CN103320491B - Salmonella and the rapid screening method of shigella in enterobacteriaceae - Google Patents
Salmonella and the rapid screening method of shigella in enterobacteriaceae Download PDFInfo
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- CN103320491B CN103320491B CN201210074335.9A CN201210074335A CN103320491B CN 103320491 B CN103320491 B CN 103320491B CN 201210074335 A CN201210074335 A CN 201210074335A CN 103320491 B CN103320491 B CN 103320491B
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- salmonella
- shigella
- enterobacteriaceae
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- rapid screening
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Abstract
The invention discloses Salmonella and the rapid screening method of shigella in a kind of enterobacteriaceae: specimen lined on maconkey agar flat board, hatch in 37 DEG C, then choose a colourless bacterium colony, a part is used for detecting oxidase active；Another part is inoculated on the filter paper bar being soaked with tryptophan, pyroglutamyl 4 methoxyl group naphthylamines and 4 methyl umbelliferone Octanoates, and moistens with a water or buffer；Inoculation bar after Shi Run is placed in moisturizing vessel hatches, and then filter paper bar is placed under Wood lamp detection fluorescence, and fluorescent positive represents the existence of C 8 esterase active；Paper slip drips the solution containing diazo colours and iron chloride, has fluorescence but invariant color represents Salmonella；I.e. unstressed configuration invariant color again represents shigella.The present invention uses mixed substrates method to detect the activity of three kinds of enzymes simultaneously, and experimental technique is simple, and substrate used is less, and cost is relatively low, quickly can filter out Salmonella and shigella from the non-pathogenic bacteria of enterobacteriaceae.
Description
Technical field
The present invention relates to Salmonella and the rapid screening method of shigella in a kind of enterobacteriaceae.
Background technology
Enterobacteriaceae is similar by a group biological character, the gram negative bacilli of amphimicrobian, oxidase negative
Composition, some of which has stronger pathogenic effects to the mankind, can cause include intestinal infection, septicemia and
Urinary tract infection is at interior many infectious disease.Salmonella and Shigella are the most very important
Pathogenic bacterium, can cause diarrhoea and gastroenteritis etc..The experimental technique of screening Salmonella and shigella is relatively at present
For complexity, and needing to use a lot of substrate, the screening to suspicious bacterium colony is the most thorough.And clinical laboratory
To be accredited as, the biochemical test of purpose is the most relatively time-consuming, cost is the highest, is therefore badly in need of a kind of from being grown in
In non-pathogenic bacteria in selectivity intestinal culture medium, rapid screening goes out the test of Salmonella and shigella
Method.
Summary of the invention
It is an object of the invention to: Salmonella and the rapid screening of shigella in a kind of enterobacteriaceae are provided
Method.The present invention uses mixed substrates method tryptophan deaminase, pyroglutamyl aminopeptidase and C-8 esterase
Activity detects simultaneously, goes out Salmonella and will with rapid screening in the non-pathogenic bacteria in culture medium
Hayes bacterium.
Technical scheme: in enterobacteriaceae, the rapid screening method of Salmonella and shigella is:
Specimen is lined on maconkey agar flat board, hatch 18-20 hour in 36 DEG C-38 DEG C, then check flat board
The colourless bacterium colony of upper growth；Choose a colourless bacterium colony, be used for detecting oxidase active by one part；Separately
A part is inoculated in and is soaked with tryptophan, pyroglutamyl-4-methoxyl group naphthylamines and 4-methyl umbelliferone ioxynil octanoic acid
On the filter paper bar of ester, and moisten with a water or buffer；Inoculation bar after Shi Run is positioned in moisturizing vessel
Hatch 2-3 hour in 36 DEG C-38 DEG C, then filter paper bar is placed under Wood lamp detection fluorescence, fluorescent positive
Represent the existence of C-8 esterase active；Paper slip drips the solution containing diazo colours and iron chloride, face
The red expression of complexion changed is not Salmonella and shigella；There is fluorescence but invariant color represents it is Salmonella；I.e. without
Fluorescence invariant color again represents it is shigella.
