CN109055513A - A kind of rapid detection method of salmonella - Google Patents

A kind of rapid detection method of salmonella Download PDF

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CN109055513A
CN109055513A CN201810390498.5A CN201810390498A CN109055513A CN 109055513 A CN109055513 A CN 109055513A CN 201810390498 A CN201810390498 A CN 201810390498A CN 109055513 A CN109055513 A CN 109055513A
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salmonella
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曾小敏
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Abstract

The invention discloses a kind of rapid detection method of salmonella, the step of the detection method are as follows: salmonella is inoculated into the culture medium of mixed protein peptone soybean meat field and cultivates to obtain bacteria suspension；Nitrine ethidium bromide solution is added to bacteria suspension under the conditions of being protected from light, is stood in dark, then uncaps and is placed on ice, expose, centrifugation is abandoned supernatant, is resuspended with ultrapure water, boiling water bath, is centrifuged, supernatant is DNA profiling；The salmonella specificity announced based on GeneBankinvAGene order is designed PCR primer using primer-design software；The sterile ultrapure water of DNA profiling will be extracted to dilute, carry out PCR amplification；" maximum second derivative method " is selected to be analyzed, with CP value judging result.It has the beneficial effect that detection method specificity, reproducible, increasing bacterium efficiency, sensitivity, accuracy rate height, can quickly identify the dead state of salmonella and the state bacterium that lives, testing cost are low.

Description

A kind of rapid detection method of salmonella
Technical field
The present invention relates to technical field of microbial detection more particularly to a kind of rapid detection methods of salmonella.
Background technique
In recent years, with the improvement of people's living standards, China's food hygiene situation is greatly improved, but food source Property pathogenic microorganisms prevalence break out accident and still appear in the newspapers repeatly, in food pathogenic bacteria pollution be food safety it is most important endanger because Element, and the main reason of food poisoning.In recent years, salmonella, listeria spp, Escherichia coli O 157, campylobacter Etc. food pathogenics have become the number one killer for endangering food safety.Wherein, salmonella is one of common food-borne pathogens, It is that one kind can parasitize in human and animal's enteron aisle, there is the common zoonosis of high risks to people and livestock birds health Pathogenic bacteria.The pathogenic bacteria can not only cause many animals disease, such as: white diarrhea, fowl typhoid, paratyphoid, necrotic enteritis, stream Produce etc., additionally it is possible to so that the mankind is suffered from the diseases such as typhoid fever, paratyphoid, septicemia, gastroenteritis, infectious diarrhea, causes to poison by food.Generation The food poisoning case that boundary various regions are induced by it every year ranks first, it is not only seriously endangered human health, and also will cause huge Huge economic loss.
Currently, the detection of salmonella mainly has bacterium isolated culture and PCR method, the former marks as the gold in laboratory Standard, needs to increase repeatedly bacterium, bacterium colony separation and a variety of biochemistry, serology identification experiment etc., complicated for operation, time-consuming, and the latter is main DNA nucleic acid based on microorganism carries out augmentation detection, and bacterium either all has DNA core under viable bacteria or dead bacterium state Acid, so as to cause pathogenic bacteria living are actually had no in the positive sample of some PCR detection, this is detected with existing traditional pathogenic bacteria Standard is inconsistent, i.e., it is micro- that existing PCR detection method and standard based on DNA cannot distinguish state living and dead state present in sample Biology becomes the bottleneck using round pcr detection pathogenic bacteria.It is based on this reason, the relevant criterion promulgated at present In, round pcr is not used as routine diagnostic method, and is used only to quickly reflect to the pathogenic bacteria being separately cultured It is fixed, the pathogenic bacteria in sample cannot directly be detected, this is all there's no one who doesn't or isn't such in developed countries such as the U.S..It therefore, is adaptation Cargo speeds passage through customs the needs tested and put in inlet and outlet inspection and quarantine, shortens detection time, increases existing molecular Biological Detection side The accuracy of method avoids the unnecessary wasting of resources, workload is effectively reduced, and develops a kind of quick detection side of salmonella Method is imperative.
