WO2020108237A1 - 一种用于检测大肠杆菌o157与非o157大肠杆菌的显色培养基 - Google Patents

一种用于检测大肠杆菌o157与非o157大肠杆菌的显色培养基 Download PDF

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WO2020108237A1
WO2020108237A1 PCT/CN2019/115520 CN2019115520W WO2020108237A1 WO 2020108237 A1 WO2020108237 A1 WO 2020108237A1 CN 2019115520 W CN2019115520 W CN 2019115520W WO 2020108237 A1 WO2020108237 A1 WO 2020108237A1
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coli
substrate
chromogenic
detecting
medium
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French (fr)
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韦献虎
吴清平
张菊梅
张淑红
卢勉飞
陈谋通
蔡芷荷
杨广珠
冯颖
陈敏玲
丁郁
王涓
张友雄
古其会
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广东省微生物研究所（广东省微生物分析检测中心）
广东环凯微生物科技有限公司
广东环凯生物科技有限公司
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/045—Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/10—Enterobacteria

Definitions

  • the invention relates to the technical field of microorganism detection, in particular to a chromogenic medium for detecting E. coli O157 and non-O157 E. coli.
  • Escherichia coli is generally regarded as an indicator bacteria for fecal contamination that is closely related to the hygienic quality, cleanliness, or risk of disease of food, water, feed, clinical and environmental samples.
  • E.coli O157 can cause asymptomatic excretion in humans, non-bloody diarrhea, hemorrhagic colitis, hemolytic uremic syndrome and even death. It is intestinal hemorrhagic (or Shiga toxin-producing)
  • EHEC or STEC The most important pathogenic serotype in .coli has become an important pathogenic bacterium that has attracted worldwide attention.
  • non-O157 E.coli includes non-O157 EHEC (or STEC) and other such as intestinal pathogenic E.coli (EPEC), enterotoxigenic E.coli (ETEC), Intestinal invasive E.coli (EIEC), intestinal agglomeration E.coli (EAEC), and other pathogenic types that require further epidemiological studies, such as diffuse adhesion E.coli (DAEC), can also cause humans The outbreak of diarrhea and other diseases also needs attention (Gomes et al., Brazilian Journal of Microbiology, 2016, 47: 3-30; Mead et al., Lancet, 1998, 352: 1207-1212; Pennington et al., Lancet ,2010,376:1428-1435; Yang et al., Archives of Microbiology, 2017,199:811-825.). Therefore, the detection of E.coli O157 and non-O157 E.coli is indispensable for ensuring food safety and other public health safety.
  • EPEC intestinal pathogenic E.coli
  • ETEC enterotoxigenic E.coli
  • Chromocult (Merck, Germany), Coli ID (bioMérieux, France), CHROMagar ECC (Chromagar, France), HiCrome ECC (Union Carbide, USA), etc.
  • These color development media are supplemented with two different enzymes ( ⁇ -galactosidase And ⁇ -glucuronidase) chromogenic substrate, can be used to detect coliform bacteria and non-O157 E.coli with ⁇ -glucuronidase activity; but on them, E.coli O157 and other large intestine The microflora have the same color and are difficult to distinguish (Manafi M., International Journal of Food Microbiology, 2000, 60:205-218.).
  • the E.coli O157 color developing medium is only suitable for the specific detection of E.coli O157, and the detection effect of non-O157 E.coli is relatively poor, such as CHROMagar O157 (Chromagar, France), Rainbow Agar O157 (Biolog Inc. , United States) and Rapid E. coli O157:H7 (Bio-Rad), etc. (Verhaegen et al., International Journal of Environmental Research and Public Health, 2015, 12:6965-6978; Fan et al., International Journal of Food Microbiology, 2018, 266: 295-300.).
  • the object of the present invention is to provide a coloring medium for detecting E. coli O157 and non-O157 E. coli.
  • a coloring medium for the detection of E. coli O157 and non-O157 E. coli It does not contain sorbitol nor neutral red, but contains two color-developing substrates that can produce different color signals without diffusion.
  • a fluorescent substrate that does not develop color but can produce a non-blue fluorescent signal without diffusion.
  • the chromogenic substrate and the fluorescent substrate include a ⁇ -glucuronidase substrate and ⁇ -galactose In addition to the glycosidase substrate, it also contains at least ⁇ -glucosidase substrate, ⁇ -N-acetylglucosaminidase substrate, and cellobiosidase substrate, which have the same or similar color or fluorescence signal.
  • a kind of substrate is a kind of substrate.
  • the color-developing substrate is an indolol-based color-developing substrate based on a 3-indolphenol derivative, and the structure of the 3-indolephenol derivative is shown in the following formula (I):
  • R in the above formula may be a mono-substituted group or a poly-substituted group.
  • the 3-indolephenol derivative is 5-bromo-4-chloro-3-indolephenol, 5-bromo-6-chloro-3-indolephenol or 6-chloro-3-indole Phenol and so on.
  • the 3-indolephenol-based indolephenol-based chromogenic substrate is 5-bromo-4-chloro-3-indolyl- ⁇ -D-glucoside, 5-bromo-6 -Chloro-3-indolyl- ⁇ -D-galactoside, 5-bromo-4-chloro-3-indolyl- ⁇ -D-glucuronide cyclohexylamine salt or 5-bromo-6- Chloro-3-indolyl- ⁇ -D-glucuronide cyclohexylamine salt.
  • the fluorescent substrate is a fluorescent substrate based on a 2-(benzothiazole-2'-yl)phenol derivative, and the 2-(benzothiazole-2'-yl)phenol derivative
  • the structure is shown in the following formula (2):
  • R1 and R2 in the above formula represent groups such as halogen atoms or alkoxy groups.
  • the 2-(benzothiazol-2'-yl)phenol derivative is 2-(benzothiazol-2'-yl)-4-bromophenol and the like.
  • the fluorescent substrate based on 2-(benzothiazol-2'-yl)phenol derivative is 2-(benzothiazol-2'-yl)-4-bromophenyl- ⁇ -D- Glucuronide, 2-(benzothiazol-2'-yl)-4-bromophenyl- ⁇ -D-galactoside or 2-(benzothiazol-2'-yl)-4-bromophenyl - ⁇ -D-glucoside.
  • the phosphor 2-(benzothiazol-2'-yl)phenol derivative is reported according to the literature [Otsubo et al. Bioorganic & Medicinal Letters, 2013, 23(7): 2245-2249] Method synthesis.
  • the synthesis method of the fluorescent substrate based on the 2-(benzothiazole-2'-yl)phenol derivative includes a two-step reaction. The specific steps are:
  • lithium hydroxide is preferred as the base; for the synthesis of other glycosides, potassium hydroxide is preferred as the base.
  • the catalyst silver oxide or cesium carbonate
  • the sugar donor acetyl halo sugar
  • the molar ratio of the catalyst: sugar donor: sugar acceptor is 1.5:1.5:1.0.
  • the coloring medium for detecting E. coli O157 and non-O157 E. coli also contains sodium dodecyl sulfate (SDS) and/or bile salt (No. 3 bile salt, sodium deoxycholate, etc.) ), components required for bacterial survival and growth (such as peptone, yeast extract, sodium chloride, etc.), agar, isopropyl- ⁇ -D-thiogalactopyranoside (IPTG), and possibly a small amount of lactose , Salicin or cellobiose, Tween 80 or Triton X-100.
  • SDS sodium dodecyl sulfate
  • bile salt No. 3 bile salt, sodium deoxycholate, etc.
  • components required for bacterial survival and growth such as peptone, yeast extract, sodium chloride, etc.
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • lactose Salicin or cellobiose
  • the concentration of the chromogenic substrate and the fluorescent substrate in the chromogenic medium are both 0.01 to 0.30 g/L.
  • the color developing medium further contains sodium lauryl sulfate 0.05-0.25g/L and/or bile salt 0-1.50g/L, peptone 1-20g/L, yeast extract powder 0-6g/ L, sodium chloride 4-8g/L, agar 10-20g/L, isopropyl- ⁇ -D-thiogalactopyranoside 0.01-0.10g/L.
  • the color development medium further contains 0.01-0.15 g/L of lactose, salicin or cellobiose.
  • the color developing medium further contains Tween 80 ⁇ 0 ⁇ 0.60mL/L or Triton X-100 ⁇ 0 ⁇ 0.20mL/L.
  • the ⁇ -glucuronidase substrate is in the form of carboxylic acid or carboxylate.
  • the coloring medium for detecting E. coli O157 and non-O157 E. coli can be used in the case of reduced input of coloring medium type selection and related processing work (such as plate preparation work before use of coloring medium, etc.) , Can still meet the multiple purposes that can be used to detect both E. coli O157 and non-O157 E. coli, and the detection specificity is relatively high.
  • Figure 1 is the fluorescent substrate 2-(benzothiazol-2'-yl)-4-bromophenyl- ⁇ -D-glucuronide, 2-(benzothiazol-2'-yl)-4-bromo Synthetic route of phenyl- ⁇ -D-galactoside or 2-(benzothiazol-2'-yl)-4-bromophenyl- ⁇ -D-glucoside.
  • Figure 2 shows the coloring medium used to detect E. coli O157 and non-O157 E. coli in Example 4 against E. coli O157 ATCC 35150, E. coli ATCC 25922, Enterobacter sakazakii ATCC 29544, and Salmonella typhimurium ATCC 14028
  • the upper right, upper left, lower left, and lower right of the tablet are E. coli O157 ATCC 35150, E. coli ATCC 25922, Enterobacter sakazakii ATCC 29544 and Salmonella typhimurium ATCC 14028,
  • A is under visible light
  • B is Under 365nm ultraviolet light
  • C is visible light and flashlight under 375nm ultraviolet light.
  • Fig. 3 shows the coloring medium used for the detection of E. coli O157 and non-O157 E. coli in Examples 4, 5, and 6 for E. coli O157, non-O157 E. coli and non-E. coli at 37°C Detection effect after 12h and 18h incubation.
