CN102121051A - Multiplex fluorescent quantitative PCR detection method for main pathogenic bacteria in aquatic product - Google Patents
Multiplex fluorescent quantitative PCR detection method for main pathogenic bacteria in aquatic product Download PDFInfo
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- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention discloses a multiplex fluorescent quantitative polymerase chain reaction (PCR) detection method for main pathogenic bacteria in an aquatic product. The method is characterized by comprising the following steps of: (1) performing enrichment culture on a sample to be detected in enrichment solution for 4 to 8 hours; (2) extracting genome DNA from the cultured sample by adopting a boiling method; (3) performing multiplex fluorescent quantitative PCR detection by using specific primers and TaqMan probes; and (4) performing result judgment according to the detected specific S amplification curve and threshold value (Ct value). Compared with the prior art, the method has the advantages that: one-tube multi-detection actual requirement can be met, and a solid technical guarantee can be provided for quick, sensitive and specific detection of vibrio parahaemolyticus, vibrio cholerae and listeria monocytogenes.
Description
Technical field
The present invention relates to the multiple fluorescence quantitative PCR detection method of Vibrio parahaemolyticus, vibrio cholerae and Listeria monocytogenes in a kind of fishery products.
Background technology
In recent years, food origin disease presents ascendant trend, and this class disease is mainly caused by food-borne pathogens, is included in the medium-term and long-term Vibrio parahaemolyticus that exists of aquatic animal or breeding environment, Listeria monocytogenes and can passes through food-borne vibrio cholerae etc.Yet, bacterial strain few owing to bacteria containing amount makes a variation greatly and the reasons such as limitation of detection method, often makes traditional detection method to detect or omission easily.Therefore, seek fast, Vibrio parahaemolyticus, vibrio cholerae and Listeria monocytogenes sensitive, that special method detects in the fishery products be significant.
The detection technique of foodborne bacterial pathogens has experienced from traditional isolation identification method, immunoassay technology, to the transformation of molecular biology method.At present, both at home and abroad to the existing many researchs of the Fast Detection Technique of Vibrio parahaemolyticus, vibrio cholerae and Listeria monocytogenes, as the patent No. is that (Granted publication number: CN1280425C), and for example application number is open " a kind of method that is used to detect Vibrio parahaemolyticus " (publication number: CN101140243A) of Chinese invention patent application of 200710046637.4 for the Chinese invention patent " method of a kind of rapid detection Vibrio parahaemolyticus " of ZL03146896.9; The application number that has at vibrio cholerae is that (publication number: CN101153332A), and for example application number is open " cholera vibrio gene quick diagnosis reagent kit and detection method thereof " (publication number: CN101403005A) of Chinese invention patent application of 200810198809.4 to 200710030446.9 Chinese patent application open " vibrio cholerae detects with primer, detection method, detection kit "; The application number that has at Listeria monocytogenes is 200610029348.9 Chinese invention patent application open " test kit of Listeria monocytogenes in a kind of rapid detection sample " (publication number: CN101113465A), see that again application number is open " Listeria monocytogenes detection kit and detection method thereof " (publication number: CN101381777A) of Chinese invention patent application of 200810201408.X; The document that the multiple fluorescence quantitative PCR aspect is also arranged in the open source literature is open " the PCR detection method of vibrio cholerae, Vibrio parahaemolyticus and Vibrio mimicus in the food " (publication number: CN1967234A) of 200610068561.0 Chinese invention patent application as application number.
Though prior art has been done a lot of researchs, but in same reaction tubes, utilization has the fluorescent PCR detector of a plurality of laser channelings, set up that a kind of multiple fluorescence quantitative PCR reaction system that detects Vibrio parahaemolyticus, vibrio cholerae and Listeria monocytogenes is not simultaneously appeared in the newspapers and pertinent literature open.
Summary of the invention
Technical problem to be solved by this invention is that a kind of multiple fluorescence quantitative PCR detection method that can detect Vibrio parahaemolyticus, vibrio cholerae and Listeria monocytogenes in fishery products simultaneously is provided at the above-mentioned state of the art.
