.

Referring to Fig. 69, similarly, when pathogenic and non-pathogenic E. coli are included in the specimen, implementing the present invention can also distinguish whether E. coli is pathogenic or not.

The severity of diseases caused by bacteria can vary greatly, especially for children, the elderly, and immune-deficient patients. The present invention has been tested by China Medical University. The tested bacterial samples are all samples directly from hospital patients. There is no need for complicated and time-consuming bacterial culture processing and confirmation of the OD600 value. The sampled samples can be targeted within 0-15 minutes. The specimens are tested immediately, achieving convenient and fast inspection effects. In contrast to the prior art, which mainly only tested a few bacteria cultivated in the laboratory, the present invention can target the actual bacterial species that are clinically in the hospital, may cause harm to the human body, and even some bacteria have developed resistance to some antibiotics. Quick and accurate analysis and identification can be made to give the patient suitable treatment. Therefore, the present invention is indeed suitable for the actual use of the current hospital.

In addition, the concentration of bacteria in the body of a patient varies from person to person, and the conditions of bacterial attack are also different. The present invention uses the peak identification method to compare with the pre-stored bacteria map, and the correct bacterial species can be compared. After identification, verify and identify the results of bacterial species identification according to the sorting results, and can be combined with a second pure water cleaning to remove other excess impurities in the specimen, avoid excessive Raman spectroscopy noise to be analyzed, and/or concentration to improve the bacterial detection of the patient The concentration of bacteria in the body increases the accuracy of the analysis results of the visibility of the bacteria test signal, and can accurately compare and identify the types of bacteria and mixed bacteria.

Furthermore, the portable Raman spectrometer system to which the present invention is applicable is an Ocean Optics Raman instrument (Ocean Optics Raman, model QEPro, SR-510) with a price of NT$1 million, which is easy to operate, small and easy to carry. , And can test the specimen immediately, not limited to the test location, it is convenient and practical to promote the widespread use in the medical hospitals, and to improve the long time of traditional medical examinations and the suffering of patients suffering from bacterial infections. Sex.

In summary, the present invention applies the portable Raman system to quickly identify bacteria, mainly using the surface-enhanced Raman spectroscopy chip 3 in conjunction with the portable Raman spectrometer system 5, without the need for bacterial culture, and immediately within 0-15 minutes The sample taken from the patient can be washed and diluted or concentrated with pure water twice to obtain the corresponding Raman spectrum to be analyzed. After that, the peak identification method is used to sort the HQI similarity and identify the entire section. Or segment-based principal component analysis, spot distribution, to identify single or multiple mixed bacterial species, drug-resistant strains and bacterial concentration, effectively improving the existing bacterial testing methods, so it can indeed achieve the purpose of the invention.

However, the above are only examples of the present invention. When the scope of implementation of the present invention cannot be limited by this, all simple equivalent changes and modifications made in accordance with the scope of the patent application of the present invention and the content of the patent specification still belong to Within the scope of the patent for the present invention.

201: Step 33: Particle 202: Step 5: Portable Raman system 203: Step 51: Pedestal 204: Step 511: substrate groove 205: Step 52: light source 206: Step 53: Spectrometer 900: sample to be tested 3: Surface enhanced Raman spectroscopy wafer 31: Substrate 311: top surface 32: Hydrophobic polymer membrane 321: rough surface

The other features and effects of the present invention will be clearly presented in the embodiments with reference to the drawings, in which: Figure 1 is a flowchart illustrating the inspection process of the existing food microbiological inspection method "inspection of Salmonella"; 2 is a flowchart illustrating an embodiment of the method for rapidly identifying bacteria using a portable Raman system according to the present invention. Steps (e) 205 and (f) 206 respectively illustrate the full-segment principal component analysis using the peak identification method , And the HQI similarity ranking and identification of the whole segment or segment to distinguish the bacteria; Figure 3 is a schematic diagram illustrating the application of the portable Raman system in this embodiment of the method for rapid identification of bacteria, using a The surface-enhanced Raman spectroscopy chip is placed in