CN106544436B - A kind of method of salmonella in detection textile - Google Patents
A kind of method of salmonella in detection textile Download PDFInfo
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- CN106544436B CN106544436B CN201611049531.5A CN201611049531A CN106544436B CN 106544436 B CN106544436 B CN 106544436B CN 201611049531 A CN201611049531 A CN 201611049531A CN 106544436 B CN106544436 B CN 106544436B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
Abstract
The invention discloses a kind of methods of salmonella in quickly detection textile, and include the following steps: step 1: sample increases bacterium；Step 2: LAMP test；Step 3: MALDI-TOF-MS technical appraisement；Step 4: result report.Detection time is substantially reduced using this method, negative result only needs 1 day, and positive test symbol only needs 3 days.This method uses LAMP technology and MALDI-TOF-MS combination of sciences, both simple, economical, has the characteristics that high throughput again, it is suitble to instant, on-site test, if on-site test is the positive, can purposive increasing sampling experiment room inspection, make supervision get up not only reaction speed it is fast, with strong points, and save the time.
Description
Technical field
The present invention relates to a kind of methods of salmonella, more particularly, to salmonella in a kind of quickly detection textile
Method.
Background technique
Salmeterol fluticasone propionate main background in textile: salmonella (Salmonella) is the disease of a kind of zoonosis
Opportunistic pathogen is enterobacteriaceae Salmonella member, is the Gram-negative enterobacteria of a kind of conditionity cytozoicus.This bacterium battalion
Support of less demanding, distributed in nature is extremely wide, can detect, can infect a variety of in food, water, soil, animal hair, fiber
Animal and people, it is most of with very strong pathogenic.The Salmonella serogroup found at present has 2500 or more, wherein being permitted
More serotype bacterium can the cross-infection between humans and animals, a variety of different clinical manifestations of humans and animals can be caused, mainly caused
Fever, gastroenteritis, diarrhea and septicemia etc. cause serious harm to the health of the mankind.In recent years with economic globalization into
The deep development of the quickening of journey and China's opening, the country, China and inlet and outlet textile quantity required increasingly increase, and take
Risk with the harms of microbe mankind increases year by year, and pathogen can not be ignored by the incoming risk in environmental pollution, port, through looking into
Data and investigation, the textile being contaminated may carry infectious extremely strong, pathogenic extremely strong salmonella.Due to Salmonella
Bacterium can be used as one important goal bacterium of sanitary inspection, at present Salmeterol fluticasone propionate in textile there are with a long history, widely distributed
Based on plate separation conventional identification, not only cumbersome, time-consuming, because of salmonella phenotypic characteristic and citrobacter, big
The more difficult resolution of intestines Escherichia, proteus, enterobacter cloacae, serum cross reaction ratio is very big, institute in the conventional way vulnerable to
Miscellaneous bacteria interference, causes false negative result, and sensibility and specificity is all impacted, does not adapt to quick detection when epidemic situation occurs.
Therefore, it is badly in need of perspective research of the progress in relation to salmonella laboratory testing method in textile at present, establishes Salmonella
The accurate rapid detection method of bacterium is prevented trouble before it happens applied to the monitoring and detection of salmonella in textile, thus to epidemic situation
Reach effective prevention and control.
The status of Salmeterol fluticasone propionate in textile: there are many methods for Salmeterol fluticasone propionate, and common mainly has traditional mark
Quasi- method, molecular biology method (amplified fragment length polymorphism technology, polymerase chain reaction technique, biochip technology, ring
Mediated isothermal amplification technology, phage splitting technology), immunological method (Enzyme-linked Immunosorbent Assay, dot enzyme-linked immuno absorption, exempt from
Epidemic disease magnetic separation technique, immunofluorescence label, automatic enzyme-labeled immunity detector), electrical impedance method etc..Traditional detection method program is numerous
It is trivial, the period is long, sensitivity is low, the development of molecular biology method, immunological method, electrical impedance method substantially increases salmonella
Detection sensitivity shortens detection time, simplifies detection program, but these methods can only be used as prescreening method, final positive
Determine that result also wants return to traditions standard method.The above detection method is widely used in field of food, and textile field
The detection method of salmonella is rarely reported.
Summary of the invention
In view of this, in view of the deficiencies of the prior art, it is an object of the present invention to provide a kind of accurate quickly detection Salmonellas
The method of bacterium, it is efficient, accurate, time saving and energy saving to have achieved the effect that.
In order to achieve the above objectives, the invention adopts the following technical scheme:
The method of salmonella, includes the following steps: in a kind of quick detection textile
Step 1: sample increases bacterium
It is sampled with sterile mode, Zengjing Granule, obtains the first enrichment liquid；
Step 2: LAMP test
2.1) preparation of DNA profiling: enrichment liquid is taken to be put into sterilizing Eppendorf pipe, at high speed freezing centrifuge centrifugation
Reason removes impurity, obtains DNA profiling；
2.2) LAMP amplified reaction:
2.2.1) amplification prepares: LAMP reaction tube number n (n=1 pipe blank control+1 required for being set according to sample size
+ 1 pipe positive control of pipe negative control+sample number), take out n LAMP reaction tube；Amplification reaction system is shown in Table 1, calculates by table 1
The usage amount of good each reagent is added in sterile centrifugation tube by the sequence of table 1, is uniformly mixed, and 2000rpm is centrifuged 10sec, to setting
N LAMP reaction tube in be separately added into 23 μ L；
1 reaction system of table
2.2.2 blank control, negative control and positive control) are set；
Blank control: DNA profiling is replaced with water；Negative control: the first enrichment liquid is replaced with water, in the way of step 2.1)
Extract the template that template DNA is reacted as the LAMP；Positive control: salmonella reference culture presses step 2.1) after increasing bacterium
Mode extracts the template that template DNA is reacted as the LAMP；
2.2.3 it) is loaded: DNA profiling, the step 2.2.2 that will be prepared in step 2.1)) resulting blank control, feminine gender
Control and positive control respectively take 2 μ L to be added in corresponding reaction tube, and reaction system is made to reach 25 μ L；Pipe lid is covered tightly, is mixed,