The prior art such as Authorization Notice No. is the Chinese invention patent of 104278102 B of CN, discloses a kind of salmonella Quadruple- PCR detection method.The detection method is by hns gene (152bp), fliM gene (418bp), invA gene (681bp) 4 pairs of primers for expanding this 4 genetic fragments are placed on one as four gene targets of PCR detection with Hut gene (495bp) A PCR reaction system establishes the Quadruple- PCR detection that salmonella is detected directly from sample by parameters adjusting and optimizing Method.Different from normal PCR, on the one hand which has the characteristics that quadruple insurance, improves the accuracy of detection and reliable Property, on the other hand its virulence factor encoding gene structure variation can also be analyzed, to grasp its variation trend, be played The effect of double gain.But this method cannot distinguish state living and dead state microorganism present in sample.
Summary of the invention
The purpose of the present invention is to provide a species specificity, reproducible, increasing bacterium efficiency, sensitivity, accuracy rate height, can be fast Speed identifies the dead state of salmonella and state bacterium living, the rapid detection method of the low work state salmonella of testing cost.
The present invention in background technique aiming at the problem that mentioning, the technical solution taken are as follows:
A kind of rapid detection method of salmonella, including Zengjing Granule, DNA profiling preparation, the design of primer and synthesis, PCR Reaction system is established, result judges, the specific steps of which are as follows:
Zengjing Granule: salmonella is inoculated into the culture medium of mixed protein peptone soybean meat field, the Zengjing Granule 6- at 35-40 DEG C 10h is to get bacteria suspension, and spare, above-mentioned mixed protein peptone is the tryptone that ratio is 1:0.8-1.2 and the mixing of fish meal protein peptone Peptone, the culture medium can provide richer carbon source, nitrogen source, inorganic salts etc. for Salmonella growth, and enriching effect is significant, makes Salmonella can reach logarithmic growth phase as early as possible, at this time bacterial structure stablize, metabolism, physiological property than more consistent and good, Defence capability is most strong, and it is maximum that nitrine ethidium bromide acts on difficulty；
DNA profiling preparation: taking culture bacteria suspension 1ml, and the nitrine ethidium bromide solution that concentration is 0.05mg/mL is added under the conditions of being protected from light (EMA), EMA final concentration is made to reach 2.0-4.0mg/L, slight oscillatory mixes, and stands 14-16min in dark, then takes bacterium solution Out, it apart from fluorescent tube 14-16cm, uncaps and is placed on ice, persistently exposing 5-10min with 600-700W tungsten halogen lamp makes nitrine ethidium bromide light Sample is taken out and is placed on revolving speed to be centrifuged 2-5min in the centrifuge of 10000-15000r/min, abandoned supernatant, use ultrapure water by solution It is resuspended 1 time, then boiling water bath 18-22min, then is placed in that temperature is 0-5 DEG C, revolving speed is in the centrifuge of 10000-15000r/min It is centrifuged 4-6min, supernatant is DNA profiling, spare, DNA insert type fluorescent dye EMA novel as one kind in the step, It can be inserted into cell wall or the incomplete dead cell DNA of cell membrane, when activating EMA with high-intensitive visible light exposure, meeting Active nitrene intermediate is generated, intermediate and the close covalent bond of DNA form compound and lose it and continue to expand energy Power, the EMA nitrene reactive intermediate to dissociate in system forms azanol in conjunction with the water in system, so that EMA be made to be consumed completely Fall, will not influence subsequent DNA cloning, and the complete cell wall of living cells and cell membrane can prevent the entrance of EMA dyestuff, due to The formation of azanol effectively prevents the influence to viable bacteria DNA cloning, directly detects so as to not have to separate viable bacteria and dead bacterium Viable bacteria, while step DNA is dissolved in ultrapure water, obtained DNA can be directly as double fluorescent PCR amplification template, and this is mentioned DNA profiling method is taken not expend reagent, easy to operate, direct, quick, acquisition amount of DNA is more, and purity also can reach test and want It asks；
The design and synthesis of primer: the salmonella specificity announced based on GeneBankinvAGene order is set using primer Meter software Primer Premier 5.0 is designed PCR primer, specific amplification salmonella sequence, primer sequence are as follows: Forward primer Salm F:5 '-ACGTTCGGGCAATTCGTT-3 ', reverse primer Salm R:5 '-CGGCAATAGCGTCACCTT- 3 ', above-mentioned primer length is appropriate, the sequence close complementary with template, be difficult to be formed between primer and primer stable dimer and Hairpin structure, extremely low in the efficiency of initiation of mismatch site, complementary series is not present in its own, itself is difficult to form hairpin structure, With the sequence of template can close complementary, high sensitivity, amplification efficiency are high, and it is anti-to be conducive to amplification less than 74 DEG C for elongating temperature The progress answered；
PCR reaction system is established: DNA profiling will be extracted with sterile ultrapure water and do 10 times of gradient dilutions, carry out PCR amplification, it is above-mentioned PCR reacts 25 μ L of total volume, includes Premix Ex Taq (Probe qPCR) 12.5 μ L, each 0.5 μ L of 10 μM of primers, ddH2O 7.5 μ L, 1 μ L, PCR response parameter of DNA profiling: 37 DEG C of 5min, 94 DEG C of initial denaturation 3min, 94 DEG C of denaturation 20s, 60 DEG C of annealing extend 45s(detects fluorescence signal), 40 circulations；
As a result judge: according to LightCycler480 fluorescence quantitative PCR instrument operation manual, " maximum second derivative method " being selected to carry out Analysis, with CP value (crossing point, refer to amplification curve generate fluorescence jumping when cycle-index) judging result, CP value≤ 35 are judged to the positive, and value >=40 CP are judged to feminine gender, and CP value should suitably increase template quantity and reform test between 35-40, the detection Method specificity, reproducible, high sensitivity, can quickly identify the dead state of salmonella and state bacterium living, and testing result meets me The existing conventional bacteria isolated culture examination criteria of state, application value with higher and promotion prospect.
