CN101824462B - Quantitative detection method of campylobacter in food - Google Patents
Quantitative detection method of campylobacter in food Download PDFInfo
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- CN101824462B CN101824462B CN2010101805160A CN201010180516A CN101824462B CN 101824462 B CN101824462 B CN 101824462B CN 2010101805160 A CN2010101805160 A CN 2010101805160A CN 201010180516 A CN201010180516 A CN 201010180516A CN 101824462 B CN101824462 B CN 101824462B
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- 241000589876 Campylobacter Species 0.000 title abstract description 4
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- 241000894006 Bacteria Species 0.000 claims abstract description 39
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention relates to the bacterium separation technology, in particular to a direct quantitative counting method of campylobacter in food. The method comprises the following steps: culturing samples on a solid CCDA culture medium containing growth promoters and six kinds of antibiotics such as polymyxin B, trimethoprim, rifampicin, actidione, cefoperazone and fungizone B; then, directly carrying out campylobacter counting and separation. Compared with a bacterium culture, separation and certification method of a traditional method, the invention has the advantages of simplicity, convenience, specificity and accuracy.
Description
Technical field
The present invention relates to bacterium separation technology, particularly to the direct quantitative method of counting of the crooked bacterium (Campylobacter) in the food.
Background technology
Crooked bacterium be gastro-enteritis in the global range main diseases because of, be main foodborne bacterial pathogens, can cause that sporadic and endemical gastro-enteritis breaks out; Particularly at the individuality of defective, like cancer patient, AIDS patient, diabetics, child and old man etc., children's below 5 years old sickness rate is the highest; In addition; (Guillain-Barre syndrome is severe complications after the particular serotype Cj infection GBS) to Guillain Barre syndrome, can cause paralysis of respiratory muscle and death; Therefore, crooked bacterium also is the emphasis that global public health is paid close attention to.In the last few years; The campylobacter infection rate is generally in rising trend all over the world; Become modal, acute bacterial infectious intestinal disease; According to the monitoring of American National food net, crooked bacterium is the highest pathogenic bacteria of sickness rate, and its case load has surpassed listeriosis, salmonellosis and shigellosis.
Crooked bacterium epidemiological study shows, handles or consumer food, and especially poultry is the most important approach that the people infects campylobacteriosis, is 500CFU (bacterium colony) to people's minimum infective dose.Crooked bacterium all is the projects that detect in public health, food safety, animal and veterinary and inspection and quarantining for import/export as the important indicator that pathogenic bacterium detect, and important society, economic implications are arranged.Crooked bacterium is facultative microaerobe, and culture condition is harsh, and conventional cultivation needs abundant nutritional condition and stable suitable gas condition, and domestic and international research is started late, and its conventional cultural method awaits further perfect.Traditional crooked bacterium detects and separates main dependence biochemical test and serum agglutination test.Because that crooked bacterium has is high to the substratum nutritional requirement, culture condition is harsh; Oxygen is all compared responsive; Can only in low-oxygen environment, grow, culture cycle adopts different testing procedures, method than characteristics such as length; Reporting the result differs greatly, and especially can't realize the direct census (detection by quantitative) to crooked bacterium in the food.Make a definite diagnosis assay and need 10～20 days at least, defective such as that the method for inspection exists is loaded down with trivial details, waste time and energy, specificity is low.Therefore, quick, accurate, easy method of detecting bacterium will be the direction of development.The separation and the quantitative analysis of setting up crooked bacterium in a kind of rapid detection food have great importance.
Summary of the invention
The object of the invention is to go out the detection method whether sample is bent fungi pollution and amount of contamination for people provide a kind of ability sharp separation and quantitative analysis.
The quantitative detecting method of the said crooked bacterium of the present invention is that sample is directly carried out separating and direct census of crooked bacterium after containing the solid CCDA culture medium culturing of microbiotic and growth stimulant.
Microbiotic comprises PXB, trimethoprim, Rifampin, cycloheximide, amphotericin B and 6 kinds of microbiotic of cefoperazone in the above CCDA solid medium.
The content of said microbiotic in substratum is:
PXB: 6.7 * 10
-3G/L;
Trimethoprim: 0.1g/L;
Rifampin: 0.1g/L;
Cycloheximide: 1g/L;
Cefoperazone: 0.128g/L;
Amphotericin B: 0.04g/L.
