CN109055514A - Single rapid detection method for increasing listeria spp viable bacteria - Google Patents
Single rapid detection method for increasing listeria spp viable bacteria Download PDFInfo
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- CN109055514A CN109055514A CN201810390524.4A CN201810390524A CN109055514A CN 109055514 A CN109055514 A CN 109055514A CN 201810390524 A CN201810390524 A CN 201810390524A CN 109055514 A CN109055514 A CN 109055514A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
The invention discloses single rapid detection method for increasing listeria spp viable bacteria, the step of the detection method are as follows: single increase in listeria spp inoculation trypticase soy agar culture medium is cultivated to obtain bacteria suspension；Culture bacteria suspension is taken, nitrine ethidium bromide solution is added under the conditions of being protected from light, is stood in dark, takes out exposure, supernatant is abandoned in centrifugation, is resuspended 1 time with ultrapure water；Boiling waterbath DNA, centrifugation, supernatant is DNA profiling；The Listeria monocytogenes announced based on GeneBankhlyAGene order is designed PCR primer using primer-design software Primer Premier 5.0；DNA profiling will be extracted and carry out PCR amplification, 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 single increasing dead state of listeria spp and the state bacterium that lives, testing cost are low.
Description
Technical field
The present invention relates to technical field of microbial detection, more particularly to single quick detection side for increasing listeria spp viable bacteria
Method.
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, single listeria spp, listeria spp, Escherichia coli O 157, curved of increasing
The food pathogenics such as curved bar bacterium have become the number one killer for endangering food safety.Wherein, single listeria spp that increases is Listeria
One kind of bacterium, single listeria spp that increases is a kind of saprophytic mattress being seen everywhere in nature, with dead and rotting organic
Object is food, and most food (meat, eggs, birds, marine product, dairy products, vegetables) are all likely to become the pollution pair of the bacterium
As.The mankind eat this bacterium pollution food after, be easy to get listeria spp disease, be mainly shown as: osteomyelitis, meningitis, myocarditis,
Leukaemia and pregnant woman's miscarriage etc., average mortality reaches 20-30%, (old man, newborn child, pregnant woman, slow in hypoimmunity crowd
Std patient) in more up to 70%, considerably beyond other all common food-borne pathogens.Single master for increasing listeria spp
Want morbid substance to have: hemolysin O is a kind of basic virulence factor of the bacterium, byhlyGene coding, main function are to mediate carefully
Bacterium dissolution；Phosphatidase, phosphatidylinositol phospholipase C (plcA) bacterium can be assisted to escape from primary phagosome, phosphatidyl gallbladder
Alkali phosphatidase (plcB) spread bacterium between host；PrfA albumen is a kind of important transcription factor, in a variety of virulence genes
Play key regulator in equipotential expression, will lead to Strain Virulence forfeiture after missing；Other morbid substances,inlA、actA、iapAlso it is important virulence gene, it is closely related with the invasiveness of bacterium, locomitivity and bacterial virulence respectively.
In recent years, more and more for single research for increasing listeria spp detection method, main includes tradition separation identification
Method, immunological method, microorganism automated detection system, nucleic acid detection method etc., detection method is gradually towards more acurrate, more
Sensitive, easier direction is developed.Currently, it is single increase the common detection method of listeria spp mainly have bacterium isolated culture and
PCR method, the former goldstandard as laboratory need to increase repeatedly bacterium, bacterium colony separation and a variety of biochemical, serology identification experiments
Deng complicated for operation, time-consuming, the DNA nucleic acid that the latter is based primarily upon microorganism carries out augmentation detection, and bacterium is either in viable bacteria
Or all there is DNA nucleic acid under dead bacterium state, so as to cause living cause a disease actually is had no in the positive sample of some PCR detection
Bacterium, this is inconsistent with existing traditional pathogenic bacteria examination criteria, i.e., existing PCR detection method and standard based on DNA cannot
State living and dead state microorganism present in sample are distinguished, the bottleneck using round pcr detection pathogenic bacteria is become.It is based on this
Reason, in the relevant criterion promulgated at present, round pcr is not used as routine diagnostic method, and is used only to having divided
Rapid identification is carried out from cultured pathogenic bacteria, the pathogenic bacteria in sample cannot directly be detected, this is in prosperities such as the U.S.
