CN106544436A - A kind of method of Salmonella in quick detection textile - Google Patents
A kind of method of Salmonella in quick detection textile Download PDFInfo
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- CN106544436A CN106544436A CN201611049531.5A CN201611049531A CN106544436A CN 106544436 A CN106544436 A CN 106544436A CN 201611049531 A CN201611049531 A CN 201611049531A CN 106544436 A CN106544436 A CN 106544436A
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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 in a kind of quick detection textile Salmonella method, comprise the steps：Step one：Sample increases bacterium；Step 2：LAMP is tested；Step 3：MALDI TOF MS technical appraisement；Step 4：As a result report.Detection time is substantially reduced using the method, negative result is only needed 1 day, and positive test symbol only needs 3 days.The method uses LAMP technology and MALDI TOF MS combination of sciences, both it is simple, economical, there is high flux feature again, it is adapted to instant, Site Detection, if Site Detection is the positive, can autotelic increasing sampling experiment room censorship, make supervision not only response speed of getting up fast, with strong points, and save the time.
Description
Technical field
The present invention relates to a kind of method of Salmonella, more particularly, to Salmonella in a kind of quick detection textile
Method.
Background technology
Salmeterol fluticasone propionate main background in textile：Salmonella (Salmonella) is the disease of a class zoonosiss
Opportunistic pathogen, is enterobacteriaceae Salmonella member, is the endotrophic Gram-negative enterobacteria of a class conditionality.This bacterium is sought
Support less demanding, distributed in nature is extremely wide, can detect, can infect various in food, water, soil, animal hair, fiber
Animal and people, it is most of have it is very strong pathogenic.The Salmonella serogroup for being found at present has more than 2500, wherein being permitted
Many serotype bacterium can the cross infection between humans and animals, the various different clinical manifestations of humans and animals can be caused, mainly caused
Heating, gastroenteritiss, diarrhoea and septicemia etc., the health for giving people class cause serious harm.In recent years as economic globalization is entered
The quickening of journey and the deep development of China's opening, the country of China increasingly increase with textile quantity required is imported and exported, and which is taken
Risk with the harms of microbe mankind increases year by year, and pathogen is can not be ignored by the incoming risk in environmental pollution, port, and Jing is looked into
Data and investigation, the textile being contaminated may carry infectious extremely strong, pathogenic extremely strong Salmonella.Due to Salmonella
Bacterium history of existence is long, widely distributed, can be used as one important goal bacterium of sanitary inspection, Salmeterol fluticasone propionate in current textile
Based on plate isolation conventional identification, not only cumbersome, time-consuming, because of Salmonella phenotypic characteristic and citrobacter, big
The more difficult resolution of intestinal Escherichia, Bacillus proteuss, enterobacter cloacae, serum cross reaction ratio are very big, and institute easily receives in the conventional way
Miscellaneous bacteria disturbs, and causes false negative result, and Sensitivity and Specificity is all impacted, it is impossible to adapt to quick detection when epidemic situation occurs.
Therefore, it is badly in need of the prospective research carried out about Salmonella test in laboratory method in textile at present, sets up Salmonella
The accurate method for quick of bacterium, is applied to the monitoring and detection of Salmonella in textile, prevents trouble before it happens, so as to epidemic situation
Reach effectively prevention and control.
The present situation of Salmeterol fluticasone propionate in textile：Salmeterol fluticasone propionate has various methods, 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
Trivial, cycle length, sensitivity are low, and molecular biology method, immunological method, the development of electrical impedance method substantially increase Salmonella
Detection sensitivity, shortens detection time, simplifies detection program, but these methods can only be final positive as prescreening method
Result of determination 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.
The content of the invention
In view of this, the purpose of the present invention is for the deficiencies in the prior art, there is provided a kind of accurate quick detection Salmonella
The method of bacterium, has reached efficient, accurate, time saving and energy saving effect.
To reach above-mentioned purpose, the present invention is employed the following technical solutions：
In a kind of quick detection textile, the method for Salmonella, comprises the steps：
Step one：Sample increases bacterium
Sampled with aseptic mode, Zengjing Granule, obtain the first enrichment liquid；
Step 2：LAMP is tested
2.1) preparation of DNA profiling：Take enrichment liquid to be put in sterilizing Eppendorf pipes, at High speed refrigerated centrifuge centrifugation
Reason, removes impurity, obtains DNA profiling；
2.2) LAMP amplified reactions：
2.2.1) amplification preparation：LAMP reaction tube number n (n=1 pipes blanks+1 according to required for sample size setting
+ 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 order of table 1, mix homogeneously, 2000rpm centrifugation 10sec, to setting
23 μ L are separately added in n fixed LAMP reaction tube；
1 reaction system of table
2.2.2 blank, negative control and positive control are set)；
Blank：DNA profiling is replaced with water；Negative control：First enrichment liquid is replaced with water, by step 2.1) in the way of
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：By step 2.1) in prepare DNA profiling, step 2.2.2) obtained by blank, feminine gender
Control and positive control are respectively taken in the corresponding reaction tube of 2 μ L additions, make reaction system reach 25 μ L；Lid is covered tightly, is mixed,
2000rpm is centrifuged 10sec；
2.2.4 machine reaction on)：By step 2.2.3) reaction tube is placed in water-bath or the real-time transmissometers of LAMP, and 65
DEG C amplification 40min；
