In-situ identification method for microbial colonies based on Raman spectrum

Abstract

The invention relates to the technical field of pathogenic bacteria identification, in particular to an in-situ identification method for microbial colonies based on Raman spectroscopy, which comprises the following steps: inoculating a sample containing pathogenic bacteria on a plate, culturing at 32-42 DEG C for 12-48 hours, performing in-situ detection on the selected colonies by using Raman spectroscopy, setting parameters as follows: the power of a laser is 250-350mW, the detection time is 1.5-2.5 s, and the detection time is 1.5-2.5 s; and the distance between the probe and the bacterial colony is fixed to be 8-12mm. Compared with a traditional method, the method has the advantages that in-situ detection is carried out on pathogenic bacterium colonies by utilizing a Raman spectrum technology, a tedious sample pretreatment process is not needed, time consumption is short, the method has the characteristics of high accuracy and high flux, and a detection result can be effectively used for assisting clinical infection diagnosis, environmental microorganism detection and the like. In addition, the detection method provided by the invention does not destroy the survival state of pathogenic bacteria, and can effectively link the analysis of subsequent related pathogenic bacteria.

Description

Technical Field

[0001] The invention relates to the technical field of pathogen identification, and in particular to an in-situ identification method for microbial colonies based on Raman spectroscopy. Background Art

[0002] Pathogen (bacterial and fungal) infections have become an important issue of global health concern. Different pathogen infections can cause different symptoms, such as mild gastrointestinal inflammation, wound infection, and severe fatal diseases, which seriously threaten human health. In clinical practice, for unknown pathogen infections, patients must receive broad-spectrum antibiotic treatment before the diagnosis results come out, which can easily lead to serious consequences such as misdiagnosis and missed diagnosis. At present, the identification of pathogens in clinical infections faces problems such as poor timeliness, empirical medication, and difficulty in efficiently responding to mixed infections, which seriously affects treatment. Therefore, in the fields of public health and medicine, it is of great significance to quickly, accurately, and high-throughput identify pathogens for targeted treatment.

[0003] At present, the technologies used for clinical diagnosis of pathogens mainly include culture method, nucleic acid detection method and mass spectrometry. Among them, microbial culture is the gold standard for clinical diagnosis of pathogens. It is simple to operate, low-cost, can detect multiple pathogens at the same time, and has high specificity and sensitivity. At present, for pathogen culture technology, technicians can observe the characteristics of colonies based on experience to directly identify the cultured colonies, but the accuracy is low and it is easy to cause misjudgment. Mass spectrometry technology is mainly based on the isolation and cultivation of pathogens, and the strategy of picking single clones for mass spectrometry analysis. Mass spectrometry detection is fast and accurate, and is widely used in clinical practice. Raman spectroscopy technology is a new method for pathogen diagnosis developed in recent years. Raman technology has the characteristics of rapid response and high sensitivity for the detection of low-abundance targets, and has application prospects in the identification of bacteria. However, mass spectrometry, sequencing or existing Raman technology cannot complete the direct identification of cultured colonies, and all require pre-treatment of pathogen cultures. There are problems such as cumbersome sample preparation, complex operation, and long time consumption, and the quality of sample processing seriously affects the accuracy of its identification results. Therefore, due to defects such as poor timeliness, low throughput and inability to be used for live bacteria analysis, the existing mass spectrometry or Raman technology is limited in clinical application, especially cannot meet the needs of grassroots hospitals.

[0004] Currently, there is still a lack of a high-throughput, rapid and accurate pathogen diagnosis technology based on in situ identification of bacterial colonies. In view of this, the present invention is proposed. Summary of the invention

[0005] In order to solve the above technical problems, the present invention proposes a method for in-situ detection of pathogenic bacteria colonies, which integrates colony positioning with in-situ detection of colony Raman spectroscopy. There is no need for sample pretreatment of pathogens, which has high accuracy and saves time and manpower.

[0006] Specifically, the technical solution of the present invention is as follows: In the first aspect, the present invention provides a method for identifying pathogens, inoculating a sample containing pathogens on a plate, culturing at 32-42°C for 12-48 hours, and using Raman spectroscopy to perform in situ detection of selected colonies, with the setting parameters being: laser power 250-350mW, detection time 1.5-2.5s, and the distance between the probe and the colony fixed at 8-12mm.

[0007] Preferably, the present invention inoculates a sample containing pathogens on a plate, cultures it at 36-38°C for 18-30 hours, and uses Raman spectroscopy to perform in-situ detection on selected colonies, with the following parameters: laser power 290-310 mW, detection time 1.8-2.2 s, and the distance between the probe and the colony fixed at 9-11 mm.

