CN111735871A - Kit for screening escherichia coli and shigella - Google Patents
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Abstract
The invention provides a kit for screening escherichia coli and shigella, and relates to the technical field of pathogen screening and detection based on mass spectrometry. The invention combines a biochemical method and a mass spectrum technology, and determines the mass-to-charge ratio of two characteristic proteins according to respective biochemical characteristics of escherichia coli and shigellam/z103.12 +/-0.1 and 89.10 +/-0.1 respectively, and the two characteristic peaks are detected by MALDI-TOF MS in combination with a biochemical kit reaction solution, so that whether a sample to be detected is escherichia coli or shigella sonnei can be accurately judged, and whether the sample is shigella sonnei or not can be determined. The kit contains biochemical reagents, reaction liquid and standard correction liquid, and the method has the advantages of high accuracy, good repeatability, high flux, low detection cost, reliable result and good application prospect.
Description
Technical Field
The invention relates to the technical field of protein mass spectrometry detection, in particular to a kit for screening escherichia coli and shigella sonnei and identifying shigella sonnei.
Background
The pathogenic escherichia coli includes 5 types, namely enterohemorrhagic escherichia coli (EHEC), pathogenic escherichia coli (EPEC), enterotoxigenic escherichia coli (ETEC), enteroinvasive escherichia coli (EIEC), and cohesive escherichia coli (EAEC), respectively. The shigella species are classified into 4 serogroups according to biochemical reaction and O antigen, namely shigella dysenteriae (group A), shigella flexneri (group B), shigella boydii (group C) and shigella sonnei (group D). Coli and shigella have close genetic relationship, are very close in genetics and similar in genetic information, so that distinguishing the four populations of the escherichia coli, the shigella and the shigella is very difficult, but has great significance. From the perspective of infectious disease control, diarrhea caused by escherichia coli belongs to other infectious diarrhea diseases in class c, and bacillary dysentery caused by shigella belongs to class b infectious diseases, and reports and treatment requirements are different. Therefore, the screening and identification of the escherichia coli and the shigella are very important, and the significance of rapid and accurate distinguishing and identification is great. The Shigella sonnei and the Shigella flexneri are main floras of isolated strains in China, the separation rate of the Shigella sonnei and the Shigella flexneri in the Shigella isolated strains in 6 years in China is 92.3%, and the two populations are rapidly distinguished, so that the method has important significance for diagnosis and prevention of Shigella flexneri diseases in China.
The distinguishing and identification of the escherichia coli and the shigella mainly depends on a traditional system biochemical method, and the shigella distinguishes the species group by adopting serology. At present, the whole genome sequencing technology, 16S rRNA and other molecular biology methods are also used for distinguishing escherichia coli from shigella and screening and identifying each serogroup of shigella. The development of high-end technology systems on the global scale provides a favorable tool for identifying and identifying bacteria. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) technology has been developed for over 10 years, application in the field of microorganism identification and identification tends to mature, and the characteristics of rapidness, accuracy, easy operation, low cost and high throughput have been accepted worldwide. However, the direct adoption of the commercialized MALDI-TOF MS technology cannot distinguish escherichia coli from shigella, which is common knowledge in the field, researchers adopt a mode of greatly increasing standard reference bacteria spectra in a commercialized system database, a mode of combining MALDI-TOF MS with ClinProTools software and a method of combining MALDI-TOF MS with artificial neural network analysis to distinguish escherichia coli from shigella, so that the recognition capability is improved compared with that of the original commercialized system, and the distinguishing capability reaches 90-94%. However, these methods are based on the ribosomal proteins of the strains themselves, and use the same sample handling as in the commercial systems, but with an improvement in the analytical methods. The shigella species are still indistinguishable by MALDI-TOF MS. And the researchers also distinguish the Song Neigongzh from the Escherichia coli by four characteristic protein peaks (5612.81 +/-8.7, 4871.12 +/-45.9, 4164.03 +/-26 and 3247.05 +/-6.9) of the Song Neigongzh, and the method adopts 24 strains of the Song Neigheng Shigella, collects 480 pictures, constructs a standard reference spectrum, and takes four specific peaks obtained by comparing the standard reference spectrum with the standard spectrum of the Escherichia coli as the standard for identifying and distinguishing the four specific peaks. However, the method has two problems, one is that the method is based on the manual operation of ribosomal protein of the Shigella sonnei by adopting a MALDI-TOF MS conventional method for distinguishing, the workload is complicated and complicated, the characteristic protein peak of the Shigella sonnei can be screened and obtained completely and efficiently by adopting the existing MALDI-TOF MS combined with the ClinProTools software method, and replaces the technology, the screening of the ribosomal protein of the Shigella sonnei has wider coverage, and another problem is that the characteristic protein peak is characterized by 5612.81 +/-8.7 and the like, the characterization mode is incorrect in the field, for example 5612.81 is the peak mass-to-charge ratio, 8.7 is the signal-to-noise ratio, it is not reasonable to add or subtract two different unit values, because the signal-to-noise ratio is not a fixed value, each spectrogram is not completely the same, and the mass-to-charge ratio (mass) cannot be added or subtracted by the signal-to-noise ratio (signal intensity ratio); the four proteins can not distinguish the Shiga sonnei from the Escherichia coli, and the Shigella species can not be distinguished.
