CN104212887A - Method for rapidly detecting salmonella - Google Patents
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- CN104212887A CN104212887A CN201410400047.7A CN201410400047A CN104212887A CN 104212887 A CN104212887 A CN 104212887A CN 201410400047 A CN201410400047 A CN 201410400047A CN 104212887 A CN104212887 A CN 104212887A
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Abstract
The invention discloses a method for rapidly detecting salmonella. The method comprises the following steps: 1, mixing an immunomagnetic bead with a liquid sample to be detected in order to capture salmonella possibly existing in the sample to be detected, wherein the immunomagnetic bead is obtained by coupling an antibody with a magnetic ball; 2, mixing the immunomagnetic bead possibly capturing the salmonella with a liquid medium, and culturing the obtained mixture under salmonella proliferation conditions; and 3, carrying out PCR amplification on thallus obtained after culturing in step 2 by using a specific primer for the salmonella, and determining whether the salmonella exists in the sample to be tested or not according to a PCR amplification product. The method has the advantages of simple and fast operation, short required time, high sensitivity and accuracy, and strong applicability, is particularly useful for large scale application popularization, and is of great significance to the food safety monitoring and supervision.
Description
Technical field
The invention belongs to biological technical field, particularly, relate to a kind of method of rapid detection Salmonellas.
Background technology
Pathogenic bacterium (Pathogenic bacteria) can cause the microorganism of disease to be called pathogenic micro-organism or pathogenic bacterium.Pathogenic micro-organism comprises bacterium, virus, spirochete, Rickettsiae, chlamydozoan, mycoplasma, fungi and actinomycetes etc.General said pathogenic bacterium refer to the bacterium in pathogenic micro-organism.Pathogenic and its virulence of bacterium, invade quantity and portal entry relevant.Although most bacterium is harmless even useful, a large portion can be caused a disease.Conditioned pathogen is only caused a disease under given conditions, bacterium can be allowed to enter blood if any wound, or when immunizing power reduces.Such as, streptococcus aureus and suis are also normal microfloras, often may reside in skin surface, nasal cavity and do not cause disease, but potentially can cause skin infections, as pneumonia (pneumonia), meningitis (meningitis) and septicemia (sepsis) etc.
Salmonellas is a kind of Gram-negative tyrothricin, does not form brood cell, can show as gastro-enteritis, enteroidea, microbemia syndrome or focal disease clinically.Threatening maximum by this bacterium is infant, old man and immunocompromised subject.In bacterial species food poisoning according to statistics in countries in the world, the normal row umber one of salmonellal food poisoning.China hinterland is also first place with Salmonellas.Current, national governments and institutions for academic research are all being devoted to the research of food-safety problem, and food-safety problem receives unprecedented concern.
The ordinary method of Salmeterol fluticasone propionate is the method that most domestic feeler mechanism is all using, key step is front increasing bacterium (8-18h), increases bacterium (18-24h), slat chain conveyor separation (18-24h), biochemical test and serological identification, because the shortcomings such as its sense cycle is long, program is complicated, required reagent is various can not meet modern measure requirement far away.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, provide a kind of can the method for rapid detection Salmonellas.
To achieve these goals, the present inventor has carried out large quantifier elimination, found that the method with the use of immune magnetic separation technique and pcr amplification is particularly conducive to the quick specific detection realizing Salmonellas.Therefore, the invention provides a kind of method of rapid detection Salmonellas, the method comprises the following steps:
(1) mixed with the testing sample of liquid form by immunomagnetic beads, to catch the Salmonellas that may exist in testing sample, described immunomagnetic beads is obtained by antibody and the coupling of magnetic ball;
(2) immunomagnetic beads that may capture Salmonellas in step (1) is mixed with liquid nutrient medium, and cultivate under gained mixture being placed in the condition that Salmonellas can be made to breed;
(3) adopting the Auele Specific Primer for Salmonellas to carry out pcr amplification to step (2) through cultivating the thalline obtained, judging the existence of Salmonellas in testing sample according to pcr amplification product.
Method of the present invention is simple and quick, and required time is short, has higher sensitivity and accuracy, and suitability is strong, is particularly conducive to large-scale application, is significant to food safety monitoring supervision.