The present invention solves the problems of the technologies described above the technical scheme that is adopted: the multiple fluorescence quantitative PCR detection method of The main pathogenic fungi in a kind of fishery products is characterized in that comprising
1. get testing sample and in enrichment liquid, increase bacterium cultivation 4～8 hours;
2. the sample after cultivating adopts boiling method extracting genomic dna;
3. utilizing specific primer and TaqMan probe to carry out multiple fluorescence quantitative PCR detects;
4. carry out result's judgement according to specificity S amplification curve after detecting and cycle values,
Wherein specific primer and the TaqMan probe of step in 3. is as follows:
5 ' of probe then will carry out distinctive mark according to the multiple fluorescence channel of the quantitative real time PCR Instrument that is adopted, and 3 ' quenching group of probe can be selected ECLIPSE or BHQ series.
The substratum that comprises following concentration in the 1. described enrichment liquid of step: peptone 8～11g/L, yeast soak powder or beef extract powder 17～18g/L, glucose 1～3g/L, and SODIUM PHOSPHATE, MONOBASIC 2～3g/L, sodium-chlor 25～35g/L, and the pH value remains on 8.1～8.5.
Reaction system when the 3. middle multiple fluorescence quantitative PCR of step detects contains Mg
2+PCR buffer 2～3 μ L, dNTP is 0.4～0.5 μ L, the Taq enzyme is 0.5～0.6 μ L, tlh, vcc, hly gene upstream and downstream primer are respectively got 6～6.5pmol, TaqMan probe at tlh, vcc, hly gene is respectively got 4～4.5pmol, and sample DNA is got 2 μ L, ddH
2O replenishes volume to 20 μ L.
Reaction parameter when the 3. middle multiple fluorescence quantitative PCR of step detects is 95 ℃ of pre-sex change 3～5min; 95 ℃ of sex change 10～15sec, 62 ℃ of annealing are also extended 45～60sec, and 40 circulations are collected fluorescent signal in 62 ℃ of annealing of each round-robin and extension stage.
At first, according to conditional filterings such as nutritional requirement, salt concn and pH values and make a kind of selective medium, can increase bacterium to three kinds of bacteriums simultaneously cultivates, secondly, go up the nucleotide sequence of announcing according to NCBI, design multiple Auele Specific Primer and probe, grope to select the suitableeest primer and probe, and carry out different fluorescein-labelled by subsequent authentication and condition.At last,, optimize multiple fluorescence quantitative PCR reaction system and reaction parameter, set up the fluorescence quantifying PCR method of the many inspections of a pipe through repetition test.
Based on main pathogenic bacteria multiple fluorescent quantificationally PCR detecting kit in the fishery products of the present invention's development, comprise following reagent:
A, TZ liquid 1 pipe are used for DNA and extract, and are stored in-20 ℃;
B, PCR reaction mixture 1 pipe, in Taq enzyme (commercial), dNTP (commercial) arranged, contain Mg
2+PCR buffer (commercial), ddH
2O, the three pairs of primers and probe (sequence is designed by the contriver, see claim book table 1) ,-20 ℃ keep in Dark Place;
C, positive control 1 pipe, (the extractive DNA of Vibrio parahaemolyticus, vibrio cholerae and Listeria monocytogenes of 1-5 * 106CFU/mL) is stored in-20 ℃ to interior dress proper concn;
D, negative control 1 pipe, the DNA that the fishery products of interior dress specific pathogen free bacterium extract after increasing bacterium with method is stored in-20 ℃.
Triple fluorescent quantitative PCR reaction system based on the present invention sets up, can adopt following specific embodiments:
The processing of sample and DNA method for extracting preferably adopt following scheme: get 25g aquatic products category sample, make the homogenate postposition with above-mentioned enrichment liquid 225mL and cultivate 4-8h for 37 ± 1 ℃.Get enrichment liquid 1.5mL after the cultivation to centrifuge tube, the centrifugal 3min of 12000rpm, and supernatant discarded; With the resuspended precipitation of the ddH2O of 1mL, the centrifugal 2min of 12000rpm, and supernatant discarded repeat this step operation once; 2 * the TZ and the ddH2O that in precipitation, add 30uL respectively, mixing is placed 30min in-20 ℃; 100 ℃ of boiling water bath 10min; Ice-water bath 10min, the centrifugal 1min of 12000rpm, supernatant liquor is required DNA.
The application of sample step, preferably adopt following scheme: the PCR reaction mixture of getting 18 μ L adds in the PCR reaction tubes, adds 2 μ L DNA extracts then respectively.In addition, add positive control dna liquid 2 μ L in the positive control hole of setting, then add negative control DNA liquid or ddH2O 2 μ L in the negative control hole.Slightly centrifugal, make the sample mixing and fall into the pipe end, can prepare to carry out the multiple fluorescence quantitative PCR reaction.
