CN105316391A - Method of detecting salmonella, shigella and staphylococcus aureus - Google Patents
Method of detecting salmonella, shigella and staphylococcus aureus Download PDFInfo
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Abstract
The invention discloses a method of detecting salmonella, shigella and staphylococcus aureus, which includes the following steps: 1) mixing a to-be-tested sample with a liquid culture medium and performing cultivation to the mixture under a condition which enables bacteria possibly existing in the to-be-test sample to be amplified to obtain an amplified sample; 2) capturing the salmonella, the shigella and the staphylococcus aureus, which possibly exist in the amplified sample, by means of immunomagnetic beads which are prepared through coupling of an antibody and magnetic spheres; 3) extracting the genome DNA of the bacteria captured in the step 2); and 4) with a specific primer aiming to the salmonella, the shigella and the staphylococcus aureus, performing multiple fluorescent quantitative PCR to the extracted genome DNA to determine the existence of the salmonella, the shigella and the staphylococcus aureus in the to-be-tested sample. The method achieves simultaneous detection of the salmonella, the shigella and the staphylococcus aureus, is high in sensitivity and is accurate in result.
Description
Technical field
The present invention relates to field of food safety, particularly, the method that one detects Salmonellas (Salmonella), Shigellae (Shigella) and streptococcus aureus (Staphyloccocusaureus) three kinds of bacterial strains is simultaneously related to.
Background technology
The food origin disease that the Salmonellas existed in food, Shigellae and streptococcus aureus cause, in ascendant trend year by year, brings very large potential threat to the health of people.The food origin disease caused in the world by Salmonellas, Shigellae and streptococcus aureus causes the massive loss of life, causes multi-million dollar to lose.The Bacterial foodborne diseases analyses of epidemiological characteristics display of a 2003-2007 China, in the food origin disease event caused by microorganism, the ratio of Salmonellas, Shigellae and streptococcus aureus is respectively 10.3%, 8.9% and 1.5%, and the food origin disease total number of events that three kinds of pathogenic bacterium cause accounts for more than 20% of 1060 food origin disease times of statistics.
Deliver to correlation detection mechanism after the means of current reply food safety accident are generally collected specimens and carry out traditional separation and Culture, carry out confirming or getting rid of after bio-chemical characteristics.This method incubation time is long, workload is large, experimental procedure is loaded down with trivial details, can not reflect data accurately in time in a lot of accident.Conventional PCR method is short for detection time, but its sensitivity is can not irrespective problem with the pollution that may bring.Immunity magnetic separation technique is by specific antibody and a certain size magnetic bead coupling, utilize the principle that specific antibody can combine with cell-surface antigens, by magnetic ball and Cell binding, under the effect of outside magnetic field, magnetic ball-cell complexes is separated from environment, reach the technology obtaining target cell, but can only realize when being used alone this technology being separated, also need to detect in conjunction with other means, and the sensitivity using immune magnetic separation technique to carry out the detection method be separated at present still has much room for improvement.Taqman probe method multiple real time fluorescence quantifying PCR technology have employed closed reaction system, adopts many group-specific primerses and probe, the augmentation detection while of carrying out for many group object bacteria, and its accuracy still has much room for improvement.
Current application immunity Magneto separate-multiple fluorescence quantitative PCR carries out three re-detections to the Salmonellas in food, Shigellae and streptococcus aureus and yet there are no report, is worth research further.
Summary of the invention
The object of the invention is to overcome existing detection method sensitivity and accuracy is not high and the defect that detects while being difficult to realize three kinds of bacterial strains, provide a kind of and there is highly sensitive and accuracy and the method for Salmonellas, Shigellae and streptococcus aureus can be detected simultaneously.