Other features and advantages of the present invention are described in detail in embodiment part subsequently.
Accompanying drawing explanation
Accompanying drawing is used to provide a further understanding of the present invention, and forms a part for specification sheets, is used from explanation the present invention, but is not construed as limiting the invention with embodiment one below.In the accompanying drawings:
Fig. 1 is part agarose gel electrophoresis result figure in embodiment 1, wherein, the electrophoresis result of M to be Marker, 1-3 be experimental group 1-3,4 is the electrophoresis result of dead bacterium group 4, and 5 is the electrophoresis result of positive controls 5, and 6 is the electrophoresis result of negative control group 6;
Fig. 2 is another part agarose gel electrophoresis result figure in embodiment 1, and wherein, M is Marker, and 1 is the electrophoresis result of negative control group 7, and 2 is the electrophoresis result of negative control group 8, and 3 is the electrophoresis result of negative control group 9, and 4 is the electrophoresis result of positive controls 5;
Fig. 3 is agarose gel electrophoresis result figure in embodiment 2, and wherein, M is Marker, and 1 and 2 is the electrophoresis result of negative control group 5 and 6, and 3 and 4 is the electrophoresis result of positive controls 3 and 4, and 5 and 6 is the electrophoresis result of experimental group 1 and 2.
Embodiment
Below the specific embodiment of the present invention is described in detail.Should be understood that, embodiment described herein, only for instruction and explanation of the present invention, is not limited to the present invention.
In the present invention, when not doing contrary explanation, the term " immunomagnetic beads " of use to refer to by linked reaction by antibodies on the surface of magnetic microsphere (or magnetic ball), the immune magnetic microsphere of formation; Term " PCR " refers to polymerase chain reaction; Term " primer " comprises at least one upstream primer and at least one downstream primer; The liquid volume used in the present invention is the numerical value at 20 DEG C.
The method of rapid detection Salmonellas provided by the invention comprises the following steps:
(1) mixed with the testing sample of liquid form by immunomagnetic beads, to catch the Salmonellas that may exist in testing sample, described immunomagnetic beads is obtained by antibody and the coupling of magnetic ball;
(2) immunomagnetic beads that may capture Salmonellas in step (1) is mixed with liquid nutrient medium, and cultivate under gained mixture being placed in the condition that Salmonellas can be made to breed;
(3) adopting the Auele Specific Primer for Salmonellas to carry out pcr amplification to step (2) through cultivating the thalline obtained, judging the existence of Salmonellas in testing sample according to pcr amplification product.
According to the present invention, the method that antibody and the coupling of magnetic ball obtain described immunomagnetic beads can be ordinary method, such as, can according to document (Liu Huirong etc., the preparation of simple and effective isolated cell Novel immune magnetic bead, Chinese public health, 2008,11:1349-1351) in method carry out.
In order to catch the Salmonellas in testing sample, described antibody be can with the antibody of Salmonellas specific binding, preferably, described antibody is be numbered the LSBIO monoclonal antibody of LS-C103073 (this antibody can be commercially available from LSBIO company, article number (Cat.#) is LS-C103073) and/or be numbered the KPL polyclonal antibody (this antibody can be commercially available from KPL company of the U.S., and article number (Cat.#) is 01-95-99) of 01-95-99.The viable bacteria health check-up adopting this preferred antibody (particularly described polyclonal antibody) can realize Salmonellas is surveyed, and further increases the actual use value of detection method of the present invention.
Under preferable case, on the magnetic ball of every milligram, the amount of the antibody of coupling is 0.05-2mg.
Under preferable case, the testing sample of the liquid form relative to every milliliter, with the weighing scale of magnetic ball, the consumption of described immunomagnetic beads is 10-25 μ g.
Described magnetic ball can be that various routine is used for the magnetic ball of immune Magneto separate, such as, can have the superparamagnetism Fe of amino or carboxyl for finishing
3o
4polystyrene complex microsphere.Under preferable case, the particle diameter of described magnetic ball is 150-200nm.