  • subscript 1 means observation under visible light
  • subscript 2 means observation under 365nm ultraviolet light
  • I is A to C color development The corresponding number of each bacteria in the medium plate.
  • Fig. 4 shows the chromogenic medium used for the detection of E. coli O157 and non-O157 E. coli in Example 7 for aerobic culture of E. coli O157, non-O157 E. coli and non-E. coli at 37°C for 12h and 18h respectively After the detection effect.
  • the left picture shows observation under visible light
  • the right picture shows observation under 365nm ultraviolet light
  • I is the corresponding number of each bacteria in the chromogenic medium plate.
  • Figure 5 shows the detection effect of some existing media on E. coli O157, non-O157 E. coli and non-E. coli after aerobic culture at 37°C for 12h and 18h, respectively.
  • D TBX agar group
  • E CHROMagar E.coli group
  • F CHROMagar O157 group
  • G Sorbitol-Macconkey medium (SMAC) group
  • subscript 1 indicates observation under visible light
  • subscript 2 indicates Observed under 365nm ultraviolet light
  • I is the corresponding number of each bacteria in the D to G chromogenic medium plate.
  • a coloring medium for detecting E. coli O157 and non-O157 E. coli which contains 10g peptone, 3g yeast extract powder, 5g sodium chloride, 0.1g SDS, 0.1g sodium deoxycholate, 0.5mL per 1000mL Temperature 80, 0.15g fluorescent substrate 2-(benzothiazol-2'-yl)-4-bromophenyl- ⁇ -D-glucuronide (can produce yellow-green fluorescence under 365 ⁇ 375nm ultraviolet light), 0.05g chromogenic substrate 5-bromo-4-chloro-3-indolyl- ⁇ -D-glucoside (can produce blue to blue-green), 0.05g chromogenic substrate 5-bromo-6-chloro-3 -Indolyl- ⁇ -D-galactoside (can produce purple-red), 0.05g IPTG, 15g agar. (Note: The color developing medium can be steam sterilized at 121°C for 15 minutes).
  • a coloring medium for detecting E. coli O157 and non-O157 E. coli each 1000mL contains 10g peptone, 3g yeast extract powder, 5g sodium chloride, 0.1g SDS, 0.1g sodium deoxycholate, 0.045g fluorescence Substrate 2-(benzothiazol-2'-yl)-4-bromophenyl- ⁇ -D-glucoside (yellow-green fluorescence can be produced under ultraviolet light at 365 ⁇ 375nm), 0.20g color substrate 5- Bromo-4-chloro-3-indolyl- ⁇ -D-glucuronide cyclohexylamine salt (can produce blue to blue green), 0.05g color substrate 5-bromo-6-chloro-3-ind Indoyl- ⁇ -D-galactoside (which can produce magenta), 0.05g IPTG, 0.01g salicin, 0.01g cellobiose, 15g agar. (Note: The chromogenic medium can also be steam sterilized at
  • a coloring medium for detecting E. coli O157 and non-O157 E. coli which contains 10g peptone, 3g yeast extract powder, 5g sodium chloride, 0.1g SDS, 0.1g sodium deoxycholate, 0.5mL per 1000mL Temperature 80, 0.045g fluorescent substrate 2-(benzothiazole-2′-yl)-4-bromophenyl- ⁇ -D-galactoside (can produce yellow-green fluorescence under 365 ⁇ 375nm ultraviolet light), 0.20 g color development substrate 5-bromo-6-chloro-3-indolyl- ⁇ -D-glucuronide cyclohexylamine salt (can produce purple red), 0.055g color development substrate 5-bromo-4- Chloro-3-indolyl- ⁇ -D-glucoside (can produce blue to blue-green), 0.025g IPTG, 0.025g lactose, 15g agar. (Note: The chromogenic medium can also be steam sterilized at 121°C
  • a coloring medium for detecting E. coli O157 and non-O157 E. coli each 1000mL contains 10g peptone, 3g yeast extract powder, 5g sodium chloride, 0.20g SDS, 0.20g sodium deoxycholate, 0.10mL koji Laton X-100, 0.15g fluorescent substrate 2-(benzothiazol-2′-yl)-4-bromophenyl- ⁇ -D-glucuronide (can produce yellow-green under 365 ⁇ 375nm ultraviolet light Fluorescence), 0.05g chromogenic substrate 5-bromo-4-chloro-3-indolyl- ⁇ -D-glucoside (can produce blue to blue-green), 0.05g chromogenic substrate 5-bromo-6- Chloro-3-indolyl- ⁇ -D-galactoside (can produce purple-red), 0.05g IPTG, 15g agar. (Note: The color developing medium can be steam sterilized at 121°C for 15 minutes).
  • a coloring medium for detecting E. coli O157 and non-O157 E. coli each 1000mL contains 1g peptone, 6g yeast extract powder, 4g sodium chloride, 0.05g SDS, 1.5g bile salt No. 3, 0.20mL koji Laton X-100, 0.05g fluorescent substrate 2-(benzothiazole-2'-yl)-4-bromophenyl- ⁇ -D-glucoside (can produce yellow-green fluorescence under 365 ⁇ 375nm ultraviolet light) , 0.20g chromogenic substrate 5-bromo-4-chloro-3-indolyl- ⁇ -D-glucuronide cyclohexylamine salt (can produce blue to blue green), 0.05g chromogenic substrate 5- Bromo-6-chloro-3-indolyl- ⁇ -D-galactoside (can produce purple-red), 0.1g IPTG, 0.15g salicin, 10g agar. (Note: The chromogenic medium can also be steam sterilize
  • a coloring medium for detecting E. coli O157 and non-O157 E. coli which contains 20g peptone, 8g sodium chloride, 0.25g SDS, 0.60mL Tween 80, 0.05g fluorescent substrate 2-(benzene) per 1000mL Thiazol-2′-yl)-4-bromophenyl- ⁇ -D-galactoside (yellow-green fluorescence can be produced under ultraviolet light at 365 ⁇ 375nm), 0.30g color substrate 5-bromo-6-chloro -3-indolyl- ⁇ -D-glucuronide cyclohexylamine salt (can produce purple red), 0.01g color substrate 5-bromo-4-chloro-3-indolyl- ⁇ -D- Glucoside (can produce blue to blue-green), 0.05g color substrate 5-bromo-4-chloro-3-indolyl- ⁇ -DN-acetylglucosamine (can produce blue to blue-green), 0.20g Color-developing substrate 5-bromo
  • the upper right, upper left, lower left, and lower right of the tablet in Figure 2 are E.coli O157:H7 ATCC 35150, E.coli ATCC 25922, Enterobacter sakazakii ATCC 29544, and Salmonella typhimurium ATCC 14028.
  • A is under visible light.
  • B is under 365nm ultraviolet light,
  • C is under visible light and flashlight under 375nm ultraviolet light.
  • the coloring medium for detecting E. coli O157 and non-O157 E. coli of Example 4 can give a good distinction between the following four pathogenic bacteria: E. coli O157:H7 ATCC 35150 (Upper right) pink and no fluorescence, E.
  • the colorimetric media for detecting E. coli O157 and non-O157 E. coli in Examples 4, 5, and 6 of the present invention were evaluated for the detection effect of E. coli O157, non-O157 E. coli, and non-E. coli, respectively.
  • the inoculation method is to use 2.5 ⁇ L pipette to suck the corresponding pure bacterial culture liquid and carefully plant 0.2 ⁇ L to the corresponding position on the plate by reverse pipetting method; aerobic culture at 37°C for 12h and 18h. Then observe under visible light and 365nm ultraviolet light. The results are shown in Figure 3.
  • A Example 4 group
  • B Example 5 group
  • C Example 6 group
  • subscript 1 indicates observation under visible light
  • subscript 2 indicates observation under 365 nm ultraviolet light
  • I is A to C
  • the corresponding numbers of the bacteria in the chromogenic medium plate, the experimental strains are: 1 intestinal agglomerated E.coli (EAEC) isolate (No. 1288-1), 2 EAEC isolate (No. 3476B3), 3 AEC isolate ( No. 3164A1), 4 intestinal invasive E. coli (EIEC) isolate (No. 2968A1), 5 EIEC isolate (No. 2987A2), 6 intestinal pathogenic E. coli (EPEC) isolate (No. 2684-2), 7 EPEC isolate (No.
  • EAEC intestinal agglomerated E.coli
  • EIEC intestinal invasive E. coli
  • EPEC intestinal pathogenic E. coli
  • E. 2627-2 8 EPEC isolate (No. 545-1), 9 Shiga toxin (or intestinal hemorrhagic) E. coli (STEC or EHEC) isolate ( No.T1 4259B3), 10STEC or EHEC isolate (No.T5 3424A1), STEC or EHEC isolates (No. T7 4274A1), STEC or EHEC isolate (No. T18 3724C1), STEC or EHEC isolates (No. T91 1964-1), STEC or EHEC isolate (No.
  • E.coli O157:H7 ATCC 35150 E.coli O157:H7NCTC 12900, E.coli O157:H7 882364, E.coli O157:H7 ATCC 43895, E.coli O157:H7 ATCC 43888, E.coli CMCC(B)44102, E.coli CMCC(B)44103, E.coli ATCC 8739, E.coli ATCC 25922, E.coli ATCC 8099, E.coli ATCC 35218, E.coli CMCC(B)44113, E.coli HK 70115, Enterobacter aerogenes CMCC (B) 45103, Enterobacter cloacae CMCC(B)45301, Klebsiella pneumoniae CMCC (B) 46117, Klebsiella pneumoniae wide clinical examination-57, Enterobacter sakazakii ATCC 29544, Enterobacter sakazakii isolate (No.10Ta), Enterobacter sakazakii isolate (No.10Ta), Enter
  • E.coli O157:H7 all showed pink and no fluorescence
  • non-O157E.coli all showed pink and yellow-green fluorescence
  • non-E.coli showed green (See Enterobacter sakazakii), blue purple (see Enterobacter aerogenes, Enterobacter cloacae, 1 strain of Klebsiella pneumoniae), metallic blue (see 1 strain of Klebsiella pneumoniae), or no color (see Salmonella typhimurium, Salmonella enteritidis, Salmonella typhi, Pseudomonas aeruginosa, Proteus), Gram-positive bacteria were inhibited (see Bacillus cereus ATCC14579).