Preferably, No. 5 cholate and 2.8-3.2g/L in Zengjing Granule step in culture medium containing 3.0-3.2g/L Cysteine, levo form and be 100:0.35-0.4, No. 5 cholate and half Guang in culture medium with the ratio of d-isomer in cysteine The addition of propylhomoserin can effectively be enriched with salmonella, inhibit the growth of nonsalmonella, play the role of preferable selective culture, mention High increasing bacterium efficiency, to be separately separated out salmonella for subsequent detection, the final inspection for improving Salmonella in Food The rate that tests the speed and accuracy rate；Simultaneously levo form and the dead bacterium of salmonella is enabled to the special proportion of d-isomer in cysteine The extent of damage of membrane structure increases, so that subsequent EMA can play work quickly through the membrane structure of dead bacterium cell With raising detection rates and and accuracy rate.
Preferably, containing 0.012-0.016% deoxysodium cholate in the extraction step of strain gene group DNA in EMA solution With ‰ carnitine of 0.022-0.025, the presence of deoxysodium cholate and carnitine can significantly change dead bacterium cell membrane table in EMA solution Surface properties, the extent of damage for increasing dead bacterium membrane structure, enable EMA quickly through the membrane structure of dead bacterium cell, in turn With DNA tertiary structure covalent bond therein, the DNA of dead bacterium is made to lose continuation amplification ability, effectively eliminates part in result Film cracks insufficient dead bacterium false positive phenomenon caused by result, the final accuracy rate for improving state Detection Methods of Salmonella living and Rate, and the dosage of EMA can be reduced, and then reduce testing cost.
Compared with prior art, the invention has the benefit that
1) detection method specificity, reproducible, sensitivity, accuracy rate are high, can quickly identify the dead state of salmonella and State bacterium living, testing cost is low, and testing result meets the existing conventional bacteria isolated culture examination criteria in China, has higher Application value and promotion prospect；
2) detection method can effectively be enriched with salmonella with culture medium, inhibit the growth of nonsalmonella, play preferably Selectivity culture effect, improve and increase bacterium efficiency, while enabling to the impaired journey of the membrane structure of the dead bacterium of salmonella Degree increases so that subsequent EMA can play a role quickly through the membrane structure of dead bacterium cell, improve detection rates and and Accuracy rate；
3) detection method loses continuation amplification ability with EMA solution energy dead cell DNA, and can effectively prevent to viable bacteria The influence of DNA cloning directly detects viable bacteria so as to not have to separate viable bacteria and dead bacterium；
4) detection method effectively eliminates part film in result with EMA solution and cracks insufficient dead bacterium caused by result False positive phenomenon, the final accuracy rate and rate for improving state Detection Methods of Salmonella living, and the dosage of EMA can be reduced, into And reduce testing cost.