According to the present invention, utilize immunomagnetic ca pture Salmonellas can be undertaken by immune magnetic separation system (such as Matrix Pathatrix immunity magnetic separation system) or magnetic frame, to described condition of catching, there is no particular limitation, preferably, in step (1), the condition of catching comprises: temperature is 35-38 DEG C, and the time is 25-35 min.
According to the present invention, in order to detect the existence of Salmonellas in testing sample exactly, need to carry out bacterium amplification process to testing sample after immune Magneto separate, thus the Salmonellas that may exist in amplification testing sample.Described liquid nutrient medium can be the substratum that this area routine is used for Salmonellas cultivation, as buffered peptone water or nutrient broth medium.The temperature of cultivating can be 35-38 DEG C.The time of cultivating is preferably 4-8h (being more preferably 4-6h).Shaking table can be utilized to carry out described cultivation, and the rotating speed of shaking table can be set to 100-200rpm usually.In the present invention, the culture obtained through culturing step can directly as the template of pcr amplification.The present invention adopts and first carries out immune Magneto separate, then multiplication culture, decreases the time needed for detection to a great extent, improves detection efficiency.
According to the present invention, pcr amplification Auele Specific Primer used is invA (the invasin protein A of Salmonellas of can increasing specifically, invasionprotein A) primer of gene (such as GenBank Accession Number:M90846.1) and/or FimY (pili Y albumen, fimbriae Y protein) gene (such as GenBank Accession Number:JQ665438.1).Wherein, as the particularly preferred a kind of embodiment of the present invention, pcr amplification Auele Specific Primer used is made up of the forward primer of sequence as shown in SEQ ID NO:1 (5 '-TCCTCCGCTCTGTCTACTTA-3 ') and the sequence reverse primer as shown in SEQ ID NO:2 (5 '-ACCGAAATATTCATTGACGTT-3 '), or is made up of the forward primer of sequence as shown in SEQ ID NO:3 (5 '-GCCGGTAAACTACACGATGA-3 ') and the sequence reverse primer as shown in SEQ ID NO:4 (5 '-GAGTTACTGAACCAACAGCT-3 ').
In the present invention, if there is the target DNA fragment that Auele Specific Primer can increase in pcr amplification product, so there is Salmonellas in interpret sample.Conventional electrophoretic detection can be adopted to analyze the DNA fragmentation that may exist in pcr amplification product, thus judge whether there is Salmonellas in testing sample, such as, agarose gel electrophoresis (AGE), the actual conditions of electrophoresis can comprise: voltage is 3-15V/cm, time is 20-40min, and the applied sample amount of pcr amplification product is 8-20 μ L.
According to method of the present invention, wherein, testing sample can at least one in food, water use for environment (comprising municipal wastewater, trade effluent, agricultural water, town water, extraordinary water etc.) and medicine.Particularly, testing sample can be obtained according to the method in standard GB/T 4789.1-2010 " national food safety standard food microbiological analysis general provisions ".
Below will be described the present invention by embodiment.In following examples, buffered peptone water is purchased from Beijing road and bridge technology limited liability company; The bacterial strain used all by commercially available, and respectively purchased from ATCC (American Type Culture preservation center), CGMCC (China General Microbiological culture presevation administrative center) and CMCC (Chinese medicine Microbiological Culture Collection administrative center); 1-ethyl-(3-dimethylamino-propyl) phosphinylidyne diimine (EDC) and N-hydroxy-succinamide (NHS) available from Sigma; The Auele Specific Primer used entrusts the synthesis of Shanghai Ying Jun Bioisystech Co., Ltd; Magneto separate carries out on magnetic frame; To be commercially available from LSBIO company, the antibody that article number (Cat.#) is LS-C103073 is as specific antibody.
Preparation embodiment 1
This is prepared embodiment 1 and uses antibody 1 to prepare method of the present invention immunomagnetic beads used.