2.2.5) preliminary observation result：By step 2.2.4) reaction tube put under black background observe, have white precipitate
Person is the positive, is feminine gender without white precipitate person；If result is feminine gender, Salmonella can not be detected in directly reporting sample, such as
As a result for positive or suspicious, proceed the experiment of step 3；
Step 3：MALDI-TOF-MS technical appraisement
3.1) secondary increasing bacterium：By step 2.2.5) preliminary observation result is that the culture 1mL of positive or suspicious sample connects
Plant in the test tube equipped with 10mL rappaport vassiliadis mdeiums, 42 DEG C of ± 1 DEG C of culture 24h ± 2h obtain the second enrichment liquid；Step is taken separately
Rapid 2.2.5) preliminary observation result is that positive or suspicious culture 1mL is inoculated in and increases bacterium equipped with 10mL selenites cystine
In the test tube of liquid, 36 DEG C of ± 1 DEG C of culture 24h ± 2h obtain the 3rd enrichment liquid；
3.2) separate：With aseptic inoculation ring picking step 3.1) in the second enrichment liquid and each 1 ring of the 3rd enrichment liquid, respectively
Streak inoculation is in a bismuth sulfite agar flat board and a salmonella color culture medium flat board；It is placed in 36 DEG C of ± 1 DEG C of constant temperature
Incubator, bismuth sulfite agar flat board culture 48h ± 2h, salmonella color culture medium flat board culture 24h ± 2h；Culture terminates
After observe colony growth situation on each flat board, a kind of situation is：Grow without suspicious or colonies typical, directly report Salmonella
Do not detect；Another kind of situation is：There is suspicious or colonies typical to grow, then suspicious described in picking or colonies typical after purification, is carried out
MALDI-TOF-MS technical appraisement confirms；On bismuth sulfite agar flat board, Salmonella colony characteristicses sepia or Lycoperdon polymorphum Vitt are to black
Color, has metallic luster sometimes, and surrounding media is in brown or black；, in celadon, surrounding media is constant or micro- for some bacterial strains
It is dimmed；Salmonella colony characteristicses on its chromogenic culture medium are judged according to the explanation of chromogenic culture medium, picking 2 or 2
Typical or suspicious bacterium colony on individual above bismuth sulfite agar flat board and salmonella color culture medium flat board, by MALDI-TOF-
The suspicious bacterium colony fingerprint chromatogram of MS technical appraisement, draws qualification result using MALDI Biotyper System analysis contrasts；
3.3) standard substance and substrate are prepared：The standard solvent of configuration standard product and substrate be volume ratio be 50% acetonitrile+
+ 2.5% trifluoroacetic acid of 47.5% water；Standard substance are prepared：The special standard substance of MALDI Biotyper System are taken out, is added
50 μ L of standard solvent；Using the mode of pipettor pressure-vaccum repeatedly, Biotyper System powder is dissolved.Room temperature place 5min it
Repeat pressure-vaccum afterwards, 13000r/min centrifugation 2min are standby；Substrate preparation：Take out the special bases of MALDI Biotyper System
Matter HCCAportioned, adds 250 μ L standard solvents, vibration to mix, until forming settled solution；
3.4) MALDI BioTyper are identified automatically：Picking bismuth sulfite agar flat board and salmonella color culture medium are flat
On plate, single bacterium colony uniform application is in target plate wad cutter, plus 1 μ L HCCA matrix solutions cover above-mentioned sample spot, dry under room temperature；
Target plate is put into into the automatic assessing instruments of MALDI BioTyper, sample during MALDI Biotyper System analyses, is gathered simultaneously
Quality spectrogram, obtains protein peak information, automatic qualification result by FlexControl3.0；
Step 4：As a result report
According to the test of step 2, PRELIMINARY RESULTS is feminine gender, can not detect Salmonella in directly reporting sample；It is preliminary to tie
Fruit is positive or suspicious sample, by the testing result of step 3, detects or do not detect Salmonella in reporting sample.
Preferably, the sampling, Zengjing Granule are to open censorship textile samples with aseptic mode, equal with sterile scissors
Even sample cutting, weighs and is added to after the sample 25g for cutting is shredded in the aseptic homogenizing bag for filling 225mL SCDLP fluid mediums,
1min～2min is patted with slap type homogenizer, is fully mixed；The sample for preparing is put into into 36 DEG C of ± 1 DEG C of constant incubators
In, Zengjing Granule 16h ± 2h obtain the first enrichment liquid.
Preferably, step 2.1) DNA profiling be prepared as take 1mLSCDLP enrichment liquids be put into 1.5mL sterilizing
In Eppendorf pipes, 10000r/min centrifugations 5min in High speed refrigerated centrifuge, suction abandon supernatant；1mL sterilizing distilled waters are added to mix
Even, 10000r/min centrifugations 5min in High speed refrigerated centrifuge, suction abandon supernatant；Then plus sterilizing distilled water 1mL is mixed, 100 are put
DEG C 5min is boiled, 10000r/min centrifugations 2min, takes supernatant as DNA profiling in High speed refrigerated centrifuge, and -20 DEG C of storages are standby
With.
Preferably, step 2.2.5) described in detection method：When deposited phenomenon is not obvious, 1000 are added in reaction tube
2 μ L of × SYBR GreenI dyestuffs, gently mix, and observe 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 not obvious, by 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, step 3.2) in prepare substrate concentration be not more than 10mg/mL.
In the present invention, commonly used equipment and material are as follows：
1) equipment and material
The basic equipment and material of 1.1 conventional microbiologicals detection.
1.2 sterile scissors, tweezers.
1.3 electronic balance：0.01g.
The aseptic homogenizing bag of 1.4 full filter screens.
1.5 slap type homogenizer：400mL.
1.6 constant incubator：36℃±1℃、42℃±1℃.
1.7 inoculating loop.
1.8 microscope slide.
1.9 micropipettor：1 μ L～20 μ L of range；200 μ L～1000 μ L of range.
1.10 sterilizing Eppendorf centrifuge tubes, LAMP reaction tubes：1.5mL.
1.11 high speed centrifuge：2000r/min～13000r/min.
1.12 water-bath：65℃±1℃.
The real-time transmissometers of 1.13 LAMP.
The automatic assessing instruments of 1.14 MALDI BioTyper.