Specific embodiment
The present invention program is described further below by embodiment:
Embodiment 1:
A kind of rapid detection method of salmonella, including Zengjing Granule, DNA profiling preparation, the design of primer and synthesis, PCR Reaction system is established, result judges, the specific steps of which are as follows:
1) Zengjing Granule: salmonella being inoculated into the culture medium of mixed protein peptone soybean meat field, the Zengjing Granule 6h at 40 DEG C, Spare up to bacteria suspension, above-mentioned mixed protein peptone is the tryptone that ratio is 1:1.2 and fish meal protein peptone mixed protein peptone, The culture medium can provide richer carbon source, nitrogen source, inorganic salts etc. for Salmonella growth, and enriching effect is significant, so that sramana Salmonella can reach logarithmic growth phase as early as possible, and bacterial structure is stablized at this time, and metabolism, physiological property are than more consistent and good, defence energy Power is most strong, and it is maximum that nitrine ethidium bromide acts on difficulty；
2) prepared by DNA profiling: taking culture bacteria suspension 1ml, it is molten that the nitrine ethidium bromide that concentration is 0.05mg/mL is added under the conditions of being protected from light Liquid (EMA) makes EMA final concentration reach 2.0mg/L, and slight oscillatory mixes, and stands 16min in dark, then takes out bacterium solution, away from From fluorescent tube 14cm, uncaps and be placed on ice, persistently exposing 5min with 700W tungsten halogen lamp makes nitrine ethidium bromide photodissociation, after sample is taken out It is placed in the centrifuge that revolving speed is 15000r/min and is centrifuged 2min, abandon supernatant, be resuspended 1 time with ultrapure water, then boiling water bath 22min, then it is placed in that temperature is 0 DEG C, revolving speed is that 4min is centrifuged in the centrifuge of 15000r/min, supernatant is DNA profiling, standby With EMA can be inserted into cell wall or cell membrane is incomplete as a kind of novel DNA insert type fluorescent dye in the step In dead cell DNA, when activating EMA with high-intensitive visible light exposure, active nitrene intermediate can be generated, intermediate with The close covalent bond of DNA forms compound and it is made to lose continuation amplification ability, the EMA nitrene reactive intermediate to dissociate in system Azanol is formed in conjunction with the water in system, so that EMA be made to be consumed completely, will not influence subsequent DNA cloning, and living cells is complete Whole cell wall and cell membrane can prevent the entrance of EMA dyestuff, due to the formation of azanol, effectively prevent expanding viable bacteria DNA The influence of increasing directly detects viable bacteria so as to not have to separate viable bacteria and dead bacterium, while step DNA is dissolved in ultrapure water, Obtained DNA can be directly as double fluorescent PCR amplification template, and the extraction DNA profiling method does not expend reagent, operation letter Single, direct, quick, acquisition amount of DNA is more, and purity also can reach test requirements document；
3) design and synthesis of primer: the salmonella specificity announced based on GeneBankinvAGene order uses primer Design software Primer Premier 5.0 is designed PCR primer, specific amplification salmonella sequence, primer sequence Are as follows: forward primer Salm F:5 '-ACGTTCGGGCAATTCGTT-3 ', reverse primer Salm R:5 '- CGGCAATAGCGTCACCTT-3 ', above-mentioned primer length is appropriate, the sequence close complementary with template, difficult between primer and primer Extremely low in the efficiency of initiation of mismatch site to form stable dimer and hairpin structure, complementary series is not present in its own, from Body is difficult to form hairpin structure, with the sequence of template can close complementary, high sensitivity, amplification efficiency are high, and elongating temperature is small In 74 DEG C, be conducive to the progress of amplified reaction；
4) PCR reaction system is established: DNA profiling will be extracted with sterile ultrapure water and do 10 times of gradient dilutions, carry out PCR amplification, on State PCR reaction 25 μ L of total volume, include Premix Ex Taq (Probe qPCR) 12.5 μ L, each 0.5 μ L of 10 μM of primers, ddH27.5 μ L of O, 1 μ L, PCR response parameter of DNA profiling: 37 DEG C of 5min, 94 DEG C of initial denaturation 3min, 94 DEG C of denaturation 20s, 60 DEG C are moved back Fire extends 45s(and detects fluorescence signal), 40 circulations；
5) result judges: according to LightCycler480 fluorescence quantitative PCR instrument operation manual, select " maximum second derivative method " into Row analysis, with CP value (crossing point refers to cycle-index when amplification curve generates fluorescence jumping) judging result, CP value ≤ 35 are judged to the positive, and value >=40 CP are judged to feminine gender, and CP value should suitably increase template quantity and reform test between 35-40, the inspection Survey method specificity, reproducible, high sensitivity, can quickly identify the dead state of salmonella and state bacterium living, and testing result meets The existing conventional bacteria isolated culture examination criteria in China, application value with higher and promotion prospect.