The finishing activated by 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide (EDC) and N-hydroxy-succinamide (NHS) there is the superparamagnetism Fe of carboxyl
3o
4polystyrene complex microsphere is (purchased from Aorun Weina New Material Science and Technology Co., Ltd., Shanghai, PM3-020 type polymkeric substance magnetic ball, concentration is 10mg/mL, particle diameter is 180nm, finishing has carboxyl functional group) use as magnetic ball, get 2mg magnetic ball, in dispersion phosphate buffer solution, in dispersion liquid, the concentration of magnetic ball is 2mg/mL.Get 0.2mg specific antibody (being dissolved in the phosphoric acid buffer of 1mL) to mix with 2mg magnetic ball, in shaking table (200r/min), 3h is kept under room temperature, then cleaned by Magneto separate, remove the specific antibody not being coupled to magnetic ball surface, adopt BioSpec-nano instrument (SHIMADZU, Japan) detect the micro-antibody of non-coupling, and the amount recording the specific antibody not being coupled to magnetic ball surface is 300ng.Then be the bovine serum albumin solution (being dissolved in phosphoric acid buffer) of 1% (w/v) by concentration, at 37 DEG C, close 30min, then cleaned by Magneto separate, wash away unnecessary bovine serum albumin solution, namely obtain immunomagnetic beads.
Embodiment 1
The present embodiment is used for the sensitivity of the inventive method, specificity and accuracy are described.
The PBS of different bacterial strains (see table 1) and 2mL is mixed to get the bacteria suspension (i.e. testing sample) of different concns, and carry out immunomagnetic ca pture, microbial culture, pcr amplification and electrophoresis detection in such a way, wherein, the testing sample of dead bacterium group is by bacteria suspension being placed in 121 DEG C, 30min carries out deactivation and obtains under the condition of 0.103MPa.
Table 1
Note: " Fig. 1-1 " represents 1 swimming lane in Fig. 1, and the rest may be inferred.
(1) immunomagnetic ca pture
Joined by bacteria suspension in the immunomagnetic beads centrifuge tube that obtains of preparation embodiment 1, mixing that turbine mixer vibrates gently, is placed in vortex mixer or shaking table, and room temperature (25 DEG C) rotates, and (or vibration) hatches 30min; Be placed on Matrix Pathatrix immunity magnetic separation system, leave standstill 30min under room temperature (25 DEG C), carefully siphon away the liquid in pipe, adaptive immune enrichment with magnetic bead body.
(2) microbial culture
Transferred to by immunomagnetic beads enrich body in 10mL buffered peptone water, vortex mixes, and is placed into constant incubator, at 36 ± 1 DEG C, cultivates 5.5h; Collect nutrient solution 5mL in Eppendorf pipe, the centrifugal 10min of 12000g, gained precipitation 1mL sterilized water is broken up, and under 12000g centrifugal 10min, abandon supernatant, precipitation is broken up with 100 μ L sterilized waters, obtains thalline stand-by (as pcr amplification template).
(3) pcr amplification
The Auele Specific Primer for described Salmonellas is used to carry out pcr amplification to cultivating the thalline obtained.The sequence of Auele Specific Primer (the invA gene of the 283bp that can increase) is as follows:
Forward primer (invAF): 5 '-TCCTCCGCTCTGTCTACTTA-3 ' (SEQ ID NO:1)
Reverse primer (invAR): 5 '-ACCGAAATATTCATTGACGTT-3 ' (SEQ ID NO:2)
PCR reaction system is in table 2:
Table 2
| Reagent | Stock solution concentration | Injection volume in 25 μ L reaction systems |
| 10 × PCR damping fluid is not (containing MgCl 2) | — | 7.8μL |
| dNTP | 2.5mmol/L | 2μL |
| MgCl 2 | 25mmol/L | 1.5μL |
| Upstream primer | 20μmol/L | 0.3μL |
| Downstream primer | 20μmol/L | 0.3μL |
| Taq enzyme | 5U/μL | 0.2μL |
| DNA profiling | 50ng/μL | 10μL |
| Distilled water | — | 2.9μL |
Pcr amplification program is in table 3:
Table 3
| 1 | 94℃ | 3min |
| 2 | 94℃ | 30s |
| 3 | 55℃ | 30s |
| 4 | 72℃ | 40s |
| 5 | Return the 2nd step | 30 circulations |
| 6 | 72℃ | 10min |
| 7 | 4℃ | Forever |
(4) electrophoresis detection of pcr amplification product
Prepare the sepharose of 2% with electrophoretic buffer (1 × TAE), adding ethidium bromide to final concentration when 58 DEG C is 0.5 μm of ol/mL; Get the pcr amplification product of 10 μ L, point sample is carried out in the sample-loading buffer mixing of respectively with 2 μ L, and do reference with DNA molecular amount marker, electrophoresis 30min under 10V/cm constant voltage, electrophoresis detection result Labworks image acquisition and analysis software record is also preserved.