To achieve these goals, the present inventor has carried out large quantifier elimination, find that the residual component etc. of extracting solution in complicated ingredient, medium component and the DNA extraction process in food is all the potential supressor of PCR reaction, directly can affect the accuracy of detected result.Find after further research, detect while can effectively realizing Salmonellas, Shigellae and streptococcus aureus with the use of the method for immune magnetic separation technique and multiple fluorescence quantitative PCR.Therefore, the invention provides a kind of method detecting Salmonellas, Shigellae and streptococcus aureus, the method comprises the following steps:
(1) testing sample is mixed with liquid nutrient medium, and cultivate under mixture being placed in the condition of the bacterium amplification that testing sample can be made to exist, obtain the sample after amplification;
(2) Salmonellas that may exist in the sample after utilizing immunomagnetic ca pture to increase, Shigellae and streptococcus aureus, described immunomagnetic beads is obtained by antibody and the coupling of magnetic ball;
(3) genomic dna of the thalline of catching in extraction step (1);
(4) Auele Specific Primer for Salmonellas, Shigellae and streptococcus aureus is adopted to carry out multiple fluorescence quantitative PCR to the genomic dna extracted, according to the existence of Salmonellas in the result determination testing sample of multiple fluorescence quantitative PCR, Shigellae and streptococcus aureus.
By technique scheme, detect while present invention achieves Salmonellas, Shigellae and streptococcus aureus, highly sensitive and result is accurate.And the inventive method can detect the Salmonellas in testing sample, Shigellae and streptococcus aureus quickly, the ability improving reply food safety accident is fast significant.
Other features and advantages of the present invention are described in detail in embodiment part subsequently.
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 unit " CFU/g " of use means with the CFU number of every gram of testing sample bacterial strain that is benchmark; The term " immunomagnetic beads " used 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 invention provides a kind of method detecting Salmonellas, Shigellae and streptococcus aureus, the method comprises the following steps:
(1) testing sample is mixed with liquid nutrient medium, and cultivate under mixture being placed in the condition of the bacterium amplification that testing sample can be made to exist, obtain the sample after amplification;
(2) Salmonellas that may exist in the sample after utilizing immunomagnetic ca pture to increase, Shigellae and streptococcus aureus, described immunomagnetic beads is obtained by antibody and the coupling of magnetic ball;
(3) genomic dna of the thalline of catching in extraction step (1);
(4) Auele Specific Primer for Salmonellas, Shigellae and streptococcus aureus is adopted to carry out multiple fluorescence quantitative PCR to the genomic dna extracted, according to the existence of Salmonellas in the result determination testing sample of multiple fluorescence quantitative PCR, Shigellae and streptococcus aureus.
According to the present invention, in order to detect the existence of three kinds of bacterial strains in testing sample exactly, need to carry out bacterium amplification process to testing sample before immune Magneto separate, thus the bacterium, particularly Salmonellas that may exist in amplification testing sample, Shigellae and streptococcus aureus.Described liquid nutrient medium can be the substratum that this area routine is used for microbial culture, as nutrient broth medium (preferably adding the nutrient broth medium of the glycine having 0.8-1.2 % by weight).The condition of described cultivation also can be conventional selection, and such as, the temperature of cultivation can be 35-38 DEG C, and the time of cultivation can be 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 sample after amplification.
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, Shigellae and streptococcus aureus, described antibody comprise respectively can with Salmonellas, the antibody that Shigellae and staphylococcus aureus specific combine, preferably, described antibody comprises Salmonellas polyclonal antibody, Shigellae polyclonal antibody and streptococcus aureus polyclonal antibody, more preferably the rabbit anti-salmonella antibody that the article number (Cat.#) purchased from Meridian company is G5V61-500 is comprised, the anti-Shigellae antibody of rabbit being B65901R purchased from the article number (Cat.#) of Meridian company and the rabbit anti-Staphylococcus aureus antibody being B65881R purchased from the article number (Cat.#) of Meridian company.
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.
Under preferable case, the magnetic ball relative to every milligram, the consumption >=0.1mg of described antibody, is more preferably 0.1-1mg.When actually operating, can, by different antibodies obtained three kinds of immunomagnetic beadses respectively, carry out again three being added in system when immune Magneto separate is caught.
Under preferable case, the sample after the amplification relative to every milliliter, with the weighing scale of magnetic ball, the consumption of described immunomagnetic beads is 0.5-5 μ g, is more preferably 2-3 μ g.