Aiming at the distinguishing and identification of escherichia coli and shigella species and the distinguishing and identification of shigella populations, the traditional biochemical method and serological method are adopted at present, and the method is complex in operation, long in time consumption and high in cost. The results of identifying escherichia coli and shigella based on MALDI-TOF MS are not clear, and the improved technical method published in the article can only achieve the highest 94% of discrimination capability; the MALDI-TOF MS technology is based on and cannot distinguish and identify shigella species, and particularly, the shigella is easy to be misdetected into escherichia coli, so that the MALDI-TOF MS technology cannot accurately distinguish the escherichia coli from the shigella, and further cannot identify whether the shigella is the shigella in Song, and inconvenience and misdiagnosis risks are brought to food safety inspection, infectious disease pathogen identification and infectious disease prevention and control work.
Therefore, when an unknown bacterium is identified by MALDI-TOF mass spectrometry to obtain that the identification result is Escherichia coli or Shigella or Escherichia coli + Shigella, the existing MALDI-TOF mass spectrometry cannot further distinguish the Escherichia coli from the Shigella, and cannot accurately identify whether the unknown bacterium is the Shigella in Song.
Disclosure of Invention
The invention aims to provide a method for rapidly, sensitively, accurately and high-flux screening escherichia coli and shigella dysenteriae and determining whether the shigella dysenteriae is based on MALDI-TOF MS technology.
The invention starts from the aspect of biochemical characteristics of microorganisms, examines the biochemical characteristic difference of escherichia coli and shigella, and discovers that the escherichia coli and the shigella can secrete a plurality of biological enzymes in the biochemical characteristic aspect, and the invention discovers that all types of escherichia coli can produce lysine decarboxylase and ornithine decarboxylase in the growth process through careful combing, screening and screening, A, B, C, D only shigella in Song produces ornithine decarboxylase (does not produce lysine decarboxylase) in the growth process, and other three groups of shigella produce neither lysine decarboxylase nor ornithine decarboxylase. Lysine decarboxylase decomposes lysine into cadaverine and carbon dioxide, and ornithine decarboxylase decomposes ornithine into putrescine and carbon dioxide.
Based on the analysis and induction of the applicant, for the known bacteria to be detected to belong to escherichia coli or shigella, if escherichia coli or shigella is further determined, the bacteria to be detected can be determined to be escherichia coli (including all types) only by detecting the charged cadaverine characteristic peak m/z103.12 +/-0.1 of the bacteria to be detected, and no matter whether the characteristic peak m/z89.10 +/-0.1 exists or not; if m/z is not detected to be 103.12 +/-0.1, the bacteria to be detected can be determined to be shigella, and therefore, the charged cadaverine 103.12 +/-0.1 is determined to be a characteristic peak for screening escherichia coli and shigella.
The applicant further finds that if the characteristic peak of the detection result is not m/z103.12 +/-0.1, and the characteristic peak m/z89.10 +/-0.1 is detected, the strain only produces ornithine decarboxylase, and the strain can be judged as the Shigella sonnei, so that charged putrescine m/z89.10 +/-0.1 is determined as the characteristic peak for screening the Shigella sonnei.
According to the biochemical characteristics of escherichia coli and shigella, namely, the escherichia coli produces lysine decarboxylase and ornithine decarboxylase, the shigella sonnei only produces ornithine decarboxylase, the characteristic peaks of corresponding decarboxylation peaks of lysine and ornithine are respectively 103.12 +/-0.1 of charged cadaverine m/z (the molar mass of the cadaverine is 103.19, the precise molecular weight detected by mass spectrometry is 103.12, the charged cadaverine m/z is 103.12 +/-0.1 and 89.10 +/-0.1 of charged putrescine m/z is 89.16 due to errors in data acquisition of different equipment, the precise molecular weight detected by mass spectrometry is 89.10, the charged putrescine m/z is 89.10 +/-0.1 due to errors in data acquisition of different equipment, and whether a sample to be detected is the escherichia coli or the shigella is judged by MALDI-TOFMS detection of the two characteristic peaks, and further determining whether the strain is Shigella sonnei.