a substrate groove of the portable Raman system, and the Raman spectrum to be analyzed is obtained by laser excitation; FIG. 4 is a Raman spectrum diagram illustrating the application of the portable Raman system. A Raman spectrum to be analyzed obtained in this embodiment of the method for rapid identification of bacteria by the Raman system, wherein the ordinate represents the relative intensity, and the abscissa represents the Raman shift; Figure 5 is a spectrum comparison analysis diagram illustrating the implementation When the present invention uses this embodiment of the method for rapidly identifying bacteria using a portable Raman system, the Raman spectra to be analyzed and the pre-stored spectrograms of bacteria species are identified by the peak identification method of the peak identification system to identify the Raman spectra to be analyzed. a single or multiple mixed acid bacteria, wherein the secretion is taken from the patient and having urethritis coli (Escherichia coli, E. coli) are subject to the test sample to be analyzed to obtain the Raman spectrum, the peak identification the method selected 600-750cm ^-1, 950-1100cm ^-1, 1400-2000cm ^-1 three sections; FIG. 6 is a comparative analysis of the spectral FIG described embodiment of the present invention is applied portable Raman system for rapid identification of bacteria method In this embodiment, the Raman spectrum to be analyzed and the spectrum of the pre-stored non-bacterial species are identified by the peak identification method of the peak identification system to identify the single or multiple mixed bacterial species corresponding to the Raman spectrum to be analyzed. urethritis patients having autocrine and E. coli (Escherichia coli, E. coli) are subject to the test sample to obtain the segment statistics for the region of the Raman spectrum ratio, peak identification method to be analyzed, HQI (i.e., to be analyzed The degree of similarity between the specific peaks of the Raman spectrum and the spectra of different types of bacteria pre-existing) is 0.89, 0.65, 0.45; Figure 7 is a Raman spectrum, illustrating that the implementation of the present invention is effective for Escherichia coli from patients with urinary tract inflammation. , E. coli ) the corresponding Raman spectra of the sample to be tested obtained by the second pure water cleaning treatment of the bacterial sample; FIG. 8 is a spectrum comparison analysis diagram, which illustrates the rapid application of the portable Raman system of the present invention In this embodiment of the method for identifying bacteria, the Raman spectrum to be analyzed and the spectrum of the pre-stored non-bacterial species are identified by the peak identification method of the peak identification system to identify the single or multiple mixed bacteria corresponding to the Raman spectrum to be analyzed Bacteria, among them, the specimens with Escherichia coli ( E. coli ) taken from patients with urinary tract inflammation have not been tested by secondary pure water to obtain the Raman spectrum to be analyzed, and the method of peak identification Perform statistical comparison of the whole segment, HQI is 0.70, 0.81, and 0.67; Figure 9 is a spectrum comparison analysis diagram, which illustrates that when implementing this embodiment of the method for rapid identification of bacteria using the portable Raman system of the present invention, it will be analyzed Raman spectra and pre-existing spectra of non-bacterial species are identified by the peak identification method of the peak identification system to identify the single or multiple mixed species corresponding to the Raman spectra to be analyzed. Among them, they are taken from patients with urinary tract inflammation and have large intestine Escherichia coli ( E. coli ) specimens are treated with secondary pure water to obtain the Raman spectrum to be analyzed, and the peak identification method is used for statistical comparison of the whole section, and the HQI is improved to 0.79, 0.71 0.62, which proves that the test sample formed by the second pure water treatment of the present invention can accurately identify single or multiple mixed bacterial species; Figure 10 is a Raman spectrum diagram illustrating the implementation of the present invention against patients with urinary tract inflammation Whether the Escherichia coli ESBL ( E. coli ESBL ) bacterial specimen has the corresponding Raman spectra obtained by the concentration process, and shows that the spectral signal of the sample to be tested after the concentration process is significantly enhanced; Figure 11 is a pull Mann spectrogram, which shows whether the corresponding Raman spectra of Proteus mirabilis ( P. mirabilis ) bacteria specimens from patients with urinary tract inflammation are obtained by the concentration process in the implementation of the present invention, and also indicates that the specimens need to be concentrated. The spectral signal of the test sample is significantly enhanced; Fig. 12 is a Raman spectrogram, illustrating the implementation of the present invention for Escherichia coli ( E. coli ) bacterial samples from patients with urinary tract inflammation. Mann spectroscopy, and shows that the spectral signal of the sample to be tested after the concentrated processing of the specimen is significantly enhanced; Figure 13 is a spectrum comparison analysis diagram, illustrating the implementation of this embodiment of the present invention using the portable Raman system to quickly identify bacteria , The Raman spectrum to be analyzed and the spectrum of the pre-stored non-bacterial species are identified by the peak identification method of the peak identification system to identify the single or multiple mixed bacterial species corresponding to the Raman spectrum to be analyzed. Among them, it is taken from urinary tract inflammation. and having a patient test sample of E. coli (Escherichia coli, E. coli) has not been subject to a concentration process to obtain the Raman spectrum, the peak identification method to be analyzed for statistical comparison region segment, as 0.66,0.69 HQI , 0.61; Figure 14 is a spectrum comparison analysis diagram, illustrating that when implementing this embodiment of the invention using the portable Raman system to quickly identify bacteria, the Raman spectrum to be analyzed and the pre-stored non-bacterial species FIG spectral peak identification method to identify the peak identification system to be analyzed Raman spectra corresponding to single or multiple mixed acid bacteria, wherein the secretion is taken from the patient and having urethritis coli (Escherichia coli, E. coli) of The sample to be tested is formed by the concentration processing of the sample to obtain the Raman spectrum to be analyzed, and the peak identification method is used for the statistical comparison of the whole section, and the HQI is increased to 086, 0.72, and 0.49, which proves that the concentration processing of the present invention is made The sample can increase the identification to accurately identify single or multiple mixed bacterial species; Figure 15 is an analysis result diagram illustrating the implementation of the present invention on two mixed bacteria from urinary tract infection patients Citrobacter freundii ( C. freundii ), Proteus mirabilis ( P. mirabilis ) bacterial specimens of the Raman spectroscopy of the whole section analysis of the drop point distribution, which can be seen from multiple data points that also contain Citrobacter freundii The body tends to gather in the same area, so a specific area range for determining whether it contains Citrobacter freundii is selected; Fig. 16 is a graph of analysis results illustrating the implementation of the present invention to determine whether it contains Proteus mirabilis ( P. mirabilis) Fig. 17 is an analysis result diagram illustrating the specific area range selected to determine whether Escherichia coli ( E. coli ) is contained in the implementation of the present invention; Fig. 18 is an analysis result diagram illustrating the selection of the implementation of the present invention Determine whether the specific area of Pseudomonas aeruginosa ( P. aeruginosa ) is contained; Figure 19 is a graph of analysis results, illustrating the implementation of the present invention to select and determine Citrobacter freundii ( C. freundii , C. freundii ), Proteus mirabilis (Proteus mirabilis, P. mirabilis), E. coli (Escherichia coli, E. coli), particular region of Pseudomonas aeruginosa (Pseudomonas aeruginosa, P aeruginosa.); Figure 20 is an analysis diagram illustrating a sample from a patient After the implementation of the present invention, it was determined that the specimens contained Citrobacter freundii ( C. freundii ) and Proteus mirabilis ( P. mirabilis ); Figure 21 is a graph of analysis results, Explain the drop point distribution of a specimen from a patient after implementing the present invention, and determine that the specimen contains Freund's citric acid Coli (Citrobacter freundii, C. freundii), Pseudomonas aeruginosa (Pseudomonas aeruginosa, P. aeruginosa), and due to the placement and distribution of segments of different strains of the determination, the present invention can indeed be verified distinguish two kinds of mixed bacteria strain; FIG. 22 is a graph of analysis results, illustrating the distribution of drop points of a specimen from a patient after implementing the present invention, and determining that the specimen contains Citrobacter freundii ( C. freundii ) and Klebsiella pneumoniae ( Klebsiella pneumoniae , K. pneumoniae ), and the different strains are determined due to the distribution of drop points, verifying that the present invention can indeed distinguish two mixed bacterial species; Figure 23 is an analysis result diagram illustrating a specimen from a patient after placement of the embodiment of the present invention, the distribution, and determines that the subject comprises E. coli (Escherichia coli, E. coli), Pseudomonas aeruginosa (Pseudomonas aeruginosa, P. aeruginosa), and due to the placement and distribution of segments of different strains of determination , To verify that the present invention can indeed distinguish two kinds of