According to the present invention, utilize immunomagnetic ca pture three kinds of thalline can be undertaken by immune magnetic separation system (such as MatrixPathatrix immunity magnetic separation system), 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 25-35min.
According to the present invention, to the not special requirement of the concrete grammar extracting genomic dna in step (2), can be the method for the extraction DNA that this area routine adopts, can use bacterial genomes DNA extraction kit, concrete operations and condition can see the specification sheetss of test kit.
According to the present invention, described multiple fluorescence quantitative PCR is triple fluorescent quantitative PCR, and described Auele Specific Primer preferably can increase the NA of Salmonellas specifically
athe primer of the ipaH gene (AccessionNumber:M76445) of gene (its nucleotide sequence is as shown in SEQIDNO:10), Shigellae and the Sa442 gene (AccessionNumber:AF033191) of streptococcus aureus.
Further preferably, the nucleotide sequence of upstream primer, downstream primer and corresponding specificity fluorescent probe that uses of multiple fluorescence quantitative PCR is as follows:
Upstream primer 1:5 '-GCTATTTTCGTCCGGCATGA-3 ' (SEQIDNO:1)
Downstream primer 1:5 '-GCGACTATCAGGTTACCGTGGA-3 ' (SEQIDNO:2)
Specificity fluorescent probe 1:5 '-TAGCCAGCGAGGTGAAAACGACAAAGG-3 ' (SEQIDNO:7)
Upstream primer 2:5 '-CTTGACCGCCTTTCCGATA-3 ' (SEQIDNO:3)
Downstream primer 2:5 '-AGCGAAAGACTGCTGTCGAAG-3 ' (SEQIDNO:4)
Specificity fluorescent probe 2:5 '-AACAGGTCGCTGCATGGCTGGAA-3 ' (SEQIDNO:8)
Upstream primer 3:5 '-TTCTTCACGACTAAATAAACGCTCA-3 ' (SEQIDNO:5)
Downstream primer 3:5 '-GGTACTACTAAAGATTATCAAGACGGCT-3 ' (SEQIDNO:6)
Specificity fluorescent probe 3:5 '-CAGAACACAATGTTTCCGATGCAACGT-3 ' (SEQIDNO:9)
Specificity fluorescent probe is the oligonucleotide that two ends are marked with reporter fluorescence group and quenching fluorescence group respectively, wherein, to the special requirement of the concrete selection of described reporter fluorescence group and quenching fluorescence group, as long as the color of the reporter fluorescence group in three species specificity fluorescent probes is different separately, and those skilled in the art can select it.Such as, described reporter fluorescence group can be FAM (6-Fluoresceincarboxylic acid (6-carboxy-fluorescein), excitation wavelength is 495nm, emission wavelength is 521nm, color is green), CY3 (3H-indole cyanine dyes or 3H-indoles cyanine dye, excitation wavelength is 550nm, emission wavelength is 570nm, color is red) and CY5 (3H-indole cyanine dyes or 3H-indoles Cyanine, excitation wavelength is 649nm, emission wavelength is 670nm, color is purple), TET (Tetrachlorofluorescein, excitation wavelength is 521nm, emission wavelength is 536nm, color is orange) or JOE (2, 7-dimethyl-4, the chloro-6-Fluoresceincarboxylic acid of 5-bis-, excitation wavelength is 520nm, emission wavelength is 548nm, color is pink colour), described quenching fluorescence group can be BHQ1 (BlackHoleQuencher1), BHQ2 (BlackHoleQuencher2) or TAMRA (6-carboxyl tetramethylrhodamin).
A preferred embodiment of the invention, specificity fluorescent probe 1 can be that 5 ' end is connected with FAM and 3 ' end is connected with the SEQIDNO:7 of BHQ1, specificity fluorescent probe 2 can be that 5 ' end is connected with CY3 and 3 ' end is connected with the SEQIDNO:8 of BHQ1, and specificity fluorescent probe 3 can be that 5 ' end is connected with CY5 and 3 ' end is connected with the SEQIDNO:9 of BHQ2.
