CN105755141A - Kit for fast detecting live Shigella in food and application of kit - Google Patents
Kit for fast detecting live Shigella in food and application of kit Download PDFInfo
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Abstract
The invention discloses a kit for detecting live Shigella in food and application of the kit.The kit comprises a PCR reaction reagent, ethidium monoazide, positive control and negative control.The kit has the advantages that the kit can distinguish the dead Shigella in the food and is high in specificity and sensitivity; the whole detection process can be completed in less than 2 hours, and the kit is especially suitable for detecting and removing the live Shigella in the food.
Description
(1) technical field
The present invention relates to a kind of nucleic acid Fast Detection Technique congratulating viable bacteria for will, be suitable for will in food is congratulated the qualitative detection of viable bacteria.
(2) background technology
Shigella dysenteriae is widely present in the whole world, especially the developing country in temperate zone, the Southern Hemisphere or subtropical countries and area.Can causing bacillary dysentery and alimentary toxicosis, the whole world is annual person-time is estimated as 1.65 hundred million because of Shigella infection, causes 1,100,000 cases death, and M & M occupies the first place of infectious diarrhea.It is the Category B notifiable disease of regulation report, one of poisoning pathogen of Major Foods in China's law on the prevention and control of infectious diseases.The source of infection of shigella dysenteriae is patient and bacillicarrier, the infection morbidity mainly through the edible food polluted by shigella dysenteriae.The multinomial food hygienic standard of China must not specify and detect shigella dysenteriae.
In current food, the detection method of shigella dysenteriae separates mainly through culture method, identifies, whole process operation is loaded down with trivial details, and length consuming time, positive rate is low.Real time fluorescent PCR method has sensitivity, specificity and stability characteristic, can well make up above-mentioned deficiency.But due to the death in food processing process of quite a few shigella dysenteriae, DNA but can be able to retain for a long time in dead bacterium, cause traditional real time fluorescent PCR method testing result for the positive, disturb the verity of detection, make the popularization and application of shigella dysenteriae real-time PCR detection technology in food be limited by very large.
Present invention application nitrine ethidium bromide (Ethidiumbromidemonoazide, EMA) can through the cell membrane of dead antibacterial, with genomic DNA covalent bond under the effect of photoactivation, the DNA of dead thalline in sample is suppressed to carry out the characteristic [4 of pcr amplification, 5], being combined with round pcr by EMA, establish EMA real-time fluorescence PCR technology and quickly detect will he viable bacteria technology in food, quick, accurate, the specific detection of congratulating viable bacteria for will in food provide scientific basis.
(3) summary of the invention
It is an object of the present invention to provide a kind of nucleic acid detection technique test kit accurate, sensitive, detection will he viable bacteria rapidly and detection method thereof.
The technical solution used in the present invention is:
The present invention provides will in a kind of quick detection food to congratulate the test kit of viable bacteria, and described test kit includes following composition:
(1) nitrine ethidium bromide: analytical pure (Ethidiumbromidemonoazide, EMA).
(2) PCR reaction reagent (preferred Takara company PremixEXTaqTM(probeqPCR), article No. DRR390A, B), including: 2 × PremixExTaqTM, upstream and downstream primer, fluorescent probe, ROX, template DNA and aseptic double-distilled water；
Forward primer (ipaHF) 5 '-CAGGCATCAGAAGGCCTTTT-3 '；
Downstream primer (ipaHR) 5 '-TCGAGGCGGAACATTTCC-3 '；
Fluorescent probe (ipaHpb) 5 '-FAM-CGGCGCTCTGCTCTCCCTGG-BHQ1-3 '；
Wherein FAM is fluorescent reporter group, and BHQ1 is fluorescent quenching group；
All of primer and probe are by the synthesis of Shanghai raw work biology company limited；
(3) positive control: containing the DNA plasmid of Shigella Gene ipaH Using fragment；
(4) negative control: aseptic double-distilled water.