2000rpm is centrifuged 10sec；
2.2.4 machine reacts on): the step 2.2.3) reaction tube being placed in water-bath or the real-time transmissometer of LAMP, 65
DEG C amplification 40min；
2.2.5) preliminary observation result: the step 2.2.4) reaction tube is set and is observed under black background, there is white precipitate
Person is the positive, and no white precipitate person is feminine gender；If result is feminine gender, it can directly report and salmonella is not detected in sample, such as
As a result it is the positive or suspicious, continues the experiment of step 3；
Step 3: MALDI-TOF-MS technical appraisement
3.1) secondary increasing bacterium: the culture 1mL that step 2.2.5) preliminary observation result is positive or suspicious sample is connect
For kind in the test tube equipped with 10mL rappaport vassiliadis mdeium, 42 DEG C ± 1 DEG C is cultivated ± 2h for 24 hours, obtains the second enrichment liquid；Separately take step
Rapid 2.2.5) preliminary observation result is that positive or suspicious culture 1mL is inoculated in equipped with 10mL selenite cystine broth
Test tube in, 36 DEG C of ± 1 DEG C of culture ± 2h for 24 hours obtain third enrichment liquid；
3.2) it separates: with the second enrichment liquid and each 1 ring of third enrichment liquid in aseptic inoculation ring picking step 3.1), difference
Streak inoculation is in a bismuth sulfite agar plate and a salmonella color culture medium plate；It is placed in 36 DEG C of ± 1 DEG C of constant temperature
Incubator, bismuth sulfite agar plate culture 48h ± 2h, salmonella color culture medium plate culture ± 2h for 24 hours；Culture terminates
After observe bacterium colony growing state on each plate, a kind of situation are as follows: the growth of no suspicious or colonies typical, directly report salmonella
It is not detected；Another situation are as follows: there is suspicious or colonies typical to grow, then suspicious described in picking or colonies typical after purification, carries out
MALDI-TOF-MS technical appraisement confirmation；Salmonella colony characteristics sepia or grey are to black on bismuth sulfite agar plate
Color, there is metallic luster sometimes, and surrounding media is in brown or black；Some bacterial strains are in celadon, and surrounding media is constant or micro-
It is dimmed；Salmonella colony characteristics on its chromogenic culture medium illustrate to be determined according to chromogenic culture medium, picking 2 or 2
Typical or suspicious bacterium colony, passes through MALDI-TOF- on a above bismuth sulfite agar plate and salmonella color culture medium plate
The suspicious bacterium colony fingerprint chromatogram of MS technical appraisement obtains qualification result using MALDI Biotyper System analysis comparison；
3.3) prepare standard items and matrix: the standard solvent of configuration standard product and matrix be volume ratio be 50% acetonitrile+
+ 2.5% trifluoroacetic acid of 47.5% water；Standard items are prepared: being taken out the dedicated standard items of MALDI Biotyper System, be added
50 μ L of standard solvent；Using the mode of pipettor pressure-vaccum repeatedly, Biotyper System powder is dissolved.Be placed at room temperature for 5min it
Pressure-vaccum is repeated afterwards, and 13000r/min is centrifuged 2min, spare；Substrate preparation: the dedicated base of MALDI Biotyper System is taken out
250 μ L standard solvents are added in matter HCCAportioned, and oscillation mixes, until forming clear solution；
3.4) MALDI BioTyper is identified automatically: picking bismuth sulfite agar plate and salmonella color culture medium are flat
Single bacterium colony is uniformly applied in target plate wad cutter on plate, is added 1 μ L HCCA matrix solution to cover above-mentioned sample spot, is dried at room temperature；
Target plate is put into the automatic assessing instrument of MALDI BioTyper, acquires sample simultaneously in MALDI Biotyper System analytic process
Quality spectrogram obtains protein peak information, automatic qualification result by FlexControl3.0；
Step 4: result report
According to the test of step 2, PRELIMINARY RESULTS is feminine gender, can directly report and salmonella is not detected in sample；Preliminary knot
Fruit is positive or suspicious sample, by the testing result of step 3, reports in sample and detects or be not detected salmonella.
Preferably, the sampling, Zengjing Granule are to open inspection textile samples with sterile mode, equal with sterile scissors
Even sample cutting is weighed after the sample 25g cut is shredded and is added in the sterile homogenizing bag for filling 225mL SCDLP fluid nutrient medium,
1min~2min is patted with slap type homogenizer, is mixed well；The sample prepared is put into 36 DEG C of ± 1 DEG C of constant incubators
In, Zengjing Granule 16h ± 2h obtains the first enrichment liquid.
Preferably, the step 2.1) DNA profiling be prepared as taking 1mLSCDLP enrichment liquid be put into 1.5mL sterilizing
In Eppendorf pipe, 10000r/min is centrifuged 5min in high speed freezing centrifuge, inhales and abandons supernatant；It is mixed that 1mL sterilizing distilled water is added
Even, 10000r/min is centrifuged 5min in high speed freezing centrifuge, inhales and abandons supernatant；Then plus sterilizing distilled water 1mL is mixed, and sets 100
DEG C 5min is boiled, 10000r/min is centrifuged 2min in high speed freezing centrifuge, takes supernatant as DNA profiling, -20 DEG C of storages are standby
With.
Preferably, step 2.2.5) described in detection method: when deposited phenomenon is unobvious, reaction tube be added 1000
2 μ L of × SYBR GreenI dyestuff, mixes gently, and observes color change immediately, and green is the positive, orange for feminine gender.
Preferably, step 2.2.5) described in detection method: when deposited phenomenon is unobvious, pass through the real-time turbidity of LAMP
Instrument generates amplification curve, and when reaction system turbidity value is more than 0.1, result judgement is the positive.
Preferably, the concentration that matrix is prepared in step 3.2) is not more than 10mg/mL.
Commonly used equipment and material are as follows in the present invention:
1) equipment and material
The basic equipment and material of 1.1 conventional microbiological detections.
1.2 sterile scissors, tweezers.
1.3 electronic balances: 0.01g.
The 1.4 complete sterile homogenizing bags of strainer.
1.5 slap type homogenizers: 400mL.
1.6 constant incubators: 36 DEG C ± 1 DEG C, 42 DEG C ± 1 DEG C.
1.7 oese.
1.8 glass slide.
1.9 micropipettors: 1 μ of μ L~20 L of range；200 μ of μ L~1000 L of range.
1.10 sterilizing Eppendorf centrifuge tubes, LAMP reaction tube: 1.5mL.
1.11 supercentrifuges: 2000r/min~13000r/min.
1.12 water-baths: 65 DEG C ± 1 DEG C.
The real-time transmissometer of 1.13 LAMP.
The 1.14 automatic assessing instruments of MALDI BioTyper.
2 culture mediums and reagent
2.1 SCDLP fluid nutrient mediums.
2.2 rappaport vassiliadis mdeium.
2.3 selenite cystine broth.
2.4 bismuth sulfite agar.
2.5 salmonella color culture medium.
2.6 sterilizing distilled waters.
2.7 10 × LAMP amplification buffer: Tris-HCl containing 200mmol/L (pH8.8), 100mmol/L ammonium sulfate,
100mmol/L potassium chloride, 20mmol/L magnesium sulfate, 1%Triton X-100.