According to the present invention, the condition optimization of multiple fluorescence quantitative PCR comprises: 94-96 DEG C of denaturation 18-22s; 94-96 DEG C of sex change 2.5-3.5s; 58-62 DEG C of annealing and extension 28-32s; 38-42 circulation.Conventional instrument can be used to carry out multiple fluorescence quantitative PCR, such as, 7500 rapid fluorescence quantitative PCR apparatus of AppliedBiosystem (ABI) company.
In the present invention, according to result (the i.e. Ct value of multiple fluorescence quantitative PCR, when namely specific fluorescent strength of signal reaches set threshold value, the reaction cycle number experienced), those skilled in the art can judge whether there is Salmonellas, Shigellae and streptococcus aureus in testing sample easily, and such as, Ct value is usually between 15 to 40, Ct value is less than 35 and is and there is bacterial strain to be detected, otherwise for not exist.
According to the present invention, described testing sample can be food (as pork).Preferably, concentration >=the 2CFU/g of Salmonellas (being preferably Salmonella typhimurium (Salmonellatyphimurium) and/or Salmonella choleraesuls (Salmonellacholeraesuis)) in sample after amplification, Shigellae (is preferably shigella flexneri (Shigellaflexneri), shigella dysenteriae (Shigelladysenteriae), at least one in Shigella bogdii (Shigellabogdii) and bacillus ceylonensis A (Shigellasonnei)) concentration >=6.8CFU/g, concentration >=the 9.6CFU/g of streptococcus aureus (Staphylococcusaureus).
Below will be described the present invention by embodiment.In following examples, nutrient broth medium (NB) is purchased from Beijing road and bridge technology limited liability company; Bacterial genomes DNA extraction kit (DP302) is purchased from Beijing Tian Gen biochemical technology company limited; 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), CMCC (Chinese medicine Microbiological Culture Collection administrative center), JCM (Japanese DSMZ), NBRC (Japanese technological assessment institute Biological Resource Center) or CICC (Chinese industrial Microbiological Culture Collection administrative center); 1-ethyl-(3-dimethylamino-propyl) phosphinylidyne diimine (EDC) and N-hydroxy-succinamide (NHS) available from Sigma; TaqManFastUniversalPCRMasterMix (2 ×) is purchased from AppliedBiosystems company; The synthesis of Shanghai Ying Jun Bioisystech Co., Ltd entrusted by the primer used and probe; Magneto separate carries out on magnetic frame; The 7500 rapid fluorescence quantitative PCR apparatus that the instrument that triple fluorescent quantitative PCR detection uses is AppliedBiosystem (ABI) company.
Preparation embodiment 1
This is prepared embodiment and adopts the immunomagnetic beads used in polyclonal antibody preparation embodiment.
(finishing has the superparamagnetism Fe of carboxyl to use 600 μ L phosphoric acid salt Tween buffer (PBST, pH=7.4) to wash 200 μ g magnetic balls successively
3o
4polystyrene complex microsphere (purchased from Shanghai Ao Run AllMag company, PM3-020 type polymkeric substance magnetic ball, concentration is 10mg/mL, and particle diameter is 180nm, and finishing has carboxyl functional group)) 3 times, remove supernatant after Magneto separate; The 15 μ L concentration adding new preparation are the EDC of 5mg/mL and 15 μ L concentration is that the NHS solution of 5mg/mL is in centrifuge tube, concussion mixing, 37 DEG C of activation 60min, wash 3 times with the PBST of 600 μ L successively, with the resuspended magnetic ball of 800 μ L phosphate buffered saline buffer (PBS, pH=7.4); Add the bacterium polyclonal antibody of 50 μ g purchased from Meridian company, 37 DEG C in conjunction with 3h; Abandoning supernatant after Magneto separate, wash 3 times successively with the PBST of 600 μ L, is the resuspended magnetic ball of bovine serum albumin (BSA) of 1 % by weight by 300 μ L concentration, closes 30min at 37 DEG C; Abandon supernatant after Magneto separate, the PBS of 600 μ L washs 4 times and namely obtains immunomagnetic beads successively, adds the PBST of 100 μ L, and 4 DEG C of preservations are stand-by.
