CN104152546A - Kit and method for simultaneously detecting salmonella, listeria monocytogenes and staphylococcus aureus - Google Patents
Kit and method for simultaneously detecting salmonella, listeria monocytogenes and staphylococcus aureus Download PDFInfo
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Abstract
The invention discloses a kit and a method for simultaneously detecting salmonella, listeria monocytogenes and staphylococcus aureus. The kit comprises upstream and downstream primers of the salmonella, listeria monocytogenes and staphylococcus aureus; specifically, the upstream and the downstream primers of the salmonella are as shown in Seq No.4 and Seq No.5 respectively; the upstream and the downstream primers of the listeria monocytogenes are as shown in Seq No.6 and Seq No.7 respectively; and the upstream and the downstream primers of the staphylococcus aureus are as shown in Seq No.8 and Seq No.9 respectively. The method is used for carrying out HRM analysis onto target genes of the salmonella, listeria monocytogenes and staphylococcus aureus respectively, and used for identifying the salmonella, listeria monocytogenes and staphylococcus aureus at the same time by utilizing a Tm value. The detecting method can be used for completing the simultaneous detection work of the three target pathogenic bacteria within 1 hour-2hours, and has the advantages of being quick, low in cost, capable of manually regulating the Tm value range, simple and convenient for interpretation, convenient to operate and capable of preventing pollution by virtue of closed-tube detection, so that the detecting speed and the detecting efficiency are greatly improved.
Description
Technical field
The present invention relates to a kind of for detect the high resolving power melting curve analysis method of Salmonellas, Listeria monocytogenes, streptococcus aureus simultaneously.Belong to technical field of molecular biology.
Background technology
Salmonellas, single increasing Li Site bacterium and streptococcus aureus are pathogenic bacterium common in food, are the main pathogenic fungi that causes food poisoning and food origin disease.Salmonellas (Salmonella) is one of a kind of common, important Zoonosis cause of disease bacterium in enterobacteriaceae, and the nutritious food such as meat, egg, milk are its main communication medias.Salmonella-pollutedly can not only cause the Animal diseases such as white dysentery, necrotic enteritis, miscarriage, can also make the mankind that typhoid fever, paratyphoid, septicemia, gastro-enteritis and food poisoning occur, cause huge financial loss to China and even the world.Listeria monocytogenes (Listeriamonocytogenes) is a kind of short and small Gram-positive sporeless bacterium, belongs to the malignant bacteria of zoonosis.It can cause people's meningitis, septicemia and miscarriage, especially pregnant woman, newborn infant, the elderly and immune deficiency patient is endangered seriously.This pathogenic agent is widely distributed in environment, and its main habitat is the feed of soil, corrupt vegetables, water drain and storage.It can tolerate low pH, hypersaline environment, and can in wide temperature range, grow.In recent years, because pollution in the food of some burst cases and consumption has Listeria monocytogenes relevant, instant food particularly, as soft cheese, ice-creams, beverage and meat product etc., the record that even has some to break out on epidemiology, because it is highly pathogenic, at home and abroad in food safety detection, day by day come into one's own.Streptococcus aureus (Staphylococcus aureus) is that a class is extensively present in the gram-positive microorganism that has height salt tolerance in air, water, soil and human and animal's movement, it can secrete multiple toxic protein, wherein staphyloentero-toxin (Staphylococcaleaterotoxi, SE) can cause streptococcus aureus food poisoning.Streptococcus aureus does not exist only in quick-frozen rice product, milk-product and meat etc. also contain the intestines toxic strain of streptococcus aureus conventionally, the acute gastroenteritis symptoms such as the food that feed is polluted by streptococcus aureus easily causes feeling sick, vomiting, stomachache, diarrhoea, serious meeting causes death.The food poisoning that streptococcus aureus causes has in recent years presented global distribution, has become the sanitarian major issue in the world.As can be seen here, these three kinds of bacteriums have similar food host, easily at same class food, occur in as milk, milk-product, poultry, meat, fruits and vegetables simultaneously, therefore set up a kind of effective method that Foodborne salmonella, Listeria monocytogenes and streptococcus aureus are detected simultaneously, to improve check speed and the accuracy to this bacterium, contaminated food is processed timely, in public health, food sanitation, animal and veterinary and port quarantine, had important meaning.