2.8 primers: according to the invA gene conserved sequence of Salmonella, a set of LAMP specific primer is designed.
Outer primer F3:5'-TCTGAGCCAATCGTTAATCTC-3'
Outer primer B3:5'-CTTAAACGGCGGTGTCTT-3'
Inner primer FIP:
5'-GCCTTCCATCAGCATTAACTGCTATGAGGTCCTGACGCAA-3'
Inner primer BIP:
5'-CATCAGAGCAACGAAGCATTGCATATCTGGTAATATGGCTGGC-3'
Ring primer LoopF:5'-TCGCTATCATGTTCTGACGG-3'
Ring primer LoopB:5'-TGGTGCAACGATGGAACA-3'
2.9 100mmol/L MgSO4。
2.10 10mmol/L deoxynucleoside triphosphate (dNTP) mixtures: every kind of nucleotide concentration 10mmol/L.
2.11 8U/ μ L Bst archaeal dna polymerases: 8U/ μ L.
2.12 developing solutions: SYBR Green I dyestuff, 1000 ×.
2.13 formic acid.
2.14 acetonitrile.
2.15 acetic acid.
2.16 trifluoroacetic acid.
2.17 standard items Bacterial Test Standard (BTS).
2.18 matrix HCCAportioned.
3 Quality-control strains: salmonella london CMCC50106, Bacterium enteritidis (hydrogen sulfide is positive) CICC21482, pig
Cholera salmonella (hydrogen sulfide is negative) CICC21493, Salmonella anatis CMCC50353, Salmonella choleraesuls (hydrogen sulfide yin
Property) ATCC10708, Salmonella choleraesuls (hydrogen sulfide is negative) ATCC12325, staphylococcus aureus ATCC29213, green pus
Bacillus ATCC10145, Listeria monocytogenes CMCC54002, Enterobacter sakazakii IQCC10403, vibrio parahemolyticus CICC
21617, Escherichia coli CMCC44102, Escherichia coli O 157 NCTC12900.
8 methods detect program
Method detection program see the table below:
The beneficial effects of the present invention are:
1. loop-mediated isothermal amplification technique (LAMP) technology is carried out instead under isothermal conditions using strand displacement archaeal dna polymerase
It answers, achievable nucleic acid amplification reaction in 40min, amplification can observe by the naked eye judgement.Detection process does not need to hold high simultaneously
Expensive instrument and equipment, simple water-bath, reaction process do not need the thermal denaturation of template, prolonged temperature cycles, expand
Volume increase object does not need cumbersome electrophoresis process, and rapid sensitive, high specificity is at low cost, is especially suitable for a large amount of, instant, scene
The primary dcreening operation of pathogenic microorganism detects, and is easy to promote and apply in base.And MALDI-TOF-MS technology is based primarily upon various bacterium sheets
The protein of body forms and molecular weight, using the distinctive protein spectrum finger-print of MALDI BioTyper Instrument measuring bacterium,
And be compared with the mass spectrogram of given data storehouse bacterium, to identify unknown bacterium.The detectable molecule of the technology
Amount range is very big, and instrumentation is simple, and scanning speed is fast, has the characteristics that high sensitivity, high specificity and repeated big, the party
Method is increasingly automated, reduces experimental error, and result is clear, unique, does not need other appended experimentals, and target plate is reusable,
Save testing cost.A kind of method of accurate quickly detection salmonella provided by the present invention be based on LAMP technology and
The characteristics of MALDI-TOF-MS technology, according to the distinctive target-gene sequence of Salmonella, for 6 isolated areas of target gene
2 pairs of design special interior outer primers start circulation strand replacement reaction using archaeal dna polymerase, right under isothermy (65 DEG C or so)
The DNA profiling that extracts carries out heat preservation 40min after one step of salmonella increases bacterium, can it is special, efficiently, quickly finish nucleic acid expansion
Increase.Product after amplification forms white precipitate, can determine that result by paired observation.DNA cloning is that negative result can be direct
Report salmonella is not detected；If DNA cloning is positive or suspect results, by enrichment liquid streak inoculation salmonella color
Plate and bismuth sulfite agar plate, typical or suspicious bacterium colony, quick using MALDI-TOF-MS technical appraisement on picking plate
Identify result.
2. 1. this method process substantially reduces detection time to method advantage, traditional negative result needs 3~7
It, positive test symbol needs 6~7 days, and rebuilding method negative result only needs 1 day, and positive test symbol only needs 3 days.
2. LAMP technology is simple, economical, it is suitble to instant, on-site test, it, can be purposive if on-site test is the positive
Increase the inspection of sampling experiment room, make supervision get up not only reaction speed it is fast, with strong points, and save the time.
3. this method is applied widely, it is not only applicable in epidemic prevention and health supervision department, can also be examined in enterprise and domestic hygiene
It is used in survey.
4. this method is easy to operate, quick, efficient, high specificity, high sensitivity, as a result observation is convenient, intuitive, is suitable for base
Layer promotes and applies.
5. loop-mediated isothermal amplification method advantage: high sensitivity (2~5 orders of magnitude higher than traditional PCR method)；Reaction
Time is short, can complete within 30~60 minutes reaction；Clinical use does not need special instrument；It is easy to operate, whether DNA or
RNA, detecting step are all that reaction solution, enzyme and template need to be mixed in reaction tube, are placed in water-bath or insulating box 63 left DEG C
Right heat preservation 30~60 minutes, visual results.
Detailed description of the invention
Fig. 1: 1~No. 30 sample LAMP amplification human visual figures；
Fig. 2: 1~8,9~16,17,18,21~26,27~No. 30 sample LAMP amplification curve diagrams；
Fig. 3: 1~8,9~16,17,18,21~26,27~No. 30 sample LAMP expand absorbance figure；
Fig. 4: 1~8,9~16,17,18,21~26,27~No. 30 sample amplification turbidity change rate figures；
Fig. 5: salmonella and its control MALDI BioTyper identify map.