In a first aspect, the invention firstly provides a characteristic protein for screening escherichia coli and shigella, the characteristic protein combination is charged cadaverine, and the characteristic peak mass-to-charge ratio of the charged cadaverinem/zComprises the following steps: 103.12 +/-0.1.
The invention provides application of the characteristic protein in constructing a kit for screening escherichia coli and shigella.
The invention also provides a characteristic protein combination for identifying the Shigella sonnei from the Shigella or escherichia coli, wherein the characteristic protein combination comprises charged cadaverine and charged putrescine, and the characteristic peak mass-to-charge ratios of the charged cadaverine and the charged putrescinem/zRespectively as follows: 103.12 +/-0.1 and 89.10 +/-0.1.
The invention provides application of the characteristic protein combination in constructing a kit for screening escherichia coli, shigella sonnei and shigella sonnei.
The application is to detect whether the bacteria to be detected have two characteristic peaks of characteristic proteins by combining a MALDI-TOF MS detection method with a biochemical method.
In a second aspect, the present invention provides a kit for screening escherichia coli and shigella sonnei and identifying shigella sonnei: the kit comprises a biochemical reagent, a reaction solution and a standard correction solution, wherein the biochemical reagent contains lysine and ornithine in a mass ratio of 1: 1-5; the reaction solution is 5-10 mg/L PBS solution of pyridoxal phosphate; the kit detects bacteria to be detected based on MALDI-TOF MS, and the detection result is that whether the bacteria to be detected have the characteristic peak mass-to-charge ratio of charged cadaverinem/zIs 103.12 +/-0.1 or the mass-to-charge ratio of the characteristic peak of the charged putrescinem/z89.10 +/-0.1 as a judgment standard; the bacteria to be detected are known to belong to escherichia coli or shigella, but specific species are not determined.
When the bacteria to be detected are detected to have a characteristic peak of the charged cadaverinem/zWhen the bacterial strain is 103.12 +/-0.1, the bacteria to be detected are escherichia coli;
when the charged cadaverine characteristic peak m/z is not detected to be 103.12 +/-0.1, the bacteria to be detected are shigella;
when the charged cadaverine characteristic peak m/z is not detected to be 103.12 +/-0.1, but the charged putrescine characteristic peak m/z is detected to be 89.10 +/-0.1, the bacteria to be detected are Shigella sonnei;
and when the characteristic peak m/z of the charged cadaverine is not detected to be 103.12 +/-0.1, and the characteristic peak m/z of the charged putrescine is not detected to be 89.10 +/-0.1, determining that the bacteria to be detected are group A, group B or group C of the shigella.
The kit of the invention contains an instruction book which records the judgment standard or records a mass spectrogram containing the characteristic protein combination and a corresponding peak map for comparison and judgment of a kit operator.
In a third aspect, the present invention provides a method for identifying non-disease diagnostic purposes of escherichia coli and shigella, comprising the steps of:
(1) inoculating the bacteria to be tested into the reaction solution, adding a biochemical reagent, and incubating; the bacteria to be detected are known to belong to escherichia coli or shigella, but are not determined to belong to escherichia coli or shigella;
(2) centrifuging, taking the supernatant to be spotted on a mass spectrometer sample target, and covering a matrix saturated solution on the sample after natural drying;
the saturated solution of the matrix is saturated solution of alpha-hydrogen-based-4-hydroxycinnamic acid in 48.75% of acetonitrile and 2.5% of trifluoroacetic acid.
(3) Collecting data by using a MALDI-TOF mass spectrometer, and giving an identification result of the bacteria to be detected according to the peak condition of the charged cadaverine characteristic peak m/z of 103.12 +/-0.1;
when the bacteria to be detected are detected to have a characteristic peak of the charged cadaverinem/zWhen the bacterial strain is 103.12 +/-0.1, the bacteria to be detected are escherichia coli;
and when the charged cadaverine characteristic peak m/z is not detected to be 103.12 +/-0.1, the bacteria to be detected are shigella.
Preferably, the present invention provides a method for identifying non-disease diagnostic purposes of escherichia coli and shigella sonnei, comprising the steps of:
(1) inoculating the bacteria to be tested into the reaction solution, adding a biochemical reagent, and incubating; the bacteria to be detected are known to belong to escherichia coli or shigella, but are not determined to belong to escherichia coli or shigella;
(2) centrifuging, taking the supernatant to be spotted on a mass spectrometer sample target, and covering a matrix saturated solution on the sample after natural drying;
the saturated solution of the matrix is saturated solution of alpha-hydrogen-based-4-hydroxycinnamic acid in 48.75% of acetonitrile and 2.5% of trifluoroacetic acid.