  • Example 5(B) all E. coli O157:H7 showed pink and no fluorescence, non-O157 E. coli all showed metallic blue and no fluorescence, non-E. coli showed pink or mauve and there was Yellow-green fluorescence (see Enterobacter aerogenes, Enterobacter cloacae and Klebsiella pneumoniae), colorless and non-fluorescent (see Salmonella, Proteus and some Pseudomonas aeruginosa), or colorless and blue-fluorescent ( (See some Pseudomonas aeruginosa), Gram-positive bacteria are inhibited (see Bacillus cereus ATCC14579).
  • Example 6(C) all E.coli O157:H7 showed colorless and beige fluorescence, non-O157E.coli all showed magenta and no obvious fluorescence, non-E.coli showed green or blue-green (See Enterobacter sakazakii, Enterobacter aerogenes, Enterobacter cloacae, Klebsiella pneumoniae), non-colorless and non-fluorescent or blue-fluorescent (see Pseudomonas aeruginosa, Proteus), Gram Positive bacteria are inhibited (see Bacillus cereus ATCC14579).
  • the effect of the coloring medium for detecting E. coli O157 and non-O157 E. coli in Example 7 of the present invention on E. coli O157, non-O157 E. coli and non-E. coli was evaluated.
  • the inoculation method is to use 2.5 ⁇ L pipette to suck the corresponding pure bacterial culture liquid and carefully plant 0.2 ⁇ L to the corresponding position on the plate by reverse pipetting method; aerobic culture at 37°C for 12h and 18h. Then observe under visible light and 365nm ultraviolet light. The results are shown in Figure 4.
  • the left picture shows observation under visible light
  • the right picture shows observation under 365nm ultraviolet light
  • I is the corresponding number of each bacterium in the chromogenic medium plate
  • the experimental strains are: 1 intestinal agglomeration E. coli (EAEC ) Isolate (No. 1288-1), 2 EAEC isolate (No. 3476B3), 3 AEC isolate (No. 3164A1), 4 intestinal invasive E. coli (EIEC) isolate (No. 2968A1), 5 EIEC isolate Strain (No. 2987A2), 6 intestinal pathogenic E. coli (EPEC) isolate (No. 2684-2), 7 EPEC isolate (No. 2627-2), 8 EPEC isolate (No.
  • E.coli O157:H7 ATCC35150 E.coli O157:H7NCTC 12900, E.coli O157:H7 882364, E.coli O157:H7 ATCC43895, E.coli O157:H7 ATCC 43888, E.coli CMCC(B)44102, E.coli CMCC(B)44103, E.coli ATCC 8739, E.coli ATCC 25922, E.coli ATCC 8099, E.coli ATCC 35218, E.coli CMCC(B)44113, E.coli HK 70115, Enterobacter aerogenes CMCC (B) 45103, Enterobacter cloacae CMCC(B)45301, Klebsiella pneumoniae CMCC (B) 46117, Klebsiella pneumoniae wide clinical examination-57, Enterobacter sakazakii ATCC29544, Enterobacter sakazakii isolate (No.10Ta), Enterobacter sakazakii isolate (No.10Ta), Enterobacter
  • the inoculation method is to use 2.5 ⁇ L pipette to suck the corresponding pure bacterial culture liquid and carefully plant 0.2 ⁇ L to the corresponding position on the plate by reverse pipetting method; aerobic culture at 37°C for 12h and 18h. Then observe under visible light and 365nm ultraviolet light. The results are shown in Figure 5.
  • D TBX agar group
  • E CHROMagar E. coli group
  • F CHROMagar O157 group
  • G Sorbitol-Macconkey medium (SMAC) group
  • subscript 1 means observation under visible light
  • subscript 2 It means observation under 365nm ultraviolet light
  • I is the number corresponding to each bacterium in the D to G chromogenic medium plate.
  • the experimental strains are: 1 intestinal aggregative E. coli (EAEC) isolate (No. 1288-1), 2 EAEC isolate (No. 3476B3), 3 AEC isolate (No. 3164A1), 4 intestinal invasive E. coli (EIEC) isolate (No. 2968A1), 5 EIEC isolate (No.
  • E. coli 6 intestinal infection Pathogenic E.coli (EPEC) isolate (No. 2684-2), 7 EPEC isolate (No. 2627-2), 8 EPEC isolate (No. 545-1), 9 Shiga toxin production (or intestinal bleeding Sex) E. coli (STEC or EHEC) isolate (No. T1 4259B3), 10 STEC or EHEC isolate (No. T5 3424A1), STEC or EHEC isolates (No. T7 4274A1), STEC or EHEC isolate (No. T18 3724C1), STEC or EHEC isolates (No. T91 1964-1), STEC or EHEC isolate (No.
  • E.coli O157:H7 ATCC 35150 E.coli O157:H7NCTC 12900, E.coli O157:H7 882364, E.coli O157:H7 ATCC 43895, E.coli O157:H7 ATCC 43888, E.coli CMCC(B)44102, E.coli CMCC(B)44103, E.coli ATCC 8739, E.coli ATCC 25922, E.coli ATCC 8099, E.coli ATCC 35218, E.coli CMCC(B)44113, E.coli HK 70115, Enterobacter aerogenes CMCC (B) 45103, Enterobacter cloacae CMCC(B)45301, Klebsiella pneumoniae CMCC (B) 46117, Klebsiella pneumoniae wide clinical examination-57, Enterobacter sakazakii ATCC 29544, Enterobacter sakazakii isolate (No.10Ta), Enterobacter sakazakii isolate (No.10Ta), Enter
  • E. coli O157:H7 On sorbitol-Macconkey medium (SMAC), E. coli O157:H7 is colorless and indistinguishable from E. sakazakii, Proteus vulgaris, Proteus mirabilis, and Pseudomonas aeruginosa, while non-O157 E.coli is indistinguishable from Enterobacter aerogenes, Enterobacter cloacae, Klebsiella pneumoniae, Salmonella typhimurium, Salmonella enteritidis, and Salmonella typhi, and it may cause some non- O157 E.coli strain red faded. It can be seen that for more E.coli O157, non-O157 E.coli and non-E. coli, the existing commercially available media TBX agar, CHROMagar E.coli, CHROMagar O157 and sorbitol-Macconkey media ( SMAC) can not give good detection specificity.