Specific embodiment
Embodiment 1
The method of salmonella, includes the following steps: in a kind of quick detection textile
Step 1: sample increases bacterium
Aseptic procedure opens inspection textile samples and is accurately weighed cut on an electronic balance with the uniform sample cutting of sterile scissors
Under sample 25g, be added to after shredding in the sterile homogenizing bag for filling 225mL SCDLP fluid nutrient medium, with slap type homogeneous
Device pats 1min~2min, mixes well；The sample prepared is put into 36 DEG C of ± 1 DEG C of constant incubators, Zengjing Granule 16h
± 2h obtains the first enrichment liquid；
Step 2: LAMP test
2.1) preparation of DNA profiling: 1mLSCDLP enrichment liquid is taken to be put into 1.5mL sterilizing Eppendorf pipe, high speed freezes
10000r/min is centrifuged 5min in centrifuge, inhales and abandons supernatant；1mL sterilizing distilled water is added to mix, in high speed freezing centrifuge
10000r/min is centrifuged 5min, inhales and abandons supernatant；Then plus sterilizing distilled water 1mL is mixed, and is set 100 DEG C and is boiled 5min, high speed freezes
10000r/min is centrifuged 2min in centrifuge, takes supernatant as DNA profiling, -20 DEG C of storages are spare；
2.2) LAMP amplified reaction:
2.2.1) amplification prepares: LAMP reaction tube number n (n=1 pipe blank control+1 required for being set according to sample size
+ 1 pipe positive control of pipe negative control+sample number), take out n LAMP reaction tube.Amplification reaction system is shown in Table 1, calculates by table 1
The usage amount of good each reagent is added in sterile centrifugation tube by the sequence of table 1, is uniformly mixed, and 2000rpm is centrifuged 10sec, to setting
N LAMP reaction tube in be separately added into 23 μ L；
1 reaction system of table
2.2.2 negative control, blank pair should all be arranged by) setting blank control, negative control and positive control, each reaction
According to and positive control；Blank control: DNA profiling is replaced with water；Negative control: sample is replaced with water, in the way of step 2.1)
Extract the template that template DNA is reacted as the LAMP；Positive control: salmonella reference culture presses step 2.1) after increasing bacterium
Mode extracts the template that template DNA is reacted as the LAMP；
2.2.3 it) is loaded: the DNA profiling prepared in step 2.1), the resulting blank control of step 2.2.2, feminine gender is right
According to respectively taking 2 μ L to be added in corresponding reaction tube with positive control, reaction system is made to reach 25 μ L；Pipe lid is covered tightly, is mixed,
2000rpm is centrifuged 10sec；
2.2.4 machine reacts on): the step 2.2.3) reaction tube being placed in water-bath or the real-time transmissometer of LAMP, 65
DEG C amplification 40min；
2.2.5) preliminary observation result: the step 2.2.4) reaction tube is set and is observed under black background, there is white precipitate
Person is the positive, and no white precipitate person is feminine gender；If result is feminine gender, it can directly report and salmonella is not detected in sample, such as
As a result it is the positive or suspicious, carries out the experiment of step 3；
Step 3: MALDI-TOF-MS technical appraisement
3.1) secondary increasing bacterium: step 2.2.5 is taken) preliminary observation result is that positive culture 1mL is inoculated in equipped with 10mL
In the test tube of rappaport vassiliadis mdeium, 42 DEG C ± 1 DEG C is cultivated ± 2h for 24 hours, obtains the second enrichment liquid；Separately take step 2.2.5) it is preliminary
Observation result is that positive culture 1mL is inoculated in the test tube equipped with 10mL selenite cystine broth, 36 DEG C ± 1 DEG C
± 2h for 24 hours is cultivated, third enrichment liquid is obtained；
3.2) it separates: the second enrichment liquid obtained by aseptic inoculation ring picking step 3.1) and each 1 ring of third enrichment liquid, respectively
Streak inoculation is in a bismuth sulfite agar plate and a salmonella color culture medium plate；It is placed in 36 DEG C of ± 1 DEG C of constant temperature
Incubator, bismuth sulfite agar plate culture 48h ± 2h, salmonella color culture medium plate culture ± 2h for 24 hours；Culture terminates
After observe bacterium colony growing state on each plate, such as grown without suspicious or colonies typical, can directly report that salmonella is not detected；
It is grown if any suspicious or colonies typical, then suspicious described in picking or colonies typical after purification, carries out MALDI-TOF-MS technology mirror
Fixed confirmation；Salmonella colony characteristics sepia or grey have metallic luster, week to black sometimes on bismuth sulfite agar plate
Culture medium is enclosed in brown or black；Some bacterial strains are in celadon, and surrounding media is constant or is slightly variable dark；Salmonella is in its colour developing
Colony characteristics illustrate to be determined according to chromogenic culture medium on culture medium, picking 2 or 2 or more bismuth sulfite agar plates
With bacterium colony typical or suspicious on salmonella color culture medium plate, pass through the suspicious bacterium colony fingerprint of MALDI-TOF-MS technical appraisement
Spectrogram obtains qualification result using MALDI Biotyper System analysis comparison；
3.3) prepare standard items and matrix: the standard solvent of configuration standard product and matrix be volume ratio be 50% acetonitrile+
+ 2.5% trifluoroacetic acid of 47.5% water；Standard items are prepared: being taken out the dedicated standard items of MALDI Biotyper System, be added
50 μ L of standard solvent.Using the mode of pipettor pressure-vaccum repeatedly, BTS powder is dissolved.It is placed at room temperature for after 5min and repeats pressure-vaccum, 13
000r/min is centrifuged 2min, spare；Substrate preparation: the dedicated matrix of MALDI Biotyper System is taken out
250 μ L standard solvents are added in HCCAportioned, and final concentration is not more than 10mg/mL, and oscillation mixes, molten until forming clarification
Liquid；
3.4) MALDI BioTyper is identified automatically: single bacterium on picking BS plate and salmonella color culture medium plate
It falls and is uniformly applied in target plate wad cutter, add 1 μ L HCCA matrix solution to cover above-mentioned sample spot, dry at room temperature.Target plate is put into
MALDI BioTyper automatic assessing instrument acquires sample mass spectrum simultaneously in MALDI Biotyper System analytic process, by
FlexControl3.0 obtains protein peak information, automatic qualification result；
Step 4: result report
According to the test of step 2, Salmeterol fluticasone propionate in preliminary judgement sample, can be direct as a result, PRELIMINARY RESULTS is feminine gender
Salmonella is not detected in report sample, as PRELIMINARY RESULTS be it is positive or suspicious, confirmed by step 3, in report sample
Detect or be not detected salmonella.