Growth stimulant comprises Sodium.alpha.-ketopropionate, Sodium Pyrosulfite and ferrous sulfate in the above CCDA solid medium.The ultimate density of growth stimulant is 0.5g/L in the substratum.
The present invention is easy, special, accurate than microbial culture, separation and the authentication method of classical way.
Description of drawings
Fig. 1 is the design sketch that contains crooked bacterium in the solid CCDA substratum direct separation food of PXB, trimethoprim, Rifampin, cycloheximide, cefoperazone and 6 kinds of microbiotic of amphotericin B and growth stimulant.
Fig. 2 is the design sketch of crooked bacterium in the common solid CCDA substratum direct separation food.
Fig. 3 is the design sketch that contains crooked bacterium in 4 kinds of antibiotic solid CCDA substratum direct separation food such as PXB, trimethoprim, Rifampin and cycloheximide.
Fig. 4 is the design sketch that contains crooked bacterium in PXB, trimethoprim, Rifampin, cycloheximide, cefoperazone and 6 kinds of antibiotic solid CCDA substratum direct separation food of amphotericin B.
Fig. 5 is the design sketch that contains crooked bacterium in the solid CCDA substratum direct separation food of PXB, trimethoprim, Rifampin, cycloheximide, cefoperazone and 6 kinds of microbiotic of amphotericin B, growth stimulant and 10% chicken serum.
Fig. 6 is the design sketch that contains crooked bacterium in the solid CCDA substratum direct separation food of PXB, trimethoprim, Rifampin, cycloheximide, cefoperazone and 6 kinds of microbiotic of amphotericin B, growth stimulant and 10% sheep whole blood.
Embodiment:
One, sample collecting, sample pretreatment
Wipe away collected specimens (poultry, pork, etc.) or directly sample is inserted Carry-Blair and transport in the substratum with disinfecting silk or cotton, in 24h, inspect by ready samples, sample collecting is followed the statistics requirement.
From transport substratum, take out sample, place the dactylethrae that contains 500 μ l sterilization PBS (pH7.2), fully soak into, 20min, vibration is for several times at interval; Take out cotton and wipe away, collect leach liquor.
Two, CCDA substratum preparation
Substratum preparation water: common zero(ppm) water boils 5-10min water oxygen gas is fully discharged, and cooling is used for the substratum preparation.
The preparation of CCDA substratum: (OXOID company, CM0739) preparing culture medium behind the autoclaving, are cooled to 50-60 ℃ and add microbiotic and growth stimulant respectively to press the CCDA product description.
6 kinds of microbiotic add, and antibiotic ultimate density is respectively in the substratum:
PXB: 6.7 * 10
-3G/L;
Trimethoprim: 0.1g/L;
Rifampin: 0.1g/L;
Cycloheximide: 1g/L;
Cefoperazone: 0.128g/L;
Amphotericin B: 0.04g/L.
Crooked bacteria growing promotor: Sodium.alpha.-ketopropionate, Sodium Pyrosulfite and ferrous sulfate respectively take by weighing 5g, add 50ml zero(ppm) water and fully dissolve, and subsequent use with 0.22 μ m membrane filtration, the ultimate density of growth stimulant is 0.5g/L in the substratum.
Prescription one: in the CCDA substratum, add PXB, trimethoprim, Rifampin, cycloheximide, cefoperazone and 6 kinds of microbiotic of amphotericin B and growth stimulant
Prescription two: common solid CCDA substratum
Prescription three: in the CCDA substratum, add PXB, trimethoprim, Rifampin and 4 kinds of microbiotic of cycloheximide
Prescription four: in the CCDA substratum, add PXB, trimethoprim, Rifampin, cycloheximide, cefoperazone and 6 kinds of microbiotic of amphotericin B
Prescription five: in the CCDA substratum, add PXB, trimethoprim, Rifampin, cycloheximide, cefoperazone and 6 kinds of microbiotic of amphotericin B, growth stimulant and 10% chicken serum
Prescription six: in the CCDA substratum, add PXB, trimethoprim, Rifampin, cycloheximide, cefoperazone and 6 kinds of microbiotic of amphotericin B, growth stimulant and 10% sheep whole blood
Three, in the food samples direct census of crooked bacterium with separate
Draw sample preparation liquid 0.1ml, evenly coat in above-mentioned six kinds of substratum, after drying, put in the 2.5L anaerobic jar, seal anaerobic jar immediately after adding aerobic in a subtle way aerogenesis bag, to cultivate 36h in 42 ℃ of incubators.