mixed bacteria; Figure 24 is a graph of analysis results, illustrating the distribution of the spot of a specimen from a patient after implementing the present invention, and it is determined that the specimen contains Citrobacter freundii (Citrobacter freundii, C. freundii), Pseudomonas aeruginosa (Pseudomonas aeruginosa, P. aeruginosa), and due to the placement and distribution of segments of different strains of the determination, the present invention can indeed be verified distinguish two kinds of mixed bacteria strain; FIG. 25 the results is a diagram illustrating the placement of a distributed after the subject embodiment of the present invention from the patient, and determines that the subject comprises E. coli (Escherichia coli, E. coli), Pseudomonas aeruginosa (Pseudomonas aeruginosa, P. aeruginosa) , And the different strains are judged due to the distribution of the drop points, verifying that the present invention can indeed distinguish two kinds of mixed bacterial strains; Figure 26 is an analysis result diagram illustrating the drop points of a specimen from a patient after implementing the present invention distribution, and determines that the subject comprises E. coli (Escherichia coli, E. coli), Pseudomonas aeruginosa (Pseudomonas aeruginosa, P. aeruginosa), and due to the placement and distribution of segments of different strains of the determination, the present invention indeed can distinguish verification Two kinds of mixed bacterial strains were obtained; Fig. 27 is an analysis result diagram, which shows that the three mixed bacterial Escherichia coli ( Escherichia coli) from patients with urinary tract inflammation , E. coli), Pseudomonas aeruginosa (Pseudomonas aeruginosa, P. aeruginosa) and coagulase-negative staphylococci (coagulase-negative staphylococci, CoNS) distribution of the subject body after the placement region segment analysis, we can identify three kinds of mixed Bacteria, among them, it is also related to different species of Acinetobacter baumannii ( Acinetobacter baumannii/calcoaceticus complex , ABC ), Enterobacter aerogenes ( E. aerogenes ) and Serratia marcensess ( Serratia marcensess , S. marcensess) ) The strains are clearly distinguished, verifying that the present invention can indeed distinguish three mixed strains; Figure 28 is a graph of analysis results, illustrating the distribution of the spot of a specimen from a patient after implementing the present invention, and it is determined that the specimen contains the large intestine coli (Escherichia coli, E. coli), Enterococcus (Entercoccus faecalis, E. faecalis) and coagulase-negative staphylococci (coagulase-negative staphylococci, CoNS) , and because the segment placement and distribution of different strains determined to verify this indeed the invention can distinguish three kinds of mixed bacteria strain; FIG. 29 is an analysis diagram illustrating an embodiment of the sample from the patient after placement of the present invention, the distribution, and determines that the subject comprises E. coli (Escherichia coli, E. coli ), Klebsiella pneumoniae ( Klebsiella pneumoniae , K. pneumoniae ) and coagulase-negative staphylococci ( CoNS ), and because of the difference in the distribution of the colonies , the strains are determined to be different, verifying that the present invention can indeed distinguish Three kinds of mixed bacterial strains are identified; Fig. 30 is an analysis result diagram illustrating the distribution of the spot of a specimen from a patient after implementing the present invention, and it is determined that the specimen contains Klebsiella pneumoniae ( Klebsiella pneumoniae , K. pneumoniae) ), Entercoccus ( Entercoccus faecalis , E. faecalis ) and coagulase-negative staphylococci ( CoNS ), and because of the difference in the distribution of the colonies , the strains are determined to be different, verifying that the present invention can indeed distinguish three mixed bacteria Bacteria; Figure 31 is a graph of analysis results, illustrating a sample from a patient Shi distribution after placement of the present invention, and determines that the subject comprises E. coli (Escherichia coli, E. coli), Pseudomonas aeruginosa (Pseudomonas aeruginosa, P. aeruginosa) and coagulase-negative staphylococci (coagulase-negative staphylococci, CoNS ), and the different strains are determined due to the distribution of the drop points, verifying that the present invention can indeed distinguish three mixed bacterial species; Figure 32 is an analysis result diagram illustrating the drop points of a specimen from a patient after implementing the present invention distribution, and determines that the subject comprises E. coli (Escherichia coli, E. coli), Klebsiella pneumoniae (Klebsiella pneumoniae, K. pneumoniae) and Proteus mirabilis (Proteus mirabilis, P. mirabilis), and due to the placement The distribution is divided and the strains are determined to be different, verifying that the present invention can indeed distinguish three mixed bacterial species; Figure 33 is an analysis result diagram illustrating the distribution of the spot of a specimen