Embodiment 2
The method of salmonella, includes the following steps: in a kind of quick detection textile
Step 1: sample increases bacterium
Aseptic procedure opens inspection textile samples and is accurately weighed cut on an electronic balance with the uniform sample cutting of sterile scissors
Under sample 25g, be added to after shredding in the sterile homogenizing bag for filling 225mL SCDLP fluid nutrient medium, with slap type homogeneous
Device pats 1min~2min, mixes well；The sample prepared is put into 36 DEG C of ± 1 DEG C of constant incubators, Zengjing Granule 16h
± 2h obtains the first enrichment liquid；
Step 2: LAMP test
2.1) preparation of DNA profiling: 1mLSCDLP enrichment liquid is taken to be put into 1.5mL sterilizing Eppendorf pipe, high speed freezes
10000r/min is centrifuged 5min in centrifuge, inhales and abandons supernatant；1mL sterilizing distilled water is added to mix, in high speed freezing centrifuge
10000r/min is centrifuged 5min, inhales and abandons supernatant；Then plus sterilizing distilled water 1mL is mixed, and is set 100 DEG C and is boiled 5min, high speed freezes
10000r/min is centrifuged 2min in centrifuge, takes supernatant as DNA profiling, -20 DEG C of storages are spare；
2.2) LAMP amplified reaction:
2.2.1) amplification prepares: LAMP reaction tube number n (n=1 pipe blank control+1 required for being set according to sample size
+ 1 pipe positive control of pipe negative control+sample number), take out n LAMP reaction tube；Amplification reaction system is shown in Table 1, calculates by table 1
The usage amount of good each reagent is added in sterile centrifugation tube by the sequence of table 1, is uniformly mixed, and 2000rpm is centrifuged 10sec, to setting
N LAMP reaction tube in be separately added into 23 μ L；
1 reaction system of table
2.2.2 negative control, blank pair should all be arranged by) setting blank control, negative control and positive control, each reaction
According to and positive control；Blank control: DNA profiling is replaced with water；Negative control: sample is replaced with water, in the way of step 2.1)
Extract the template that template DNA is reacted as the LAMP；Positive control: salmonella reference culture presses step 2.1) after increasing bacterium
Mode extracts the template that template DNA is reacted as the LAMP；
2.2.3 it) is loaded: the DNA profiling prepared in step 2.1), the resulting blank control of step 2.2.2, feminine gender is right
According to respectively taking 2 μ L to be added in corresponding reaction tube with positive control, reaction system is made to reach 25 μ L；Pipe lid is covered tightly, is mixed,
2000rpm is centrifuged 10sec；
2.2.4 machine reacts on): the step 2.2.3) reaction tube being placed in water-bath or the real-time transmissometer of LAMP, 65
DEG C amplification 40min；
2.2.5) preliminary observation result: the step 2.2.4) reaction tube is set and is observed under black background, there is white precipitate
Person is the positive, and no white precipitate person is feminine gender；When deposited phenomenon is unobvious, 1000 × SYBR Green I is added in reaction tube
2 μ L of dyestuff, mixes gently, and observes color change immediately, and green is the positive, orange for feminine gender；If result is feminine gender, can be direct
Report sample in salmonella is not detected, as result be the positive or it is suspicious, carry out following subsequent experimental；
Step 3: MALDI-TOF-MS technical appraisement
3.1) secondary increasing bacterium is that positive sample carries out secondary increasing bacterium to preliminary observation result, takes step 2.2.5) it is preliminary
Observation result is that positive culture 1mL is inoculated in the test tube equipped with 10mL rappaport vassiliadis mdeium, 42 DEG C of ± 1 DEG C of cultures
± 2h for 24 hours obtains the second enrichment liquid；Separately taking step 2.2.5) preliminary observation result is that positive culture 1mL is inoculated in and is equipped with
In the test tube of 10mL selenite cystine broth, 36 DEG C ± 1 DEG C is cultivated ± 2h for 24 hours, obtains third enrichment liquid；
3.2) it separates: the second enrichment liquid obtained by aseptic inoculation ring picking step 3.1) and each 1 ring of third enrichment liquid, respectively
Streak inoculation is in a bismuth sulfite agar plate and a salmonella color culture medium plate；It is placed in 36 DEG C of ± 1 DEG C of constant temperature
Incubator, bismuth sulfite agar plate culture 48h ± 2h, salmonella color culture medium plate culture ± 2h for 24 hours；Culture terminates
After observe bacterium colony growing state on each plate, such as grown without suspicious or colonies typical, can directly report that salmonella is not detected；
It is grown if any suspicious or colonies typical, then suspicious described in picking or colonies typical after purification, carries out MALDI-TOF-MS technology mirror
Fixed confirmation；Salmonella colony characteristics sepia or grey have metallic luster, week to black sometimes on bismuth sulfite agar plate
Culture medium is enclosed in brown or black；Some bacterial strains are in celadon, and surrounding media is constant or is slightly variable dark；Salmonella is in its colour developing
Colony characteristics illustrate to be determined according to chromogenic culture medium on culture medium, picking 2 or 2 or more bismuth sulfite agar plates
With bacterium colony typical or suspicious on salmonella color culture medium plate, pass through the suspicious bacterium colony fingerprint of MALDI-TOF-MS technical appraisement
Spectrogram obtains qualification result using MALDI Biotyper System analysis comparison；
3.3) prepare standard items and matrix: the standard solvent of configuration standard product and matrix be volume ratio be 50% acetonitrile+
+ 2.5% trifluoroacetic acid of 47.5% water；Standard items are prepared: being taken out the dedicated standard items of MALDI Biotyper System, be added
50 μ L of standard solvent.Using the mode of pipettor pressure-vaccum repeatedly, BTS powder is dissolved.It is placed at room temperature for after 5min and repeats pressure-vaccum, 13
000r/min is centrifuged 2min, spare；Substrate preparation: the dedicated matrix of MALDI Biotyper System is taken out
HCCAportioned, 250 its maximum concentration of μ L standard solvent of addition are 10mg/mL, and oscillation mixes, until forming clear solution；
3.4) MALDI BioTyper is identified automatically: on picking bismuth sulfite agar and salmonella color culture medium plate
Single bacterium colony is uniformly applied in target plate wad cutter, is added 1 μ LHCCA matrix solution to cover above-mentioned sample spot, is dried at room temperature；By target
Plate is put into the automatic assessing instrument of MALDI BioTyper, acquires sample mass spectrum simultaneously in MALDI BiotyperSystem analytic process
Figure, obtains protein peak information, automatic qualification result by FlexControl3.0；
Step 4: result report
According to the test of step 2, Salmeterol fluticasone propionate in preliminary judgement sample, can be direct as a result, PRELIMINARY RESULTS is feminine gender
Salmonella is not detected in report sample, as PRELIMINARY RESULTS be it is positive or suspicious, confirmed by step 3, in report sample
Detect or be not detected salmonella.