Country is all there's no one who doesn't or isn't such.Therefore, it speeds passage through customs the needs tested and put to adapt to cargo in inlet and outlet inspection and quarantine, when shortening detection
Between, increase the accuracy of existing molecular biology for detection, avoids the unnecessary wasting of resources, workload is effectively reduced, grind
It is imperative to study carefully single rapid detection method for increasing listeria spp viable bacteria out.
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 single increasing dead state of listeria spp and state bacterium living, and the low work state list of testing cost increases the quick detection side of listeria spp
Method.
The present invention in background technique aiming at the problem that mentioning, the technical solution taken are as follows:
Single rapid detection method for increasing listeria spp viable bacteria, specifically includes the following steps:
Step 1: single listeria spp that increases being inoculated into trypticase soy agar culture medium, the Zengjing Granule 22- at 33-37 DEG C
25h is to get bacteria suspension, spare, half Guang ammonia of No. 5 cholate and 2.8-3.2g/L in above-mentioned culture medium containing 2.6-3.0g/L
Acid, levo form and be 100:0.35-0.4 with the ratio of d-isomer in cysteine, No. 5 cholate and cysteine in culture medium
Addition can effectively be enriched with single increasing listeria spp, inhibit non-single growth for increasing listeria spp, play preferable selective culture
Effect improves and increases bacterium efficiency, is used for subsequent detection to be separately separated out single listeria spp that increases, final to improve in food
Single detection rates and accuracy rate for increasing listeria spp；Levo form and can with the special proportion of d-isomer in cysteine simultaneously
So that the extent of damage of single membrane structure for increasing the dead bacterium of listeria spp increases, so that subsequent EMA can be quickly through
The membrane structure of dead bacterium cell and play a role, improve detection rates and and accuracy rate, the culture medium can increase listeria spp to be single
Growth provides richer carbon source, nitrogen source, inorganic salts etc., and enriching effect is significant, and single listeria spp that increases is reached as early as possible
Logarithmic growth phase, bacterial structure is stablized at this time, and metabolism, physiological property are than more consistent and good, and defence capability is most strong, nitrine bromine second
It is maximum that ingot acts on difficulty；
Step 2: culture bacteria suspension 1ml is taken, the nitrine ethidium bromide solution (EMA) that concentration is 0.05mg/mL is added under the conditions of being protected from light,
EMA final concentration is set to reach 2.0-4.0mg/L, slight oscillatory mixes, and 14-16min is stood in dark, then takes out bacterium solution, away from
It 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 photodissociation, will
Sample, which takes out to be placed in the centrifuge that revolving speed is 10000-15000r/min, is centrifuged 2-5min, abandons supernatant, is resuspended 1 with ultrapure water
Secondary, spare, EMA can be inserted into cell wall or cell membrane is endless as a kind of novel DNA insert type fluorescent dye in the step
In whole dead cell DNA, when activating EMA with high-intensitive visible light exposure, active nitrene intermediate can be generated, it is intermediate
Body and the close covalent bond of DNA form compound and it are made to lose continuation amplification ability, in the EMA nitrene activity dissociated in system
Mesosome forms azanol in conjunction with the water in system, so that EMA be made to be consumed completely, will not influence subsequent DNA cloning, and living thin
The complete cell wall of born of the same parents and cell membrane can prevent the entrance of EMA dyestuff from effectively preventing due to the formation of azanol to viable bacteria
The influence of DNA cloning directly detects viable bacteria so as to not have to separate viable bacteria and dead bacterium；
Step 3: boiling water bath 18-22min, then be placed in temperature be 0-5 DEG C, revolving speed be in the centrifuge of 10000-15000r/min from
Heart 4-6min, supernatant is DNA profiling, spare, and step DNA is dissolved in ultrapure water, and obtained DNA can be directly as dual
Fluorescent PCR expands template, and the extraction DNA profiling method does not expend reagent, easy to operate, direct, quick, obtains amount of DNA more