The cysteine of No. 5 cholate and 2.8g/L in above-mentioned Zengjing Granule step in culture medium containing 3.2g/L, half Guang Levo form and be 100:0.4 with the ratio of d-isomer in propylhomoserin, the addition of No. 5 cholate and cysteine can be effectively rich in culture medium Collect salmonella, inhibit the growth of nonsalmonella, plays the role of preferable selective culture, improve and increase bacterium efficiency, so as to Salmonella is separately separated out for subsequent detection, the final detection rates and accuracy rate for improving Salmonella in Food；Together When cysteine in levo form and enable to the impaired of the membrane structure of the dead bacterium of salmonella with the special proportion of d-isomer Degree increases so that subsequent EMA can play a role quickly through the membrane structure of dead bacterium cell, improve detection rates and And accuracy rate.
Contain 0.012% deoxysodium cholate and 0.025 ‰ meat in the extraction step of above-mentioned bacterial strains genomic DNA in EMA solution Alkali, the presence of deoxysodium cholate and carnitine can significantly change that dead bacterium cell membrane surface property, to increase dead bacterium thin in EMA solution The extent of damage of after birth structure enables EMA quickly through the membrane structure of dead bacterium cell, so with DNA three-level knot therein Structure covalent bond, makes the DNA of dead bacterium lose continuation amplification ability, effectively eliminates part film in result and cracks insufficient dead bacterium pair As a result false positive phenomenon caused by, the final accuracy rate and rate for improving state Detection Methods of Salmonella living, and EMA can be reduced Dosage, and then reduce testing cost.
Embodiment 2:
A kind of rapid detection method of salmonella, including Zengjing Granule, DNA profiling preparation, the design of primer and synthesis, PCR Reaction system is established, result judges, the specific steps of which are as follows:
1) Zengjing Granule: salmonella is inoculated into the culture medium of mixed protein peptone soybean meat field, the Zengjing Granule at 35 DEG C 10h is to get bacteria suspension, and spare, above-mentioned mixed protein peptone is the tryptone that ratio is 1:0.8 and fish meal protein peptone mixed protein Peptone；
2) prepared by DNA profiling: taking culture bacteria suspension 1ml, it is molten that the nitrine ethidium bromide that concentration is 0.05mg/mL is added under the conditions of being protected from light Liquid (EMA) makes EMA final concentration reach 4.0mg/L, and slight oscillatory mixes, and stands 14min in dark, then takes out bacterium solution, away from It from fluorescent tube 16cm, uncaps and is placed on ice, persistently exposing 10min with 600W tungsten halogen lamp makes nitrine ethidium bromide photodissociation, and sample is taken out It is placed in the centrifuge that revolving speed is 10000r/min and is centrifuged 5min, abandon supernatant, be resuspended 1 time with ultrapure water, then boiling water bath 18min, then it is placed in that temperature is 5 DEG C, revolving speed is that 6min is centrifuged in the centrifuge of 10000r/min, supernatant is DNA profiling, standby With；
3) design and synthesis of primer: the salmonella specificity announced based on GeneBankinvAGene order uses primer Design software Primer Premier 5.0 is designed PCR primer, specific amplification salmonella sequence, primer sequence Are as follows: forward primer Salm F:5 '-ACGTTCGGGCAATTCGTT-3 ', reverse primer Salm R:5 '- CGGCAATAGCGTCACCTT-3'；
4) PCR reaction system is established: DNA profiling will be extracted with sterile ultrapure water and do 10 times of gradient dilutions, carry out PCR amplification, on State PCR reaction 25 μ L of total volume, include Premix Ex Taq (Probe qPCR) 12.5 μ L, each 0.5 μ L of 10 μM of primers, ddH27.5 μ L of O, 1 μ L, PCR response parameter of DNA profiling: 37 DEG C of 5min, 94 DEG C of initial denaturation 3min, 94 DEG C of denaturation 20s, 60 DEG C are moved back Fire extends 45s(and detects fluorescence signal), 40 circulations；
5) result judges: according to LightCycler480 fluorescence quantitative PCR instrument operation manual, select " maximum second derivative method " into Row analysis, with CP value (crossing point refers to cycle-index when amplification curve generates fluorescence jumping) judging result, CP value ≤ 35 are judged to the positive, and value >=40 CP are judged to feminine gender, and CP value should suitably increase template quantity and reform test between 35-40.