The Protocols in Molecular Biology that domestic and international application detects in foodborne bacterial pathogens at present, is mainly divided three classes: regular-PCR technology, Fluorescence PCR assay and biochip technology.Method for gene chip detection efficiency is high, but technology is also immature, and false positive rate and false negative rate are all difficult to control, and cost is higher, at present also in conceptual phase.Regular-PCR method and technology is ripe, also the earliest for the detection of foodborne bacterial pathogens, but need to carry out aftertreatment to PCR product, very easily causes PCR product pollution, and has certain non-specific amplification.Fluorescent PCR is on the basis of regular-PCR, adds a specific fluorescent probe when adding a pair of Auele Specific Primer in amplification reaction system again, detects the technology of target nucleotide sequences with the fluorescent PCR detector of Real-Time Monitoring.But there is not yet the method that simultaneously detects pathogenic bacterium common in above-mentioned food at present.
Summary of the invention
The object of this invention is to provide a kind of for detect the method for Salmonellas, Listeria monocytogenes, streptococcus aureus simultaneously.Based on above-mentioned purpose, the invention provides following technical scheme:
Detect a test kit for Salmonellas, Listeria monocytogenes and streptococcus aureus simultaneously, comprise the upstream and downstream primer of above-mentioned three kinds of bacterium, specific as follows:
Mentioned reagent box also comprises 10 * buffer, MgCl
2, dNTP, BSA, 20 * Evagreen, Taq enzyme, Taq enzyme antibody and water.
This test kit is the test kit of 25 μ L reaction systems, and each component concentration is specific as follows:
A method that simultaneously detects Salmonellas, Listeria monocytogenes and streptococcus aureus, comprises the steps:
(1) testing sample is cultivated in the mixed culture medium of Salmonellas, Listeria monocytogenes and streptococcus aureus, choose respectively invA, hly, Sa442 is the goal gene of Salmonellas (Salmonella), Listeria monocytogenes (Listeriamonocytogenes) and streptococcus aureus (Staphylococcus aureus), design and invA, the supporting upstream and downstream primer of hly, Sa442, carry out DNA extraction to the product after above-mentioned mixed culture; Wherein, primer sequence is as follows:
(2) adopt pcr amplification primer to carry out pcr amplification to the mixing masterplate DNA of said extracted; Pcr amplification product is carried out to high resolving power liquation, and program is: 95 ℃ of 15s, and 60 ℃ of 1min, 95 ℃ of 15s, 60 ℃ of 15s, melting speed is 0.2 ℃/s;
(3) by high resolving power melting curve method, testing sample is identified, used T
mvalue is as interpretation standard, as the T recording
mvalue is 79.38 ± 0.5 ℃, and when 82.54 ± 0.5 ℃ and 77.36 ± 0.5 ℃, corresponding bacterial strain is respectively Salmonellas, Listeria monocytogenes and streptococcus aureus.
The reaction system of described pcr amplification is: cumulative volume is 25 μ L, and every 25 μ L PCR systems comprise 10 * buffer2.5 μ L, the Taq enzyme 0.5 μ L of concentration 5U/ μ L, the T aq antibody 0.5 μ L of concentration 1 μ g/ μ L, the dNTP2.5 μ L of 10mM, the MgCl of concentration 25mM
23 μ L, the BSA0.2 μ L that concentration is 20mg/ml, each 0.5 μ L of the upstream primer of concentration 10 μ M and downstream primer, mixes masterplate DNA2 μ L, and 1 μ L fluorescence dye EvaGreen, spends nucleic acid water polishing to 25 μ L.
Described PCR response procedures is: 95 ℃ of 10min; 95 ℃ of 10s, 60 ℃ of 45s, 40 circulations.
Described mixed culture medium by weight, comprise 0.010 part of 17 parts of Tryptoness, 3 parts of peptones, 2.5 parts of SODIUM PHOSPHATE, MONOBASIC, 2.5 parts of glucose, 1.0 parts of Vitamin C2s, 1000 parts of distilled water, 2.5 parts of Sodium.alpha.-ketopropionates, 5.0 parts, N.F,USP MANNITOL, 20.0 parts, sodium-chlor, 2.0 parts of lithium chlorides, 0.00015 part of potassium tellurite and nalidixic acid, pH value 7.2-7.3.
Described culture condition is: 35-38 ℃, 100-300rpm are cultivated 12-36h.
Present method by choosing respectively invA, hly, Sa442 is the goal gene of Salmonellas, Listeria monocytogenes and streptococcus aureus, design and invA, the supporting upstream and downstream primer of hly, Sa442, the mixed culture medium of Salmonellas, Listeria monocytogenes and streptococcus aureus is carried out to DNA extraction, pcr amplification and HRM and analyze, when realizing three kinds of object bacterium, detect.Goal gene, primer sequence and product T
mvalue is in Table 1.