(3) Collecting data by using a MALDI-TOF mass spectrometer, and giving an identification result of bacteria to be detected according to peak conditions of corresponding characteristic peaks m/z of 103.12 +/-0.1 and 89.10 +/-0.1 of charged cadaverine and charged putrescine;
when the charged cadaverine characteristic peak m/z is not detected to be 103.12 +/-0.1, but the charged putrescine characteristic peak m/z is detected to be 89.10 +/-0.1, the bacteria to be detected are Shigella sonnei;
and when the characteristic peak m/z of the charged cadaverine is not detected to be 103.12 +/-0.1, and the characteristic peak m/z of the charged putrescine is not detected to be 89.10 +/-0.1, determining that the bacteria to be detected are group A, group B or group C of the shigella.
In the identification method of the present invention, in step (1), the bacterial sample to be tested is preferably a liquid medium-cultured bacterium, more preferably a liquid medium-cultured logarithmic growth phase bacterium.
The bacteria cultured by the solid culture medium are used for detection, and the detection of whether the bacteria are escherichia coli or shigella and whether the bacteria are sonnei shiga can also be realized, but the target peak intensity is lower than that of liquid culture, and certain requirements are provided for the performance of MALDI-TOF MS mass spectrum.
The reaction solution is 5-10 mg/L PBS solution of pyridoxal phosphate; the biochemical reagent contains lysine and ornithine in a mass ratio of 1: 1-5; the inoculation amount of the bacteria sample to be detected inoculated to the reaction liquid is that the turbidity of the inoculated reaction liquid is 0.2-1.0 McLee unit, and the mass-volume ratio of the biochemical reagent to the reaction liquid is 4-12:1 (mg/ml).
In the identification system of the present invention, the incubation in step (1) is carried out at 28 to 42 ℃ for 1 to 8 hours, preferably at 37 ℃ for 4 hours.
The mass spectrometer in the step (2) is a MALDI-TOF MS mass spectrometer, data are collected in a reflection mode or a linear mode, the collection range is 0-200 Da, and the total collection frequency is adjusted to enable the total intensity of a spectrogram to be larger than 10000; standard calibration solutions (Bruker) were used to control the calibration apparatus to a mass deviation of less than 300 ppm.
Based on the understanding of the technical personnel in the field, the method for identifying or identifying the common intestinal pathogenic bacteria such as escherichia coli and shigella sonnei can also be applied to the fields of food safety detection, environmental detection and the like, so that the method for identifying the escherichia coli and the shigella sonnei also has the application in non-disease diagnosis.
Therefore, the invention also provides a method for identifying Shigella sonnei, which comprises the following steps:
(1) inoculating a bacterial sample to be detected into the reaction solution, adding a biochemical reagent, and incubating;
(2) centrifuging, taking the supernatant to be spotted on a mass spectrometer sample target, and covering a matrix saturated solution on the sample after natural drying;
(3) data are collected by a mass spectrometer, identification results of bacteria to be detected are given according to peak conditions of corresponding decarboxylation characteristic peaks m/z of lysine and ornithine of 103.12 +/-0.1 and 89.10 +/-0.1, and when the characteristic peak m/z of charged cadaverine of 103.12 +/-0.1 is not detected but the characteristic peak m/z of charged putrescine of 89.10 +/-0.1 is detected, the bacteria to be detected are Shigella sonnei.
The kit is adopted to carry out screening and identification on 109 strains of bacteria, including 79 strains of escherichia coli, including enterohemorrhagic escherichia coli, pathogenic escherichia coli, enterotoxigenic escherichia coli, enteroinvasive escherichia coli and cohesive escherichia coli; 30 Shigella species including Shigella dysenteriae, Shigella flexneri, Shigella boydii and Shigella sonnei. The culture mode is liquid culture, the culture time in the reaction is 2-4 hours, and the data acquisition mode is a reflection mode. The results show that 99 strains of escherichia coli are correctly identified, all the shigellas in 30 strains of shigella sonnei are correctly screened and detected, and the sensitivity and the accuracy of the detection kit are both 100%. Because the Shigella species in China mainly comprises Song-Neng Shigella and Fu-Shi Shigella, if the strain isolated in China is identified not to be Song-Neng Shigella by the method, the strain is suggested to be Shigella flexneri, and only further confirmation is needed.