  • SMAC sorbitol-Maccon

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Abstract

公开了一种用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基。所述的显色培养基含有两种能产生不同颜色信号的显色底物和一种不显色但能产生非蓝色荧光信号的荧光底物；显色底物和荧光底物中除了含有β-葡萄糖醛酸苷酶底物和β-半乳糖苷酶底物外，还至少含有β-葡萄糖苷酶底物、β-N-乙酰氨基葡萄糖苷酶底物、纤维二糖苷酶底物这三种所产颜色或荧光信号相同或相近底物中的一种底物。提供的显色培养基对于更多的E.coli O157、non-O157 E.coli以及非大肠杆菌，均能给出良好的检测特异性。

Description

一种用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基 技术领域
本发明涉及微生物检测技术领域，具体涉及一种用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基。
背景技术
大肠杆菌(Escherichia coli，E.coli)被普遍视为与食品、水、饲料、临床和环境样本卫生质量、清洁程度或致病风险密切相关的粪便污染指示菌。其中，E.coli O157可导致人类无症状的排泌、非血性腹泻、出血性结肠炎、溶血性尿毒综合症甚至死亡等不同后果的病症，是肠道出血性(或产志贺毒素)E.coli(EHEC或STEC)中最主要的致病血清型，已成为引起世界范围内广泛关注的一种重要致病菌。然而，除了E.coli O157之外，non-O157 E.coli包括non-O157 EHEC(或STEC)以及其它如肠道致病性E.coli(EPEC)、产肠毒素E.coli(ETEC)、肠道侵袭性E.coli(EIEC)、肠道集聚性E.coli(EAEC)以及其它仍需进一步流行病学研究的致病型如弥散粘附性E.coli(DAEC)等也能导致人类腹泻等病症爆发，也是需要引起重视的(Gomes et al.,Brazilian Journal of Microbiology,2016,47:3-30；Mead et al.,Lancet,1998,352:1207-1212；Pennington et al.,Lancet,2010,376:1428-1435；Yang et al.,Archives of Microbiology,2017,199:811-825.)。因此，检测E.coli O157与non-O157 E.coli，对于保障食品安全等公共卫生安全是必不可少的。
尽管目前已有多种关于检测E.coli的显色培养基被发展出来，但是它们仍然难以满足这样的“一款多用”目的，即在相对较高特异地检测E.coli O157与non-O157 E.coli上均可应用。例如，TBX琼脂(Tryptone Bile X-glucuronide agar)已被国际标准(ISO 16649-3:2015和ISO/TS 13136:2012)推荐应用于计数具有β-葡萄糖醛酸苷酶(β-glucuronidase)活性的E.coli与分离包括无β-葡萄糖醛酸苷酶活性E.coli O157在内等STEC，但它只含有X-glucuronide一种显色底物，只能使具有β-葡萄糖醛酸苷酶活性的non-O157 E.coli显蓝绿色，而不具有β-葡萄糖醛酸苷酶活性的E.coli O157与很多种杂菌均显无色，导致E.coli O157难以被辨别，使检测、鉴定工作量大幅度增加。Chromocult(Merck，德国)、Coli ID(bioMérieux，法国)、CHROMagar ECC(Chromagar，法国)、HiCrome ECC(Union Carbide，美国)等这些显色培养基添加了两种不同酶(β-半乳糖苷酶与β-葡萄糖醛酸苷酶)显色底物，可用于检测大肠菌群和具有β-葡萄糖醛酸苷酶活性的non-O157 E.coli；但在它们上面，E.coli O157与其它大肠菌群均显相同的颜色而难以被区分(Manafi M.,International Journal of Food Microbiology,2000,60:205-218.)。而E.coli O157显色培养基又只适用于特异性检测E.coli O157，对non-O157 E.coli的检测效果比较差，例如CHROMagar O157(Chromagar，法国)、Rainbow Agar  O157(Biolog Inc.，美国)和Rapid E.coli O157:H7(Bio-Rad)等等(Verhaegen et al.,International Journal of Environmental Research and Public Health,2015,12:6965-6978；Fan et al.,International Journal of Food Microbiology,2018,266:295-300.)。
发明内容
本发明的目的在于提供一种用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基。
本发明通过以下技术方案来实现：
一种用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基，不含有山梨醇、也不含有中性红，但含有两种能产生无扩散性的不同颜色信号的显色底物和一种不显色但能产生无扩散性的非蓝色荧光信号的荧光底物，所述的显色底物和荧光底物中除了含有β-葡萄糖醛酸苷酶底物和β-半乳糖苷酶底物外，还至少含有β-葡萄糖苷酶底物、β-N-乙酰氨基葡萄糖苷酶底物、纤维二糖苷酶底物这三种所产颜色或荧光信号相同或相近底物中的一种底物。
优选地，所述的显色底物为基于3-吲哚酚衍生物的吲哚酚基显色底物，所述的3-吲哚酚衍生物的结构如下式(I)所示：
Figure PCTCN2019115520-appb-000001
上式中的R可以为单取代基团，也可为多取代基团。
优选地，所述的3-吲哚酚衍生物为5-溴-4-氯-3-吲哚酚、5-溴-6-氯-3-吲哚酚或6-氯-3-吲哚酚等。
优选地，所述的基于3-吲哚酚衍生物的吲哚酚基显色底物为5-溴-4-氯-3-吲哚基-β-D-葡萄糖苷、5-溴-6-氯-3-吲哚基-β-D-半乳糖苷、5-溴-4-氯-3-吲哚基-β-D-葡萄糖醛酸苷环己胺盐或5-溴-6-氯-3-吲哚基-β-D-葡萄糖醛酸苷环己胺盐。
优选地，所述的荧光底物为基于2-(苯并噻唑-2'-基)苯酚衍生物的的荧光底物，所述的2-(苯并噻唑-2'-基)苯酚衍生物的结构如下式(2)所示：
Figure PCTCN2019115520-appb-000002
上式中的R1和R2代表卤原子或烷氧基等基团。
优选地，所述的2-(苯并噻唑-2'-基)苯酚衍生物为2-(苯并噻唑-2'-基)-4-溴苯酚等。
优选地，所述的基于2-(苯并噻唑-2'-基)苯酚衍生物的荧光底物为2-(苯并噻唑-2'-基)-4-溴苯基-β-D-葡萄糖醛酸苷、2-(苯并噻唑-2'-基)-4-溴苯基-β-D-半乳糖苷或2-(苯并噻唑-2'-基)-4-溴苯基-β-D-葡萄糖苷。
优选地，所述的荧光物2-(苯并噻唑-2'-基)苯酚衍生物是根据文献[Otsubo et al.Bioorganic & Medicinal Chemistry Letters,2013,23(7):2245-2249]报道的方法合成。而基于2-(苯并噻唑-2'-基)苯酚衍生物的荧光底物的合成方法包括两步反应，具体步骤为：
S1：以2-(苯并噻唑-2'-基)苯酚衍生物为糖基受体，相应的乙酰卤代糖为糖基供体，氧化银或碳酸铯为催化剂，乙腈为溶剂，进行糖基化反应，得到含保护基团乙酰基的目标糖苷底物；
S2：将得到的含保护基团乙酰基的目标糖苷底物置于含氢氧化钾或氢氧化锂的甲醇和四氢呋喃混合体系中进行脱保护基反应，得到目标荧光底物。
上述合成方法中，对于相应的β-葡萄糖醛酸苷合成时，优选氢氧化锂作为碱；而对于其它糖苷合成时，优选氢氧化钾作为碱。
优选地，所述的催化剂(氧化银或碳酸铯)与糖基供体(乙酰卤代糖)在投料摩尔量上均多于糖基受体(2-(苯并噻唑-2'-基)苯酚衍生物)，优选，所述的催化剂：糖基供体：糖基受体的投料摩尔比为1.5:1.5:1.0。
优选地，本发明用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基，还含有十二烷基硫酸钠(SDS)和(或)胆盐(3号胆盐、脱氧胆酸钠等)、细菌存活和生长所需的成分(如蛋白胨、酵母提取粉、氯化钠等)、琼脂、异丙基-β-D-硫代吡喃半乳糖苷(IPTG)以及可能含有的少量乳糖、水杨苷或纤维二糖、吐温80或曲拉通X-100。需要说明的是，通过适当地控制显色培养基中SDS和胆盐的用量等措施，可更好地抑制背景菌的生长，从而提高对O157/non-O157 E.coli的选择性。
优选地，所述的显色培养基中显色底物和荧光底物的浓度均为0.01～0.30g/L。
优选地，所述的显色培养基还含有十二烷基硫酸钠0.05～0.25g/L和/或胆盐0～1.50g/L、蛋白胨1～20g/L、酵母提取粉0～6g/L、氯化钠4～8g/L、琼脂10～20g/L、异丙基-β-D-硫代吡喃半乳糖苷0.01～0.10g/L。
优选地，所述的显色培养基还含有乳糖、水杨苷或纤维二糖0.01～0.15g/L。
优选地，所述的显色培养基还含有吐温80 0～0.60mL/L或曲拉通X-100 0～0.20mL/L。
所述的β-葡萄糖醛酸苷酶底物为羧酸形式或羧酸盐形式。
与现有技术相比，本发明的优势在于：
本发明用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基可以在显色培养基种类选择以及相关处理工作(如显色培养基使用前的平板制备工作等)上投入减少的情况下，仍然可以满足既 能用于检测E.coli O157、也能用于检测非O157 E.coli的多重目的，并且检测的特异性相对较高。
附图说明
图1为荧光底物2-(苯并噻唑-2'-基)-4-溴苯基-β-D-葡萄糖醛酸苷、2-(苯并噻唑-2'-基)-4-溴苯基-β-D-半乳糖苷或2-(苯并噻唑-2'-基)-4-溴苯基-β-D-葡萄糖苷的合成路线。
图2为实施例4的用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基对E.coli O157 ATCC 35150、E.coli ATCC 25922、阪崎肠杆菌ATCC 29544和鼠伤寒沙门氏菌ATCC 14028四种不同致病菌在37℃下有氧培养18h后的检测效果。其中，平板中右上方、左上方、左下方、右下方依次为E.coli O157 ATCC 35150、E.coli ATCC 25922、阪崎肠杆菌ATCC 29544和鼠伤寒沙门氏菌ATCC 14028，A为可见光下，B为365nm紫外光下，C为可见光和手电筒375nm紫外光下。