2 culture medium and reagent
2.1 SCDLP fluid 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 buffers：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 primer：According to the invA gene conserved sequences of Salmonella, a set of LAMP specific primers are 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 triphosphates (dNTP) mixture：Every kind of nucleotide concentration 10mmol/L.
2.11 8U/ μ L Bst archaeal dna polymerases：8U/μL.
2.12 nitrite ion：SYBR Green I dyestuffs, 1000 ×.
2.13 formic acid.
2.14 acetonitrile.
2.15 acetic acid.
2.16 trifluoroacetic acid.
2.17 standard substance Bacterial Test Standard (BTS).
2.18 substrate HCCAportioned.
3 Quality-control strains:Salmonella london CMCC50106, Salmonella enteritidis (hydrogen sulfide is positive) CICC21482, pig
Cholera Salmonella (hydrogen sulfide is negative) CICC21493, Salmonella anatis CMCC50353, Salmonella choleraesuls (hydrogen sulfide the moon
Property) ATCC10708, Salmonella choleraesuls (hydrogen sulfide negative) ATCC12325, staphylococcus aureuses ATCC29213, green pus
Bacillus ATCC10145, Listeria monocytogenes CMCC54002, Enterobacter sakazakii IQCC10403, vibrio parahaemolyticus CICC
21617th, escherichia coli CMCC44102, Escherichia coli O 157 NCTC12900.
8 methods detect program
Method detection program see the table below：
The invention has the beneficial effects as follows：
1. loop-mediated isothermal amplification technique (LAMP) technology is carried out instead under isothermal conditions using strand displacement archaeal dna polymerase
Should, nucleic acid amplification reaction can be completed in 40min, amplification can observe by the naked eye judgement.Detection process need not be held high simultaneously
Expensive instrument and equipment, simple water-bath, course of reaction do not need the thermal denaturation of template, prolonged temperature cycles, expand
Volume increase thing does not need loaded down with trivial details electrophoresis process, rapid sensitive, high specificity, low cost to be especially suitable for a large amount of, instant, scene
The primary dcreening operation detection of pathogenic microorganism, it is easy in basic unit's popularization and application.And MALDI-TOF-MS technologies are based primarily upon various antibacterial sheets
The protein composition of body and molecular weight, using the distinctive protein spectrum finger printing of MALDI BioTyper Instrument measuring antibacterials,
And be compared with the mass spectrum of given data storehouse antibacterial, so as to identify to unknown antibacterial.The detectable molecule of the technology
Amount scope is very big, and instrumentation is simple, and scanning speed is fast, with sensitivity is high, high specificity and the features such as big repeatability, should
Method is increasingly automated, reduces experimental error, and its result is clearly, uniquely, it is not necessary to other appended experimentals, and target plate is repeatable to be made
With saving testing cost.A kind of method of accurate quick detection Salmonella provided by the present invention is based on LAMP technology
The characteristics of with MALDI-TOF-MS technologies, according to the distinctive target-gene sequence of Salmonella, for 6 independent zones of target gene
2 pairs of special interior outer primers are designed in domain, start circulation strand replacement reaction using archaeal dna polymerase, under isothermy (65 DEG C or so)
Increasing the DNA profiling that extracts after bacterium to one step of Salmonella carries out insulation 40min, you 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
Flat board and bismuth sulfite agar flat board, typical on picking flat board or suspicious bacterium colony are quick using MALDI-TOF-MS technical appraisement
Identify result.
2. 1. the method flow process substantially reduces detection time to method advantage, and traditional negative result needs 3～7
My god, positive test symbol needs 6～7 days, and rebuilding method negative result is only needed 1 day, and positive test symbol only needs 3 days.
2. LAMP technology is simple, economical, is adapted to instant, Site Detection, if Site Detection is the positive, you can autotelic
Sampling experiment room censorship is increased, is made supervision not only response speed of getting up fast, with strong points, and is saved the time.
3. this method is applied widely, is not only suitable for epidemic prevention and health supervision department, can also examine in enterprise and domestic hygiene
Used in survey.
4. the method is simple to operate, quick, efficient, and high specificity, sensitivity are high, as a result observes convenient, directly perceived, suitable base
Layer popularization and application.
5. loop-mediated isothermal amplification method advantage：Sensitivity high (than high 2～5 orders of magnitude of traditional PCR method)；Reaction
Time is short, can just complete within 30～60 minutes reaction；Clinical practice does not need special instrument；It is simple to operate, whether DNA or
RNA, detecting step are that reactant liquor, enzyme and template need to be mixed in reaction tube, are placed in 63 left DEG C in water-bath or calorstat
Right insulation 30～60 minutes, visual results.
Description of the drawings
Fig. 1：1～No. 30 sample LAMP amplification human visual figure；
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 rate of change figures；
Fig. 5：Salmonella and its control MALDI BioTyper identification collection of illustrative plates.