The bacterium polyclonal antibody being respectively G5V61-500, B65901R and B65881R by article number (Cat.#) with upper type is adopted to prepare three kinds of immunomagnetic beadses respectively, by three kinds of immunomagnetic beads balanced mix during use.
Embodiment 1
The present embodiment is used for illustrating in the inventive method the specificity of primer and the corresponding specificity fluorescent probe used.
(1) microbial culture
Respectively different bacterial strains (see table 1,37 strain bacterial strains, comprise 16 strain object bacteria and 21 strain non-targeted bacterium) is added in the NB of 5mL, cultivate lower 24 hours in 37 DEG C.
(2) DNA extraction
High speed low temperature centrifugal machine (Eppendorf5804R whizzer) is used to be undertaken centrifugal at 12000rpm, at 4 DEG C by the strain culture that 1mL step (1) obtains, after abandoning supernatant, bacterial genomes DNA extraction kit is used to extract genomic dna, as the DNA profiling of step (3).
(3) triple fluorescent quantitative PCR detects (RT-PCR detection)
Triple fluorescent quantitative PCR reaction system is the TaqManFastUniversalPCRMasterMix (2 ×) of 20 μ L:10 μ L, (5 ' end is connected with FAM and 3 ' end is connected with BHQ1 and the probe 1 μ L of nucleotide sequence as shown in SEQIDNO:7 for each 0.5-1 μ L of three species specificity fluorescent probes (2.5 μMs), 5 ' end is connected with CY3 and 3 ' end is connected with BHQ1 and the probe 0.5 μ L of nucleotide sequence as shown in SEQIDNO:8, 5 ' end is connected with CY5 and 3 ' end is connected with BHQ2 and the probe 0.5 μ L of nucleotide sequence as shown in SEQIDNO:9), three kinds of primer (10 μMs) 0.5-1 μ L (upstream primer and downstream primer primer 1 μ L respectively as shown in SEQIDNO:1 and SEQIDNO:2, upstream primer and the downstream primer primer 0.5 μ L respectively as shown in SEQIDNO:3 and SEQIDNO:4, upstream primer and the downstream primer primer 0.5 μ L respectively as shown in SEQIDNO:5 and SEQIDNO:6), DNA profiling 1 μ L, moisturizing to 20 μ L.Reaction conditions: 95 DEG C of denaturation 20s; 95 DEG C of sex change 3s; 60 DEG C of annealing and extension 30s; 40 circulations.
The result of triple fluorescent quantitative PCR is as shown in table 1, and wherein, "+" represents that result is positive, and "-" represents that result is negative.
Table 1
Note: Salm represents Salmonellas, Shig represents Shigellae, and Stap represents streptococcus aureus
Embodiment 2
The present embodiment is used for the sensitivity of the inventive method, specificity and accuracy are described.
Learn from else's experience national standard method (GB/T4789.4-2010 (national food safety standard-food microbiological analysis-Salmonellas inspection), GB/T4789.5-2012 (national food safety standard-food microbiological analysis-Shigellae inspection) and GB/T4789.10-2010 (national food safety standard-food microbiological analysis-Staphylococcus aureus inspection)) to verify be that 67 parts of pork sample averages that three kinds of bacterial strains are feminine gender are divided into 7 groups, and inoculated certain density three kinds of bacterium respectively (see table 2, 7th group is blank group), and carry out microbial culture in such a way, immunity Magneto separate is caught, DNA extraction and RT-PCR detect:
(1) microbial culture
Get different pork sample 25g respectively to add in the improvement NB (add and have the glycine of 1 % by weight) of 225mL and obtain 250mL system, in 37 DEG C, cultivate 5 hours under 150rpm.