[0008] Preferably, the present invention inoculates a sample containing pathogens on a plate, cultures it at 37°C for 24 hours, and uses Raman spectroscopy to perform in-situ detection of selected colonies, with the following parameters set: laser power 300mW, detection time 2s, and the distance between the probe and the colony fixed at 10mm.

[0009] The pathogen identification method provided by the present invention can be used for the detection of various bacteria and fungi. In a more specific and preferred embodiment, the present invention detects Staphylococcus aureus and Acinetobacter baumannii with good results.

[0010] Preferably, after the in-situ detection of the present invention is completed, the collected data is imported into NGSLabSpec software, and the data analysis is performed after the baseline is automatically balanced to obtain a Raman spectrum detection spectrum.

[0011] Preferably, when the Raman spectrum detection spectrum is substantially as follows Figure 4 When shown, the selected colony is judged to be an Acinetobacter baumannii colony.

[0012] Preferably, when the Raman spectrum detection spectrum is substantially as follows Figure 6 When shown, the selected colony is judged to be a Staphylococcus aureus colony.

[0013] Furthermore, the pathogen identification method described in the present invention is a pathogen identification method for purposes other than disease diagnosis or treatment (such as environmental microbial detection).

[0014] Beneficial effects: The present invention provides an in-situ identification method for microbial colonies based on Raman spectroscopy. A sample containing pathogens is inoculated on a plate, cultured at 32-42°C for 12-48 hours, and the selected colonies are detected in situ using Raman spectroscopy. The parameters are set as follows: laser power 250-350mW, detection time 1.5-2.5s, and the distance between the probe and the colony is fixed at 8-12mm. Compared with the traditional method, the present invention proposes to use Raman spectroscopy technology to detect pathogen colonies in situ, without the need for cumbersome sample pretreatment process, short time consumption, high accuracy and high throughput, and the detection results can be effectively used to assist clinical infection diagnosis, environmental microbial detection, etc. In addition, the detection method provided by the present invention will not destroy the survival state of pathogens, and can effectively connect the subsequent analysis of related pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be described below.

[0016] Figure 1 1 is the colony location of different pathogens in the examples of the present invention.

[0017] Figure 2 Spectra of Raman spectroscopy detection of different pathogenic bacteria colonies in the embodiments of the present invention.

[0018] Figure 3 The figure shows the culture colony positioning of Acinetobacter baumannii in the embodiment of the present invention.

[0019] Figure 4 This is an in situ Raman spectroscopy detection spectrum of the cultured colonies of Acinetobacter baumannii in the embodiment of the present invention.

[0020] Figure 5 Figure 2 shows the localization of cultured colonies of Staphylococcus aureus in an embodiment of the present invention.

[0021] Figure 6 This is an in-situ Raman spectrum detection spectrum of the cultured colonies of Staphylococcus aureus in an embodiment of the present invention.

[0022] Figure 7 This is the fitting result of the spectrum collected five times continuously for Staphylococcus aureus in the embodiment of the present invention. DETAILED DESCRIPTION

[0023] The present invention provides an in situ identification method for microbial colonies based on Raman spectroscopy. Compared with traditional methods, the present invention proposes to use Raman spectroscopy technology to perform in situ detection of pathogenic bacteria colonies, which does not require cumbersome sample pretreatment process, is time-saving and has the characteristics of high accuracy and high throughput. The detection results can be effectively used to assist clinical infection diagnosis, environmental microbial detection, etc.

[0024] In one of the more specific and preferred embodiments provided by the present invention, the identification method comprises the following steps: 1) Colony location of pathogens: Inoculate the sample containing pathogens on a plate and culture it at 37°C for 24 hours. Single colonies with different morphologies can be observed on the plate and selected for Raman spectroscopy detection.

[0025] 2) Use a handheld Raman spectrometer to perform in situ detection and analysis of the selected colonies.

[0026] 3) Output the detected colony Raman spectrum data to obtain the Raman spectrum of the pathogen.

[0027] The present invention integrates colony positioning and Raman spectroscopy in-situ detection to identify pathogenic microorganisms, without the need for pre-treatment of cultured pathogens, saving time and manpower, with high accuracy and high throughput, and without destroying the survival state of bacteria. The present invention proposes the integration of pathogen colony positioning and colony Raman spectroscopy in-situ detection technology, which can be used as a new method for accurate identification of pathogens, and can be used for rapid identification of colonies in clinical culture specimens, rapid auxiliary diagnosis of pathogenic infections, and rapid identification of pathogenic culture clones in samples related to food safety and environmental testing. In addition, the method proposed by the present invention can be used in a synchronous in-situ Raman spectroscopy acquisition and detection scheme for multiple colonies cultured on a plate.