Specific embodiment
Embodiment 1
In a kind of quick detection textile, the method for Salmonella, comprises the steps：
Step one：Sample increases bacterium
Aseptic procedure opens censorship textile samples, with the uniform sample cutting of sterile scissors, accurately weighs and cut on electronic balance
Under sample 25g, be added to after shredding in the aseptic homogenizing bag for filling 225mL SCDLP fluid mediums, use slap type homogenizing
Device pats 1min～2min, fully mixes；The sample for preparing is put in 36 DEG C of ± 1 DEG C of constant incubators, Zengjing Granule 16h
± 2h, obtains the first enrichment liquid；
Step 2：LAMP is tested
2.1) preparation of DNA profiling：Take 1mLSCDLP enrichment liquids to be put in 1.5mL sterilizing Eppendorf pipes, freeze at a high speed
10000r/min centrifugations 5min in centrifuge, suction abandon supernatant；1mL sterilizing distilled waters are added to mix, in High speed refrigerated centrifuge
10000r/min is centrifuged 5min, and supernatant is abandoned in suction；Then plus sterilizing distilled water 1mL is mixed, put 100 DEG C and boil 5min, freeze at a high speed
In centrifuge, 10000r/min centrifugations 2min, takes supernatant as DNA profiling, and -20 DEG C of storages are standby；
2.2) LAMP amplified reactions：
2.2.1) amplification preparation：According to sample size setting required for LAMP reaction tube number n (n=1 pipe blanks+
+ 1 pipe positive control of 1 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 order of table 1, mix homogeneously, 2000rpm centrifugation 10sec, to setting
N LAMP reaction tube in be separately added into 23 μ L；
1 reaction system of table
2.2.2 blank, negative control and positive control are set), it is right that negative control, blank all should be set per secondary response
According to and positive control；Blank：DNA profiling is replaced with water；Negative control：Sample is replaced with water, by step 2.1) in the way of
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：By step 2.1) in the DNA profiling, blank obtained by step 2.2.2, negative right for preparing
Add in corresponding reaction tube according to 2 μ L respectively being taken with positive control, make reaction system reach 25 μ L；Lid is covered tightly, is mixed,
2000rpm is centrifuged 10sec；
2.2.4 machine reaction on)：By step 2.2.3) reaction tube is placed in water-bath or the real-time transmissometers of LAMP, and 65
DEG C amplification 40min；
2.2.5) preliminary observation result：By step 2.2.4) reaction tube put under black background observe, have white precipitate
Person is the positive, is feminine gender without white precipitate person；If result is feminine gender, Salmonella can not be detected in directly reporting sample, such as
As a result for positive or suspicious, carry out the experiment of step 3；
Step 3：MALDI-TOF-MS technical appraisement
3.1) secondary increasing bacterium：Taking 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 of ± 1 DEG C of culture 24h ± 2h obtain the second enrichment liquid；Step 2.2.5 is taken separately) it is preliminary
Observation result is that positive culture 1mL is inoculated in the test tube equipped with 10mL selenite cystine broths, 36 DEG C ± 1 DEG C
Culture 24h ± 2h, obtains the 3rd enrichment liquid；
3.2) separate：With aseptic inoculation ring picking step 3.1) obtained by the second enrichment liquid and each 1 ring of the 3rd enrichment liquid, respectively
Streak inoculation is in a bismuth sulfite agar flat board and a salmonella color culture medium flat board；It is placed in 36 DEG C of ± 1 DEG C of constant temperature
Incubator, bismuth sulfite agar flat board culture 48h ± 2h, salmonella color culture medium flat board culture 24h ± 2h；Culture terminates
After observe colony growth situation on each flat board, such as grow without suspicious or colonies typical, can directly report that Salmonella is not detected；
Grow if any suspicious or colonies typical, then suspicious described in picking or colonies typical after purification, carries out MALDI-TOF-MS technology mirror
It is fixed to confirm；On bismuth sulfite agar flat board, Salmonella colony characteristicses sepia or Lycoperdon polymorphum Vitt have metallic luster sometimes to black, week
It is in brown or black to enclose culture medium；, in celadon, surrounding media is constant or micro- dimmed for some bacterial strains；Salmonella develops the color at which
In culture medium, colony characteristicses are judged according to the explanation of chromogenic culture medium, 2 or more than 2 bismuth sulfite agar flat boards of picking
Typical with salmonella color culture medium flat board or suspicious bacterium colony, by the suspicious bacterium colony fingerprint of MALDI-TOF-MS technical appraisement
Spectrogram, draws qualification result using MALDI Biotyper System analysis contrasts；
3.3) standard substance and substrate are prepared：The standard solvent of configuration standard product and substrate be volume ratio be 50% acetonitrile+
+ 2.5% trifluoroacetic acid of 47.5% water；Standard substance are prepared：The special standard substance of MALDI Biotyper System are taken out, is added
50 μ L of standard solvent.Using the mode of pipettor pressure-vaccum repeatedly, BTS powder is dissolved.Room temperature place 5min after repeat pressure-vaccum, 13
000r/min is centrifuged 2min, standby；Substrate preparation：Take out the special substrate of MALDI Biotyper System
HCCAportioned, adds 250 μ L standard solvents, its final concentration to be not more than 10mg/mL, and vibration is mixed, molten until forming clarification
Liquid；
3.4) MALDI BioTyper are identified automatically：It is single on picking BS flat boards and salmonella color culture medium flat board
Bacterium colony uniform application is in target plate wad cutter, plus 1 μ L HCCA matrix solutions cover above-mentioned sample spot, dry under room temperature.Target plate is put
Enter the automatic assessing instruments of MALDI BioTyper, during MALDI Biotyper System analyses, gather sample mass spectrum simultaneously,
Protein peak information, automatic qualification result are obtained by FlexControl3.0；
Step 4：As a result report
According to the test of step 2, Salmeterol fluticasone propionate result in preliminary judgement sample, PRELIMINARY RESULTS are feminine gender, can be direct
Salmonella is not detected in report sample, such as PRELIMINARY RESULTS is positive or suspicious, is confirmed by step 3, in report sample
Detection does not detect Salmonella.