图3为实施例4、5、6的用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基对E.coli O157、non-O157 E.coli以及非大肠杆菌在37℃下分别有氧培养12h、18h后的检测效果。其中，A：实施例4组，B：实施例5组，C：实施例6组；下标1表示在可见光下观察，下标2表示在365nm紫外灯光下观察，Ⅰ为A至C显色培养基平板中各菌对应的编号。
图4为实施例7的用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基对E.coli O157、non-O157 E.coli以及非大肠杆菌在37℃下分别有氧培养12h、18h后的检测效果。其中，左图表示在可见光下观察，右图表示在365nm紫外灯光下观察；Ⅰ为显色培养基平板中各菌对应的编号。
图5为一些已有培养基对E.coli O157、non-O157 E.coli以及非大肠杆菌在37℃下分别有氧培养12h、18h后的检测效果。其中，D：TBX琼脂组，E：CHROMagar E.coli组，F：CHROMagar O157组；G：山梨醇-麦康凯培养基(SMAC)组，下标1表示在可见光下观察，下标2表示在365nm紫外灯光下观察；Ⅰ为D至G显色培养基平板中各菌对应的编号。
具体实施方式
以下实施例是对本发明的进一步说明，而不是对本发明的限制。
实施例1 2-(苯并噻唑-2'-基)-4-溴苯基-β-D-葡萄糖醛酸苷的合成
(1)糖基化反应
将0.612g(2.00mmol)荧光物2-(苯并噻唑-2'-基)-4-溴苯酚(1)、0.695g(3.00mmol,1.5e.q.)氧化银、1.192g(3.00mmol,1.5e.q.)三乙酰基-α-D-溴代葡萄糖醛酸甲酯(2a)置于含2.0g 4A分子筛的16mL干燥乙腈中，并于氩气氛围、避光和室温条件下搅拌反应32h。然后，将反应混合物用装填有致密的300-400目柱层层析硅胶层的砂芯漏斗抽滤，用二氯甲烷和乙酸乙酯混合液(v/v＝2/1)洗涤，合并洗脱液并减压旋蒸去除溶剂，依次用无水乙醇、乙酸乙酯重结晶，抽滤，干燥后可得约0.348g(产率约28％)含保护基的目标糖苷底物(3a)。
或者，将0.306g(1.00mmol)荧光物2-(苯并噻唑-2'-基)-4-溴苯酚(1)、0.652g(2.00mmol)碳酸铯置于10mL干燥乙腈中搅拌10min后，加入0.596g(1.50mmol)三乙酰基-α-D-溴代葡萄糖醛酸甲酯(2a)，室温下搅拌反应2h。然后，将反应混合物用装填有致密的300-400目柱层层析硅胶层的砂芯漏斗抽滤，用二氯甲烷和乙酸乙酯混合液(v/v＝2/1)洗涤，合并洗脱液并减压旋蒸去除溶剂，依次用无水乙醇、乙酸乙酯重结晶，抽滤，干燥可得到约0.137g(产率约22％)含保护基的目标糖苷底物(结构式见式3a)。
Figure PCTCN2019115520-appb-000003
1H-NMR(500MHz,CDCl 3):δ＝8.65(s,1H),8.10(d,J＝8.0Hz,1H),7.94(d,J＝8.0Hz,1H),7.55(d,J＝8.7Hz,1H),7.51(t,J＝7.6Hz,1H),7.41(t,J＝7.5Hz,1H),7.11(d,J＝8.8Hz,1H),5.52(t,J＝8.0Hz,1H),5.45(t,J＝9.3Hz,1H),5.39(d,J＝7.5Hz,1H),5.35(t,J＝9.3Hz,1H),4.27(d,J＝9.4Hz,1H),3.71(s,3H),2.06(s,3H),2.05(s,3H),1.81(s,3H)ppm. 13C-NMR(126MHz,CDCl 3):δ＝170.12,169.29,166.59,160.77,153.03,151.80,136.10,134.35,132.49,126.37,125.41,125.19,123.14,121.39,116.95,116.55,98.97,72.85,72.08,71.10,68.70,53.11,20.61,20.55,20.52ppm.HRMS(ESI):m/z[M+H] +Calcd.for C 26H 25BrNO 10S:622.0377；found:622.0375。
(2)脱保护基反应
取0.200g(0.32mmol)上述步骤(1)所得含保护基的目标糖苷底物(3a)置于12mL无水的甲醇和四氢呋喃混合溶剂(v/v＝1/2)中，冰水浴冷却下加入8mg(0.32mmol,1.0e.q.)LiOH，于2～8℃下搅拌反应6h，然后加入Amberlite IR-120(H +)氢型阳离子交换树脂中和，过滤，将滤液于40±2℃水浴下减压旋蒸去除溶剂后，加入12mL甲醇和四氢呋喃混合溶剂(v/v＝1/1)，冰水浴冷却后，加入0.96mL LiOH(0.5mol/L)水溶液，于冰水浴下搅拌反应6h，然后加入6mL四氢呋喃，并加入Amberlite IR-120(H +)氢型阳离子交换树脂调pH值至4～5，过滤，将滤液置于30～35℃水浴下减压旋蒸去除有机溶剂，再真空冷冻干燥，然后加入二氯甲烷充分搅拌，过滤，再将固体置于二氯甲烷中充分搅拌，过滤，重复3次后，用乙腈洗涤，干燥后得到约79mg(产率约51％)最终目标产物2-(苯并噻唑-2'-基)-4-溴苯基-β-D-葡萄糖醛酸苷(结构式见式4a)。
Figure PCTCN2019115520-appb-000004
1H-NMR(500MHz,DMSO-d 6):δ＝8.55(s,1H),8.13(d,J＝8.1Hz,1H),8.10(d,J＝8.0Hz,1H),7.73(d,J＝8.4Hz,1H),7.57(t,J＝7.2Hz,1H),7.48(t,J＝6.7Hz,1H),7.38(d,J＝8.5Hz,1H),5.55(d,J＝4.5Hz,1H),5.46(d,J＝7.5Hz,1H),5.39(s,1H),4.03(d,J＝9.5Hz,1H),3.65–3.68(m,1H),3.45(t,J＝9.3Hz,1H),3.42–3.35(m,2H)ppm. 13C-NMR(126MHz,DMSO-d 6):δ＝169.98,160.68,153.49,151.34,135.92,134.39,130.63,126.45,125.38,123.69,122.76,121.87,117.22,113.94,99.77,76.02,75.45,72.89,71.25ppm.HRMS(ESI):m/z[M+H] +Calcd.for C 19H 17BrNO 7S:481.9904；found:481.9906。
上述实施例的目标荧光底物2-(苯并噻唑-2'-基)-4-溴苯基-β-D-葡萄糖醛酸苷(4a)的合成路线见图1。
实施例2 2-(苯并噻唑-2'-基)-4-溴苯基-β-D-半乳糖苷的合成
2-(苯并噻唑-2'-基)-4-溴苯基-β-D-半乳糖苷(4b)的合成路线见图1，具体步骤如下：
(1)糖基化反应
将0.306g(1.00mmol)荧光物2-(苯并噻唑-2′-基)-4-溴苯酚(1)、0.652g(2.00mmol)碳酸铯置于10mL干燥乙腈中搅拌10min后，加入0.617g(1.50mmol)四乙酰基-α-D-溴代半乳糖(2b)，室温下搅拌反应2h。过滤，二氯甲烷洗涤滤渣，合并滤液，将滤液置于40～45℃水浴下加压旋蒸去除溶剂，用无水乙醇重结晶，干燥可得到约0.600g(产率约94％)含保护基的目标糖苷底物(结构式见式3b)。
Figure PCTCN2019115520-appb-000005
1H-NMR(500MHz,CDCl 3):δ＝8.69(d,J＝2.5Hz,1H),8.13(d,J＝8.0Hz,1H),7.94(d,J＝8.0Hz,1H),7.55(dd,J＝8.6,2.3Hz,1H),7.52(d,J＝7.5Hz,1H),7.42(t,J＝7.5Hz,1H),7.12(d,J＝8.8Hz,1H),5.72(dd,J＝10.0,8.5Hz,1H),5.49(d,J＝3.0Hz,1H),5.30(d,J＝8.0Hz,1H),5.14(dd,J＝ 10.2,3.2Hz,1H),4.28–4.21(m,1H),4.20–4.13(m,2H),2.22(s,3H),2.08(s,3H),2.02(s,3H),1.81(s,3H)ppm. 13C NMR(126MHz,CDCl 3):δ＝170.43,170.33,170.23,169.43,161.11,153.31,151.74,136.19,134.41,132.64,126.56,125.56,125.10,123.22,121.48,116.88,116.45,99.38,71.66,71.41,68.69,66.92,61.51,20.86,20.81,20.70ppm。
(2)脱保护基反应
将0.500g(0.78mmol)上述步骤(1)所得含保护基的目标糖苷底物(3b)置于15mL无水的甲醇/四氢呋喃混合溶剂(v/v＝1/1)中，加入11.8μL氢氧化钾甲醇溶液(1.0mol/L；0.15e.q.)，室温下搅拌反应1h。然后于40～45℃水浴中旋蒸去除溶剂，然后加入二氯甲烷搅拌，抽滤，用二氯甲烷洗涤多次，接着用冷水洗涤2次，最后用少量乙腈洗涤2次，干燥后可得到约0.364g(产率约99％)最终目标产物2-(苯并噻唑-2'-基)-4-溴苯基-β-D-半乳糖苷(结构式见式4b)。
Figure PCTCN2019115520-appb-000006
1H-NMR(500MHz,DMSO-d 6):δ＝8.55(s,1H),8.12(d,J＝8.0Hz,1H),8.10(d,J＝8.5Hz,1H),7.70(dd,J＝9.0,2.0Hz,1H),7.56(t,J＝7.5Hz,1H),7.47(t,J＝7.3Hz,1H),7.36(d,J＝9.0Hz,1H),5.23(s,1H),5.22(s,1H),5.01(d,J＝5.5Hz,1H),4.70(t,J＝5.2Hz,1H),4.66(d,J＝4.0Hz,1H),3.94(dd,J＝14.1,8.1Hz,1H),3.76(s,1H),3.71(t,J＝5.5Hz,1H),3.61–3.47(m,3H)ppm. 13C-NMR(126MHz,DMSO):δ＝160.85,153.98,151.34,135.98,134.30,130.46,126.38,125.28,123.54,122.69,121.81,117.42,113.52,100.96,75.82,73.51,70.10,68.02,60.30ppm。
实施例3 2-(苯并噻唑-2'-基)-4-溴苯基-β-D-葡萄糖苷的合成
2-(苯并噻唑-2'-基)-4-溴苯基-β-D-葡萄糖苷(4c)的合成路线见图1，具体步骤如下：
(1)糖基化反应
将0.306g(1.00mmol)荧光物2-(苯并噻唑-2′-基)-4-溴苯酚(1)、0.652g(2.00mmol)碳酸铯置于10mL干燥乙腈中搅拌10min后，加入0.617g(1.50mmol)四乙酰基-α-D-溴代葡萄糖(2c)，室温下搅拌反应2h。过滤，二氯甲烷洗涤滤渣，合并滤液，将滤液置于40～45℃水浴下加压旋蒸去除溶剂，用无水乙醇重结晶，干燥可得到约0.523g(产率约82％)含保护基的目标糖苷底物(结构式见式3c)。
Figure PCTCN2019115520-appb-000007