Embodiment 3
The method of salmonella, includes the following steps: in a kind of quick detection textile
Step 1: sample increases bacterium
Aseptic procedure opens inspection textile samples and is accurately weighed cut on an electronic balance with the uniform sample cutting of sterile scissors
Under sample 25g, be added to after shredding in the sterile homogenizing bag for filling 225mLSCDLP fluid nutrient medium, use slap type homogenizer
1min~2min is patted, is mixed well；The sample prepared is put into 36 DEG C of ± 1 DEG C of constant incubators, Zengjing Granule 16h ±
2h obtains the first enrichment liquid；
Step 2: LAMP test
2.1) preparation of DNA profiling: 1mLSCDLP enrichment liquid is taken to be put into 1.5mL sterilizing Eppendorf pipe, high speed freezes
10000r/min is centrifuged 5min in centrifuge, inhales and abandons supernatant；1mL sterilizing distilled water is added to mix, in high speed freezing centrifuge
10000r/min is centrifuged 5min, inhales and abandons supernatant；Then plus sterilizing distilled water 1mL is mixed, and is set 100 DEG C and is boiled 5min, high speed freezes
10000r/min is centrifuged 2min in centrifuge, takes supernatant as DNA profiling, -20 DEG C of storages are spare；
2.2) LAMP amplified reaction:
2.2.1) amplification prepares: LAMP reaction tube number n (n=1 pipe blank control+1 required for being set according to sample size
+ 1 pipe positive control of pipe negative control+sample number), take out n LAMP reaction tube；Amplification reaction system is shown in Table 1, calculates by table 1
The usage amount of good each reagent is added in sterile centrifugation tube by the sequence of table 1, is uniformly mixed, and 2000rpm is centrifuged 10sec, to setting
N LAMP reaction tube in be separately added into 23 μ L；
1 reaction system of table
2.2.2 blank control, negative control and positive control) are set
Negative control, blank control and positive control should all be arranged in each reaction, blank control: replacing DNA profiling with water；
Negative control: sample is replaced with water, the template that template DNA is reacted as the LAMP is extracted in the way of step 2.1)；It is positive
Control: salmonella reference culture extracts the template that template DNA is reacted as the LAMP after increasing bacterium in the way of step 2.1)；
2.2.3 it) is loaded: the DNA profiling prepared in step 2.1), the resulting blank control of step 2.2.2, feminine gender is right
According to respectively taking 2 μ L to be added in corresponding reaction tube with positive control, reaction system is made to reach 25 μ L；Pipe lid is covered tightly, is mixed,
2000rpm is centrifuged 10sec；
2.2.4 machine reacts on): the step 2.2.3) reaction tube being placed in water-bath or the real-time transmissometer of LAMP, 65
DEG C amplification 40min；
2.2.5) preliminary observation result: the step 2.2.4) reaction tube is set and is observed under black background, there is white precipitate
Person is the positive, and no white precipitate person is feminine gender；Preferably, step 2.2.5) described in detection method: when deposited phenomenon is unknown
When aobvious, amplification curve is generated by the real-time transmissometer of LAMP, when reaction system turbidity value is more than 0.1, result judgement is the positive；
If result is feminine gender, it can directly report and salmonella is not detected in sample, if result is positive or suspicious, progress step 3
Experiment；
Step 3: MALDI-TOF-MS technical appraisement
3.1) secondary increasing bacterium, takes step 2.2.5) preliminary observation result is that positive culture 1mL is inoculated in equipped with 10mL
In the test tube of rappaport vassiliadis mdeium, 42 DEG C ± 1 DEG C is cultivated ± 2h for 24 hours, obtains the second enrichment liquid；Separately take step 2.2.5) it is preliminary
Observation result is that positive culture 1mL is inoculated in the test tube equipped with 10mL selenite cystine broth, 36 DEG C ± 1 DEG C
± 2h for 24 hours is cultivated, third enrichment liquid is obtained；
3.2) it separates: the second enrichment liquid obtained by aseptic inoculation ring picking step 3.1) and each 1 ring of third enrichment liquid, respectively
Streak inoculation is in a bismuth sulfite agar plate and a salmonella color culture medium plate；It is placed in 36 DEG C of ± 1 DEG C of constant temperature
Incubator, bismuth sulfite agar plate culture 48h ± 2h, salmonella color culture medium plate culture ± 2h for 24 hours；Culture terminates
After observe bacterium colony growing state on each plate, such as grown without suspicious or colonies typical, can directly report that salmonella is not detected；
It is grown if any suspicious or colonies typical, then suspicious described in picking or colonies typical after purification, carries out MALDI-TOF-MS technology mirror
Fixed confirmation；Salmonella colony characteristics sepia or grey have metallic luster, week to black sometimes on bismuth sulfite agar plate
Culture medium is enclosed in brown or black；Some bacterial strains are in celadon, and surrounding media is constant or is slightly variable dark；Salmonella is in its colour developing
Colony characteristics illustrate to be determined according to chromogenic culture medium on culture medium, picking 2 or 2 or more bismuth sulfite agar plates
With bacterium colony typical or suspicious on salmonella color culture medium plate, pass through the suspicious bacterium colony fingerprint of MALDI-TOF-MS technical appraisement
Spectrogram obtains qualification result using MALDI Biotyper System analysis comparison；
3.3) prepare standard items and matrix: the standard solvent of configuration standard product and matrix be volume ratio be 50% acetonitrile+
+ 2.5% trifluoroacetic acid of 47.5% water；Standard items are prepared: taking out the dedicated standard items of MALDI Biotyper System
50 μ L of standard solvent is added in (Bacterial Test Standard, BTS).Use the mode of pipettor pressure-vaccum repeatedly, dissolution
BTS powder；It is placed at room temperature for 5min and repeats pressure-vaccum later, 13000r/min is centrifuged 2min, spare；Substrate preparation: MALDI is taken out
250 μ L standard solvents are added in the dedicated matrix HCCAportioned of Biotyper System, and concentration is not more than 10mg/mL,
Oscillation mixes, until forming clear solution；
3.4) MALDI BioTyper is identified automatically: single bacterium on picking BS plate and salmonella color culture medium plate
It falls and is uniformly applied in target plate wad cutter, add 1 μ L HCCA matrix solution to cover above-mentioned sample spot, dry at room temperature.Target plate is put into
MALDI BioTyper automatic assessing instrument acquires sample mass spectrum simultaneously in MALDI Biotyper System analytic process, by
FlexControl3.0 obtains protein peak information, automatic qualification result；
Step 4: result report
According to the test of step 2, Salmeterol fluticasone propionate in preliminary judgement sample, can be direct as a result, PRELIMINARY RESULTS is feminine gender
Salmonella is not detected in report sample, as PRELIMINARY RESULTS be it is positive or suspicious, confirmed by step 3, in report sample
Detect or be not detected salmonella.