Embodiment 2
In a kind of quick detection textile, the method for Salmonella, comprises the steps：
Step one：Sample increases bacterium
Aseptic procedure opens censorship textile samples, with the uniform sample cutting of sterile scissors, accurately weighs and cut on electronic balance
Under sample 25g, be added to after shredding in the aseptic homogenizing bag for filling 225mL SCDLP fluid mediums, use slap type homogenizing
Device pats 1min～2min, fully mixes；The sample for preparing is put in 36 DEG C of ± 1 DEG C of constant incubators, Zengjing Granule 16h
± 2h, obtains the first enrichment liquid；
Step 2：LAMP is tested
2.1) preparation of DNA profiling：Take 1mLSCDLP enrichment liquids to be put in 1.5mL sterilizing Eppendorf pipes, freeze at a high speed
10000r/min centrifugations 5min in centrifuge, suction abandon supernatant；1mL sterilizing distilled waters are added to mix, in High speed refrigerated centrifuge
10000r/min is centrifuged 5min, and supernatant is abandoned in suction；Then plus sterilizing distilled water 1mL is mixed, put 100 DEG C and boil 5min, freeze at a high speed
In centrifuge, 10000r/min centrifugations 2min, takes supernatant as DNA profiling, and -20 DEG C of storages are standby；
2.2) LAMP amplified reactions：
2.2.1) amplification preparation：According to sample size setting required for LAMP reaction tube number n (n=1 pipe blanks+
+ 1 pipe positive control of 1 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 order of table 1, mix homogeneously, 2000rpm centrifugation 10sec, to setting
N LAMP reaction tube in be separately added into 23 μ L；
1 reaction system of table
2.2.2 blank, negative control and positive control are set), it is right that negative control, blank all should be set per secondary response
According to and positive control；Blank：DNA profiling is replaced with water；Negative control：Sample is replaced with water, by step 2.1) in the way of
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：By step 2.1) in the DNA profiling, blank obtained by step 2.2.2, negative right for preparing
Add in corresponding reaction tube according to 2 μ L respectively being taken with positive control, make reaction system reach 25 μ L；Lid is covered tightly, is mixed,
2000rpm is centrifuged 10sec；
2.2.4 machine reaction on)：By step 2.2.3) reaction tube is placed in water-bath or the real-time transmissometers of LAMP, and 65
DEG C amplification 40min；
2.2.5) preliminary observation result：By step 2.2.4) reaction tube put under black background observe, have white precipitate
Person is the positive, is feminine gender without white precipitate person；When deposited phenomenon is not obvious, 1000 × SYBR Green I are added in reaction tube
2 μ L of dyestuff, gently mix, and observe color change immediately, and green is the positive, orange for feminine gender；If result is feminine gender, can be direct
Salmonella is not detected in report sample, such as result is positive or suspicious, carries 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 mdeiums, 42 DEG C of ± 1 DEG C of cultures
24h ± 2h, obtains the second enrichment liquid；Step 2.2.5 is taken separately) preliminary observation result is that positive culture 1mL is inoculated in and is equipped with
In the test tube of 10mL selenite cystine broths, 36 DEG C of ± 1 DEG C of culture 24h ± 2h obtain the 3rd enrichment liquid；
3.2) separate：With aseptic inoculation ring picking step 3.1) obtained by the second enrichment liquid and each 1 ring of the 3rd enrichment liquid, respectively
Streak inoculation is in a bismuth sulfite agar flat board and a salmonella color culture medium flat board；It is placed in 36 DEG C of ± 1 DEG C of constant temperature
Incubator, bismuth sulfite agar flat board culture 48h ± 2h, salmonella color culture medium flat board culture 24h ± 2h；Culture terminates
After observe colony growth situation on each flat board, such as grow without suspicious or colonies typical, can directly report that Salmonella is not detected；
Grow if any suspicious or colonies typical, then suspicious described in picking or colonies typical after purification, carries out MALDI-TOF-MS technology mirror
It is fixed to confirm；On bismuth sulfite agar flat board, Salmonella colony characteristicses sepia or Lycoperdon polymorphum Vitt have metallic luster sometimes to black, week
It is in brown or black to enclose culture medium；, in celadon, surrounding media is constant or micro- dimmed for some bacterial strains；Salmonella develops the color at which
In culture medium, colony characteristicses are judged according to the explanation of chromogenic culture medium, 2 or more than 2 bismuth sulfite agar flat boards of picking
Typical with salmonella color culture medium flat board or suspicious bacterium colony, by the suspicious bacterium colony fingerprint of MALDI-TOF-MS technical appraisement
Spectrogram, draws qualification result using MALDI Biotyper System analysis contrasts；
3.3) standard substance and substrate are prepared：The standard solvent of configuration standard product and substrate be volume ratio be 50% acetonitrile+
+ 2.5% trifluoroacetic acid of 47.5% water；Standard substance are prepared：The special standard substance of MALDI Biotyper System are taken out, is added
50 μ L of standard solvent.Using the mode of pipettor pressure-vaccum repeatedly, BTS powder is dissolved.Room temperature place 5min after repeat pressure-vaccum, 13
000r/min is centrifuged 2min, standby；Substrate preparation：Take out the special substrate of MALDI Biotyper System
HCCAportioned, adds 250 its Cmax of μ L standard solvents to be 10mg/mL, and vibration is mixed, until forming settled solution；
3.4) MALDI BioTyper are identified automatically：On picking bismuth sulfite agar and salmonella color culture medium flat board
Single bacterium colony uniform application is in target plate wad cutter, plus 1 μ LHCCA matrix solutions cover above-mentioned sample spot, dry under room temperature；By target
Plate is put into the automatic assessing instruments of MALDI BioTyper, gathers sample mass spectrum during MALDI BiotyperSystem analyses simultaneously
Figure, obtains protein peak information, automatic qualification result by FlexControl3.0；
Step 4：As a result report
According to the test of step 2, Salmeterol fluticasone propionate result in preliminary judgement sample, PRELIMINARY RESULTS are feminine gender, can be direct
Salmonella is not detected in report sample, such as PRELIMINARY RESULTS is positive or suspicious, is confirmed by step 3, in report sample
Detection does not detect Salmonella.