1H-NMR(500MHz,DMSO-d 6):δ＝8.52(d,J＝2.5Hz,1H),8.11(d,J＝8.0Hz,1H),8.05(d,J＝8.0Hz,1H),7.81(dd,J＝9.0,2.5Hz,1H),7.60–7.56(m,1H),7.51–7.48(m,1H),7.35(d,J＝9.0Hz,1H),5.99(d,J＝8.0Hz,1H),5.43(t,J＝9.5Hz,1H),5.36(dd,J＝9.5,8.0Hz,1H),5.10(t,J＝9.5Hz,1H),4.35(ddd,J＝10.0,5.5,2.5Hz,1H),4.21(dd,J＝12.5,5.5Hz,1H),4.11(dd,J＝12.4,2.3Hz,1H),2.03,1.99,1.98,1.88(4×s,4×3H)ppm. 13C-NMR(126MHz,DMSO-d 6):δ＝170.42,170.14,169.79,169.54,160.44,153.20,151.80,135.97,135.05,131.51,127.13,126.20,124.41,123.43,122.02,118.17,115.43,97.18,72.87,71.64,71.20,68.11,61.94,20.93,20.88,20.85,20.76ppm。
(2)脱保护基反应
将0.300g(0.47mmol)上述步骤(1)所得含保护基的目标糖苷底物(3c)置于9mL无水的甲醇/四氢呋喃混合溶剂(v/v＝1/1)中，加入7.0μL氢氧化钾甲醇溶液(1.0mol/L；0.15e.q.)，室温下搅拌反应1h。然后于40～45℃水浴中旋蒸去除溶剂，然后加入二氯甲烷搅拌，抽滤，用二氯甲烷洗涤多次，接着用冷水洗涤2次，最后用少量乙腈洗涤2次，干燥后可得到约0.219g(产率约99％)最终目标产物2-(苯并噻唑-2'-基)-4-溴苯基-β-D-葡萄糖苷(结构式见式4c)。
Figure PCTCN2019115520-appb-000008
1H-NMR(500MHz,DMSO-d 6):δ＝8.55(d,J＝2.5Hz,1H),8.11(dd,J＝12.3,8.0Hz,2H),7.70(dd,J＝9.0,3.0Hz,1H),7.58–7.54(m,1H),7.49–7.45(m,1H),7.36(d,J＝9.5Hz,1H),5.42(d,J＝5.5Hz,1H),5.29(d,J 1,2＝7.5Hz,1H),5.26(s,1H),5.13(d,J＝3.5Hz,1H),4.62(t,J＝5.3Hz,1H),3.71(dd,J＝10.5,5.5Hz,1H),3.62(dd,J＝13.7,8.2Hz,1H),3.53–3.45(m,2H),3.37(d,J＝9.5Hz,1H),3.26(dt,J＝13.2,6.7Hz,1H)ppm. 13C-NMR(126MHz,DMSO-d 6):δ＝161.31,154.34,151.81,136.44,134.79,130.95,126.86,125.76,124.04,123.17,122.32,117.88,114.10,100.71,77.72,77.31,73.63,69.98,61.01ppm。
实施例4
一种用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基，其每1000mL含有10g蛋白胨、3g酵母提取粉、5g氯化钠、0.1g SDS、0.1g脱氧胆酸钠、0.5mL吐温80、0.15g荧光底物2-(苯并噻唑-2'-基)-4-溴苯基-β-D-葡萄糖醛酸苷(于365～375nm紫外光下能产黄绿色荧光)、0.05g显色底物5-溴-4-氯-3-吲哚基-β-D-葡萄糖苷(能产蓝至蓝绿色)、0.05g显色底物5-溴-6-氯-3-吲哚基-β-D-半乳糖苷(能产紫红色)、0.05g IPTG、15g琼脂。(注：该显色培养基可以于121℃下高温蒸汽灭菌15min)。
实施例5
一种用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基，其每1000mL含有10g蛋白胨、3g酵母提取粉、5g氯化钠、0.1g SDS、0.1g脱氧胆酸钠、0.045g荧光底物2-(苯并噻唑-2'-基)-4-溴苯基-β-D-葡萄糖苷(于365～375nm紫外光下能产黄绿色荧光)、0.20g显色底物5-溴-4-氯-3-吲哚基-β-D-葡萄糖醛酸苷环己胺盐(能产蓝至蓝绿色)、0.05g显色底物5-溴-6-氯-3-吲哚基-β-D-半乳糖苷(能产紫红色)、0.05g IPTG、0.01g水杨苷、0.01g纤维二糖、15g琼脂。(注：该显色培养基也可以于121℃下高温蒸汽灭菌15min)。
实施例6
一种用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基，其每1000mL含有10g蛋白胨、3g酵母提取粉、5g氯化钠、0.1g SDS、0.1g脱氧胆酸钠、0.5mL吐温80、0.045g荧光底物2-(苯并噻唑-2′-基)-4-溴苯基-β-D-半乳糖苷(于365～375nm紫外光下能产黄绿色荧光)、0.20g显色底物5-溴-6-氯-3-吲哚基-β-D-葡萄糖醛酸苷环己胺盐(能产紫红色)、0.055g显色底物5-溴-4-氯-3-吲哚基-β-D-葡萄糖苷(能产蓝至蓝绿色)、0.025g IPTG、0.025g乳糖、15g琼脂。(注：该显色培养基也可以于121℃下高温蒸汽灭菌15min)。
实施例7
一种用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基，其每1000mL含有10g蛋白胨、3g酵母提取粉、5g氯化钠、0.20g SDS、0.20g脱氧胆酸钠、0.10mL曲拉通X-100、0.15g荧光底物2-(苯并噻唑-2′-基)-4-溴苯基-β-D-葡萄糖醛酸苷(于365～375nm紫外光下能产黄绿色荧光)、0.05g显色底物5-溴-4-氯-3-吲哚基-β-D-葡萄糖苷(能产蓝至蓝绿色)、0.05g显色底物5-溴-6-氯-3-吲哚基-β-D-半乳糖苷(能产紫红色)、0.05g IPTG、琼脂15g。(注：该显色培养基可以于121℃下高温蒸汽灭菌15min)。
实施例8
一种用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基，其每1000mL含有1g蛋白胨、 6g酵母提取粉、4g氯化钠、0.05g SDS、1.5g 3号胆盐、0.20mL曲拉通X-100、0.05g荧光底物2-(苯并噻唑-2'-基)-4-溴苯基-β-D-葡萄糖苷(于365～375nm紫外光下能产黄绿色荧光)、0.20g显色底物5-溴-4-氯-3-吲哚基-β-D-葡萄糖醛酸苷环己胺盐(能产蓝至蓝绿色)、0.05g显色底物5-溴-6-氯-3-吲哚基-β-D-半乳糖苷(能产紫红色)、0.1g IPTG、0.15g水杨苷、10g琼脂。(注：该显色培养基也可以于121℃下高温蒸汽灭菌15min)。
实施例9
一种用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基，其每1000mL含有20g蛋白胨、8g氯化钠、0.25g SDS、0.60mL吐温80、0.05g荧光底物2-(苯并噻唑-2′-基)-4-溴苯基-β-D-半乳糖苷(于365～375nm紫外光下能产黄绿色荧光)、0.30g显色底物5-溴-6-氯-3-吲哚基-β-D-葡萄糖醛酸苷环己胺盐(能产紫红色)、0.01g显色底物5-溴-4-氯-3-吲哚基-β-D-葡萄糖苷(能产蓝至蓝绿色)、0.05g显色底物5-溴-4-氯-3-吲哚基-β-D-N-乙酰氨基葡萄糖苷(能产蓝至蓝绿色)、0.20g显色底物5-溴-4-氯-3-吲哚基-β-D-纤维二糖苷(能产蓝至蓝绿色)、0.15g乳糖、0.15g纤维二塘、20g琼脂。(注：该显色培养基也可以于121℃下高温蒸汽灭菌15min)。
实施例10
评价本发明实施例4的用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基对E.coli O157:H7 ATCC 35150、E.coli ATCC 25922、阪崎肠杆菌ATCC 29544和鼠伤寒沙门氏菌ATCC 14028这四种不同致病菌的检测效果，使用1μL蓝色接种环蘸取相应的纯菌培养液并划线接种于显色培养基中，于37℃下有氧培养18h。然后分别在可见光、365nm紫外光、可见光和手电筒375nm紫外光下观察显色培养基对四种不同致病菌的区分度，结果见图2。
图2平板中右上方、左上方、左下方、右下方依次为E.coli O157:H7 ATCC 35150、E.coli ATCC 25922、阪崎肠杆菌ATCC 29544和鼠伤寒沙门氏菌ATCC 14028，A为可见光下，B为365nm紫外光下，C为可见光和手电筒375nm紫外光下。如图2所示，实施例4的用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基对以下这四种致病菌能给出良好的区分度：E.coli O157:H7 ATCC 35150(右上方)显粉色且无荧光，E.coli ATCC 25922(左上方)显粉色且有黄绿色荧光，阪崎肠杆菌ATCC 29544(左下方)显蓝绿色且无荧光，鼠伤寒沙门氏菌ATCC 14028(右下方)显无色且无荧光。
实施例11
分别评价本发明实施例4、5、6的用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基对E.coli O157、non-O157 E.coli以及非大肠杆菌的检测效果。接种方法为使用2.5μL移液枪吸取相应的纯菌培养液并通过反向移液法小心点种0.2μL至平板上相应的位置；于37℃下有氧培养12h、18 h。然后分别在可见光、365nm紫外灯光下观察。结果见图3。
图3中，A：实施例4组，B：实施例5组，C：实施例6组；下标1表示在可见光下观察，下标2表示在365nm紫外灯光下观察；Ⅰ为A至C显色培养基平板中各菌对应的编号，实验菌株有：①肠道集聚性E.coli(EAEC)分离株(No.1288-1)，②EAEC分离株(No.3476B3)，③AEC分离株(No.3164A1)，④肠道侵袭性E.coli(EIEC)分离株(No.2968A1)，⑤EIEC分离株(No.2987A2)，⑥肠道致病性E.coli(EPEC)分离株(No.2684-2)，⑦EPEC分离株(No.2627-2)，⑧EPEC分离株(No.545-1)，⑨产志贺毒素(或肠道出血性)E.coli(STEC或EHEC)分离株(No.T1 4259B3)，⑩STEC或EHEC分离株(No.T5 3424A1)，
Figure PCTCN2019115520-appb-000009
STEC或EHEC分离株(No.T7 4274A1)，
Figure PCTCN2019115520-appb-000010
STEC或EHEC分离株(No.T18 3724C1)，
Figure PCTCN2019115520-appb-000011
STEC或EHEC分离株(No.T91 1964-1)，
Figure PCTCN2019115520-appb-000012
STEC或EHEC分离株(No.T111 1122-3)，
Figure PCTCN2019115520-appb-000013
E.coli O157:H7 ATCC 35150，
Figure PCTCN2019115520-appb-000014
E.coli O157:H7NCTC 12900，
Figure PCTCN2019115520-appb-000015
E.coli O157:H7 882364，
Figure PCTCN2019115520-appb-000016
E.coli O157:H7 ATCC 43895，
Figure PCTCN2019115520-appb-000017
E.coli O157:H7 ATCC 43888，
Figure PCTCN2019115520-appb-000018
E.coli CMCC(B)44102，
Figure PCTCN2019115520-appb-000019
E.coli CMCC(B)44103，
Figure PCTCN2019115520-appb-000020