If the fragment that AGE electrophoresis result display pcr amplification obtains 283bp (can be observed obvious band, positive), so there is Salmonellas in interpret sample, result is as depicted in figs. 1 and 2: experimental group 1-3 and positive controls 5 all present the band of obvious 283bp, and dead bacterium group 4 and negative control group 6-9 do not have obvious band.
As can be seen from Fig. 1 and Fig. 2, the preferred embodiment of the present invention can detect Salmonellas viable bacteria body specifically.
Embodiment 2
The detection of Salmonellas is carried out according to the method for embodiment 1, unlike, the sample of use is as shown in table 4, and the sequence of the Auele Specific Primer (the FimY gene of the 526bp that can increase) of pcr amplification use is as follows:
Forward primer (FimYF): 5 '-GCCGGTAAACTACACGATGA-3 ' (SEQ ID NO:3)
Reverse primer (FimYR): 5 '-GAGTTACTGAACCAACAGCT-3 ' (SEQ ID NO:4)
Table 4
Note: " Fig. 3-1 " represents 1 swimming lane in Fig. 3, and the rest may be inferred.
Electrophoresis result is as shown in Figure 3: experimental group 1-2 and positive controls 3-4 presents the band of obvious 526bp, and negative control group 5 and 6 does not have obvious band.As can be seen from Fig. 3 also, method of the present invention can be used in detecting Salmonellas specifically, exactly.
Embodiment 3
The present embodiment is used for illustrating that method of the present invention is in the practical application detecting pork sample.
Learning from else's experience, (it is that three kinds of bacterial strains are 20 parts of negative fresh pork sample 25g that GB/T4789.4-2010 (national food safety standard-food microbiological analysis-Salmonellas inspection) verifies to national standard method, immunomagnetic ca pture, microbial culture, pcr amplification and electrophoresis detection is carried out respectively according to the method recorded in embodiment 1, result shows, 20 parts of negative pork samples of Salmonellas are all negative by this inventive method detected result, consistent with classical culture protocols detected result.
Above-mentioned 20 parts of fresh pork samples are polluted with Salmonella typhimurium (ATCC14028) (10 ° of cfu), adopt the method recorded in embodiment 1 to carry out immunomagnetic ca pture, microbial culture, pcr amplification and electrophoresis detection again, result is all positive.
Embodiment 4
The detection of Salmonellas is carried out according to the method recorded in embodiment 1, unlike, the testing sample used in embodiment 1 is replaced with 50 parts of fresh pork samples purchased from Ge great supermarket, Beijing, and adopts the method for GB/T4789.4-2010 (national food safety standard-food microbiological analysis-Salmonellas inspection) to verify respectively.
Result shows, and GB method detects positive 20 examples of Salmonellas, and method of the present invention is also positive to the detected result of these 20 increment product, and two kinds of methods are feminine gender to the detected result of Salmonellas in all the other samples.
Show the detected result of the 50 parts of fresh pork samples bought from Ge great supermarket, Beijing area, compare GB method, the inventive method has very high sensitivity, specificity and accuracy for the detection of Salmonellas.
Embodiment 5
The detection of Salmonellas is carried out according to the method recorded in embodiment 1, unlike, the testing sample used in embodiment 1 is replaced with 50 parts of fresh chicken meat samples purchased from Ge great supermarket, Beijing, and the monoclonal antibody used in embodiment 1 is replaced with 01-95-99 polyclonal antibody (purchased from KPL company, article number (Cat.#) is 01-95-99).The method of GB/T4789.4-2010 (national food safety standard-food microbiological analysis-Salmonellas inspection) is adopted to verify respectively.