Embodiment 3
In a kind of quick detection textile, the method for Salmonella, comprises the steps：
Step one：Sample increases bacterium
Aseptic procedure opens censorship textile samples, with the uniform sample cutting of sterile scissors, accurately weighs and cut on electronic balance
Under sample 25g, be added to after shredding in the aseptic homogenizing bag for filling 225mLSCDLP fluid mediums, use slap type homogenizer
1min～2min is patted, is fully mixed；The sample for preparing is put in 36 DEG C of ± 1 DEG C of constant incubators, Zengjing Granule 16h ±
2h, obtains the first enrichment liquid；
Step 2：LAMP is tested
2.1) preparation of DNA profiling：Take 1mLSCDLP enrichment liquids to be put in 1.5mL sterilizing Eppendorf pipes, freeze at a high speed
10000r/min centrifugations 5min in centrifuge, suction abandon supernatant；1mL sterilizing distilled waters are added to mix, in High speed refrigerated centrifuge
10000r/min is centrifuged 5min, and supernatant is abandoned in suction；Then plus sterilizing distilled water 1mL is mixed, put 100 DEG C and boil 5min, freeze at a high speed
In centrifuge, 10000r/min centrifugations 2min, takes supernatant as DNA profiling, and -20 DEG C of storages are standby；
2.2) LAMP amplified reactions：
2.2.1) amplification preparation：LAMP reaction tube number n (n=1 pipes blanks+1 according to required for sample size setting
+ 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 order of table 1, mix homogeneously, 2000rpm centrifugation 10sec, to setting
N LAMP reaction tube in be separately added into 23 μ L；
1 reaction system of table
2.2.2 blank, negative control and positive control are set)
Per secondary response, negative control, blank and positive control, blank all should be set：DNA profiling is replaced with water；
Negative control：With water replace sample, by step 2.1) in the way of extract the template that template DNA is reacted as the LAMP；It is positive
Control：Salmonella reference culture increase bacterium after by step 2.1) in the way of extract the template that template DNA is reacted as the LAMP；
2.2.3) it is loaded：By step 2.1) in the DNA profiling, blank obtained by step 2.2.2, negative right for preparing
Add in corresponding reaction tube according to 2 μ L respectively being taken with positive control, make reaction system reach 25 μ L；Lid is covered tightly, is mixed,
2000rpm is centrifuged 10sec；
2.2.4 machine reaction on)：By step 2.2.3) reaction tube is placed in water-bath or the real-time transmissometers of LAMP, and 65
DEG C amplification 40min；
2.2.5) preliminary observation result：By step 2.2.4) reaction tube put under black background observe, have white precipitate
Person is the positive, is feminine gender without white precipitate person；Preferably, step 2.2.5) described in detection method：When deposited phenomenon is failed to understand
When aobvious, amplification curve is generated by the real-time transmissometers of LAMP, when reaction system turbidity value is more than 0.1, result judgement is the positive；
If result is feminine gender, Salmonella in directly reporting sample, can not be detected, such as result is positive or suspicious, carries out 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 of ± 1 DEG C of culture 24h ± 2h obtain the second enrichment liquid；Step 2.2.5 is taken separately) it is preliminary
Observation result is that positive culture 1mL is inoculated in the test tube equipped with 10mL selenite cystine broths, 36 DEG C ± 1 DEG C
Culture 24h ± 2h, obtains the 3rd enrichment liquid；
3.2) separate：With aseptic inoculation ring picking step 3.1) obtained by the second enrichment liquid and each 1 ring of the 3rd enrichment liquid, respectively
Streak inoculation is in a bismuth sulfite agar flat board and a salmonella color culture medium flat board；It is placed in 36 DEG C of ± 1 DEG C of constant temperature
Incubator, bismuth sulfite agar flat board culture 48h ± 2h, salmonella color culture medium flat board culture 24h ± 2h；Culture terminates
After observe colony growth situation on each flat board, such as grow without suspicious or colonies typical, can directly report that Salmonella is not detected；
Grow if any suspicious or colonies typical, then suspicious described in picking or colonies typical after purification, carries out MALDI-TOF-MS technology mirror
It is fixed to confirm；On bismuth sulfite agar flat board, Salmonella colony characteristicses sepia or Lycoperdon polymorphum Vitt have metallic luster sometimes to black, week
It is in brown or black to enclose culture medium；, in celadon, surrounding media is constant or micro- dimmed for some bacterial strains；Salmonella develops the color at which
In culture medium, colony characteristicses are judged according to the explanation of chromogenic culture medium, 2 or more than 2 bismuth sulfite agar flat boards of picking
Typical with salmonella color culture medium flat board or suspicious bacterium colony, by the suspicious bacterium colony fingerprint of MALDI-TOF-MS technical appraisement
Spectrogram, draws qualification result using MALDI Biotyper System analysis contrasts；
3.3) standard substance and substrate are prepared：The standard solvent of configuration standard product and substrate be volume ratio be 50% acetonitrile+
+ 2.5% trifluoroacetic acid of 47.5% water；Standard substance are prepared：Take out the special standard substance of MALDI Biotyper System
(Bacterial Test Standard, BTS), adds 50 μ L of standard solvent.Using the mode of pipettor pressure-vaccum repeatedly, dissolving
BTS powder；Room temperature repeats pressure-vaccum after placing 5min, 13000r/min centrifugation 2min are standby；Substrate preparation：Take out MALDI
Biotyper System special substrate HCCAportioned, adds 250 μ L standard solvents, its concentration to be not more than 10mg/mL,
Vibration is mixed, until forming settled solution；
3.4) MALDI BioTyper are identified automatically：Single bacterium on picking BS flat boards and salmonella color culture medium flat board
Uniform application fall in target plate wad cutter, plus 1 μ L HCCA matrix solutions cover above-mentioned sample spot, dry under room temperature.Target plate is put into
The automatic assessing instruments of MALDI BioTyper, gather sample mass spectrum simultaneously during MALDI Biotyper System analyses, by
FlexControl3.0 obtains protein peak information, automatic qualification result；
Step 4：As a result report
According to the test of step 2, Salmeterol fluticasone propionate result in preliminary judgement sample, PRELIMINARY RESULTS are feminine gender, can be direct
Salmonella is not detected in report sample, such as PRELIMINARY RESULTS is positive or suspicious, is confirmed by step 3, in report sample
Detection does not detect Salmonella.