E.coli ATCC 8739，
Figure PCTCN2019115520-appb-000021
E.coli ATCC 25922，
Figure PCTCN2019115520-appb-000022
E.coli ATCC 8099，
Figure PCTCN2019115520-appb-000023
E.coli ATCC 35218，
Figure PCTCN2019115520-appb-000024
E.coli CMCC(B)44113，
Figure PCTCN2019115520-appb-000025
E.coli HK 70115，
Figure PCTCN2019115520-appb-000026
产气肠杆菌CMCC(B)45103，
Figure PCTCN2019115520-appb-000027
阴沟肠杆菌CMCC(B)45301，
Figure PCTCN2019115520-appb-000028
肺炎克雷伯氏菌CMCC(B)46117，
Figure PCTCN2019115520-appb-000029
肺炎克雷伯氏菌广临检-57，
Figure PCTCN2019115520-appb-000030
阪崎肠杆菌ATCC 29544，
Figure PCTCN2019115520-appb-000031
阪崎肠杆菌分离株(No.10Ta)，
Figure PCTCN2019115520-appb-000032
阪崎肠杆菌分离株(No.2864C1)，
Figure PCTCN2019115520-appb-000033
阪崎肠杆菌分离株(No.1541A1-1)，
Figure PCTCN2019115520-appb-000034
鼠伤寒沙门氏菌ATCC 14028，
Figure PCTCN2019115520-appb-000035
鼠伤寒沙门氏菌分离株(No.81-8)，
Figure PCTCN2019115520-appb-000036
鼠伤寒沙门氏菌分离株(No.97-1)，
Figure PCTCN2019115520-appb-000037
肠炎沙门氏菌CMCC(B)50335，
Figure PCTCN2019115520-appb-000038
伤寒沙门氏菌CMCC(B)50071，
Figure PCTCN2019115520-appb-000039
普通变性杆菌CMCC(B)49027，
Figure PCTCN2019115520-appb-000040
奇异变性杆菌CMCC(B)49005，
Figure PCTCN2019115520-appb-000041
铜绿假单胞菌CMCC(B)10104，
Figure PCTCN2019115520-appb-000042
铜绿假单胞菌ATCC 15442，
Figure PCTCN2019115520-appb-000043
铜绿假单胞菌ATCC 27853，
Figure PCTCN2019115520-appb-000044
铜绿假单胞菌ATCC 9027，
Figure PCTCN2019115520-appb-000045
铜绿假单胞菌GIM 1.46，
Figure PCTCN2019115520-appb-000046
蜡样芽胞杆菌ATCC14579。
如图3所示，在实施例4(A)中，E.coli O157:H7全部显粉色且无荧光，non-O157 E.coli全部显粉色且有黄绿色荧光，non-E.coli显绿色(见阪崎肠杆菌)、蓝紫色(见产气肠杆菌、阴沟肠杆菌、1株肺炎克雷伯菌)、金属蓝色(见1株肺炎克雷伯菌)、或不显色(见鼠伤寒沙门氏菌、肠炎沙门氏菌、伤寒沙门氏菌、铜绿假单胞菌、变形杆菌)，革兰氏阳性菌受抑制(见蜡样芽胞杆菌ATCC14579)。
在实施例5(B)中，E.coli O157:H7全部显粉色且无荧光，non-O157 E.coli全部显金属蓝色且无荧光，non-E.coli显粉色或淡紫红色且有黄绿色荧光(见产气肠杆菌、阴沟肠杆菌和肺炎克雷伯菌)、无色且无荧光(见沙门氏菌、变形杆菌和部分铜绿假单胞菌)、或无色且有蓝色荧光(见部分铜绿假单胞菌)，革兰氏阳性菌受抑制(见蜡样芽胞杆菌ATCC 14579)。
在实施例6(C)中，E.coli O157:H7全部显无色且有米黄色荧光，non-O157 E.coli全部显紫红色且无明显荧光，non-E.coli显绿色或蓝绿色(见阪崎肠杆菌、产气肠杆菌、阴沟肠杆菌、肺炎克雷伯菌)、不显色且无荧光或有蓝色荧光(见部分铜绿假单胞菌、变形杆菌)，革兰氏阳性菌受抑制(见蜡样芽胞杆菌ATCC 14579)。
由此可见，对于更多的E.coli O157、non-O157 E.coli以及非大肠杆菌，实施例4、5和6的用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基也都能给出良好的检测特异性。
实施例12
评价本发明实施例7的用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基对E.coli O157、non-O157 E.coli以及非大肠杆菌的检测效果。接种方法为使用2.5μL移液枪吸取相应的纯菌培养液并通过反向移液法小心点种0.2μL至平板上相应的位置；于37℃下有氧培养12h、18h。然后分别在可见光、365nm紫外灯光下观察。结果见图4。
图4中，左图表示在可见光下观察，右图表示在365nm紫外灯光下观察；Ⅰ为显色培养基平板中各菌对应的编号，实验菌株有：①肠道集聚性E.coli(EAEC)分离株(No.1288-1)，②EAEC分离株(No.3476B3)，③AEC分离株(No.3164A1)，④肠道侵袭性E.coli(EIEC)分离株(No.2968A1)，⑤EIEC分离株(No.2987A2)，⑥肠道致病性E.coli(EPEC)分离株(No.2684-2)，⑦EPEC分离株(No.2627-2)，⑧EPEC分离株(No.545-1)，⑨产志贺毒素(或肠道出血性)E.coli(STEC或EHEC)分离株(No.T1 4259B3)，⑩STEC或EHEC分离株(No.T5 3424A1)，
Figure PCTCN2019115520-appb-000047
STEC或EHEC分离株(No.T7 4274A1)，
Figure PCTCN2019115520-appb-000048
STEC或EHEC分离株(No.T18 3724C1)，
Figure PCTCN2019115520-appb-000049
STEC或EHEC分离株(No.T91 1964-1)，
Figure PCTCN2019115520-appb-000050
STEC或EHEC分离株(No.T111 1122-3)，
Figure PCTCN2019115520-appb-000051
E.coli O157:H7 ATCC35150，
Figure PCTCN2019115520-appb-000052
E.coli O157:H7NCTC 12900，
Figure PCTCN2019115520-appb-000053
E.coli O157:H7 882364，
Figure PCTCN2019115520-appb-000054
E.coli O157:H7 ATCC43895，
Figure PCTCN2019115520-appb-000055
E.coli O157:H7 ATCC 43888，
Figure PCTCN2019115520-appb-000056
E.coli CMCC(B)44102，
Figure PCTCN2019115520-appb-000057
E.coli CMCC(B)44103，
Figure PCTCN2019115520-appb-000058
E.coli ATCC 8739，
Figure PCTCN2019115520-appb-000059
E.coli ATCC 25922，
Figure PCTCN2019115520-appb-000060
E.coli ATCC 8099，
Figure PCTCN2019115520-appb-000061
E.coli ATCC 35218，
Figure PCTCN2019115520-appb-000062
E.coli CMCC(B)44113，
Figure PCTCN2019115520-appb-000063
E.coli HK 70115，
Figure PCTCN2019115520-appb-000064
产气肠杆菌CMCC(B)45103，
Figure PCTCN2019115520-appb-000065
阴沟肠杆菌CMCC(B)45301，
Figure PCTCN2019115520-appb-000066
肺炎克雷伯氏菌CMCC(B)46117，
Figure PCTCN2019115520-appb-000067
肺炎克雷伯氏菌广临检-57，
Figure PCTCN2019115520-appb-000068
阪崎肠杆菌ATCC29544，
Figure PCTCN2019115520-appb-000069
阪崎肠杆菌分离株(No.10Ta)，
Figure PCTCN2019115520-appb-000070
阪崎肠杆菌分离株(No.2864C1)，
Figure PCTCN2019115520-appb-000071
阪崎肠杆菌分离株(No.1541A1-1)，
Figure PCTCN2019115520-appb-000072
鼠伤寒沙门氏菌ATCC 14028，
Figure PCTCN2019115520-appb-000073
鼠伤寒沙门氏菌分离株(No.81-8)，
Figure PCTCN2019115520-appb-000074
鼠伤寒沙门氏菌分离株(No.97-1)，
Figure PCTCN2019115520-appb-000075
肠炎沙门氏菌CMCC(B)50335，
Figure PCTCN2019115520-appb-000076
伤寒沙门氏菌CMCC(B)50071，
Figure PCTCN2019115520-appb-000077
普通变性杆菌CMCC(B)49027，
Figure PCTCN2019115520-appb-000078
奇异变性杆菌CMCC(B)49005，
Figure PCTCN2019115520-appb-000079
铜绿假单胞菌CMCC(B)10104，
Figure PCTCN2019115520-appb-000080
铜绿假单胞菌ATCC 15442，
Figure PCTCN2019115520-appb-000081
铜绿假单胞菌ATCC 27853，
Figure PCTCN2019115520-appb-000082
铜绿假单胞菌ATCC 9027，
Figure PCTCN2019115520-appb-000083
铜绿假单胞菌GIM 1.46，
Figure PCTCN2019115520-appb-000084
蜡样芽胞杆菌ATCC 14579。
如图3和图4所示，对比实施例4和实施例7可知，通过适当地控制显色培养基中SDS和胆盐的用量等措施，可更好地抑制背景菌的生长，从而提高对O157/non-O157 E.coli的选择性。
实施例13
分别评价TBX琼脂、CHROMagar E.coli、CHROMagar O157、山梨醇-麦康凯培养基这四种现有的市售培养基对E.coli O157、non-O157 E.coli以及非大肠杆菌的检测效果。接种方法为使用2.5μL移液枪吸取相应的纯菌培养液并通过反向移液法小心点种0.2μL至平板上相应的位置；于37℃下有氧培养12h、18h。然后分别在可见光、365nm紫外灯光下观察。结果见图5。