(2) immune Magneto separate is caught
By three kinds of immunomagnetic beads (samples after the amplification relative to every milliliter that the culture of acquisition obtains with preparation embodiment 1, with the weighing scale of magnetic ball, the consumption of each immunomagnetic beads is 0.8 μ g) mix and be placed on immune magnetic separation system and catch 30min at 37 DEG C.Finally obtain thalline-bead complexes suspension that final volume is 1.5mL.
(3) DNA extraction
High speed low temperature centrifugal machine (Eppendorf5804R whizzer) is used to be undertaken centrifugal at 12000rpm, at 4 DEG C by thalline-bead complexes suspension, after abandoning supernatant, bacterial genomes DNA extraction kit is used to extract genomic dna, as the DNA profiling of step (3).
(4) triple fluorescent quantitative PCR detects (RT-PCR detection)
With reference to the step (3) of embodiment 1.
Table 2
Note: mixed bacterium obtains according to Salmonella typhimurium (ATCC14028), shigella flexneri (ATCC12022) and streptococcus aureus (ATCC25923) balanced mix; Salm represents Salmonellas, and Shig represents Shigellae, and Stap represents streptococcus aureus
With reference to the relevant evaluation standard that ISO16140 compares about micro-biological process, 67 increment product, are used to the sensitivity of calculating the inventive method, specificity and accuracy.Concrete grammar is as follows: the sample of vaccination target bacterium, and the RT-PCR detected result of this object bacteria is positive, is designated as the positive; Do not have the sample of vaccination target bacterium, the RT-PCR detected result of this object bacteria is negative, is designated as feminine gender; There is no the sample of vaccination target bacterium, but the RT-PCR detected result of this object bacteria is positive, is designated as positive deviation; The sample of vaccination target bacterium, but the RT-PCR detected result of this object bacteria is negative, is designated as negative deviation.Inoculation is used to have the sample of object bacteria to account for this object bacteria RT-PCR detected result also for the ratio of positive sample carrys out the sensitivity of method for expressing; Use and do not have the sample of vaccination target bacterium to account for this object bacteria RT-PCR detected result also for the ratio of negative sample carrys out the specificity of method for expressing; The ratio that the sample that all inoculation situations are consistent with RT-PCR detected result accounts for all samples is designated as sensitivity, i.e. sensitivity=[detecting positive sum/(detecting positive sum+negative deviation sum)] × 100%, specificity=[the negative sum of detection/(detecting negative total+positive deviation sum)] × 100%, accuracy=[(detecting the negative sum of positive sum+detection)/total number of samples] × 100%.Result is as shown in table 3.
Table 3
Note:
a, SE=[PA/ (PA+ND)] × 100%; SP=[NA/ (NA+PD)] × 100%; AC=[(PA+NA)/N] × 100%
As can be seen from Table 3, only in streptococcus aureus, find a routine negative deviation in all detections, and three kinds of bacterium all there is no positive deviation to detect.
Embodiment 3
The present embodiment is used for illustrating that method of the present invention is in the practical application detecting pork sample.
The sample used in embodiment 1 is replaced with 151 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), GB/T4789.5-2012 (national food safety standard-food microbiological analysis-Shigellae inspection) and GB/T4789.10-2010 (national food safety standard-food microbiological analysis-Staphylococcus aureus inspection) to carry out tracing detection respectively.
Result shows, and GB method detects positive 2 examples of Salmonellas, and method of the present invention is also positive to the detected result of these 2 increment product, and two kinds of methods are feminine gender to the detected result of Salmonellas in all the other samples.Two kinds of methods all do not detect Shigellae.GB method detects S. aureus-positive 8 example, and the detected result of method of the present invention is 9 examples, and wherein 1 example is positive deviation: GB method is detected as feminine gender, method test positive of the present invention.
Show the detected result of 151 the fresh pork samples bought from Ge great supermarket, Beijing area, compare GB method, the inventive method all reaches 100% for the detection sensitivity of Salmonellas and Shigellae, specificity and accuracy; Although find a routine negative deviation in streptococcus aureus testing process, the detection sensitivity of streptococcus aureus, specificity and accuracy are all more than 97%.