The cysteine of No. 5 cholate and 3.2g/L in above-mentioned Zengjing Granule step in culture medium containing 3.0g/L, half Guang Levo form and be 100:0.35 with the ratio of d-isomer in propylhomoserin.
Contain 0.016% deoxysodium cholate and 0.022 ‰ meat in the extraction step of above-mentioned bacterial strains genomic DNA in EMA solution Alkali.
Embodiment 3:
A kind of rapid detection method of salmonella, including Zengjing Granule, DNA profiling preparation, the design of primer and synthesis, PCR Reaction system is established, result judges, the specific steps of which are as follows:
1) Zengjing Granule: salmonella being inoculated into the culture medium of mixed protein peptone soybean meat field, the Zengjing Granule 8h at 37 DEG C, Spare up to bacteria suspension, above-mentioned mixed protein peptone is the tryptone that ratio is 1:1 and fish meal protein peptone mixed protein peptone；
2) prepared by DNA profiling: taking culture bacteria suspension 1ml, it is molten that the nitrine ethidium bromide that concentration is 0.05mg/mL is added under the conditions of being protected from light Liquid (EMA) makes EMA final concentration reach 3.0mg/L, and slight oscillatory mixes, and stands 15min in dark, then takes out bacterium solution, away from From fluorescent tube 15cm, uncaps and be placed on ice, persistently exposing 8min with 650W tungsten halogen lamp makes nitrine ethidium bromide photodissociation, after sample is taken out It is placed in the centrifuge that revolving speed is 12000r/min and is centrifuged 4min, abandon supernatant, be resuspended 1 time with ultrapure water, then boiling water bath 20min, then it is placed in that temperature is 2 DEG C, revolving speed is that 5min is centrifuged in the centrifuge of 12000r/min, supernatant is DNA profiling, standby With；
3) design and synthesis of primer: the salmonella specificity announced based on GeneBankinvAGene order uses primer Design software Primer Premier 5.0 is designed PCR primer, specific amplification salmonella sequence, primer sequence Are as follows: forward primer Salm F:5 '-ACGTTCGGGCAATTCGTT-3 ', reverse primer Salm R:5 '- CGGCAATAGCGTCACCTT-3'；
4) PCR reaction system is established: DNA profiling will be extracted with sterile ultrapure water and do 10 times of gradient dilutions, carry out PCR amplification, on State PCR reaction 25 μ L of total volume, include Premix Ex Taq (Probe qPCR) 12.5 μ L, each 0.5 μ L of 10 μM of primers, ddH27.5 μ L of O, 1 μ L, PCR response parameter of DNA profiling: 37 DEG C of 5min, 94 DEG C of initial denaturation 3min, 94 DEG C of denaturation 20s, 60 DEG C are moved back Fire extends 45s(and detects fluorescence signal), 40 circulations；
5) result judges: according to LightCycler480 fluorescence quantitative PCR instrument operation manual, select " maximum second derivative method " into Row analysis, with CP value (crossing point refers to cycle-index when amplification curve generates fluorescence jumping) judging result, CP value ≤ 35 are judged to the positive, and value >=40 CP are judged to feminine gender, and CP value should suitably increase template quantity and reform test between 35-40.
The cysteine of No. 5 cholate and 3.0g/L in above-mentioned Zengjing Granule step in culture medium containing 3.1g/L, half Guang Levo form and be 100:0.38 with the ratio of d-isomer in propylhomoserin.
Contain 0.015% deoxysodium cholate and 0.024 ‰ meat in the extraction step of above-mentioned bacterial strains genomic DNA in EMA solution Alkali.