The invention adopts a method combining a biochemical method and a mass spectrometry technology to determine characteristic peaks for distinguishing and identifying escherichia coli and shigella sonnei and other shigella sonnei, constructs a detection kit and an identification method for quickly, highly-flux and accurately distinguishing escherichia coli and shigella sonnei by MALDI-TOF MS, has the identification accuracy of 100 percent, and realizes the identification and detection of two strains and specific flora in the strains by one-time detection. The identification process has the advantages of short time consumption, high accuracy, easy operation and low cost, no biological safety risk in the sample preparation process, high-throughput, rapid and economic ideal pathogen detection standards, and very important practical significance for clinical diagnosis, disease monitoring, epidemiological investigation, public health emergency treatment, food safety evaluation and environmental pollution detection. The invention discloses a method for distinguishing types of escherichia coli, shigella and shigella domestic isolates, and aims to develop a mass spectrum screening technology of domestic independent intellectual property rights and provide a novel technical support for infectious disease prevention and control.
Drawings
FIG. 1 shows the characteristic peak intensities of E.coli strains after liquid culture and solid culture in a reaction solution. A1 and A2 are characteristic peak diagrams of the same strain after liquid and solid culture respectively; b1 and B2 are characteristic peak patterns of the same strain after liquid and solid culture.
FIG. 2 shows the optimization of the culture time of different E.coli strains in the reaction solution.
FIG. 3 is a graph of the effect of data acquisition mode on characteristic peaks. A1 and A2 are data collected in a reflection mode and a linear mode of the same strain; b1 and B2 are data collected in a reflection mode and a linear mode of the same strain.
Detailed Description
The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.
Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The reagents used in the examples are commercially available.
The reaction solution described in the following examples is a PBS solution of pyridoxal phosphate at 5-10 mg/L; the biochemical reagent contains lysine and ornithine in a mass ratio of 1: 1-5. The mass-volume ratio of the biochemical reagent to the reaction solution is 4-12:1 (mg/ml).
Example 1 determination of characteristic proteins for screening Escherichia coli and Shigella
The invention starts from the aspect of microbial biochemical characteristics, inspects the biochemical characteristic difference of escherichia coli and shigella, explores different types of enzymes of escherichia coli and shigella, and screens suitable enzyme reactions, aiming at searching the difference of escherichia coli and shigella by comparing the molecular weights of compounds before and after the enzyme reactions. The intracellular/extracellular enzymes of Escherichia coli and Shigella species mainly include oxidases, reductases, synthetases, transferases, decarboxylases, dehydrogenases, deaminases, etc. After repeated experiments and verification, the invention determines the selection principle of the characteristic marker enzyme for screening escherichia coli and shigella dysenteriae as follows: (1) the substrate and the product have certain molecular weight difference; (2) can be metabolized to the outside of bacteria, the enzyme reaction is relatively simple, the condition is mild, and the operation is easy; (3) the product is stable and the hydrogenation peak is not disturbed by the matrix peak.
After experimental screening is carried out by combining the principles, dehydrogenase, deaminase, deacetylase and decarboxylase are determined as candidate characteristic enzymes. However, the research finds that the dehydrogenase is not easy to adopt due to small molecular weight difference between the substrate and the product; the enzyme reaction of the deaminase is that amino groups are removed after water is added into a substrate, the molecular weight difference between the substrate and a product is only 1 Da, and the difference is extremely small and is not easy to adopt. For deacetylases, such as escherichia coli containing N-acetylglucosamine deacetylase, glucosamine can be generated after deacetylation, and can be utilized by bacteria and is difficult to detect; the substrate and product of the decarboxylase reaction have a molecular weight difference of 44 Da, and the enzyme is an inducible intracellular enzyme, when the external environment of the bacterium is appropriate, the enzyme reaction is induced to occur, protons of specific amino acids are consumed, the decarboxylation product is transported to the outside of the cell by a transporter, and new substrate is exchanged again.
Escherichia coli contains lysine decarboxylase, ornithine decarboxylase, arginine decarboxylase, glutamic acid decarboxylase, and the like. Shigella contains arginine decarboxylase, ornithine decarboxylase, etc. Through comprehensive analysis, the decarboxylase is the best choice, and four amino acid decarboxylases are the alternatives (lysine decarboxylase, arginine decarboxylase, glutamic acid decarboxylase and arginine decarboxylase). Wherein, the hydrogenation peaks of the products of arginine decarboxylase and glutamic acid decarboxylase are overlapped with the background interference peak and can not be identified. Thus, lysine decarboxylase and ornithine decarboxylase were chosen. Shigella does not contain lysine decarboxylase, and the hydrogenation peak of the decarboxylation product can be monitored through the enzymatic reaction, so that the distinction between escherichia coli and Shigella is realized. The ornithine decarboxylase is specific to the Shigella sonnei, and can be used for monitoring the hydrogenation peak of the ornithine decarboxylation product and distinguishing the Shigella sonnei from other Shigella sonnei.