Response procedures is provided with, and preferably adopts following scheme: under the multi-channel detection pattern, use different colours flag F AM, TAMRA, three fluorescence channels of TEXAS RED respectively, and testing sample is put into corresponding position.It is as follows that response procedures is set: 95 ℃ of pre-sex change 3min; 95 ℃ of sex change 15sec, 62 ℃ of annealing are also extended 1min, and 40 circulations are collected fluorescent signal in 62 ℃ of annealing of each round-robin and extension stage.
Determination methods preferably adopts following scheme: judge according to specificity S curve in the triple fluorescent quantitative PCR reaction and corresponding threshold size whether Vibrio parahaemolyticus, vibrio cholerae and listerisa monocytogenes in mjme are arranged in the test sample as a result.Produce and Ct value 〉=40 for no S curve, result of determination is negative, can directly report not detect; Ct value≤35, this sample result of decidable is positive; Ct value＞35 and＜40, the suggestion sample reform.Ct value 〉=40 of reforming are negative, otherwise positive.
Compared with prior art, the invention has the advantages that: can realize the actual needs of the many inspections of a pipe, for above-mentioned Vibrio parahaemolyticus, vibrio cholerae and Listeria monocytogenes fast, sensitive, special detecting provide great technical guarantee.Simultaneously, can be applied in the daily Micro biological Tests, make significant contribution for improving aspects such as food hygiene quality, promotion China food export.
Description of drawings
Fig. 1 detects Vibrio parahaemolyticus, vibrio cholerae and the Listeria monocytogenes amplification curve diagram of positive and negative sample for utilizing the detection kit that the present invention relates to;
Fig. 2 detects the Vibrio parahaemolyticus amplification curve diagram of positive and negative sample for utilizing the detection kit that the present invention relates to;
Fig. 3 detects the vibrio cholerae amplification curve diagram of positive and negative sample for utilizing the detection kit that the present invention relates to;
Fig. 4 detects the Listeria monocytogenes amplification curve diagram of positive and negative sample for utilizing the detection kit that the present invention relates to;
Fig. 5 detects Vibrio parahaemolyticus, the vibrio cholerae amplification curve diagram of positive and negative sample for utilizing the detection kit that the present invention relates to;
Fig. 6 detects Vibrio parahaemolyticus, the Listeria monocytogenes amplification curve diagram of positive and negative sample for utilizing the detection kit that the present invention relates to;
Fig. 7 detects vibrio cholerae, the Listeria monocytogenes amplification curve diagram of positive and negative sample for utilizing the detection kit that the present invention relates to;
The interference experimental study amplification curve diagram that Fig. 8 carries out for the detection kit that utilization the present invention relates to;
The sensitivity test amplification curve diagram that Fig. 9 carries out for the detection kit that utilization the present invention relates to;
Figure 10 is the pairing canonical plotting of Fig. 9;
Figure 11 is the amplification curve diagram of A sample gained.
Figure 12 is the amplification curve diagram of B sample gained.
Embodiment
Embodiment describes in further detail the present invention below in conjunction with accompanying drawing.
Examples of implementation: the detection of peeled shrimp (A, two samples of B), specifically according to following steps:
1. get 25g peeled shrimp sample, mix with the 225mL enrichment liquid respectively after shredding, put 37 ± 1 ℃ and cultivate 4-6h;
2. get the 1.5mL enrichment liquid respectively to centrifuge tube, the centrifugal 3min of 12000rpm abandons supernatant;
3. use the resuspended precipitation of ddH2O of 1mL, the centrifugal 2min of 12000rpm, and abandon supernatant, repetitive operation is once;
4. boiling method, the genomic dna of extracting A, B sample;
5. the PCR reaction mixture of getting 18 μ L adds in the PCR reaction tubes, adds 2 μ L DNA then respectively;
6. after being provided with by aforementioned program, the PCR reaction tubes being put into quantitative real time PCR Instrument detect;
7. analyze the Ct value of specificity S curve and testing sample, carry out the result and judge.