More, purity also can reach test requirements document；
Step 4: the Listeria monocytogenes announced based on GeneBankhlyAGene order uses primer-design software Primer
Premier 5.0 is designed PCR primer, primer sequence are as follows: upstream primer hly A1:5 '-CCTAAGACGCCAATCGAA-
3 ', upstream primer hly A2:5 '-AAGCGCTTGCAACTGCTC-3 ', above-mentioned primer length is appropriate, close with the sequence of template
Complementation is difficult to form stable dimer and hairpin structure between primer and primer, extremely low in the efficiency of initiation of mismatch site,
Complementary series is not present in itself, itself is difficult to form hairpin structure, with the sequence of template can close complementary, high sensitivity, expansion
Increasing Efficiency is high, and elongating temperature is conducive to the progress of amplified reaction less than 74 DEG C；
Step 5: DNA profiling will be extracted and carry out PCR amplification, 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, ddH27.5 μ L of O, 1 μ L, PCR response parameter of DNA profiling: 94 DEG C of initial denaturations
3min, 94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 80s, 5 circulations, 56 DEG C of annealing 30s, 72 DEG C of extension 80s, 25
Circulation, 72 DEG C of many extension 10min, the specificity of step two primer is good, non-false positive；
Step 6: according to LightCycler480 fluorescence quantitative PCR instrument operation manual, " maximum second derivative method " being selected to be divided
Analysis, with CP value (crossing point refers to cycle-index when amplification curve generates fluorescence jumping) judging result, value≤35 CP
It is judged to the positive, value >=40 CP are judged to feminine gender, and CP value should suitably increase template quantity and reform test, the detection side between 35-40
Method specificity, reproducible, high sensitivity can quickly identify single increasing dead state of listeria spp and state bacterium living, testing result symbol
Close the existing conventional bacteria isolated culture examination criteria in China, application value with higher and promotion prospect.
Preferably, containing ‰ meat of 0.012-0.016% deoxysodium cholate and 0.022-0.025 in step 2 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 list living and increasing listeria spp detection method, and can
The dosage of EMA is reduced, and then reduces testing cost.
For optimisation technique scheme, the measure taken further include: with the dextrorotation meat containing 2.2-2.4% in carnitine in step 2
Alkali, the photodissociation that the dextrorotation carnitine which contains special proportion can accelerate EMA take off rate, improve reactive intermediate containing nitrene and exist
Dispersibility in water, and then sufficiently activate and form azanol in conjunction with water and inactivate, make EMA thoroughly inactivate no longer with DNA molecular knot
It closes, avoids EMA from increasing the influence of the extracting genome DNA amount of listeria spp to state list living, guarantee the extracted amount of DNA, and then make
The DNA cloning that the state that must live list increases listeria spp is gone on smoothly, the final sensitivity and accuracy rate for improving detection method.
Compared with prior art, the invention has the benefit that 1) detection method is specific, reproducible, spirit
Sensitivity, accuracy rate are high, can quickly identify single increasing dead state of listeria spp and state bacterium living, testing cost is low, and testing result meets
The existing conventional bacteria isolated culture examination criteria in China, application value with higher and promotion prospect；2) present invention inspection
Survey method can effectively be enriched with single increasing listeria spp with culture medium, inhibit non-single growth for increasing listeria spp, improve and increase bacterium
Efficiency, while the extent of damage of single membrane structure for increasing the dead bacterium of listeria spp being enabled to increase, 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
Influence to viable bacteria DNA cloning directly detects viable bacteria so as to not have to separate viable bacteria and dead bacterium；4) detection method
Part film in result, which is effectively eliminated, with EMA solution cracks insufficient dead bacterium false positive phenomenon caused by result, it is final to improve
State list living increases the accuracy rate and rate of listeria spp detection method, and can reduce the dosage of EMA, so reduce detection at
This.