Further, positive control of the present invention is the fragment of the Shigella Gene ipaH Using sequence containing long 319bp, and the nucleotides sequence of described fragment is classified as shown in SEQIDNO.1:
ctcacatggaacaatctccggaaaaccctcctggtccatcaggcatcagaaggccttttcgataatgataccggcgctctgctctccctgggcagggaaatgttccgcctcgaaattctggaggacattgcccgggataaagtcagaactctccattttgtggatgagatagaagtctacctggccttccagaccatgctcgcagagaaacttcagctctccactgccgt。
Further, positive control of the present invention is prepared as follows:
With comprise amplification region Shigella Gene ipaH Using fragment (SEQIDNO.1 shown in) for template, by two peripheral primers (periphery, upstream primer: 5 '-agctgtgaggaccgtgtcgcg-3 '；Downstream peripheral primer 5 '-catagaaacgcatttccttc-3 ') carry out pcr amplification acquisition amplified production；Utilize PCR purification kit (Promega) that pcr amplification product is purified；Amplified production after purification is built the complete plasmid sequence containing genes of interest by T-easy plasmid transfection test kit (Promega), with spectrophotometer, the plasmid of institute's extracting is surveyed A280Quantitatively and be diluted to 1 copy/μ l, it is thus achieved that positive control ,-20 DEG C of preservations.
The present invention also provides for will in a kind of quick detection food and congratulates the application in detection food will he viable bacteria of the test kit of viable bacteria, described application is: first extract detected sample DNA as template DNA with nitrine ethidium bromide, then PCR reaction reagent is adopted to react, select FAM fluoroscopic examination, baseline adjustment takes the fluorescence signal of 3～15 circulations, sets threshold line with threshold line just above the peak of normal negative control；If testing sample fluorescence growth curve exceedes threshold line, and increasing in good logarithm, CT value is less than or equal to 35, it is judged that for the positive, if CT value is more than 35 or without CT value, is then judged as feminine gender.
Specifically, the application that in quick detection food of the present invention, the test kit of will he viable bacteria is congratulated in viable bacteria in detection food will is:
A) sample EMA pre-treatment
Weigh 10g sample to the aseptic homogenizing bag filling 90mL physiological saline solution, 1min～2min is patted with slap type homogenizer, make even liquid, 1000r/min, centrifugal 5min, take supernatant, 3000r/min, centrifugal 10min, goes supernatant, precipitation to add after physiological saline solution 1mL dissolves and moves to 1.5mLeppendorf pipe (EP pipe)；If bacterium culture, then directly draw 1mL to 1.5mLEP and manage；Adding the EMA of final concentration of 30 μ g/mL, after 8min is placed at dark place, in 650W Halogen light illumination 8min, sample cell is placed on from light source 15～20cm place, and during illumination, EP pipe is placed on ice, in order to avoid sample temperature is too high；After repeating the placement of dark place, photo-irradiation treatment several times with condition, for DNA nucleic acid extraction after precipitating 3 times with normal saline；
B) DNA nucleic acid extraction: by above-mentioned precipitate, dissolves with 200 μ l sterile distilled waters, puts the centrifugal 5min of boiling water bath 10min, 10000rpm, sucts clear to another centrifuge tube, and-40 DEG C save backup, and are template DNA；
C) PCR reaction: overall reaction system 20 μ L, including 2 × PremixExTaqTM10 μ L, upstream and downstream primer (10 μm of ol/L) each 0.4 μ L, probe (10 μm of ol/L) 0.8 μ L, ROX0.2 μ L, template DNA is respectively 5.0 μ L, uses ddH2O supplies；
D) reaction condition: 95 DEG C of 30s of denaturation, 95 DEG C of 10s of degeneration, anneal 60 DEG C of 40s, 40 circulations；Setting fluoroscopic examination point, fluoroscopic examination model selection FAM fluorescence at 60 DEG C of places, baseline adjustment takes the fluorescence signal of 3～15 circulations, sets threshold line with threshold line just above the peak of normal negative control；If testing sample fluorescence growth curve exceedes threshold line, and increases in good logarithm, CT value is less than or equal to 35, it is judged that for the positive；If CT value is more than 35 or without CT value, then it is judged as feminine gender.
The present invention compared with prior art, has the following advantages and effect:
1. in quick detection food of the present invention, will is congratulated viable bacteria test kit and is only detected shigella dysenteriae alive.
2. specificity is good, highly sensitive, and step is simple, repeatable high；
3. response speed is fast, and single sample is from sample process to completing detection, less than 2h；
4. whole amplification and detection process need not open PCR pipe lid, decrease the chance that amplified production pollutes.
(4) accompanying drawing explanation
Fig. 1 is sensitivity Detection result figure, A is the curve chart of 7 kinds of concentration shigella dysenteriae bacteria suspension this method detection；B is CT value and bacteria concentration log-linear graph of a relation to be checked.