[0028] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] The endpoints and any values ​​of the ranges disclosed in this specification are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0030] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "specific implementations", or "some specific implementations" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the embodiment of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0031] In the examples provided in this specification, if no specific techniques or conditions are specified, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0032] Example 1 This embodiment provides a method for in-situ identification of microbial colonies based on Raman spectroscopy, which integrates colony positioning and in-situ detection of Raman spectroscopy to identify pathogenic microorganisms.

[0033] (1) Colony positioning: The sample containing pathogens is inoculated on a plate and cultured at 37°C for 24 hours. Single colonies with different morphologies can be observed on the plate, and colonies with specific morphological characteristics are selected for Raman spectroscopy detection. The colony positioning of different pathogens is shown in Figure 2. Figure 1 As shown, the left one is Candida albicans, the middle one is Helicobacter pylori, and the right one is Talaromyces marneffei.

[0034] (2) In situ Raman spectroscopy detection and analysis of bacterial colonies A handheld Raman spectrometer was used to set the parameters of laser power to 300mW, detection time to 2s, and the distance between the probe and the colony to 10mm for detection. After the detection was completed, the data collected by the Raman spectrometer was imported into the NGSLabSpec software, and the relevant data analysis was performed after the baseline was automatically balanced. Figure 1 The Raman spectra of different pathogenic bacteria colonies identified in the experiment are shown in Figure 2. Figure 2 shown. Figure 2 In the figure, the horizontal axis is the Raman shift, and the unit is wave number (cm -1 ), the vertical axis is the response intensity, the unit is au.

[0035] (3) Raman spectroscopy analysis results of different pathogens According to the above operation procedures (1) and (2), in situ Raman spectroscopy data collection and analysis were performed on the cultured colonies of Staphylococcus aureus and Acinetobacter baumannii. Figure 3 As shown, the in situ Raman spectroscopy detection spectrum of the cultured colonies of Acinetobacter baumannii is as follows Figure 4 The location of Staphylococcus aureus colonies is shown in Figure 5 As shown, the in situ Raman spectroscopy detection spectrum of the cultured colony of Staphylococcus aureus is shown in Figure 6 shown.

[0036] The above results show that: the technology of the present invention can obtain specific colony in situ Raman characteristic spectra for Gram-positive bacteria Staphylococcus aureus and Gram-negative bacteria Acinetobacter baumannii without sample pretreatment, and the technology of the present invention can be used to identify different pathogens.

[0037] Example 2 The present invention performs continuous in-situ spectral data acquisition on the cultured Staphylococcus aureus colonies according to the above operation procedures (1) and (2), fits the spectra acquired multiple times, and compares them with the spectra obtained in Example 1 (3). Figure 7 The spectrum fitting results were collected 5 times continuously for Staphylococcus aureus.

[0038] The results show that for the same pathogen, the in situ Raman spectra obtained by the technology of the present invention have good consistency after fitting and are similar to the spectrum obtained in Example 1 (3), indicating that the technology has high accuracy.

[0039] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for identifying pathogens, characterized in that: The sample containing pathogens was inoculated on a plate and cultured at 32-42°C for 12-48 hours. The selected colonies were detected in situ using Raman spectroscopy. The parameters were set as follows: laser power 250-350mW, detection time 1.5-2.5s, and the distance between the probe and the colony was fixed at 8-12mm.

2. The method for identifying pathogens according to claim 1, characterized in that: The sample containing pathogens was inoculated on a plate and cultured at 36-38°C for 18-30 hours. The selected colonies were detected in situ using Raman spectroscopy with the following parameters: laser power 290-310mW, detection time 1.8-2.2s, and the distance between the probe and the colony was fixed at 9-11mm.

3. The method for identifying pathogens according to claim 2, characterized in that: The sample containing pathogens was inoculated on a plate and cultured at 37°C for 24 hours. The selected colonies were detected in situ using Raman spectroscopy. The parameters were set as follows: laser power 300 mW, detection time 2 s, and the distance between the probe and the colony was fixed at 10 mm.

4. The method for identifying pathogens according to claim 1, characterized in that: After the in-situ detection is completed, the collected data is imported into the NGSLabSpec software, and the data analysis is performed after the baseline is automatically balanced to obtain the Raman spectrum detection spectrum.

5. The method for identifying pathogens according to claim 4, characterized in that: When the Raman spectrum detection spectrum is substantially as shown in FIG4 , the selected colony is determined to be an Acinetobacter baumannii colony.

6. The method for identifying pathogens according to claim 4, characterized in that: When the Raman spectrum detection spectrum is substantially as shown in FIG6 , the selected colony is determined to be a Staphylococcus aureus colony.

7. The method for identifying pathogens according to any one of claims 1 to 6, characterized in that: The pathogen identification method is a pathogen identification method for purposes other than disease diagnosis or treatment.

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