Specific embodiment
The present invention program is described further below by embodiment:
Embodiment 1:
Single rapid detection method for increasing listeria spp viable bacteria, specifically includes the following steps:
Step 1: single listeria spp that increases being inoculated into trypticase soy agar culture medium, the Zengjing Granule 22h at 37 DEG C, i.e.,
Bacteria suspension, spare, the cysteine of No. 5 cholate and 2.8g/L in above-mentioned culture medium containing 3.0g/L are left in cysteine
It revolves body and is 100:0.4 with the ratio of d-isomer, the addition of No. 5 cholate and cysteine, which can be effectively enriched with, in culture medium single increases Lee
This special Salmonella inhibits non-single growth for increasing listeria spp, plays the role of preferable selective culture, improves and increase bacterium efficiency,
To be separately separated out single listeria spp that increases for subsequent detection, the detection for singly increasing listeria spp in food is finally improved
Rate and accuracy rate；Levo form and enable to that single to increase listeria spp dead with the special proportion of d-isomer in cysteine simultaneously
The extent of damage of the membrane structure of bacterium increases, so that subsequent EMA can be sent out quickly through the membrane structure of dead bacterium cell
The effect of waving, improve detection rates and and accuracy rate, the culture medium can for it is single increase listeria spp grow provide richer carbon source,
Nitrogen source, inorganic salts etc., enriching effect is significant, enables single listeria spp that increases to reach logarithmic growth phase as early as possible, at this time bacterium knot
Structure is stablized, and than more consistent and good, defence capability is most strong for metabolism, physiological property, and it is maximum that nitrine ethidium bromide acts on difficulty；
Step 2: culture bacteria suspension 1ml is taken, the nitrine ethidium bromide solution (EMA) that concentration is 0.05mg/mL is added under the conditions of being protected from light,
EMA final concentration is set to reach 2.0mg/L, slight oscillatory mixes, and stands 16min in dark, then takes out bacterium solution, apart from fluorescent tube
14cm uncaps and is placed on ice, and persistently exposing 5min with 700W tungsten halogen lamp makes nitrine ethidium bromide photodissociation, and sample taking-up is placed on and is turned
It is centrifuged 2min in the centrifuge that speed is 15000r/min, abandons supernatant, is resuspended 1 time with ultrapure water, spare, EMA conduct in the step
A kind of novel DNA insert type fluorescent dye can be inserted into cell wall or the incomplete dead cell DNA of cell membrane, when with height
When the visible light exposure of intensity activates EMA, active nitrene intermediate, intermediate and the close covalent bond shape of DNA can be generated
It is set to lose continuation amplification ability at compound, the EMA nitrene reactive intermediate to dissociate in system is in conjunction with the water in system
Azanol is formed, so that EMA be made to be consumed completely, will not influence subsequent DNA cloning, and the complete cell wall of living cells and cell
Film can prevent the entrance of EMA dyestuff, due to the formation of azanol, the influence to viable bacteria DNA cloning be effectively prevented, so as to not
Viable bacteria is directly detected with viable bacteria and dead bacterium is separated；
Step 3: 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
Liquid is DNA profiling, spare, and step DNA is dissolved in ultrapure water, and obtained DNA can be directly as double fluorescent PCR amplification mould
Plate, and the extraction DNA profiling method does not expend reagent, easy to operate, direct, quick, acquisition amount of DNA is more, and purity is also reachable
To test requirements document；
Step 4: the Listeria monocytogenes announced based on GeneBankhlyAGene order uses primer-design software Primer
Premier 5.0 is designed PCR primer, primer sequence are as follows: upstream primer hly A1:5 '-CCTAAGACGCCAATCGAA-
3 ', upstream primer hly A2:5 '-AAGCGCTTGCAACTGCTC-3 ', above-mentioned primer length is appropriate, close with the sequence of template
Complementation is difficult to form stable dimer and hairpin structure between primer and primer, extremely low in the efficiency of initiation of mismatch site,
Complementary series is not present in itself, itself is difficult to form hairpin structure, with the sequence of template can close complementary, high sensitivity, expansion
Increasing Efficiency is high, and elongating temperature is conducive to the progress of amplified reaction less than 74 DEG C；
Step 5: DNA profiling will be extracted and carry out PCR amplification, 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, ddH27.5 μ L of O, 1 μ L, PCR response parameter of DNA profiling: 94 DEG C of initial denaturations
3min, 94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 80s, 5 circulations, 56 DEG C of annealing 30s, 72 DEG C of extension 80s, 25
Circulation, 72 DEG C of many extension 10min, the specificity of step two primer is good, non-false positive；
Step 6: according to LightCycler480 fluorescence quantitative PCR instrument operation manual, " maximum second derivative method " being selected to be divided
Analysis, with CP value (crossing point refers to cycle-index when amplification curve generates fluorescence jumping) judging result, value≤35 CP
It is judged to the positive, value >=40 CP are judged to feminine gender, and CP value should suitably increase template quantity and reform test, the detection side between 35-40
Method specificity, reproducible, high sensitivity can quickly identify single increasing dead state of listeria spp and state bacterium living, testing result symbol
Close the existing conventional bacteria isolated culture examination criteria in China, application value with higher and promotion prospect.