Therefore, the invention induces that all types of Escherichia coli produce lysine decarboxylase and ornithine decarboxylase, and only Song Neighur produces ornithine decarboxylase (does not produce lysine decarboxylase) in the Shigella strains of the four groups of ABCD. Lysine decarboxylase and ornithine decarboxylase encountering lysine and ornithine can decompose them into cadaverine (molar mass 103.19, mass spectrometrically detected precise molecular weight of 103.12) and carbon dioxide, putrescine (molar mass 89.16, molar mass spectrometrically detected precise molecular weight of 89.10) and carbon dioxide, respectively. Therefore, as long as m/z103.12 +/-0.1 is detected, whether m/z89.10 +/-0.1 exists or not can be determined to be escherichia coli (including all types), and if m/z103.12 +/-0.1 is not detected, the escherichia coli can be determined to be shigella, so that the charged cadaverine is determined to be a characteristic peak for screening escherichia coli and shigella. If m/z is not 103.12 +/-0.1, and m/z is 89.10 +/-0.1, the strain only produces ornithine decarboxylase, and the strain can be judged as the Shigella sonnei, so that charged putrescine m/z89.10 +/-0.1 is determined as the characteristic peak for screening the Shigella sonnei.
Example 2 screening of Escherichia coli and Shigella and identification of Shiga in Song
1. Determination of Strain culture method
The invention optimizes and selects the culture condition before the strain (escherichia coli or shigella) enters the reaction solution. The bacterial culture comprises solid culture and liquid culture. Inoculating escherichia coli on an LB culture medium for overnight culture at 37 ℃, scraping lawn PBS, cleaning for 1 time, inoculating the lawn PBS into a reaction solution, culturing for 4 hours at 37 ℃, centrifuging, taking 1 microliter of supernatant to spot on a sample target, naturally drying, covering 1 microliter of CHCA matrix saturated solution (saturated solution of alpha-hydrogen-4-hydroxycinnamic acid in 48.75% acetonitrile and 2.5% trifluoroacetic acid), and collecting data by MALDI-TOF MS after drying.
Escherichia coli and Shigella sonnei were inoculated in liquid LB medium, respectively, and cultured overnight on a shaker at 37 ℃. Centrifuging to take precipitation bacteria, washing 1 time by PBS, inoculating the precipitation bacteria into reaction liquid, adding a biochemical reagent into the reaction liquid according to the mass-to-volume ratio of 1:8mg/ml, culturing for 2 hours at 37 ℃, centrifuging to take 1 mu l of supernatant, spotting the supernatant on a sample target, naturally drying, covering 1 mu l of CHCA matrix saturated solution, drying, and collecting data by adopting a MALDI-TOF MS reflection mode, wherein the collection mass range is 0-200 Da. The mass spectrum data acquisition adopts the same laser intensity and the same spectrogram accumulation times.
The results show that under the same reaction conditions and reaction time and the same collection conditions, the peak intensity obtained by the liquid culture strain sample is far greater than that obtained by the solid culture strain, and the abundance ratio of all the peaks of the characteristic peaks 103.12 +/-0.1 and 89.10 +/-0.1 is also far greater than that of the solid culture sample, so that even if the collection times and the laser intensity are increased for the solid culture sample alone, the situation cannot be improved (the characteristic peak intensity graph after the culture of escherichia coli in different culture media is shown in figure 1), and the shigella is in the same situation. Therefore, liquid culture of the strain to logarithmic growth phase before culture in the reaction solution is the optimum culture method determined by the present invention.
2. Optimization of incubation time
Culturing 7 strains of Escherichia coli and Song Neighur Shiga to logarithmic phase by the liquid culture method, centrifuging, washing with PBS for 1 time, inoculating to the reaction solution, culturing at 37 deg.C for 6 hr, taking out 200 μ l suspension every 1 hr, centrifuging, and collecting supernatant. And (3) respectively taking 1 microliter of supernatant of 42 samples obtained at six time points of 1, 2, 3, 4, 5 and 6 hours, spotting the 1 microliter of supernatant on a sample target, naturally drying, covering 1 microliter of CHCA matrix saturated solution, and collecting data by adopting a MALDI-TOF MS reflection mode after drying. And adjusting the total acquisition frequency to enable the total intensity of the spectrogram to be more than 10000 and the mass deviation to be less than 300 ppm.
The experimental results show that: as the culture time is prolonged, the signal-to-noise ratios of the characteristic peaks 103.12. + -. 0.1 and 89.10. + -. 0.1 gradually increase, reach the highest values at 4 hours, and then start to decrease. The detection requirement can be met after the liquid culture is carried out for 1 hour, a relatively ideal peak value is reached after 2 hours, the optimized result of the culture time of the escherichia coli reaction liquid is shown in a figure 2, and the shigella is under the same condition.