As Figure 11 and shown in Figure 12, the amplification curve atlas analysis according to A, B two samples among the embodiment exists Vibrio parahaemolyticus and vibrio cholerae simultaneously in the A sample, have Vibrio parahaemolyticus, vibrio cholerae, Listeria monocytogenes in the B sample simultaneously.Tlh, vcc gene have produced specific S type amplification curve in the A sample, and the hly gene does not then have specific amplification, show in this A sample and contain Vibrio parahaemolyticus and vibrio cholerae simultaneously, do not have Listeria monocytogenes.Tlh, vcc, hly three genes all produce tangible S type amplification curve in the B sample, show to contain Vibrio parahaemolyticus, vibrio cholerae and Listeria monocytogenes in this sample simultaneously.
The multiple fluorescence quantitative PCR detection kit comprises following reagent:
E, TZ liquid 1 pipe are used for DNA and extract, and are stored in-20 ℃;
F, PCR reaction mixture 1 pipe, in Taq enzyme (commercial), dNTP (commercial) arranged, contain Mg
2+PCR buffer (commercial), ddH
2O, the three pairs of primers and probe (sequence is designed by the contriver, see claim book table 1) ,-20 ℃ keep in Dark Place;
G, positive control 1 pipe, (the extractive DNA of Vibrio parahaemolyticus, vibrio cholerae and Listeria monocytogenes of 1-5 * 106CFU/mL) is stored in-20 ℃ to interior dress proper concn;
H, negative control 1 pipe, the DNA that the fishery products of interior dress specific pathogen free bacterium extract after increasing bacterium with method is stored in-20 ℃.
Triple fluorescent quantitative PCR reaction system based on the present invention sets up, can adopt following specific embodiments:
The processing of sample and DNA method for extracting preferably adopt following scheme: get 25g aquatic products category sample, make the homogenate postposition with above-mentioned enrichment liquid 225mL and cultivate 4-8h for 37 ± 1 ℃.Get enrichment liquid 1.5mL after the cultivation to centrifuge tube, the centrifugal 3min of 12000rpm, and supernatant discarded; With the resuspended precipitation of the ddH2O of 1mL, the centrifugal 2min of 12000rpm, and supernatant discarded repeat this step operation once; 2 * the TZ and the ddH2O that in precipitation, add 30uL respectively, mixing is placed 30min in-20 ℃; 100 ℃ of boiling water bath 10min; Ice-water bath 10min, the centrifugal 1min of 12000rpm, supernatant liquor is required DNA.
The application of sample step, the PCR reaction mixture of getting 18 μ L adds in the PCR reaction tubes, adds 2 μ L DNA extracts then respectively.In addition, add positive control dna liquid 2 μ L in the positive control hole of setting, then add negative control DNA liquid or ddH2O 2 μ L in the negative control hole.Slightly centrifugal, make the sample mixing and fall into the pipe end, can prepare to carry out the multiple fluorescence quantitative PCR reaction.
Response procedures is provided with, and under the multi-channel detection pattern, uses different colours flag F AM, TAMRA, three fluorescence channels of TEXASRED respectively, and testing sample is put into corresponding position.It is as follows that response procedures is set: 95 ℃ of pre-sex change 3min; 95 ℃ of sex change 15sec, 62 ℃ of annealing are also extended 1min, and 40 circulations are collected fluorescent signal in 62 ℃ of annealing of each round-robin and extension stage.
Determination methods judges according to specificity S curve in the triple fluorescent quantitative PCR reaction and corresponding threshold size whether Vibrio parahaemolyticus, vibrio cholerae and listerisa monocytogenes in mjme are arranged in the test sample as a result.Produce and Ct value 〉=40 for no S curve, result of determination is negative, can directly report not detect; Ct value≤35, this sample result of decidable is positive; Ct value＞35 and＜40, the suggestion sample reform.Ct value 〉=40 of reforming are negative, otherwise positive.
As shown in Figure 1, the X-coordinate of amplification curve is reaction cycle number (Cycle Number), and ordinate zou is the pairing fluorescent value of different cycle numbers (Delta Rn).Tlh, vcc, the trigenic amplification curve of hly all present specific S type in the positive control; And tlh, vcc in the negative control, the trigenic amplification curve of hly is straight.Tlh, vcc, hly three genes all present tangible S type amplification curve in the sample, show to contain Vibrio parahaemolyticus, vibrio cholerae and Listeria monocytogenes in this sample simultaneously.