Embodiment 4
The present embodiment is used for further illustrating the sensitivity of the inventive method.
The detection of Salmonellas, Shigellae and streptococcus aureus is carried out according to the method for embodiment 2, unlike, in pork sample, inoculate the Salmonellas of gradient dilution, Shigellae and streptococcus aureus, after microbial culture step, the final concentration of Salmonellas, Shigellae and streptococcus aureus is respectively 0.2-2000CFU/g, 0.68-6800CFU/g and 0.96-9600CFU/g (being recorded by the method for plate count).Result shows, and the detection limit of Salmonellas, Shigellae and streptococcus aureus is respectively 2.0CFU/g, 6.8CFU/g and 9.6CFU/g.
Comparative example 1
The detection of Salmonellas, Shigellae and streptococcus aureus is carried out according to the method for embodiment 4, unlike, do not carry out the step of immune Magneto separate, and directly carry out DNA extraction and multiple fluorescence quantitative PCR, result shows, and the detection limit of Salmonellas, Shigellae and streptococcus aureus is respectively 400CFU/g, 80CFU/g and 4700CFU/g.
As can be seen from the above embodiments, method of the present invention detects while can realize Salmonellas, Shigellae and streptococcus aureus quickly and easily.In addition, the result of comparing embodiment 4 and comparative example 1 can be found out, the present invention adopts immune magnetic separation technique to coordinate the method for multiple fluorescence quantitative PCR to the detection limit of Salmonellas, Shigellae and streptococcus aureus all at below 10CFU/g, and significantly lower than the detection limit being used alone multiple fluorescence quantitative PCR method, illustrate that the sensitivity of method of the present invention is 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 (10)
1. detect a method for Salmonellas, Shigellae and streptococcus aureus, the method comprises the following steps:
(1) testing sample is mixed with liquid nutrient medium, and cultivate under mixture being placed in the condition of the bacterium amplification that testing sample can be made to exist, obtain the sample after amplification;
(2) Salmonellas that may exist in the sample after utilizing immunomagnetic ca pture to increase, Shigellae and streptococcus aureus, described immunomagnetic beads is obtained by antibody and the coupling of magnetic ball;
(3) genomic dna of the thalline of catching in extraction step (1);
(4) Auele Specific Primer for Salmonellas, Shigellae and streptococcus aureus is adopted to carry out multiple fluorescence quantitative PCR to the genomic dna extracted, according to the existence of Salmonellas in the result determination testing sample of multiple fluorescence quantitative PCR, Shigellae and streptococcus aureus.
2. method according to claim 1, wherein, described antibody comprises Salmonellas polyclonal antibody, Shigellae polyclonal antibody and streptococcus aureus polyclonal antibody.
3. method according to claim 1, wherein, the particle diameter of described magnetic ball is 150-200nm.
4. according to the method in claim 1-3 described in any one, wherein, the magnetic ball relative to every milligram, the consumption >=0.1mg of described antibody.
5. method according to claim 1, wherein, the sample after the amplification relative to every milliliter, with the weighing scale of magnetic ball, the consumption of described immunomagnetic beads is 0.5-5 μ g.
6. method according to claim 1, wherein, in step (1), the condition of catching comprises: temperature is 35-38 DEG C, and the time is 25-35min.
7. method according to claim 1, wherein, described multiple fluorescence quantitative PCR is triple fluorescent quantitative PCR, and described Auele Specific Primer is the NA of Salmonellas of can increasing specifically
athe primer of the ipaH gene of gene, Shigellae and the Sa442 gene of streptococcus aureus.
8. method according to claim 7, wherein, the nucleotide sequence of the upstream primer that multiple fluorescence quantitative PCR uses, downstream primer and corresponding specificity fluorescent probe is as follows:
Upstream primer 1 is as shown in SEQIDNO:1, and downstream primer 1 is as shown in SEQIDNO:2, and specificity fluorescent probe 1 is as shown in SEQIDNO:7;
Upstream primer 2 is as shown in SEQIDNO:3, and downstream primer 2 is as shown in SEQIDNO:4, and specificity fluorescent probe 2 is as shown in SEQIDNO:8;
Upstream primer 3 is as shown in SEQIDNO:5, and downstream primer 3 is as shown in SEQIDNO:6, and specificity fluorescent probe 3 is as shown in SEQIDNO:9.