Fig. 2 is specificity experiments result figure.
(5) detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited to that:
The composition of embodiment 1 test kit of the present invention and preparation
(1) PCR reaction reagent (preferred Takara company PremixEXTaqTM(probeqPCR), article No. DRR390A, B).
(2) nitrine ethidium bromide: analytical pure.
(3) specific primer and fluorescent probe sequence are as follows:
Forward primer (ipaHF) 5 '-CAGGCATCAGAAGGCCTTTT-3 '
Downstream primer (ipaHR) 5 '-TCGAGGCGGAACATTTCC-3 '
Fluorescent probe (ipaHpb) 5 '-FAM-CGGCGCTCTGCTCTCCCTGG-BHQ1-3 '
Wherein FAM is fluorescent reporter group, and BHQ1 is fluorescent quenching group.
(4) positive control: containing the DNA plasmid of long 319bp Shigella Gene ipaH Using fragment (shown in SEQIDNO.1)；Described positive control is prepared as follows:
With comprise amplification region Shigella Gene ipaH Using fragment (SEQIDNO.1 shown in) for template, by two peripheral primers (periphery, upstream primer: 5 '-agctgtgaggaccgtgtcgcg-3 '；Downstream peripheral primer 5 '-catagaaacgcatttccttc-3 ') carry out pcr amplification acquisition amplified production；Utilize PCR purification kit (Promega) that pcr amplification product is purified；Amplified production after purification is built the complete plasmid sequence containing genes of interest by T-easy plasmid transfection test kit (Promega), with spectrophotometer, the plasmid of institute's extracting is surveyed A280Quantitatively and be diluted to 1 copy/μ l, it is thus achieved that positive control ,-20 DEG C of preservations.
(5) negative control: aseptic double-distilled water.
Embodiment 2 concrete grammar of test kit of the present invention detection shigella dysenteriae viable bacteria nucleic acid
(1) sample EMA pre-treatment
Weigh 10g sample to the aseptic homogenizing bag filling 90mL normal saline, 1min～2min is patted with slap type homogenizer, make even liquid, 1000r/min, centrifugal 5min, take supernatant, 3000r/min, centrifugal 10min, goes supernatant, precipitation to add after physiological saline solution 1mL dissolves and moves to 1.5mLeppendorf pipe (EP pipe).If bacterium culture, then directly draw 1mL to 1.5mLEP and manage.Adding the EMA of final concentration of 30 μ g/mL, after 8min is placed at dark place, in 650W Halogen light illumination 8min, sample cell is placed on from light source 15～20cm place, and during illumination, EP pipe is placed on ice, in order to avoid sample temperature is too high.After repeating the placement of dark place, photo-irradiation treatment several times with condition, for DNA nucleic acid extraction after precipitating 3 times with normal saline.
(2) DNA nucleic acid extraction: by above-mentioned precipitate, dissolves with 200 μ l sterile distilled waters, puts the centrifugal 5min of boiling water bath 10min, 10000rpm, sucts clearly to another centrifuge tube, be template DNA, and-40 DEG C save backup；
(3) overall reaction system: overall reaction system 20 μ L, including 2 × PremixExTaqTM10 μ L, upstream and downstream primer (10 μm of ol/L) each 0.4 μ L, probe (10 μm of ol/L) 0.8 μ L, ROX0.2 μ L, template DNA is 5.0 μ L, uses ddH2O supplies.
(4) reaction condition: 95 DEG C of 30s of denaturation, 95 DEG C of 10s of degeneration, anneal 60 DEG C of 40s, 40 circulations.Fluoroscopic examination point is set at 60 DEG C of places.Fluoroscopic examination model selection FAM fluorescence, baseline adjustment takes the fluorescence signal of 3～15 circulations, sets threshold line with threshold line just above the peak of normal negative control；If testing sample fluorescence growth curve exceedes threshold line, and increases in good logarithm, CT value is less than or equal to 35, it is judged that for the positive.If CT value is more than 35 or without CT value, then it is judged as feminine gender.