from a patient after implementing the present invention, and determining its detection comprises Escherichia (Escherichia coli, E. coli), Proteus mirabilis (Proteus mirabilis, P. mirabilis) and coagulase-negative staphylococci (coagulase-negative staphylococci, CoNS) , and because of the placement of strain distribution on the segments determined Different, verify that the present invention can indeed distinguish three kinds of mixed bacteria; Figure 34 is a graph of analysis results, illustrating the distribution of the spot of a specimen from a patient after implementing the present invention, and it is determined that the specimen contains Klebsiella pneumonia coli (Klebsiella pneumoniae, K. pneumoniae), Enterobacter (Enterobacter cloacae, E. cloacae) and Pseudomonas aeruginosa (Pseudomonas aeruginosa, P. aeruginosa), and due to the placement and distribution of segments of different strains of the determination, the present invention does verify Three kinds of mixed bacteria can be distinguished; Fig. 35 is an analysis result diagram illustrating the distribution of the spot of a specimen from a patient after implementing the present invention, and it is determined that the specimen contains Klebsiella pneumoniae ( Klebsiella pneumoniae , K . pneumoniae), Pseudomonas aeruginosa (Pseudomonas aeruginosa, P. aeruginosa) and VRE (vancomycin resistant enterococ ci , VRE ), and the different strains are judged due to the distribution of the drop points, verifying that the present invention can indeed distinguish three mixed bacterial species; Figure 36 is an analysis result diagram illustrating a specimen from a patient after implementing the present invention the placement distribution, and determines that the subject comprises E. coli (Escherichia coli, E. coli), Enterococcus (Entercoccus faecalis, E. faecalis) and coagulase-negative staphylococci (coagulase-negative staphylococci, CoNS) , and because falling The point distribution is divided to determine that the strains are different, verifying that the present invention can indeed distinguish three mixed bacterial species; Figure 37 is a Raman spectrum comparison diagram, illustrating that the implementation of the present invention dilutes the specimens from peritonitis patients into multiple when the test sample to be analyzed in the obtained Raman spectrum, wherein the sample containing the non-multidrug resistant Staphylococcus aureus (Methicillin - sensitive Staphylococcus aureus, MSSA ); FIG. 38 is a Raman spectrum comparison diagram illustrating the embodiment In the present invention, the Raman spectra to be analyzed are obtained when a bacterial specimen from a peritonitis patient is diluted into a plurality of specimens to be tested, wherein the specimen contains Methicillin - resistant Staphylococcus aureus ( MRSA ) Fig. 39 is a graph of analysis results, illustrating the distribution of drop points obtained by full-segment analysis using the Raman spectra of Fig. 37 and Fig. 38 to be analyzed, which can identify multi-drug resistant Staphylococcus aureus ( Methicillin - resistant Staphylococcus aureus , MRSA ) and non-multidrug-resistant Staphylococcus aureus ( Methicillin - sensitive Staphylococcus aureus , MSSA ); Figure 40 is a comparison of Raman spectra, regarding the difference in urinary tract of Escherichia coli ESBL ( E. coli ESBL ) bacterial vaginitis patients subject; FIG. 41 is a comparison of FIG Raman spectrum, urethritis patients secrete different subject bacteria on E. coli (Escherichia coli, E. coli) of; FIG. 42 is an analysis diagram illustrating the use of FIG. 40, FIG. 41 to be analyzed for the placement of a Raman spectrum distribution obtained by the analysis region segments to identify drug resistance on E. coli (Escherichia coli ESBL, E. coli ESBL ), E. coli (Escherichia coli, E. coli); Figure 43 is about drug-resistant Klebsi Raman spectra of different urethritis patients' bacterial specimens of Klebsiella pneumoniae ESBL ( Klebsiella pneumoniae ESBL , K. pneumoniae ESBL ) to be analyzed; Figure 44 is about the difference of Klebsiella pneumoniae ( Klebsiella pneumoniae , K. pneumoniae ) The Raman spectra of the bacterial specimens of patients with urinary tract inflammation to be analyzed; Figure 45 is a graph of analysis results, illustrating the distribution of drop points obtained from the analysis of the whole section of the Raman spectra to be analyzed in Figure 43 and Figure 44 to identify the relevant resistance Klebsiella pneumoniae (Klebsiella pneumoniae ESBL, K. pneumoniae ESBL ), Klebsiella pneumoniae (Klebsiella pneumoniae, K. pneumoniae); FIG. 46 is an on Enterobacteriaceae (Enterobacter cloacae, E. cloacae) of The to-be-analyzed Raman spectra of the bacterial specimens of different urinary tractitis patients; Figure 47 is a to - be-analyzed analysis of the bacterial specimens of different urinary tract inflammation patients with drug-resistant Enterobacter cloacae ( Carbapenem-Resistant Enterobacter cloacae , CR E. cloacae ) Fig. 48 is an analysis result diagram, illustrating the drop point distribution obtained from the analysis of the whole section of the Raman spectrum to be