3. Difference analysis of mass spectrometry data acquisition modes
Culturing two strains of escherichia coli to a logarithmic growth phase by adopting a liquid culture method, centrifuging, washing by PBS once, inoculating the escherichia coli to a reaction solution, culturing at 37 ℃ for 4 hours, centrifuging, taking 1 microliter of supernatant to spot on a sample target, naturally drying, covering 1 microliter of CHCA matrix saturated solution, and collecting data by adopting a MALDI-TOF MS reflection mode and a linear mode after drying. And adjusting the total acquisition frequency to enable the total intensity of the spectrogram to be more than 10000 and the mass deviation to be less than 300 ppm.
The experimental results show that: under the same acquisition condition, the characteristic peak 103.12 +/-0.1 is 2 to 5 times higher than that of a linear mode in a reflection mode, and the characteristic peak and an interference peak adjacent to the characteristic peak of 1 Da can be separated in the reflection mode, so that the characteristic peak is easier to accurately identify; under the linear mode, the resolution ratio is lower than that of the reflection mode, so that the peak width is larger, the resolution ratio of the interference peak to the adjacent 1 Da is not as good as that of the reflection mode, but the identification of the characteristic peak can be realized, the identification result is not influenced, the influence of different mass spectrum data acquisition modes of escherichia coli on the characteristic peak is shown in fig. 3, and shigella is under the same condition. The user needs to combine the conditions of the equipment, and if the equipment has the reflection mode, the data collection in the reflection mode is recommended to be preferentially selected.
Example 3 evaluation of actual test Effect of the kit
The kit is adopted to carry out screening and identification on 109 strains of bacteria, including 79 strains of escherichia coli, including enterohemorrhagic escherichia coli, pathogenic escherichia coli, enterotoxigenic escherichia coli, enteroinvasive escherichia coli and cohesive escherichia coli; 30 Shigella species including Shigella dysenteriae, Shigella flexneri, Shigella boydii and Shigella sonnei. The culture mode is liquid culture, the culture time in the reaction is 2 hours, and the data acquisition mode is a reflection mode. The results are given in the following table:
the results show that 99 strains of escherichia coli are correctly identified, 12 strains of shigella sonnei in 30 strains of shigella sonnei are correctly screened and detected, the rest 18 strains of shigella sonnei are also correctly identified as shigella, the detection results are consistent with the detection results of the traditional biochemical method, and the sensitivity and the accuracy of the detection kit are both 100%. Because the Shigella species in China are mainly Song-Neng Shigella and Fu-Shi Shigella, if the strain isolated in China is identified not to be Song-Neng Shigella by the method, the strain is suggested to be Shigella flexneri, and only further confirmation is needed. The method has the advantages of short analysis time, high accuracy, easy operation, low cost and no biological safety risk in the sample preparation process. Therefore, the invention is a practical detection technology for screening escherichia coli and shigella and screening and identifying shigella in Song, solves the bottleneck problem that the screening and identifying cannot be carried out based on MALDI-TOF MS at present, and provides a key technical support for diagnosis of diseases related to escherichia coli and shigella and prevention and control of epidemic situations.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. The characteristic protein for screening escherichia coli and shigella is characterized in that the characteristic protein combination is charged cadaverine, and the charged cadaverine has a characteristic peak mass-to-charge ratiom/zComprises the following steps: 103.12 +/-0.1.
2. The use of the characteristic protein of claim 1 in the construction of a kit for screening escherichia coli and shigella spp.
3. A characteristic protein combination for identifying Shigella sonnei from Shigella or Escherichia coli is characterized in that the characteristic protein combination is charged cadaverine and charged putrescine, and the charged cadaverine and charged putrescine have characteristic peak mass-to-charge ratiosm/zRespectively as follows: 103.12 +/-0.1 and 89.10 +/-0.1.
4. The use of the combination of characteristic proteins of claim 3 in the construction of kits for screening Escherichia coli, Shigella species and Shigella sonnei species.
5. A kit for screening Escherichia coli and Shigella sonnei and identifying Shigella sonnei is characterized by comprising a biochemical reagent, a reaction solution and a standard correction solution, wherein the biochemical reagent contains lysine and ornithine in a mass ratio of 1: 1-5; the reaction solution is 5-10 mg/L PBS solution of pyridoxal phosphate;
the kit detects bacteria to be detected based on MALDI-TOF MS, and the detection result is judged by whether the bacteria to be detected has the characteristic protein of claim 1 or the characteristic protein combination of claim 3, and the characteristic peak mass-to-charge ratio of the charged cadaverine is judgedm/zIs 103.12 +/-0.1 or the mass-to-charge ratio of the characteristic peak of the charged putrescinem/z89.10 +/-0.1 as a judgment standard; the bacteria to be detected are known to belong to escherichia coli or shigella, but specific species are not determined.