Result such as Fig. 1-shown in Figure 6; It is best that Fig. 1 (the solid CCDA substratum that contains 6 kinds of microbiotic such as PXB, trimethoprim, Rifampin, cycloheximide, cefoperazone and amphotericin B and growth stimulant) separates in the food effect of crooked bacterium, and varied bacteria growing quantity seldom, crooked bacteria growing state is good, help counting and separate.And the assorted bacterium quantity of Fig. 2 (not containing microbiotic and growth stimulant), Fig. 3 (containing 4 kinds of microbiotic) growth is many, can't separate and counting crooked bacterium; The crooked bacterium of Fig. 4 (contain 6 kinds of microbiotic, do not contain growth stimulant) obtains separating, but the bacterial growth state difference is difficult to count, counts inaccurate; Fig. 5 (containing 6 kinds of microbiotic, growth stimulant and 10% chicken serum), the crooked bacteria growing state of Fig. 6 (containing 6 kinds of microbiotic, growth stimulant and 10% sheep whole blood) are good, but assorted bacterium is many, also be unfavorable for directly to the counting of crooked bacterium with separate.
With platinum earrings picking translucent, greyish white or greyish-green, the little lawn of metalluster is arranged, be inoculated in once more that to contain antibiotic CCDA dull and stereotyped, repeat 2-3 time, until obtaining single pure culture bacterium colony.
The further evaluation of strain isolated can be adopted national standard method and PCR method.
Claims (1)
1. the quantitative detecting method of crooked bacterium in the food is characterized in that sample is directly carried out separating and counting of crooked bacterium after containing the solid CCDA culture medium culturing of microbiotic and growth stimulant;
Antibiotic content is in the above CCDA solid medium:
PXB: 6.7 * 10
-3G/L;
Trimethoprim: 0.1 g/L;
Rifampin: 0.1 g/L;
Cycloheximide: 1 g/L;
Cefoperazone: 0.128 g/L;
Amphotericin B: 0.04 g/L;
Growth stimulant is in the above CCDA solid medium: Sodium.alpha.-ketopropionate, Sodium Pyrosulfite and ferrous sulfate respectively take by weighing 5 g; Adding 50 ml zero(ppm) water fully dissolves; Subsequent use with 0.22 μ m membrane filtration, the ultimate density of growth stimulant is 0.5 g/L in the substratum.
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| CN104195239A (en) * | 2014-08-15 | 2014-12-10 | 中山鼎晟生物科技有限公司 | Qualitative detection kit and detection method for Campylobacter jejuni in food |
| CN104372077A (en) * | 2014-09-20 | 2015-02-25 | 中山鼎晟生物科技有限公司 | Detection kit and detection method of food pathogenic bacteria |
| CN104278087A (en) * | 2014-09-20 | 2015-01-14 | 中山鼎晟生物科技有限公司 | Detection kit and detection method for Campylobacter jejuni in pork |
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| US6368847B1 (en) * | 1999-06-03 | 2002-04-09 | The United States Of America, As Represented By The Secretary Of Agriculture | Selective media for recovery and enumeration of campylobacters |
| CN1598544A (en) * | 2004-08-13 | 2005-03-23 | 汕头大学 | Method for detecting jejunum arcuation bacterial |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1096819A (en) * | 1993-06-23 | 1994-12-28 | 铜陵市卫生防疫站 | A kind of long-acting preservative jejunum campylobacter colony culture medium and preparation method |
| US6368847B1 (en) * | 1999-06-03 | 2002-04-09 | The United States Of America, As Represented By The Secretary Of Agriculture | Selective media for recovery and enumeration of campylobacters |
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| 杨毓环等.福建省2003- 2005 年食品中空肠和结肠弯曲菌的监测与分析.《中国食品卫生杂志》.2007,第19卷(第1期),第15-18页. * |
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