Principle of the present invention is to utilize the combination situation of high-resolution fusion curve analytical technology Real-Time Monitoring temperature-rise period double center chain DNA saturated fluorescence dyestuff and pcr amplification product, without using specific probe can realize the melting curve mutation analysis of nucleic acid, thus the difference of reflection properties of nucleic acids.When saturated fluorescence dyestuff Evagreen and DNA double chain combination, send fluorescence; When DNA double chain discharges, fluorescent signal sharply weakens, if rising temperature makes DNA sex change, the temperature of take is mapped to fluorescent signal as X-coordinate, can obtain melting curve, wherein the temperature of 50%DNA molecule generation sex change is called melting point temperature (melting temperature, T
mvalue).Because the Tm value of each bacterial classification is different, therefore can utilize the specific T of bacterial classification
mvalue reaches the object that three kinds of target bacterial classifications are detected.
Compared with prior art, tool of the present invention has the following advantages:
The present invention adopts high-resolution fusion curve analytical technology (High Resolution Melting, HRM) to Salmonellas, Listeria monocytogenes and streptococcus aureus detect simultaneously, the method is not being calculated DNA extraction process in the situation that consuming time, PCR 40-90min consuming time left and right, HRM analyzes 10-30min, therefore detection method of the present invention can complete testing in three kinds of object pathogenic bacterium in 1～2h, have fast, low-cost, can manual shift Tm value scope, interpretation is easy, easy to operate and stopped pipe detects the advantages such as pollution that prevent, make in HRM-PCR Multiple detection system, reaction conditions is consistent, method high specificity, highly sensitive, reproducible, and be applicable to Salmonellas in artificial contaminated bacteria samples and natural microbiological contamination sample, in the time of Listeria monocytogenes and three kinds of food-borne pathogens of streptococcus aureus, detect, judge exactly the infection of different bacterium, the speed and the efficiency that detect have greatly been improved.
Accompanying drawing explanation
Fig. 1 is the high resolving power melting curve figure that embodiment 2HRM-PCR detects Salmonellas;
Fig. 2 is the high resolving power melting curve figure that embodiment 2HRM-PCR detects Listeria monocytogenes;
Fig. 3 is the high resolving power melting curve figure that embodiment 2HRM-PCR detects streptococcus aureus;
Fig. 4 is the high resolving power melting curve figure that embodiment 3HRM-PCR detects Salmonellas, Listeria monocytogenes and streptococcus aureus simultaneously.
Embodiment
When can carrying out Salmonellas, Listeria monocytogenes, streptococcus aureus to Marketing pork sample, the inventive method detects.For measuring method of the present invention can be more clearly described, below test method of the present invention is done with detailed explanation.Primer sequence of the present invention is in Table 1.
Embodiment 1
The foundation of multiple HRM-PCR detection system and optimization
1. the selection of goal gene and primer
Choose conservative gene fragment (referring to Seq No.1,2,3) and the corresponding Auele Specific Primer of three kinds of bacterial classifications, primer is synthetic by the raw work in Shanghai.Primer sequence is in Table 1.
Table 1 primer sequence
Note:F, upstream primer; R, downstream primer
2. the preparation of mixed culture medium and DNA extraction
The reference culture of three kinds of bacterial classifications is inoculated in mixed culture medium SSL and (comprises 17 parts of Tryptoness, 3 parts of peptones, 2.5 parts of SODIUM PHOSPHATE, MONOBASIC, 2.5 parts of glucose, 1.0 parts of Vitamin C2s, 1000 parts of distilled water, 2.5 parts of Sodium.alpha.-ketopropionates, 5.0 parts, N.F,USP MANNITOL, 20.0 parts, sodium-chlor, 2.0 parts of lithium chlorides, 0.00015 part of potassium tellurite, 0.010 part of nalidixic acid, pH value 7.2-7.3), 37 ℃ of 200rpm cultivate 24h, carry out DNA extraction.The extracting method of Salmonellas, Listeria monocytogenes and streptococcus aureus DNA carries out according to the working specification of precious biotech firm DNA extraction test kit.Get nutrient solution 1mL and carry out 10 times of gradient dilutions, choose 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6totally 6 extent of dilution are as serial positive template, extract respectively genomic nucleic acids, with the primer in above-mentioned detection sequence area, carry out pcr amplification respectively again, and get wherein person between Ct value 24-27, be mixed into Salmonellas, Listeria monocytogenes and streptococcus aureus three gang mould versions.