Method stability test:
One, prepare sample
1, sample is collected
Totally 20 parts of sample for collecting the sample that different fibers form and different purposes, comprising: 2 parts of the new cotton of various colors,
New 1 part of diablement fort, 1 part of silk goods, 1 part of knitting wool clothes material, 1 part of cotton thread, 1 part of woven dacron, 1 part of blended plain cloth, 1 part of garrha,
1 part of oilcloth, 1 part of canvas, 1 part of poplin cloth cloth, 1 part of denim, 1 part of corduroy, 1 part of mull-chiffon, 1 part of silks and satins cloth, polyamide cloth 1
Part, 1 part of soft plain-weave silk fabric cloth, every a sample is dried after water rinses be fitted into and do high pressure in a sterile sampler bag and go out by 2 parts of velvet
Bacterium processing.Respectively number 1,2,3,4 ..., 20, it is each number in sample type it is random.Separately collect 10 traditional technique in measuring sun
Property sample, comprising: jeans 1, hospital's sheet 2,2, hospital's pillowcase, 1 set of clothing of nurses, hotel quilt cover 1, hotel hair
2, towel, couchette sheet 1, respectively number 21,22,23,24 ... 30.
2, sample making
Take quality control standard bacterial strain salmonella london CMCC50106, Bacterium enteritidis (hydrogen sulfide is positive)
CICC21482, Salmonella choleraesuls (hydrogen sulfide is negative) CICC21493, Salmonella anatis CMCC50353, hog cholera sramana
Salmonella (hydrogen sulfide is negative) ATCC10708, Salmonella choleraesuls (hydrogen sulfide is negative) ATCC12325, staphylococcus aureus
ATCC29213, Pseudomonas aeruginosa ATCC10145, Listeria monocytogenes CMCC54002, Enterobacter sakazakii IQCC10403, secondary haemolysis
Property vibrios CICC 21617, Escherichia coli CMCC44102, Escherichia coli O 157 NCTC12900, be inoculated with 300mL nutrient meat respectively
It is brought back to life in soup, the meat soup after culture respectively takes 10ml to be mixed, and mixed bacteria liquid is inoculated with plate, through plate count, salmonella
Original bacterial concentration be 5 × 109CFU/ml, after 10 times of doubling dilutions, it is artificial to choose high concentration bacterium solution (5 × 105CFU/ml)
1~No. 8 sample placement overnight is polluted, 9~No. 18 sample placements overnight of low concentration bacterium solution (5CFU/ml) artificial contamination are taken.
Two, it tests
1~No. 30 sample is detected according to rebuilding method simultaneously.Single bacterium is isolated to LAMP detection positive findings
It falls, using MALDI-TOF MS technical appraisement, final 1~No. 8,9~No. 18,21~No. 30 samples whole detection salmonellas,
It is consistent with expected results.Concrete outcome is shown in Table 2.Fig. 1 lists 1~No. 30 sample LAMP amplification human visual figure, Fig. 2 column
1~No. 30 sample LAMP amplification curve diagram is gone out, Fig. 3 lists 1~No. 30 sample LAMP amplification absorbance figure, and Fig. 4 lists 1
~No. 30 sample LAMP expand turbidity change rate figure, and Fig. 5 lists salmonella and its control MALDI BioTyper qualification figure
Spectrum.
2 1-30 sample detection situation of table
As can be seen from Table 2, artificial addition either high concentration horizontal (5 × 105CFU/ml) or low concentration is horizontal
(5CFU/ml), LAMP can be expected to detect, and after selective enrichment, MALDI-TOF-MS identification accuracy rate also reaches
100%, detection time is substantially reduced using this method, negative result only needs 1 day, and positive test symbol only needs 3 days.It should
Method uses LAMP technology and MALDI-TOF-MS combination of sciences, not only simply, it is economical, but also have the characteristics that high throughput, it is suitable it is instant,
On-site test, if on-site test be the positive, can purposive increasings sampling experiment room inspection, make supervise get up not only to react
Speed is fast, with strong points, and saves the time.
Finally, it is stated that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, this field is common
Other modifications or equivalent replacement that technical staff makes technical solution of the present invention, without departing from technical solution of the present invention
Spirit and scope, be intended to be within the scope of the claims of the invention.
Claims (5)
1. a kind of method of salmonella in detection textile, characterized by the following steps:
Step 1: sample increases bacterium
Inspection textile samples are opened with sterile mode, with the uniform sample cutting of sterile scissors, are weighed after the sample 25g cut shreds
It is added in the sterile homogenizing bag for filling 225mLSCDLP fluid nutrient medium, pats 1min~2min with slap type homogenizer, fill
Divide and mixes；The sample prepared is put into 36 DEG C of ± 1 DEG C of constant incubators, Zengjing Granule 16h ± 2h obtains the first enrichment liquid；
Step 2: LAMP test
2.1) preparation of DNA profiling: enrichment liquid is taken to be put into sterilizing Eppendorf pipe, high speed freezing centrifuge centrifugal treating is removed
Decontamination obtains DNA profiling；
2.2) LAMP amplified reaction:
2.2.1) amplification prepares: LAMP reaction tube number n required for being set according to sample size takes out n LAMP reaction tube；Expand
Increase reaction system and be shown in Table 1, the usage amount of each reagent is calculated by table 1, is added in sterile centrifugation tube by the sequence of table 1, mixing is equal
Even, 2000rpm is centrifuged 10sec, is separately added into 23 μ L into n LAMP reaction tube of setting；Wherein, the LAMP reaction tube
Number+1 pipe positive control of+1 pipe negative control of n=1 pipe blank control+sample number；
1 reaction system of table
2.2.2 blank control, negative control and positive control) are set；
Blank control: DNA profiling is replaced with water；Negative control: the first enrichment liquid is replaced with water, is extracted in the way of step 2.1)
The template that template DNA is reacted as the LAMP；Positive control: salmonella reference culture presses step after increasing bacterium
2.1) mode extracts the template that template DNA is reacted as the LAMP；
2.2.3 it) is loaded: DNA profiling, the step 2.2.2 that will be prepared in step 2.1)) resulting blank control, negative control
It respectively takes 2 μ L to be added in corresponding reaction tube with positive control, reaction system is made to reach 25 μ L；Pipe lid is covered tightly, is mixed, 2000rpm
It is centrifuged 10sec；
2.2.4 machine reacts on): the step 2.2.3) reaction tube being placed in water-bath or the real-time transmissometer of LAMP, 65 DEG C of expansions
Increase 40min；
2.2.5) preliminary observation result: the step 2.2.4) reaction tube is set and is observed under black background, having white precipitate, person is
The positive, no white precipitate person are feminine gender；If result is feminine gender, it can directly report and salmonella is not detected in sample, such as result