Method stability test：
First, prepare sample
1st, collect sample
Totally 20 parts of the sample of the sample and different purposes of different fiber compositions is collected, including：2 parts of the new cotton of shades of colour,
New 1 part of diablement fort, 1 part of silk, 1 part of knitting wool clothes material, 1 part of cotton thread, 1 part of woven dacron, 1 part of blending 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 fabric 1
Part, 1 part of soft plain-weave silk fabric cloth, 2 parts of velvet do high pressure during one aseptic sampler bag of every a sample loading is dried Jing after water rinsing and go out
Bacterium is processed.Respectively numbering 1,2,3,4 ..., 20, in each numbering, sample type is random.10 traditional technique in measuring sun are collected separately
The sample of property, including：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 numbering 21,22,23,24 ... 30.
2nd, sample making
Take quality control standard bacterial strain salmonella london CMCC50106, Salmonella 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 aureuses
ATCC29213, bacillus pyocyaneus ATCC10145, Listeria monocytogenes CMCC54002, Enterobacter sakazakii IQCC10403, secondary haemolysis
Property vibrio CICC 21617, escherichia coli CMCC44102, Escherichia coli O 157 NCTC12900, respectively be inoculated with 300mL nutrient meats
Bring back to life in soup, the meat soup after culture respectively takes 10ml and mixed, and mixed bacteria liquid is inoculated with flat board, Jing plate counts, Salmonella
Original bacterial concentration be 5 × 109CFU/ml, after 10 times of doubling dilutions, choose high concentration bacterium solution (5 × 105CFU/ml) artificial
The placement overnight of 1～No. 8 sample of pollution, takes the placement overnight of 9～No. 18 samples of low concentration bacterium solution (5CFU/ml) artificial contamination.
2nd, test
Simultaneously 1～No. 30 sample is detected according to rebuilding method.Single bacterium is isolated to LAMP detection positive findingses
Fall, using MALDI-TOF MS technical appraisement, final 1～No. 8,9～No. 18, the whole detection Salmonellas of 21～No. 30 samples,
It is consistent with expected resultss.Concrete outcome is shown in Table 2.Fig. 1 lists 1～No. 30 sample LAMP amplification human visual figure, Fig. 2 row
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 rate of change figure, and Fig. 5 lists Salmonella and its control MALDI BioTyper qualification figures
Spectrum.
2 1-30 sample detection situations of table
As can be seen from Table 2, it is artificial to add either high concentration level (5 × 105CFU/ml) or low concentration level
(5CFU/ml), LAMP can be expected detection, and after selective enrichment, MALDI-TOF-MS identifications accuracy rate also reaches
100%, detection time is substantially reduced using the method, negative result is only needed 1 day, and positive test symbol only needs 3 days.Should
Method uses LAMP technology and MALDI-TOF-MS combination of sciences, both simply, it is economical, there is high flux feature again, be adapted to immediately,
Site Detection, if Site Detection is the positive, you can autotelic increasing sampling experiment room censorship, makes supervision get up not only and reacts
Speed is fast, with strong points, and saves the time.
Finally illustrate, only to illustrate technical scheme and unrestricted, this area is common for above example
Other modifications or equivalent that technical staff is made to technical scheme, without departing from the technology of the present invention side
The spirit and scope of case, all should cover in the middle of scope of the presently claimed invention.
Claims (6)
1. in a kind of quick detection textile Salmonella method, it is characterised in that：Comprise the steps：
Step one：Sample increases bacterium
Sampled with aseptic mode, Zengjing Granule, obtain the first enrichment liquid；
Step 2：LAMP is tested
2.1) preparation of DNA profiling：Take enrichment liquid to be put in sterilizing Eppendorf pipes, High speed refrigerated centrifuge centrifugal treating is removed
Decontamination, obtains DNA profiling；
2.2) LAMP amplified reactions：
2.2.1) amplification preparation：(+1 pipe of n=1 pipes blank is cloudy for LAMP reaction tube number n according to required for sample size setting
Property control+1 pipe positive control+sample number), take out n LAMP reaction tube；Amplification reaction system is shown in Table 1, calculates respectively by table 1
The usage amount of reagent, is added in sterile centrifugation tube by the order of table 1, mix homogeneously, 2000rpm centrifugation 10sec, to the n of setting
23 μ L are separately added in individual LAMP reaction tubes；
1 reaction system of table
2.2.2 blank, negative control and positive control are set)；
Blank：DNA profiling is replaced with water；Negative control：With water replace the first enrichment liquid, by step 2.1) in the way of extract
The template that template DNA is reacted as the LAMP；Positive control：By step 2.1 after Salmonella reference culture increasing bacterium) in the way of
Extract the template that template DNA is reacted as the LAMP；
2.2.3) it is loaded：By step 2.1) in prepare DNA profiling, step 2.2.2) obtained by blank, negative control
2 μ L are respectively taken with positive control to add in corresponding reaction tube, makes reaction system reach 25 μ L；Lid is covered tightly, is mixed, 2000rpm
Centrifugation 10sec；
2.2.4 machine reaction on)：By step 2.2.3) reaction tube is placed in water-bath or the real-time transmissometers of LAMP, 65 DEG C of expansions
Increase 40min；
2.2.5) preliminary observation result：By step 2.2.4) reaction tube put under black background observe, person is white precipitate
The positive, is feminine gender without white precipitate person；If result is feminine gender, Salmonella, such as result can not be detected in directly reporting sample
For positive or suspicious, proceed the experiment of step 3；
Step 3：MALDI-TOF-MS technical appraisement