Contain 0.016% deoxysodium cholate and 0.022 ‰ carnitines in above-mentioned steps 2 in EMA solution, deoxygenates gallbladder in EMA solution
The impaired journey that the presence of sour sodium and carnitine can significantly change dead bacterium cell membrane surface property, increase dead bacterium membrane structure
Degree, enable EMA quickly through the membrane structure of dead bacterium cell, and then with DNA tertiary structure covalent bond therein, make dead bacterium
DNA lose continuation amplification ability, effectively eliminate in result part film and crack insufficient dead bacterium false positive caused by result
Phenomenon, the final accuracy rate and rate for improving state list increasing listeria spp detection method living, and the dosage of EMA can be reduced, into
And reduce testing cost.
The dextrorotation carnitine in carnitine containing 2.4% is used in above-mentioned steps 2, which contains the dextrorotation carnitine energy of special proportion
The photodissociation for enough accelerating EMA takes off rate, improves the dispersibility of reactive intermediate containing nitrene in water, and then sufficiently activate in conjunction with water
It forms azanol and inactivates, inactivate EMA thoroughly no longer in conjunction with DNA molecular, EMA is avoided to increase the base of listeria spp to state list living
Because of a group influence for DNA extracted amount, guarantee the extracted amount of DNA so that state list living increase the DNA cloning of listeria spp smoothly into
Row, the final sensitivity and accuracy rate for improving detection method.
Embodiment 2:
Single rapid detection method for increasing listeria spp viable bacteria, specifically includes the following steps:
Step 1: single listeria spp that increases being inoculated into trypticase soy agar culture medium, the Zengjing Granule 25h at 33 DEG C, i.e.,
Bacteria suspension, spare, the cysteine of No. 5 cholate and 3.2g/L in above-mentioned culture medium containing 2.6g/L are left in cysteine
It revolves body and is 100:0.35 with the ratio of d-isomer；
Step 2: culture bacteria suspension 1ml is taken, the nitrine ethidium bromide solution (EMA) that concentration is 0.05mg/mL is added under the conditions of being protected from light,
EMA final concentration is set to reach 4.0mg/L, slight oscillatory mixes, and stands 14min in dark, then takes out bacterium solution, apart from fluorescent tube
16cm uncaps and is placed on ice, and persistently exposing 10min with 600W tungsten halogen lamp makes nitrine ethidium bromide photodissociation, and sample taking-up is placed on
It is centrifuged 5min in the centrifuge that revolving speed is 10000r/min, abandons supernatant, is resuspended 1 time with ultrapure water, it is spare；
Step 3: 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
Liquid is DNA profiling, spare；
Step 4: the Listeria monocytogenes announced based on GeneBankhlyAGene order uses primer-design software Primer
Premier 5.0 is designed PCR primer, primer sequence are as follows: upstream primer hly A1:5 '-CCTAAGACGCCAATCGAA-
3 ', upstream primer hly A2:5 '-AAGCGCTTGCAACTGCTC-3 '；
Step 5: DNA profiling will be extracted and carry out PCR amplification, 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, ddH27.5 μ L of O, 1 μ L, PCR response parameter of DNA profiling: 94 DEG C of initial denaturations
3min, 94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 80s, 5 circulations, 56 DEG C of annealing 30s, 72 DEG C of extension 80s, 25
Circulation, 72 DEG C of many extension 10min；
Step 6: according to LightCycler480 fluorescence quantitative PCR instrument operation manual, " maximum second derivative method " being selected to be divided
Analysis, with CP value (crossing point refers to cycle-index when amplification curve generates fluorescence jumping) judging result, value≤35 CP
It is 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.