It is the positive or suspicious, continues the experiment of step 3；
Step 3: MALDI-TOF-MS technical appraisement
3.1) secondary increasing bacterium
The culture 1mL that step 2.2.5) preliminary observation result is positive or suspicious sample is inoculated in equipped with 10mL magnesium chloride
In the test tube of malachite green bouillon, 42 DEG C ± 1 DEG C is cultivated ± 2h for 24 hours, obtains the second enrichment liquid；Separately take step 2.2.5) preliminary observation knot
Fruit is that positive or suspicious culture 1mL is inoculated in the test tube equipped with 10mL selenite cystine broth, 36 DEG C ± 1 DEG C
± 2h for 24 hours is cultivated, third enrichment liquid is obtained；
3.2) it separates: with the second enrichment liquid and each 1 ring of third enrichment liquid in aseptic inoculation ring picking step 3.1), crossing respectively
It is inoculated in a bismuth sulfite agar plate and a salmonella color culture medium plate；It is placed in 36 DEG C of ± 1 DEG C of constant temperature incubations
Case, bismuth sulfite agar plate culture 48h ± 2h, salmonella color culture medium plate culture ± 2h for 24 hours；It is seen after culture
Examine bacterium colony growing state on each plate, a kind of situation are as follows: the growth of no suspicious or colonies typical directly reports that salmonella is not examined
Out；Another situation are as follows: there is suspicious or colonies typical to grow, then suspicious described in picking or colonies typical after purification, carries out
MALDI-TOF-MS technical appraisement confirmation；Salmonella colony characteristics sepia or grey are to black on bismuth sulfite agar plate
Color, there is metallic luster sometimes, and surrounding media is in brown or black；Some bacterial strains are in celadon, and surrounding media is constant or micro-
It is dimmed；Salmonella colony characteristics on its chromogenic culture medium illustrate to be determined according to chromogenic culture medium, picking 2 or 2
Typical or suspicious bacterium colony, passes through MALDI-TOF- on a above bismuth sulfite agar plate and salmonella color culture medium plate
The suspicious bacterium colony fingerprint chromatogram of MS technical appraisement obtains qualification result using MALDIBiotyper System analysis comparison；
3.3) prepare standard items and matrix: the standard solvent of configuration standard product and matrix be+47.5% water of 50% acetonitrile of volume ratio+
2.5% trifluoroacetic acid；Standard items are prepared: taking out the dedicated standard items of MALDI Biotyper System, standard solvent 50 is added
μL；Using the mode of pipettor pressure-vaccum repeatedly, Biotyper System powder is dissolved；It is placed at room temperature for 5min and repeats pressure-vaccum later,
13000r/min is centrifuged 2min, spare；Substrate preparation: the dedicated matrix of MALDI Biotyper System is taken out
250 μ L standard solvents are added in HCCAportioned, and oscillation mixes, until forming clear solution；
3.4) MALDI BioTyper is identified automatically: on picking bismuth sulfite agar plate and salmonella color culture medium plate
Single bacterium colony is uniformly applied in target plate wad cutter, is added 1 μ L HCCA matrix solution to cover above-mentioned sample spot, is dried at room temperature；By target
Plate is put into the automatic assessing instrument of MALDI BioTyper, acquires sample matter simultaneously in MALDI Biotyper System analytic process
Spectrogram obtains protein peak information, automatic qualification result by FlexControl3.0；
Step 4: result report
According to the test of step 2, PRELIMINARY RESULTS is feminine gender, can directly report and salmonella is not detected in sample；PRELIMINARY RESULTS is
Positive or suspicious sample is reported in sample by the testing result of step 3 and is detected or be not detected salmonella；
Wherein, step 2.2.1) described in primer are as follows:
Outer primer F3:5'-TCTGAGCCAATCGTTAATCTC-3'
Outer primer B3:5'-CTTAAACGGCGGTGTCTT-3'
Inner primer FIP:5'-GCCTTCCATCAGCATTAACTGCTATGAGGTCCTGACGCAA-3'
Inner primer BIP:5'-CATCAGAGCAACGAAGCATTGCATATCTGGTAATATGGCTGGC-3'
Ring primer LoopF:5'-TCGCTATCATGTTCTGACGG-3'
Ring primer LoopB:5'-TGGTGCAACGATGGAACA-3'.
2. the method for salmonella in a kind of detection textile according to claim 1, it is characterised in that: step 2.1) institute
That states DNA profiling is prepared as taking 1mLSCDLP enrichment liquid to be put into 1.5mL sterilizing Eppendorf pipe, in high speed freezing centrifuge
10000r/min is centrifuged 5min, inhales and abandons supernatant；1mL sterilizing distilled water is added to mix, 10000r/min in high speed freezing centrifuge
It is centrifuged 5min, inhales and abandons supernatant；Then plus sterilizing distilled water 1mL is mixed, and is set 100 DEG C and is boiled 5min, in high speed freezing centrifuge
10000r/min is centrifuged 2min, takes supernatant as DNA profiling, -20 DEG C of storages are spare.
3. the method for salmonella in a kind of detection textile according to claim 1, it is characterised in that: step 2.2.5)
Described in detection method: when deposited phenomenon is unobvious, reaction tube be added 1000 × SYBR GreenI dyestuff, 2 μ L, gently
It mixes, observes color change immediately, green is the positive, orange for feminine gender.
4. the method for salmonella in a kind of detection textile according to claim 1, it is characterised in that: step 2.2.5)
Described in detection method: when deposited phenomenon is unobvious, amplification curve is generated by the real-time transmissometer of LAMP, works as reaction system
When turbidity value is more than 0.1, result judgement is the positive.
5. the method for salmonella in a kind of detection textile according to claim 1, it is characterised in that: in step 3.3)
The concentration for preparing matrix is not more than 10mg/mL.
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| CN101045943A (en) * | 2007-04-26 | 2007-10-03 | 上海交通大学 | Process of PCR detecting salmonella with added amplifying internal standard |
| CN101149355A (en) * | 2007-11-09 | 2008-03-26 | 东北农业大学 | Method for detecting cow's milk salmonella |
| CN102424842A (en) * | 2011-12-21 | 2012-04-25 | 中国人民解放军疾病预防控制所 | Salmonella LAMP (loop-mediated isothermal amplification) detection method, and special primer and kit thereof |
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| CN101045943A (en) * | 2007-04-26 | 2007-10-03 | 上海交通大学 | Process of PCR detecting salmonella with added amplifying internal standard |
| CN101149355A (en) * | 2007-11-09 | 2008-03-26 | 东北农业大学 | Method for detecting cow's milk salmonella |
| CN102424842A (en) * | 2011-12-21 | 2012-04-25 | 中国人民解放军疾病预防控制所 | Salmonella LAMP (loop-mediated isothermal amplification) detection method, and special primer and kit thereof |
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