In preceding method, the suitable dip amount of filter paper is: be soaked with 0.2-0.3ml on the filter paper of 1 square inch
Containing tryptophan, pyroglutamyl-4 methoxyl group naphthylamines and each 40-180 of 4-methyl umbelliferone Octanoate
The 20mM Tris buffer of microgram, pH is 7.5.
Filter paper used by preceding method is Whatman 3 (Whatman Ltd.Maidstone England).
Diazo colours used by preceding method are the peony GBC salt of 90-100 μ g/ml, iron chloride used
Solution concentration is 12-13mg/ml.
Laboratory test results of the present invention see table:
Note: * may be negative, but mostly generally is positive.
The present invention uses mixed substrates method to three kinds of enzymes (tryptophan deaminase, pyroglutamyl aminopeptidase and C-8 ester
Enzyme) activity detect simultaneously.
The suitable substrates of phenylalanine deaminase reaction is phenylalanine；The suitable substrates of tryptophan deaminase reaction
It it is tryptophan；The developer of the two reaction is iron chloride.
The suitable substrates of pyroglutamyl aminopeptidase reaction includes pyroglutamyl-α/β naphthylamines
(pyroglutamyl-alpha/beta naphthylamide), pyroglutamyl-4-methoxyl group naphthylamines and Jiao Gu
Aminoacyl paranitroanilinum (pyroglutamyl paranitroanalide).
The suitable substrates of C-8 Esterase reaction includes that fluorescein Octanoate, 4-methyl umbelliferone ioxynil are pungent
Acid esters, neighbour/p-nitrophenyl Octanoate, indoxyl Octanoate, naphthalene Octanoate and
4-methoxy naphthalene Octanoate.
Compared with prior art: the present invention uses mixed substrates method to detect the activity of three kinds of enzymes simultaneously,
Experimental technique is simple, and substrate used is less, and cost is relatively low, can be quickly from the non-pathogenic bacteria of enterobacteriaceae
In filter out Salmonella and shigella.
Detailed description of the invention
Embodiments of the invention 1: specimen lined on maconkey agar flat board, hatch 18 hours in 37 DEG C,
Then the colourless bacterium colony of growth on flat board is checked；Choose a colourless bacterium colony, be used for detecting oxygen by one part
Change enzymatic activity；Another part is inoculated in and is soaked with tryptophan, pyroglutamyl-4-methoxyl group naphthylamines and 4-methylumbelliferyl
On the filter paper bar of ketone Octanoate, and moisten with a water or buffer.The suitable dip amount of filter paper is:
The filter paper of 1 square inch is soaked with 0.2ml and contains tryptophan, pyroglutamyl-4 methoxyl group naphthylamines and 4-first
The 20mM Tris buffer (pH 7.5) of each 100 micrograms of base umbelliferone Octanoate, filter paper used
It is Whatman 3 (Whatman Ltd.Maidstone England).Inoculation bar after Shi Run is positioned over
Moisturizing vessel are hatched 2 hours in 37 DEG C, then filter paper bar is placed under Wood lamp detection fluorescence, fluorescence sun
Property represents the existence of C-8 esterase active；Paper slip drips containing the diazo colours (peony of 90 μ g/ml
GBC salt) and the solution of 12mg/ml iron chloride, color reddens → nonsalmonella and shigella；There is fluorescence
But invariant color → Salmonella；I.e. unstressed configuration invariant color → shigella again.
Embodiments of the invention 2: specimen lined on maconkey agar flat board, hatch 20 hours in 36 DEG C,
Then the colourless bacterium colony of growth on flat board is checked；Choose a colourless bacterium colony, be used for detecting oxygen by one part
Change enzymatic activity；Another part is inoculated in and is soaked with tryptophan, pyroglutamyl-4-methoxyl group naphthylamines and 4-methylumbelliferyl
On the filter paper bar of ketone Octanoate, and moisten with a water or buffer.The suitable dip amount of filter paper is:
The filter paper of 1 square inch is soaked with 0.3ml and contains tryptophan, pyroglutamyl-4 methoxyl group naphthylamines and 4-first
The 20mM Tris buffer (pH 7.5) of each 160 micrograms of base umbelliferone Octanoate, filter paper used
It is Whatman 3.Inoculation bar after Shi Run is positioned in moisturizing vessel hatches 3 hours in 36 DEG C, then
Filter paper bar is placed under Wood lamp detection fluorescence, and fluorescent positive represents the existence of C-8 esterase active；At paper slip
Above drip containing diazo colours (the peony GBC salt of 100 μ g/ml) and the solution of 13mg/ml iron chloride,
Color reddens → nonsalmonella and shigella；There is fluorescence but invariant color → Salmonella；I.e. unstressed configuration is again
Invariant color → shigella.