analyzed in Fig. 46 and Fig. 47 to identify Enterobacter cloacae ( E. cloacae ), Drug-resistant Enterobacter cloacae ( Carbapenem-Resistant Enterobacter cloacae , CR E. cloacae ); Figure 49 is a Raman spectrogram of different urethritis patients' bacterial specimens of Enterobacter aerogenes ( E. aerogenes ) for analysis Fig. 50 is a Raman spectrogram for analysis of different urinary tract inflammation patient bacterial specimens about drug -resistant Enterobacter aerogenes ( CREA ); Fig. 51 is a graph of analysis results, which is illustrated from Fig. 49 , Figure 50 is the Raman spectroscopy to be analyzed for the full range analysis of the drop point distribution, to identify the Enterobacter aerogenes ( Enterobacter aerogenes , E. aerogenes ), drug -resistant Enterobacter aerogenes ( Carbapenem-Resistant Enterobacter aerogenes , CREA ); Figure 52 is about Citrobacter freundii ( Citr Obacter freundii , C. freundii ), the to-be-analyzed Raman spectra of different bacterial specimens of patients with urinary tract inflammation ; Figure 53 is about the difference of drug -resistant Citrobacter freundii ( Carbapenem-Resistant Citrobacter freundii , CR C. freundii ) The Raman spectra of the bacterial specimens of patients with urinary tract inflammation to be analyzed; Fig. 54 is a graph of analysis results, illustrating the drop point distribution obtained from the Raman spectra of Figs. Citrobacter freundii ( C. freundii ), drug -resistant Citrobacter freundii ( Carbapenem-Resistant Citrobacter freundii , CR C. freundii ); Figure 55 is about the drug-resistant Proteus mirabilis ESBL ( P. mirabilis) The unanalyzed Raman spectra of different bacterial specimens from patients with urinary tract inflammation ( ESBL ); Figure 56 is a Raman spectra to be analyzed of different bacterial specimens from urinary tract inflammation patients with Proteus mirabilis ( P. mirabilis ) Figure; Figure 57 is an analysis result diagram illustrating the drop point distribution obtained from the full range analysis of the Raman spectra in Figure 55 and Figure 56 to identify drug-resistant Proteus mirabilis ESBL ( P. mirabilis ESBL ) , Proteus mirabilis ( P. mirabilis ); Figure 58 is a Raman spectrogram for analysis of bacterial specimens of urinary tract inflammation patients with drug -resistant Pseudomonas aeruginosa ( MDRPA ); Figure 59 It is a Raman spectrogram of Pseudomonas aeruginosa ( P. aeruginosa ) of different urinary tract inflammation patients' bacterial specimens to be analyzed; Fig. 60 is a graph of the analysis results, which illustrates the analysis of Pseudomonas aeruginosa ( P. aeruginosa ) Raman spectroscopy analysis section for placement region distribution obtained regarding the identification of resistance to Pseudomonas aeruginosa (multidrug-resistant Pseudomonas aeruginosa, MDRPA ), Pseudomonas aeruginosa (Pseudomonas aeruginosa, P. aeruginosa); FIG. 61 is an on boydii Acinetobacter baumannii/calco aceticus complex ( ABC ) of different urinary tract inflammation patients' bacterial specimens to be analyzed Raman spectra; Figure 62 is about the urinary tract of multidrug-resistant Acinetobacter baumannii/calcoaceticus complex ( MDR-ABC ) The Raman spectra of the bacterial specimens of patients with inflammation to be analyzed; Fig. 63 is a graph of analysis results, illustrating the distribution of the drop points obtained from the analysis of the Raman spectra of Fig. 61 and Fig. 62 in the whole section to identify the drug resistance Acinetobacter baumannii ( multidrug-resistant Acinetobacter baumannii/calcoaceticus complex , MDR-ABC ) and Acinetobacter baumannii/calcoaceticus complex ( ABC ); Figure 64 is about the difference between Entercoccus faecalis ( E. faecalis ) urethritis patients to be secreted bacterial specimen analyzing Raman spectra; on FIG. 65 is a VRE (vancomycin resistant enterococci, VRE) bacteria of different patients to be the subject of Raman spectrum analysis of secretion FIG urethritis; Figure 66 is an analysis result diagram illustrating the distribution of drop points obtained from the full-segment analysis of the Raman spectra of Figure 64 and Figure 65 to identify enterococcus ( Entercoccus faecalis , E. faecalis ) and vancomycin-resistant enterococcus ( Vancomycin resistant enterococci , VRE ); Figure 67 is a Raman spectrogram, illustrating the implementation of the present invention to analyze the test Escherichia coli with different concentrations of 10 times, 100 times, 1000 times and 10000 times diluted with pure water for the second time. Raman spectrum; Fig. 68 is a graph of analysis results, illustrating the drop point distribution obtained by the analysis of the whole section with the Raman spectrum to be analyzed in Fig. 67, which proves that the implementation of the present invention can identify different concentrations of E. coli to be tested; and Fig. 69 It is a graph of analysis results, which illustrates the distribution of drop points obtained by taking pathogenic and non-pathogenic E. coli specimens to implement the present invention, verifying that the present invention can further distinguish whether E. coli is pathogenic or not.