图5中，D：TBX琼脂组，E：CHROMagar E.coli组，F：CHROMagar O157组；G：山梨醇-麦康凯培养基(SMAC)组，下标1表示在可见光下观察，下标2表示在365nm紫外灯光下观察；Ⅰ为D至G显色培养基平板中各菌对应的编号，实验菌株有：①肠道集聚性E.coli(EAEC)分离株(No.1288-1)，②EAEC分离株(No.3476B3)，③AEC分离株(No.3164A1)，④肠道侵袭性E.coli(EIEC)分离株(No.2968A1)，⑤EIEC分离株(No.2987A2)，⑥肠道致病性E.coli(EPEC)分离株(No.2684-2)，⑦EPEC分离株(No.2627-2)，⑧EPEC分离株(No.545-1)，⑨产志贺毒素(或肠道出血性)E.coli(STEC或EHEC)分离株(No.T1 4259B3)，⑩STEC或EHEC分离株(No.T5 3424A1)，
Figure PCTCN2019115520-appb-000085
STEC或EHEC分离株(No.T7 4274A1)，
Figure PCTCN2019115520-appb-000086
STEC或EHEC分离株(No.T18 3724C1)，
Figure PCTCN2019115520-appb-000087
STEC或EHEC分离株(No.T91 1964-1)，
Figure PCTCN2019115520-appb-000088
STEC或EHEC分离株(No.T111 1122-3)，
Figure PCTCN2019115520-appb-000089
E.coli O157:H7 ATCC 35150，
Figure PCTCN2019115520-appb-000090
E.coli O157:H7NCTC 12900，
Figure PCTCN2019115520-appb-000091
E.coli O157:H7 882364，
Figure PCTCN2019115520-appb-000092
E.coli O157:H7 ATCC 43895，
Figure PCTCN2019115520-appb-000093
E.coli O157:H7 ATCC 43888，
Figure PCTCN2019115520-appb-000094
E.coli CMCC(B)44102，
Figure PCTCN2019115520-appb-000095
E.coli CMCC(B)44103，
Figure PCTCN2019115520-appb-000096
E.coli ATCC 8739，
Figure PCTCN2019115520-appb-000097
E.coli ATCC 25922，
Figure PCTCN2019115520-appb-000098
E.coli ATCC 8099，
Figure PCTCN2019115520-appb-000099
E.coli ATCC 35218，
Figure PCTCN2019115520-appb-000100
E.coli CMCC(B)44113，
Figure PCTCN2019115520-appb-000101
E.coli HK 70115，
Figure PCTCN2019115520-appb-000102
产气肠杆菌CMCC(B)45103，
Figure PCTCN2019115520-appb-000103
阴沟肠杆菌CMCC(B)45301，
Figure PCTCN2019115520-appb-000104
肺炎克雷伯氏菌CMCC(B)46117，
Figure PCTCN2019115520-appb-000105
肺炎克雷伯氏菌广临检-57，
Figure PCTCN2019115520-appb-000106
阪崎肠杆菌ATCC 29544，
Figure PCTCN2019115520-appb-000107
阪崎肠杆菌分离株(No.10Ta)，
Figure PCTCN2019115520-appb-000108
阪崎肠杆菌分离株(No.2864C1)，
Figure PCTCN2019115520-appb-000109
阪崎肠杆菌分离株(No.1541A1-1)，
Figure PCTCN2019115520-appb-000110
鼠伤寒沙门氏菌ATCC 14028，
Figure PCTCN2019115520-appb-000111
鼠伤寒沙门氏菌分离株(No.81-8)，
Figure PCTCN2019115520-appb-000112
鼠伤寒沙门氏菌分离株(No.97-1)，
Figure PCTCN2019115520-appb-000113
肠炎沙门氏菌CMCC(B)50335，
Figure PCTCN2019115520-appb-000114
伤寒沙门氏菌CMCC(B)50071，
Figure PCTCN2019115520-appb-000115
普通变性杆菌CMCC(B)49027，
Figure PCTCN2019115520-appb-000116
奇异变性杆菌CMCC(B)49005，
Figure PCTCN2019115520-appb-000117
铜绿假单胞菌CMCC(B)10104，
Figure PCTCN2019115520-appb-000118
铜绿假单胞菌ATCC 15442，
Figure PCTCN2019115520-appb-000119
铜绿假单胞菌ATCC 27853，
Figure PCTCN2019115520-appb-000120
铜绿假单胞菌ATCC 9027，
Figure PCTCN2019115520-appb-000121
铜绿假单胞菌GIM 1.46，
Figure PCTCN2019115520-appb-000122
蜡样芽胞杆菌ATCC 14579。
如图5所示，在TBX琼脂和CHROMagar E.coli上，除了E.coli ATCC 8739不显色或蓝绿色较淡外，其余non-O157 E.coli均呈现明显的蓝绿色，而E.coli O157:H7与全部non-E.coli菌株均显无色而难以区分。在CHROMagar O157上，E.coli O157:H7显淡紫红色，鼠伤寒沙门氏菌、伤寒沙门 氏菌显粉色，而其余non-O157 E.coli与产气肠杆菌、阴沟肠杆菌、肺炎克雷伯氏菌以及阪崎肠杆菌均显金属蓝或蓝绿色而难以区分。在山梨醇-麦康凯培养基(SMAC)上，E.coli O157:H7与阪崎肠杆菌、普通变形杆菌、奇异变性杆菌以及铜绿假单胞菌都显无色而难以区分，而non-O157 E.coli与产气肠杆菌、阴沟肠杆菌、肺炎克雷伯氏菌、鼠伤寒沙门氏菌、肠炎沙门氏菌以及伤寒沙门氏菌都显红色而难以区分，并且随着培养时间的延长，也会导致部分non-O157 E.coli菌株红色褪去。由此可见，对于更多的E.coli O157、non-O157 E.coli以及非大肠杆菌，现有的市售培养基TBX琼脂、CHROMagar E.coli、CHROMagar O157和山梨醇-麦康凯培养基(SMAC)均不能给出良好的检测特异性。
以上仅是本发明的优选实施方式，应当指出的是，上述优选实施方式不应视为对本发明的限制，本发明的保护范围应当以权利要求所限定的范围为准。对于本技术领域的普通技术人员来说，在不脱离本发明的精神和范围内，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (10)

  1. 一种用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基，其特征在于，所述的显色培养基含有两种能产生无扩散性的不同颜色信号的显色底物和一种不显色但能产生无扩散性的非蓝色荧光信号的荧光底物，所述的显色底物和荧光底物中除了含有β-葡萄糖醛酸苷酶底物和β-半乳糖苷酶底物外，还至少含有β-葡萄糖苷酶底物、β-N-乙酰氨基葡萄糖苷酶底物、纤维二糖苷酶底物这三种所产颜色或荧光信号相同或相近底物中的一种底物。
  2. 根据权利要求1所述的用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基，其特征在于，所述的显色底物为基于3-吲哚酚衍生物的吲哚酚基显色底物，所述的3-吲哚酚衍生物的结构如下式(I)所示：
    Figure PCTCN2019115520-appb-100001
    其中，R为单取代基团或多取代基团。
  3. 根据权利要求2所述的用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基，其特征在于，所述的基于3-吲哚酚衍生物的吲哚酚基显色底物为5-溴-4-氯-3-吲哚基-β-D-葡萄糖苷、5-溴-6-氯-3-吲哚基-β-D-半乳糖苷、5-溴-4-氯-3-吲哚基-β-D-葡萄糖醛酸苷环己胺盐或5-溴-6-氯-3-吲哚基-β-D-葡萄糖醛酸苷环己胺盐。
  4. 根据权利要求1所述的用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基，其特征在于，所述的荧光底物为基于2-(苯并噻唑-2'-基)苯酚衍生物的荧光底物，所述的2-(苯并噻唑-2'-基)苯酚衍生物的结构如下式(2)所示：
    Figure PCTCN2019115520-appb-100002
    其中，R1、R2代表卤原子或烷氧基基团。
  5. 根据权利要求4所述的用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基，其特征在于，所述的基于2-(苯并噻唑-2'-基)苯酚衍生物的的荧光底物为2-(苯并噻唑-2'-基)-4-溴苯基-β-D-葡萄糖醛酸苷、2-(苯并噻唑-2'-基)-4-溴苯基-β-D-半乳糖苷或2-(苯并噻唑-2'-基)-4-溴苯基-β-D-葡萄糖苷。
  6. 根据权利要求1所述的用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基，其特征在于，所述的显色培养基中显色底物和荧光底物的浓度均为0.01～0.30g/L。
  7. 根据权利要求6所述的用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基，其特征在于，所述的显色培养基还含有十二烷基硫酸钠0.05～0.25g/L和/或胆盐0～1.50g/L、蛋白胨1～20g/L、酵母提取粉0～6g/L、氯化钠4～8g/L、琼脂10～20g/L、异丙基-β-D-硫代吡喃半乳糖苷0.01～0.10g/L。
  8. 根据权利要求7所述的用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基，其特征在于，所述的显色培养基还含有乳糖、水杨苷或纤维二糖0.01～0.15g/L。
  9. 根据权利要求7或8所述的用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基，其特征在于，所述的显色培养基还含有吐温80 0～0.60mL/L或者曲拉通X-100 0～0.20mL/L。
  10. 根据权利要求1所述的用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基，其特征在于，所述的β-葡萄糖醛酸苷酶底物为羧酸形式或羧酸盐形式。
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