3.1) secondary increasing bacterium
By step 2.2.5) preliminary observation result is that the culture 1mL of positive or suspicious sample is inoculated in equipped with 10mL magnesium chlorides
In the test tube of malachite green bouillon, 42 DEG C of ± 1 DEG C of culture 24h ± 2h obtain the second enrichment liquid；Step 2.2.5 is taken separately) preliminary observation knot
Fruit is inoculated in the test tube equipped with 10mL selenite cystine broths for positive or suspicious culture 1mL, 36 DEG C ± 1 DEG C
Culture 24h ± 2h, obtains the 3rd enrichment liquid；
3.2) separate：With aseptic inoculation ring picking step 3.1) in the second enrichment liquid and each 1 ring of the 3rd enrichment liquid, rule respectively
It is inoculated in a bismuth sulfite agar flat board and a salmonella color culture medium flat board；It is placed in 36 DEG C of ± 1 DEG C of constant temperature culture
Case, bismuth sulfite agar flat board culture 48h ± 2h, salmonella color culture medium flat board culture 24h ± 2h；Culture is seen after terminating
Colony growth situation on each flat board is examined, a kind of situation is：Grow without suspicious or colonies typical, directly report Salmonella is not examined
Go out；Another kind of situation is：There is suspicious or colonies typical to grow, then suspicious described in picking or colonies typical after purification, is carried out
MALDI-TOF-MS technical appraisement confirms；On bismuth sulfite agar flat board, Salmonella colony characteristicses sepia or Lycoperdon polymorphum Vitt are to black
Color, has metallic luster sometimes, and surrounding media is in brown or black；, in celadon, surrounding media is constant or micro- for some bacterial strains
It is dimmed；Salmonella colony characteristicses on its chromogenic culture medium are judged according to the explanation of chromogenic culture medium, picking 2 or 2
Typical or suspicious bacterium colony on individual above bismuth sulfite agar flat board and salmonella color culture medium flat board, by MALDI-TOF-
The suspicious bacterium colony fingerprint chromatogram of MS technical appraisement, draws qualification result using MALDI Biotyper System analysis contrasts；
3.3) standard substance and substrate are prepared：The standard solvent of configuration standard product and substrate be+47.5% water of 50% acetonitrile of volume ratio+
2.5% trifluoroacetic acid；Standard substance are prepared：The special standard substance of MALDI Biotyper System are taken out, standard solvent 50 is added
μL；Using the mode of pipettor pressure-vaccum repeatedly, Biotyper System powder is dissolved；Room temperature repeats pressure-vaccum after placing 5min,
13000r/min is centrifuged 2min, standby；Substrate preparation：Take out the special substrate of MALDI Biotyper System
HCCAportioned, adds 250 μ L standard solvents, vibration to mix, until forming settled solution；
3.4) MALDI BioTyper are identified automatically：On picking bismuth sulfite agar flat board and salmonella color culture medium flat board
Single bacterium colony uniform application is in target plate wad cutter, plus 1 μ L HCCA matrix solutions cover above-mentioned sample spot, dry under room temperature；By target
Plate is put into the automatic assessing instruments of MALDI BioTyper, gathers sample matter during MALDI Biotyper System analyses simultaneously
Spectrogram, obtains protein peak information, automatic qualification result by FlexControl3.0；
Step 4：As a result report
According to the test of step 2, PRELIMINARY RESULTS is feminine gender, can not detect Salmonella in directly reporting sample；PRELIMINARY RESULTS is
Positive or suspicious sample, by the testing result of step 3, detects in reporting sample or does not detect Salmonella.
2. in a kind of quick detection textile according to claim 1 Salmonella method, it is characterised in that：It is described to take
Sample, Zengjing Granule are to open censorship textile samples with aseptic mode, with the uniform sample cutting of sterile scissors, weigh the sample cut
25g is added to after shredding in the aseptic homogenizing bag for filling 225mLSCDLP fluid mediums, pats 1min with slap type homogenizer
～2min, fully mixes；The sample for preparing is put in 36 DEG C of ± 1 DEG C of constant incubators, Zengjing Granule 16h ± 2h, obtains
One enrichment liquid.
3. in a kind of quick detection textile according to claim 1 Salmonella method, it is characterised in that：Step
2.1) DNA profiling be prepared as take 1mLSCDLP enrichment liquids be put into 1.5mL sterilizing Eppendorf pipe in, freeze at a high speed from
10000r/min centrifugations 5min in scheming, suction abandon supernatant；1mL sterilizing distilled waters are added to mix, in High speed refrigerated centrifuge
10000r/min is centrifuged 5min, and supernatant is abandoned in suction；Then plus sterilizing distilled water 1mL is mixed, put 100 DEG C and boil 5min, freeze at a high speed
In centrifuge, 10000r/min centrifugations 2min, takes supernatant as DNA profiling, and -20 DEG C of storages are standby.
4. in a kind of quick detection textile according to claim 1 Salmonella method, it is characterised in that：Step
2.2.5 the detection method described in)：When deposited phenomenon is not obvious, 1000 × SYBR GreenI dyestuffs, 2 μ is added in reaction tube
L, gently mixes, and observes color change immediately, and green is the positive, orange for feminine gender.
5. in a kind of quick detection textile according to claim 1 Salmonella method, it is characterised in that：Step
2.2.5 the detection method described in)：When deposited phenomenon is not obvious, amplification curve is generated by the real-time transmissometers of LAMP, when anti-
When answering system turbidity value more than 0.1, result judgement is the positive.
6. in a kind of quick detection textile according to claim 1 Salmonella method, it is characterised in that：Step
3.2) concentration that substrate is prepared in is not more than 10mg/mL.
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