6. The kit of claim 5, wherein the test bacteria have a characteristic peak for charged cadaverine when detectedm/ zWhen the bacterial strain is 103.12 +/-0.1, the bacteria to be detected are escherichia coli;
when the charged cadaverine characteristic peak m/z is not detected to be 103.12 +/-0.1, the bacteria to be detected are shigella;
when the charged cadaverine characteristic peak m/z is not detected to be 103.12 +/-0.1, but the charged putrescine characteristic peak m/z is detected to be 89.10 +/-0.1, the bacteria to be detected are Shigella sonnei;
and when the charged cadaverine characteristic peak m/z is not detected to be 103.12 +/-0.1, and the charged putrescine characteristic peak m/z is not detected to be 89.10 +/-0.1, determining that the bacteria to be detected are group A, group B or group C of Shigella sonnei.
7. A method for identifying non-disease diagnosis purposes of Escherichia coli and Shigella, which is characterized by comprising the following steps:
(1) inoculating the bacteria to be tested into the reaction solution, adding a biochemical reagent, and incubating; the bacteria to be detected are known to belong to escherichia coli or shigella, but are not determined to belong to escherichia coli or shigella;
(2) centrifuging, taking the supernatant to be spotted on a mass spectrometer sample target, and covering a matrix saturated solution on the sample after natural drying;
(3) collecting data by using a MALDI-TOF mass spectrometer, and giving an identification result of bacteria to be detected according to peak conditions of corresponding characteristic peaks m/z of 103.12 +/-0.1 and 89.10 +/-0.1 of charged cadaverine and charged putrescine;
when the bacteria to be detected are detected to have a characteristic peak of the charged cadaverinem/zWhen the bacterial strain is 103.12 +/-0.1, the bacteria to be detected are escherichia coli;
when the charged cadaverine characteristic peak m/z is not detected to be 103.12 +/-0.1, the bacteria to be detected are shigella;
when the charged cadaverine characteristic peak m/z is not detected to be 103.12 +/-0.1, but the charged putrescine characteristic peak m/z is detected to be 89.10 +/-0.1, the bacteria to be detected are Shigella sonnei;
and when the charged cadaverine characteristic peak m/z is not detected to be 103.12 +/-0.1, and the charged putrescine characteristic peak m/z is not detected to be 89.10 +/-0.1, determining that the bacteria to be detected are group A, group B or group C of Shigella sonnei.
8. The identification method according to claim 7, wherein in the step (1), the bacterial sample to be tested is a bacterium cultured in a liquid medium to logarithmic growth phase; and/or
The reaction solution is 5-10 mg/L PBS solution of pyridoxal phosphate; the biochemical reagent contains lysine and ornithine in a mass ratio of 1: 1-5; and/or
The inoculation amount of the bacteria sample to be detected inoculated into the reaction solution is that the mass-volume ratio of the Mains unitized reagent to the reaction solution after the reaction solution is inoculated is 0.2-1.0, and/or,
the incubation in step (1) is carried out at 28-42 ℃ for 1-8 hours, preferably at 37 ℃ for 4 hours.
9. The identification method according to any one of claims 7 to 8, wherein the mass spectrometer is a MALDI-TOF MS mass spectrometer, data are collected in a reflection or linear mode, the collection range is 0-200 Da, and the total collection frequency is adjusted to make the total intensity of a spectrogram greater than 10000; and a standard correction liquid quality control correction instrument is adopted, so that the quality deviation is less than 300 ppm.
10. A method for identifying Shigella sonnei is characterized by comprising the following steps:
(1) inoculating a bacterial sample to be detected into the reaction solution, adding a biochemical reagent, and incubating; the bacteria to be detected are known to belong to escherichia coli or shigella, but specific species are not determined;
(2) centrifuging, taking the supernatant to be spotted on a mass spectrometer sample target, and covering a matrix saturated solution on the sample after natural drying;
(3) collecting data by adopting a mass spectrometer, and giving an identification result of the bacteria to be detected according to peak conditions of corresponding characteristic peaks m/z of 103.12 +/-0.1 and 89.10 +/-0.1 of charged cadaverine and putrescine;
and when the charged cadaverine characteristic peak m/z is not detected to be 103.12 +/-0.1, but the charged putrescine characteristic peak m/z is detected to be 89.10 +/-0.1, the bacteria to be detected are Shigella sonnei.
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