As shown in Figure 2, the amplification curve of tlh gene presents specific S type in the sample, shows in this sample and contains Vibrio parahaemolyticus.
As shown in Figure 3, the amplification curve of vcc gene presents specific S type in the sample, shows in this sample and contains vibrio cholerae.
As shown in Figure 4, the amplification curve of hly gene presents specific S type in the sample, shows in this sample and contains Listeria monocytogenes.
As shown in Figure 5, the amplification curve of tlh gene presents specific S type in the sample, shows in this sample and contains Vibrio parahaemolyticus.
As shown in Figure 6, the amplification curve of tlh, hly gene presents specific S type in the sample, shows to contain Vibrio parahaemolyticus, Listeria monocytogenes in this sample simultaneously.
As shown in Figure 7, the amplification curve of vcc, hly gene presents specific S type in the sample, shows to contain vibrio cholerae, Listeria monocytogenes in this sample simultaneously.
As shown in Figure 8, among the figure with vibrio cholerae: Listeria monocytogenes: it is example that the DNA ratio of Vibrio parahaemolyticus equals 104: 102: 1.Show that the pairing fluorescent signal of high density bacterium is collected centering, the pairing fluorescent signal of lower concentration bacterium is collected noiseless effect.
As shown in Figure 9, the PCR reaction of the positive gradient dilution that contains Vibrio parahaemolyticus, vibrio cholerae and Listeria monocytogenes simultaneously being carried out.
As shown in figure 10, X-coordinate is fluorescence threshold (Ct value), and ordinate zou is the logarithmic value of initiate dna concentration, and the facies relationship number average of typical curve is more than 0.99 as can be seen for regression equation, and linear relationship is good.
Sequence table
＜110〉Zhoushan Entry-exit Inspection and Quarantine Bureau
＜120〉the multiple fluorescence quantitative PCR detection method of The main pathogenic fungi in the fishery products
<160>9
<210>1
<211>20
<212>DNA
<213>vibrio?parahaemolyticus
<400>1
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agaagttagc?gtctcgaaca?ag
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cgagttcatc?aaggcacaag?cga
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taccgctacttagccgccaa?cactca
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<400>7
ttagcttggg?aatggtggag
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ttcggataaa?gcgtagtgcc
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ttgatgaccg?gaacttacca?cttgtga
Claims (4)
1. the multiple fluorescence quantitative PCR detection method of The main pathogenic fungi in the fishery products is characterized in that comprising
1. get testing sample and in enrichment liquid, increase bacterium cultivation 4～8 hours;
2. the sample after cultivating adopts boiling method extracting genomic dna;
3. utilizing specific primer and TaqMan probe to carry out multiple fluorescence quantitative PCR detects;
4. carry out result's judgement according to specificity S amplification curve after detecting and cycle values,
Wherein specific primer and the TaqMan probe of step in 3. is as follows:
5 ' of probe then will carry out distinctive mark according to the multiple fluorescence channel of the quantitative real time PCR Instrument that is adopted, and 3 ' quenching group of probe can be selected ECLIPSE or BHQ series.
2. detection method according to claim 1, it is characterized in that comprising in the 1. described enrichment liquid of step the substratum of following concentration: peptone 8～11g/L, yeast soaks powder or beef extract powder 17～18g/L, glucose 1～3g/L, SODIUM PHOSPHATE, MONOBASIC 2～3g/L, sodium-chlor 25～35g/L, and the pH value remains on 8.1～8.5.
3. detection method according to claim 1 is characterized in that the reaction system when multiple fluorescence quantitative PCR detected during step 3. contains Mg
2+PCR buffer 2～3 μ L, dNTP is 0.4～0.5 μ L, the Taq enzyme is 0.5～0.6 μ L, tlh, vcc, hly gene upstream and downstream primer are respectively got 6～6.5pmol, TaqMan probe at tlh, vcc, hly gene is respectively got 4～4.5pmol, and sample DNA is got 2 μ L, ddH
2O replenishes volume to 20 μ L.
4. detection method according to claim 1 is characterized in that the reaction parameter when multiple fluorescence quantitative PCR detected during step 3. is 95 ℃ of pre-sex change 3～5min; 95 ℃ of sex change 10～15sec, 62 ℃ of annealing are also extended 45～60sec, and 40 circulations are collected fluorescent signal in 62 ℃ of annealing of each round-robin and extension stage.
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