Embodiment 3 sensitivity of shigella dysenteriae viable bacteria in test kit of the present invention detection food
It is 2.2 × 10 to concentration respectively according to the method for embodiment 25Cfu/ reaction, 2.2 × 104Cfu/ reaction, 2.2 × 103Cfu/ reaction, 2.2 × 102Cfu/ reaction, 2.2 × 101Cfu/ reaction, 2.2cfu/ reaction, 0.22cfu/ react 7 kinds of concentration shigella dysenteriae bacteria suspensions this method and detect, and result CT value and bacteria concentration logarithm to be checked have good linear relation, CT=32.10-3.19 × log (bacterium amount), correlation coefficient (R2) it is 0.994.Result is shown in Fig. 1, and the minimal detectable concentration of method reaches 2.2cfu/ reaction.
Embodiment 4 specificity of shigella dysenteriae viable bacteria in test kit of the present invention detection food
According to the method for embodiment 2,3 strain shigella dysenteriae type strains, 28 strain shigella dysenteriae separation strains and 24 strain Salmonellas, 11 strain Enterobacter sakazakiis, 24 strain vibrio parahaemolyticus, 26 strain Listeria monocytogenes, 10 strain Escherichia coli, 3 strains, gold-coloured staphylococci bacteria suspension are detected.Result is shown in Fig. 2, and result shows, 3 strain shigella dysenteriae type strains and 28 strain shigella dysenteriae separation strain CT values minimum 15.04, the highest by 26.54, and the CT value of the 98 non-shigella dysenteriaes of strain is all higher than 35 or without CT value (in straight line).
Embodiment 5 stability of shigella dysenteriae viable bacteria in test kit of the present invention detection food
According to the method for embodiment 2, shigella dysenteriae reference culture (CMCC51573), 2 strain shigella dysenteriae separation strains, 1 strain salmonella (CMCC50013) and 1 strain escherichia coli type strain (ATCC25922) are continuously repeated 5 times every other day and carry out testing result by this method, 5 kinds of antibacterials all can be appropriately determined, except 2 kinds of negative bacterium partial results are without CT value, cannot calculate outside the coefficient of variation, the coefficient of variation of all the other 3 strain bacterium CT values equal < 5%.
Embodiment 6 test kit of the present invention detection food analog sample detection
Take 15 parts of cooked meat products, 15 portions of beverages, in each title or absorption 25g to 225mLGN enrichment liquid, wherein every class sample has 4 parts to the addition of shigella dysenteriae (bacteria concentration is about 10CFU/mL, 100CFU/mL, 1000CFU/mL, 10000CFU/mL) alive, 4 parts with the addition of dead shigella dysenteriae (bacteria concentration is about 103～104CFU/mL), 2 parts not only with the addition of dead shigella dysenteriae (bacteria concentration is about 103～104CFU/mL), but also had added shigella dysenteriae (bacteria concentration is about 102～103CFU/mL) alive, and 5 parts of samples are not added with any bacterium.After increasing bacterium 5h with GN enrichment liquid, respectively inhale 1mL, detect according to the method for embodiment 2.Synchronize to carry out normal isolation culture by " inspection of national food safety standard food microbiological examination shigella " (GB4789.5-2012) method.4 parts with the addition of shigella dysenteriae alive and 2 parts and not only with the addition of dead shigella dysenteriae, but also the shigella dysenteriae sample of work that adds as a result, are the positive by 2 kinds of method testing results, and all the other samples are feminine gender.This method testing result is completely the same with GB/T4789.5-2013 isolated culture method.
Claims (7)
1. in a quick detection food, will congratulates the test kit of viable bacteria, it is characterised in that described test kit includes following composition:
(1) nitrine ethidium bromide；
(2) PCR reaction reagent, including: 2 × PremixExTaqTM, forward primer, downstream primer, fluorescent probe, ROX, template DNA and aseptic double-distilled water；
Described forward primer ipaHF:5 '-CAGGCATCAGAAGGCCTTTT-3 '；
Described downstream primer ipaHR:5 '-TCGAGGCGGAACATTTCC-3 '；
Described fluorescent probe ipaHpb:5 '-FAM-CGGCGCTCTGCTCTCCCTGG-BHQ1-3 '；
Wherein FAM is fluorescent reporter group, and BHQ1 is fluorescent quenching group；
(3) positive control: containing the DNA plasmid of shigella ipaH gene；
(4) negative control: aseptic double-distilled water.