Contain 0.012% deoxysodium cholate and 0.025 ‰ carnitines in above-mentioned steps 2 in EMA solution, contains 2.2% in carnitine
Dextrorotation carnitine.
Embodiment 3:
Single rapid detection method for increasing listeria spp viable bacteria, specifically includes the following steps:
Step 1: single listeria spp that increases is inoculated into trypticase soy agar culture medium, at 35 DEG C Zengjing Granule for 24 hours, i.e.,
Bacteria suspension, spare, the cysteine of No. 5 cholate and 3.0g/L in above-mentioned culture medium containing 2.8g/L are left in cysteine
It revolves body and is 100:0.37 with the ratio of d-isomer；
Step 2: culture bacteria suspension 1ml is taken, the nitrine ethidium bromide solution (EMA) that concentration is 0.05mg/mL is added under the conditions of being protected from light,
EMA final concentration is set to reach 3.0mg/L, slight oscillatory mixes, and stands 15min in dark, then takes out bacterium solution, apart from fluorescent tube
15cm uncaps and is placed on ice, and persistently exposing 8min with 650W tungsten halogen lamp makes nitrine ethidium bromide photodissociation, and sample taking-up is placed on and is turned
It is centrifuged 4min in the centrifuge that speed is 12000r/min, abandons supernatant, is resuspended 1 time with ultrapure water, it is spare；
Step 3: 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
Liquid is DNA profiling, spare；
Step 4: the Listeria monocytogenes announced based on GeneBankhlyAGene order uses primer-design software Primer
Premier 5.0 is designed PCR primer, primer sequence are as follows: upstream primer hly A1:5 '-CCTAAGACGCCAATCGAA-
3 ', upstream primer hly A2:5 '-AAGCGCTTGCAACTGCTC-3 '；
Step 5: DNA profiling will be extracted and carry out PCR amplification, 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, ddH27.5 μ L of O, 1 μ L, PCR response parameter of DNA profiling: 94 DEG C of initial denaturations
3min, 94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 80s, 5 circulations, 56 DEG C of annealing 30s, 72 DEG C of extension 80s, 25
Circulation, 72 DEG C of many extension 10min；
Step 6: according to LightCycler480 fluorescence quantitative PCR instrument operation manual, " maximum second derivative method " being selected to be divided
Analysis, with CP value (crossing point refers to cycle-index when amplification curve generates fluorescence jumping) judging result, value≤35 CP
It is 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.
Contain 0.015% deoxysodium cholate and 0.024 ‰ carnitines in above-mentioned steps 2 in EMA solution, contains 2.3% in carnitine
Dextrorotation carnitine.
Embodiment 4:
Viable bacteria and dead bacterial examination are surveyed
Single increasing listeria spp bacteria suspension and dead bacterium bacteria suspension are prepared respectively, and test group uses the detection of embodiment 3 and embodiment 4
Method is detected, and 1 detection method of control group is without EMA processing, other steps and embodiment 3 are identical, 2 detection method of control group
EMA in do not contain deoxysodium cholate and carnitine, other steps and embodiment 3 are identical, and control group 3 is existing traditional thin using China
Bacterium is separately cultured standard method SN/T 0973-2010 and " singly increases Listeria bacterial examination in inlet and outlet meat, meat products and other food
Survey method " and inspection and quarantine professional standard SN/T 1870-2007 " pathogenic bacteria detection method real-time PCR methodology in food " progress
Detection.