Claims (4)
1. Salmonella and the rapid screening side of shigella in the enterobacteriaceae of a non-diseases diagnostic purpose
Method, it is characterised in that: specimen is lined on maconkey agar flat board, hatch 18-20 in 36 DEG C-38 DEG C
Hour, then check the colourless bacterium colony of growth on flat board；Choose a colourless bacterium colony, one part is used
In detection oxidase active；Another part be inoculated in be soaked with tryptophan, pyroglutamyl-4-methoxyl group naphthylamines and
On the filter paper bar of 4-methyl umbelliferone Octanoate, and moisten with a water or buffer；After Shi Run
Inoculation bar be positioned in moisturizing vessel and hatch 2-3 hour in 36 DEG C-38 DEG C, then filter paper bar is placed in
Detecting fluorescence under Wood lamp, fluorescent positive represents the existence of C-8 esterase active；Paper slip drips and contains
Having the solution of diazo colours and iron chloride, color reddens represents it is not Salmonella and shigella；Have glimmering
Light but invariant color represents it is Salmonella；I.e. unstressed configuration invariant color again represents it is shigella.
2. congratulate according to Salmonella in the enterobacteriaceae of the non-diseases diagnostic purpose described in claim 1 and will
The rapid screening method of Salmonella, it is characterised in that: the dip amount of filter paper is: at the filter paper of 1 square inch
On be soaked with 0.2-0.3ml and contain tryptophan, pyroglutamyl-4 methoxyl group naphthylamines and 4-methyl umbelliferone iodobenzene
The 20mM Tris buffer of nitrile caprylate each 40-180 microgram, pH is 7.5.
3. congratulate according to Salmonella in the enterobacteriaceae of the non-diseases diagnostic purpose described in claim 1 and will
The rapid screening method of Salmonella, it is characterised in that: filter paper used is Whatman 3.
4. congratulate according to Salmonella in the enterobacteriaceae of the non-diseases diagnostic purpose described in claim 1 and will
The rapid screening method of Salmonella, it is characterised in that: diazo colours used are the dark red of 90-100 μ g/ml
Color GBC salt, ferric chloride solution concentration used is 12-13mg/ml.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210074335.9A CN103320491B (en) | 2012-03-20 | 2012-03-20 | Salmonella and the rapid screening method of shigella in enterobacteriaceae |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201210074335.9A CN103320491B (en) | 2012-03-20 | 2012-03-20 | Salmonella and the rapid screening method of shigella in enterobacteriaceae |
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| Publication Number | Publication Date |
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| CN103320491A CN103320491A (en) | 2013-09-25 |
| CN103320491B true CN103320491B (en) | 2016-08-17 |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101395278A (en) * | 2006-02-28 | 2009-03-25 | 生物梅里埃公司 | Method for identifying at least two groups of microorganisms |
| CN101974641A (en) * | 2010-11-19 | 2011-02-16 | 南京市产品质量监督检验院 | Multiple fluorescence quantitative PCR method for simultaneous and fast detecting three types of pathogenic bacteria in food |
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2012
- 2012-03-20 CN CN201210074335.9A patent/CN103320491B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101395278A (en) * | 2006-02-28 | 2009-03-25 | 生物梅里埃公司 | Method for identifying at least two groups of microorganisms |
| CN101974641A (en) * | 2010-11-19 | 2011-02-16 | 南京市产品质量监督检验院 | Multiple fluorescence quantitative PCR method for simultaneous and fast detecting three types of pathogenic bacteria in food |
Non-Patent Citations (1)
| Title |
|---|
| Evaluation of methods for isolation of Salmonella species using modified semisolid Rappaport-Vassiliadis medium and Salmonella-Shigella agar;Ruiz Gomez J等;《Eur J Clin Microbiol Infect Dis.》;19981130;第17卷(第11期);791-793 * |
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