9. the method according to claim 1,7 or 8, wherein, the condition of multiple fluorescence quantitative PCR comprises: 94-96 DEG C of denaturation 18-22s; 94-96 DEG C of sex change 2.5-3.5s; 58-62 DEG C of annealing and extension 28-32s; 38-42 circulation.
10. method according to claim 1, wherein, the concentration >=2CFU/g of Salmonellas in the sample after amplification, the concentration >=6.8CFU/g of Shigellae, the concentration >=9.6CFU/g of streptococcus aureus.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410283173.9A CN105316391A (en) | 2014-06-23 | 2014-06-23 | Method of detecting salmonella, shigella and staphylococcus aureus |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107164475A (en) * | 2017-05-24 | 2017-09-15 | 昆明理工大学 | The IC LAMP detection primers group of Shigella and its application |
| CN107746893A (en) * | 2017-10-25 | 2018-03-02 | 昆明理工大学 | The IC LAMP detection primers group of salmonella and its application |
| CN108570512A (en) * | 2018-06-12 | 2018-09-25 | 福建省农业科学院畜牧兽医研究所 | Staphylococcus aureus, Escherichia coli, the primer of salmonella triple fluorescent quantitative PCR detections, probe and method foundation |
| CN108841977A (en) * | 2018-07-16 | 2018-11-20 | 中山出入境检验检疫局检验检疫技术中心 | A kind of method of pathogenic bacteria in quick measurement milk powder |
| CN110724753A (en) * | 2019-11-19 | 2020-01-24 | 武汉尚码生物科技有限公司 | Method for rapidly detecting multiple bacteria in food, liquid phase chip and kit |
| CN111206069A (en) * | 2019-10-25 | 2020-05-29 | 舟山市食品药品检验检测研究院 | Method for rapidly capturing three pathogenic bacteria in cosmetics by using nano immunomagnetic beads |
| CN115010184A (en) * | 2022-04-19 | 2022-09-06 | 北京农学院 | Staphylococcus aureus recombinase polymerase amplification detection method based on immunomagnetic beads |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107164475A (en) * | 2017-05-24 | 2017-09-15 | 昆明理工大学 | The IC LAMP detection primers group of Shigella and its application |
| CN107746893A (en) * | 2017-10-25 | 2018-03-02 | 昆明理工大学 | The IC LAMP detection primers group of salmonella and its application |
| CN108570512A (en) * | 2018-06-12 | 2018-09-25 | 福建省农业科学院畜牧兽医研究所 | Staphylococcus aureus, Escherichia coli, the primer of salmonella triple fluorescent quantitative PCR detections, probe and method foundation |
| CN108841977A (en) * | 2018-07-16 | 2018-11-20 | 中山出入境检验检疫局检验检疫技术中心 | A kind of method of pathogenic bacteria in quick measurement milk powder |
| CN111206069A (en) * | 2019-10-25 | 2020-05-29 | 舟山市食品药品检验检测研究院 | Method for rapidly capturing three pathogenic bacteria in cosmetics by using nano immunomagnetic beads |
| CN111206069B (en) * | 2019-10-25 | 2023-05-23 | 舟山市食品药品检验检测研究院 | Method for rapidly capturing three pathogenic bacteria in cosmetics by utilizing nano immunomagnetic beads |
| CN110724753A (en) * | 2019-11-19 | 2020-01-24 | 武汉尚码生物科技有限公司 | Method for rapidly detecting multiple bacteria in food, liquid phase chip and kit |
| CN115010184A (en) * | 2022-04-19 | 2022-09-06 | 北京农学院 | Staphylococcus aureus recombinase polymerase amplification detection method based on immunomagnetic beads |
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