Embodiment 4:
A kind of rapid detection method of salmonella, including Zengjing Granule, DNA profiling preparation, the design of primer and synthesis, PCR Reaction system is established, result judges, the specific steps of which are as follows:
1) Zengjing Granule: salmonella being inoculated into the culture medium of mixed protein peptone soybean meat field, the Zengjing Granule 8h at 37 DEG C, Spare up to bacteria suspension, above-mentioned mixed protein peptone is the tryptone that ratio is 1:1 and fish meal protein peptone mixed protein peptone；
2) prepared by DNA profiling: taking culture bacteria suspension 1ml, it is molten that the nitrine ethidium bromide that concentration is 0.05mg/mL is added under the conditions of being protected from light Liquid (EMA) makes EMA final concentration reach 3.0mg/L, and slight oscillatory mixes, and stands 15min in dark, then takes out bacterium solution, away from From fluorescent tube 15cm, uncaps and be placed on ice, persistently exposing 8min with 650W tungsten halogen lamp makes nitrine ethidium bromide photodissociation, after sample is taken out It is placed in the centrifuge that revolving speed is 12000r/min and is centrifuged 4min, abandon supernatant, be resuspended 1 time with ultrapure water, then boiling water bath 20min, then it is placed in that temperature is 2 DEG C, revolving speed is that 5min is centrifuged in the centrifuge of 12000r/min, supernatant is DNA profiling, standby With；
3) design and synthesis of primer: the salmonella specificity announced based on GeneBankinvAGene order uses primer Design software Primer Premier 5.0 is designed PCR primer, specific amplification salmonella sequence, primer sequence Are as follows: forward primer Salm F:5 '-ACGTTCGGGCAATTCGTT-3 ', reverse primer Salm R:5 '- CGGCAATAGCGTCACCTT-3'；
4) PCR reaction system is established: DNA profiling will be extracted with sterile ultrapure water and do 10 times of gradient dilutions, carry out PCR amplification, on State PCR reaction 25 μ L of total volume, include Premix Ex Taq (Probe qPCR) 12.5 μ L, each 0.5 μ L of 10 μM of primers, ddH27.5 μ L of O, 1 μ L, PCR response parameter of DNA profiling: 37 DEG C of 5min, 94 DEG C of initial denaturation 3min, 94 DEG C of denaturation 20s, 60 DEG C are moved back Fire extends 45s(and detects fluorescence signal), 40 circulations；
5) result judges: according to LightCycler480 fluorescence quantitative PCR instrument operation manual, select " maximum second derivative method " into Row analysis, with CP value (crossing point refers to cycle-index when amplification curve generates fluorescence jumping) judging result, CP value ≤ 35 are judged to the positive, and value >=40 CP are judged to feminine gender, and CP value should suitably increase template quantity and reform test between 35-40.
The cysteine of No. 5 cholate and 3.0g/L in above-mentioned Zengjing Granule step in culture medium containing 3.1g/L, half Guang Levo form and be 100:0.38 with the ratio of d-isomer in propylhomoserin.
Contain 0.015% deoxysodium cholate and 0.024 ‰ meat in the extraction step of above-mentioned bacterial strains genomic DNA in EMA solution Alkali, in carnitine containing 2.36% dextrorotation carnitine, the dextrorotation carnitine that above-mentioned carnitine contains special proportion can accelerate the photodissociation of EMA De- rate improves the dispersibility of reactive intermediate containing nitrene in water, and then sufficiently activates and form azanol in conjunction with water and inactivate, It inactivates EMA thoroughly no longer in conjunction with DNA molecular, avoids influence of the EMA to the extracting genome DNA amount of state salmonella living, Guarantee the extracted amount of DNA, so that the DNA cloning of state salmonella living is gone on smoothly, it is final to improve the sensitive of detection method Degree and accuracy rate.
Embodiment 5:
Viable bacteria and dead bacterial examination are surveyed
Prepare salmonella bacteria suspension and dead bacterium bacteria suspension respectively, test group using the detection method of embodiment 3 and embodiment 4 into Row detection, 1 detection method of control group is without EMA processing, other steps and embodiment 3 are identical, the EMA of 2 detection method of control group In do not contain deoxysodium cholate and carnitine, other steps and embodiment 3 are identical, and control group 3 is using the existing traditional bacterium in China point From training status method SN/T 0973-2010 " inlet and outlet meat, meat products and other Detection Methods of Salmonella in Food " and Inspection and quarantine professional standard SN/T 1870-2007 " pathogenic bacteria detection method real-time PCR methodology in food " is detected.
The result shows that test group and control group testing result are consistent for salmonella viable bacteria, it is detection sun Property；There is phase reverse-examination in bacterium dead for salmonella, test group, 3 testing result of control group and SN/T 0973-2010 testing result It surveys as a result, and control group 1 and SN/T 0973-2010 testing result (pathogenic bacteria are not detected) are consistent.Illustrate adding for EMA Enter to identify the dead state of salmonella and state bacterium living.
Embodiment 6:
The detection of artificial contamination's poultry sample
The diseased regions samples such as liver spleen, ovary, fallopian tubal are acquired from the illness poultry of clinically doubtful bacterium infection, are received altogether Collect 60 parts of samples.