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Claims (6)

A method for rapidly identifying bacteria using a portable Raman system, including: (a) Place at least one surface-enhanced Raman spectroscopy chip in at least one of the plurality of spaced substrate grooves of the portable Raman system. The at least one surface-enhanced Raman spectroscopy chip has a substrate and a forming The hydrophobic polymer film on the top surface of the substrate, and most of the metal particles distributed in the hydrophobic polymer film with a size range of nanometers, wherein the hydrophobic polymer film is selected from the following group Is formed of at least one of the group of polymer materials: polyimide, sol-gel, maleic acid, polyvinylidene fluoride, polyethylene, polypropylene, polyamide, polystyrene, polycarbonate, and Polymethyl methacrylate, wherein the sol-gel is composed of methyltrimethoxysilane, tetramethoxysilane and octadecyltrimethoxysilane with a weight composition ratio of 10:2:1. The particles are Formed from at least one of the following groups: gold, silver, graphene, and copper; (b) A sample taken from a human body is directly selected from the method of washing and diluting with pure water twice, and concentrating without bacterial culture, and processing it into at least one sample to be tested with different bacterial concentrations; (c) immediately placing the at least one sample to be tested on the at least one surface-enhanced Raman spectroscopy chip so that the at least one sample to be tested directly contacts the hydrophobic polymer film and is affected by the metal particles; (d) irradiating the at least one sample to be tested placed on the at least one surface-enhanced Raman spectroscopy chip with the light source of the portable Raman spectrometer system to obtain at least one Raman spectrum to be analyzed respectively; and (e) Use the peak identification method to compare the at least one Raman spectrum to be analyzed and most pre-stored spectra corresponding to different bacterial species. When the comparison similarity exceeds the preset value, it is determined to correspond to the The sample to be tested for the Raman spectrum to be analyzed contains bacteria corresponding to the spectrum. The requested item is an application of the portable Raman system for rapid identification of bacteria, wherein the step (e) in the x-axis is also distinguished 600-750cm ^-1, 950-1100cm ^-1, 1400-2000cm ^-1 The three segments are sorted by similarity to determine the type of bacteria contained in the sample to be tested. The method for rapidly identifying bacteria using a portable Raman system as described in claim 1, further comprising a step (f) of using the to-be-analyzed Raman spectrum to perform a principal component analysis of the whole section to obtain a drop point distribution to identify at least one At least one of bacterial species, bacterial resistance and bacterial concentration. The method for rapidly identifying bacteria using a portable Raman system as described in claim 1, wherein the specimens taken from the human body in step (b) include, but are not limited to, urine, ascites, blood, plasma, serum, sputum, Cerebrospinal fluid, pleural fluid, or tissue fluid. The method for rapidly identifying bacteria using a portable Raman system according to claim 1, wherein in step (c), an amount of 3-10 μl is placed on the at least one surface enhanced Raman spectroscopy wafer. The method for rapidly identifying bacteria using a portable Raman system as described in claim 1, wherein in step (d), laser light with a wavelength of 785 nm is used as a light source to illuminate the sample to be tested to obtain the Raman spectrum to be analyzed.

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