The result shows that increasing listeria spp viable bacteria for single, test group and control group testing result are consistent, and are inspection
It is positive out；Increase the dead bacterium of listeria spp, test group, 3 testing result of control group and SN/T 0973-2010 testing result for single
There is opposite testing result, and control group 1 and SN/T 0973-2010 testing result (pathogenic bacteria are not detected) are consistent.It says
The addition of bright EMA can identify single increasing dead state of listeria spp and state bacterium living.
Embodiment 5:
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. single rapid detection method for increasing listeria spp viable bacteria, it is characterised in that: the detection method specifically includes following step
It is rapid:
Step 1: single listeria spp that increases is inoculated into the trypticase soy agar culture medium containing No. 5 cholate and cysteine
To get bacteria suspension, the cysteine contains d-isomer for culture；
Step 2: taking culture bacteria suspension, the nitrine ethidium bromide solution containing deoxysodium cholate and carnitine is added under the conditions of being protected from light
(EMA), slight oscillatory mix, dark in stand, then by bacterium solution take out expose, after be centrifuged, abandon supernatant, with ultrapure water weight
It is 1 time outstanding；
Step 3: boiling waterbath DNA, centrifugation, supernatant is DNA profiling；
Step 4: the Listeria monocytogenes announced based on GeneBankhlyAGene order uses primer-design software Primer
Premier 5.0 is designed PCR primer；
Step 5: DNA profiling will be extracted and carry out PCR amplification；
Step 6: according to LightCycler480 fluorescence quantitative PCR instrument operation manual, " maximum second derivative method " being selected to be divided
Analysis, with CP value judging result.
2. single rapid detection method for increasing listeria spp viable bacteria according to claim 1, it is characterised in that: the step
The cysteine of No. 5 cholate and 2.8-3.2g/L in 1 in culture medium containing 3.0-3.2g/L, levo form in the cysteine
With with the ratio of d-isomer be 100:0.35-0.4.
3. single rapid detection method for increasing listeria spp viable bacteria according to claim 1, it is characterised in that: the step
Bacteria suspension 1ml in 2, EMA solution concentration are 0.05mg/mL, the final concentration of 2.0-4.0mg/L of EMA in bacteria suspension.
4. single rapid detection method for increasing listeria spp viable bacteria according to claim 1, it is characterised in that: the step
Contain ‰ carnitine of 0.012-0.016% deoxysodium cholate and 0.022-0.025 in 2 in EMA solution.
5. single rapid detection method for increasing listeria spp viable bacteria according to claim 1, it is characterised in that: the carnitine
In the dextrorotation carnitine containing 2.2-2.4%.
6. single rapid detection method for increasing listeria spp viable bacteria according to claim 1, it is characterised in that: the step
14-16min is stood in 2 in dark, exposes 5-10min.
7. single rapid detection method for increasing listeria spp viable bacteria according to claim 1, it is characterised in that: the step
Primer sequence in 4 are as follows: upstream primer hly A1:5 '-CCTAAGACGCCAATCGAA-3 ', upstream primer hly A2:5 '-
AAGCGCTTGCAACTGCTC-3’。
8. single rapid detection method for increasing listeria spp viable bacteria according to claim 1, it is characterised in that: described to state step
In rapid 5 PCR react 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 of DNA profiling.
9. single rapid detection method for increasing listeria spp viable bacteria according to claim 1, it is characterised in that: the step
PCR response parameter in 5: 94 DEG C of initial denaturation 3min, 94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 80s, 5 recycle, and 56
DEG C annealing 30s, 72 DEG C of extensions 80s, 25 circulations, 72 DEG C of extension 10min eventually.
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| CN111621581A (en) * | 2020-06-28 | 2020-09-04 | 河南大学 | Standard plasmid freeze-dried powder for detecting listeria monocytogenes |
| CN111719007A (en) * | 2020-08-06 | 2020-09-29 | 南方医科大学 | Listeria monocytogenes nucleic acid detection plasmid DNA reference sample, preparation method and application thereof |
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