The processing of clinical sample: it from the internal organs such as liver, spleen, ovary, the fallopian tubal for taking 25g to be detected chicken are clinically collected into, grinds Sample is added to sterile increasing bacterium bag after broken, is detected using 3 method of embodiment, is then verified using conventional method, ties The fruit goodness of fit is 100%.Illustrate that the detection method accuracy rate of embodiment 3 is high.
Routine operation in operating procedure of the invention is well known to those skilled in the art, herein without repeating.
Technical solution of the present invention is described in detail in embodiment described above, it should be understood that the above is only For specific embodiments of the present invention, it is not intended to restrict the invention, all any modifications made in spirit of the invention, Supplement or similar fashion substitution etc., should all be included in the protection scope of the present invention.

Claims (9)

1. a kind of rapid detection method of salmonella, including Zengjing Granule, DNA profiling preparation, the design of primer and synthesis, PCR reaction system is established, result judges, it is characterised in that: the Zengjing Granule step are as follows: salmonella is inoculated into egg mix Zengjing Granule is carried out in the culture medium of white peptone soybean meat field to get bacteria suspension, and No. 5 cholate and half Guang ammonia are contained in the culture medium Acid.
2. a kind of rapid detection method of salmonella according to claim 1, it is characterised in that: contain in the culture medium Have the cysteine of No. 5 cholate and 2.8-3.2g/L of 3.0-3.2g/L, in the cysteine levo form and with d-isomer Ratio is 100:0.35-0.4.
3. a kind of rapid detection method of salmonella according to claim 1, it is characterised in that: the mixed protein peptone For ratio be 1:0.8-1.2 tryptone and fish meal protein peptone mixed protein peptone.
4. a kind of rapid detection method of salmonella according to claim 1, it is characterised in that: the DNA profiling system Standby step are as follows: take culture bacteria suspension, nitrine ethidium bromide solution (EMA) is added under the conditions of being protected from light, slight oscillatory mixes, quiet in dark 14-16min is set, then takes out bacterium solution, apart from fluorescent tube 14-16cm, uncaps and is placed on ice, persistently exposed with 600-700W tungsten halogen lamp Light 5-10min makes nitrine ethidium bromide photodissociation, is centrifuged after sample is taken out, and abandons supernatant, is resuspended 1 time with ultrapure water, then boiling water bath 18-22min, centrifugation, supernatant is DNA profiling.
5. a kind of rapid detection method of salmonella according to claim 4, it is characterised in that: the bacteria suspension is 1ml, EMA solution concentration are 0.05mg/mL, the final concentration of 2.0-4.0mg/L of EMA in bacteria suspension.
6. a kind of rapid detection method of salmonella according to claim 4, it is characterised in that: the strain gene group Contain ‰ carnitine of 0.012-0.016% deoxysodium cholate and 0.022-0.025 in the extraction step of DNA in EMA solution.
7. a kind of rapid detection method of salmonella according to claim 1, it is characterised in that: the design of the primer With synthesis step are as follows: the salmonella specificity announced based on GeneBankinvAGene order uses primer-design software Primer Premier 5.0 is designed PCR primer, specific amplification salmonella sequence, primer sequence are as follows: forward direction is drawn Object Salm F:5 '-ACGTTCGGGCAATTCGTT-3 ', reverse primer Salm R:5 '-CGGCAATAGCGTCACCTT-3 '.
8. a kind of rapid detection method of salmonella according to claim 1, it is characterised in that: the PCR reactant It is establishment step are as follows: DNA profiling will be extracted with sterile ultrapure water and do 10 times of gradient dilutions, carry out PCR amplification, above-mentioned PCR reaction 25 μ L of total volume includes Premix Ex Taq (Probe qPCR) 12.5 μ L, each 0.5 μ L of 10 μM of primers, ddH2O 7.5μL、 1 μ L, PCR response parameter of DNA profiling: 37 DEG C of 5min, 94 DEG C of initial denaturation 3min, 94 DEG C of denaturation 20s, 60 DEG C of annealing extend 45s(inspection Survey fluorescence signal), 40 circulations.
9. a kind of rapid detection method of salmonella according to claim 1, it is characterised in that: the result judgement step Suddenly are as follows: according to LightCycler480 fluorescence quantitative PCR instrument operation manual, " maximum second derivative method " is selected to be analyzed, with CP value (crossing point refers to cycle-index when amplification curve generates fluorescence jumping) judging result, value≤35 CP are judged to The positive, value >=40 CP are judged to feminine gender, and CP value should suitably increase template quantity and reform test between 35-40.
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Application publication date: 20181221