2. quickly detect will in food as claimed in claim 1 and congratulate the test kit of viable bacteria, it is characterised in that described PCR reaction reagent forms: 2 × PremixExTaqTM10 μ L, 10 μm of each 0.4 μ L of ol/L upstream and downstream primer, 10 μm of ol/L probes 0.8 μ L, ROX0.2 μ L, template DNA 5.0 μ L, use ddH2O complements to 20 μ L.
3. quickly detect will in food as claimed in claim 1 and congratulate the test kit of viable bacteria, it is characterised in that described positive control is the fragment of the shigella ipaH gene order containing long 319bp, and the nucleotides sequence of described fragment is classified as shown in SEQIDNO.1.
4. quickly detect will in food as claimed in claim 1 and congratulate the test kit of viable bacteria, it is characterised in that described positive control is prepared as follows:
With comprise amplification region shigella ipaH genetic fragment for template, by two peripheral primers carry out pcr amplification obtain amplified production；Utilize PCR purification kit that pcr amplification product is purified；Amplified production after purification is built the complete plasmid sequence containing genes of interest by T-easy plasmid transfection test kit, with spectrophotometer, the plasmid of institute's extracting quantitatively and is diluted to 1 copy/μ l, it is thus achieved that positive control ,-20 DEG C of preservations；Periphery, upstream primer: 5 '-agctgtgaggaccgtgtcgcg-3 '；Downstream peripheral primer 5 '-catagaaacgcatttccttc-3 '.
5. quickly detect will in food described in a claim 1 and congratulate the application in detection food will he viable bacteria of the test kit of viable bacteria.
6. apply as claimed in claim 5, it is characterized in that described application is: first with nitrine ethidium bromide pre-treatment detected sample DNA as template DNA, then PCR reaction reagent is adopted to react, select FAM fluoroscopic examination, baseline adjustment takes the fluorescence signal of 3～15 circulations, sets threshold line with threshold line just above the peak of normal negative control；If testing sample fluorescence growth curve exceedes threshold line, and increasing in good logarithm, CT value is less than or equal to 35, it is judged that for the positive, if CT value is more than 35 or without CT value, is then judged as feminine gender.
7. apply as claimed in claim 6, it is characterised in that described application is:
A) sample nitrine ethidium bromide pre-treatment
Weigh detected sample to the aseptic homogenizing bag filling physiological saline solution, pat homogenizing 1min～2min, make even liquid, 1000r/min, centrifugal 5min, takes supernatant, 3000r/min, centrifugal 10min, goes supernatant, precipitation to add and moves to EP pipe after physiological saline solution dissolves；If bacterium culture, being then directly drawn to EP and manage, add the nitrine ethidium bromide of final concentration of 30 μ g/mL, after 8min is placed at dark place, in 650W Halogen light illumination 8min, sample cell is placed on from light source 15～20cm place, and during illumination, EP pipe is placed on ice,；After repeating the placement of dark place, photo-irradiation treatment several times with condition, for DNA nucleic acid extraction after precipitating 3 times with normal saline；
B) DNA nucleic acid extraction: dissolved by above-mentioned precipitate sterile distilled water, puts the centrifugal 5min of boiling water bath 10min, 10000rpm, sucts clear to another centrifuge tube, and-40 DEG C save backup, and are template DNA；
C) PCR reaction: overall reaction system 20 μ L, including 2 × PremixExTaqTM10 μ L, 10 μm of ol/L forward primer, each 0.4 μ L of downstream primer, 10 μm of ol/L probes 0.8 μ L, ROX0.2 μ L, template DNA 5.0 μ L, use ddH2O supplies；
D) reaction condition: 95 DEG C of 30s of denaturation, 95 DEG C of 10s of degeneration, anneal 60 DEG C of 40s, 40 circulations；Setting fluoroscopic examination point, fluoroscopic examination model selection FAM fluorescence at 60 DEG C of places, baseline adjustment takes the fluorescence signal of 3～15 circulations, sets threshold line with threshold line just above the peak of normal negative control；If testing sample fluorescence growth curve exceedes threshold line, and increases in good logarithm, CT value is less than or equal to 35, it is judged that for the positive；If CT value is more than 35 or without CT value, then it is judged as feminine gender.
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| CN117969228A (en) * | 2024-04-02 | 2024-05-03 | 成都翼泰生物科技有限公司 | Bacterial liquid diluent, preparation method, reference, kit and application thereof |
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Application publication date: 20160713 |