Result shows, and GB method detects positive 23 examples of Salmonellas, and method of the present invention is also positive to the detected result of these 23 increment product, and two kinds of methods are feminine gender to the detected result of Salmonellas in all the other samples.
Also show the detected result of the 50 parts of fresh chicken meat samples bought from Ge great supermarket, Beijing area, compare GB method, the inventive method has very high sensitivity, specificity and accuracy for the detection of Salmonellas.
As can be seen from the above embodiments, method of the present invention can realize the rapid detection of Salmonellas, and sensitivity and accuracy are all higher.
More than describe the preferred embodiment of the present invention in detail; but the present invention is not limited to the detail in above-mentioned embodiment, within the scope of technical conceive of the present invention; can carry out multiple simple variant to technical scheme of the present invention, these simple variant all belong to protection scope of the present invention.
It should be noted that in addition, each concrete technical characteristic described in above-mentioned embodiment, in reconcilable situation, can be combined by any suitable mode, in order to avoid unnecessary repetition, the present invention illustrates no longer separately to various possible array mode.
In addition, also can carry out arbitrary combination between various different embodiment of the present invention, as long as it is without prejudice to thought of the present invention, it should be considered as content disclosed in this invention equally.
Claims (9)
1. a method for rapid detection Salmonellas, is characterized in that, the method comprises the following steps:
(1) mixed with the testing sample of liquid form by immunomagnetic beads, to catch the Salmonellas that may exist in testing sample, described immunomagnetic beads is obtained by antibody and the coupling of magnetic ball;
(2) immunomagnetic beads that may capture Salmonellas in step (1) is mixed with liquid nutrient medium, and cultivate under gained mixture being placed in the condition that Salmonellas can be made to breed;
(3) adopting the Auele Specific Primer for Salmonellas to carry out pcr amplification to step (2) through cultivating the thalline obtained, judging the existence of Salmonellas in testing sample according to pcr amplification product.
2. method according to claim 1, wherein, described antibody is be numbered the LSBIO monoclonal antibody of LS-C103073 and/or be numbered the KPL polyclonal antibody of 01-95-99.
3. method according to claim 1, wherein, on the magnetic ball of every milligram, the amount of the antibody of coupling is 0.05-2mg.
4. method according to claim 1, wherein, described magnetic ball is the superparamagnetism Fe that finishing has amino or carboxyl
3o
4polystyrene complex microsphere.
5. according to the method in claim 1-4 described in any one, wherein, the testing sample of the liquid form relative to every milliliter, with the weighing scale of magnetic ball, the consumption of described immunomagnetic beads is 10-25 μ g.
6. method according to claim 1, wherein, the condition of catching in step (1) comprises: temperature is 35-38 DEG C, and the time is 25-35min.
7. method according to claim 1, wherein, the time of cultivating in step (2) is 4-8h.
8. method according to claim 1, wherein, the Auele Specific Primer that in step (3), pcr amplification is used is can increase specifically the invA gene of Salmonellas and/or the primer of FimY gene.
9. the method according to claim 1 or 8, wherein, pcr amplification Auele Specific Primer used is made up of the forward primer of sequence as shown in SEQ ID NO:1 and the reverse primer of sequence as shown in SEQ ID NO:2, or is made up of the forward primer of sequence as shown in SEQ ID NO:3 and the reverse primer of sequence as shown in SEQ ID NO:4.
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| CN108085377A (en) * | 2017-12-29 | 2018-05-29 | 北京和益源生物技术有限公司 | The detection method of salmonella under a kind of high background |
| CN108254559A (en) * | 2018-01-03 | 2018-07-06 | 北京市理化分析测试中心 | The method of quick detection microorganism |
| CN110904256A (en) * | 2019-12-19 | 2020-03-24 | 内蒙古自治区农牧业科学院 | Primer group for simultaneously detecting salmonella, pasteurella multocida, staphylococcus aureus and avibacterium paragallinarum and application |
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| 叶明强等: "《一种快速检测沙门氏菌的新方法研究》", 《食品科技》 * |
| 张东方等: "《免疫磁捕获-实时荧光PCR快速检测鸡肉中沙门氏菌》", 《食品与发酵工业》 * |
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|---|---|
| CN106702016A (en) | 2017-05-24 |
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