3.PCR System Design and optimization
Amplimer mixes masterplate to three and carries out pcr amplification, and instrument is ABI7500 (American AB I company), and band HRM module, is optimized amplification system, and optimizing process is as follows:
The optimization of 3.1 primer concentrations is in reaction system, Salmonellas, single primer concentration that increases Li Site bacterium and streptococcus aureus are done to detect multiple proportions serial dilution from 0.1 μ mol/L to 0.6 μ mol/L respectively, by the analysis comparison of test-results, determine that best primer final concentration is 0.2 μ mol/L.
Under the constant prerequisite of the optimization of 3.2 magnesium ion concentrations other condition in reaction system, by MgCl
2concentration from 1mmol/L to 4mmol/L, with 0.5mmol/L, increase progressively, through repeatedly repeating to test selected 3mmol/L, be the magnesium ion concentration in test kit reaction system.
The optimization of 3.3Taq archaeal dna polymerase consumption is by comparing the optimization experiment result of Taq enzyme dosage (in the Unit of unit), and selected 0.5 μ L is as the consumption of Taq enzyme in test kit reaction system.
The optimization of 3.4dNTPs concentration is by being used the dNTPs of different concns to detect, and after comprehensive assessment, selecting concentration is that the dNTP dosage of 10mM is that 2.5 μ L are as the usage quantity of dNTPs in test kit reaction system.
3.5 melt 4 kinds of different 0.1 ℃/s of melting speed of optimized choice of speed, and 0.2 ℃/s, 0.4 ℃/s and 0.8 ℃/s detect, and finally select the 0.2 ℃/s of melting speed that melting curve resolving power is the highest.
Utilize above-mentioned primer and Evagreen to carry out the foundation of reaction system, finally determine that the Fluorescence PCR system adopting is 25 μ L systems, required each component and respective concentration are in Table 2.
HRM reaction system after table 2 is optimized
Note: a. is when Fluorescence PCR volume is different, and each reagent should be adjusted in proportion.
B. the instrument using is different, reaction parameter should be appropriately adjusted.
PCR response procedures is: 95 ℃ of 10min; 95 ℃ of 10s, 60 ℃ of 45s, 40 circulations.Negative control group (making masterplate with stoning sour water) and three positive controls (using respectively Salmonellas, Listeria monocytogenes, streptococcus aureus genomic dna as masterplate) are set.
In multi-PRC reaction system, add Taq enzyme antibody and contribute to stability and the activity of Taq enzyme, avoid the prejudice that increases, can start by Low Temperature Thermal.
4.HRM analyzes (instrument adopts the authorized equipment with HRM module, and this experiment employing equipment is ABI7500) condition: 95 ℃ of 15s, 60 ℃ of 1min, 95 ℃ of 15s, 60 ℃ of 15s, select the melting speed of 0.2 ℃/s, can obtain the HRM curve that resolving power is higher, curve is more smooth.
5. interpretation standard
Use Tm value as interpretation standard.Can when design, people be the T that widens three kinds of object bacterium
mbe worth, avoid using the complicated calculations of software, increase visible difference, avoid parallax error.The theory of three kinds of object bacterium and actual T
mvalue is in Table 3:
Under the reaction system of above-mentioned optimization and supporting primer, carry out repetition test, collection of statistical data, finally determines the T of Salmonellas, Listeria monocytogenes, streptococcus aureus
mvalue is respectively 79.32 ± 0.142 ℃, 82.52 ± 0.142 ℃ and 77.25 ± 0.147 ℃.
Table 3 target t bacteria
mvalue
Embodiment 2
Choose above-mentioned three pairs of primer pairs, by the good HRM reaction system of above-mentioned optimization and response procedures, respectively three kinds of object bacterium are detected simultaneously.
(1) sample
Utilize the HRM detection method that the present invention sets up to detect Salmonellas, Listeria monocytogenes, streptococcus aureus and other 10 strain negative control bacterial strains.Take distilled water as blank, three positive controls are set, with the positive masterplate of genomic dna of Salmonellas, Listeria monocytogenes, streptococcus aureus, using stoning sour water as blank group respectively.
(2) PCR primer sequence is in Table 1.
(3) sampling and DNA extraction
By Salmonellas, Listeria monocytogenes, streptococcus aureus and 10 kinds of non-target bacterial classification (pseudomonas, Escherichia coli O 157, Shigellaes, Enterobacter sakazakii, colitis Yersinia, legionella, Bacillus proteus, enterococcus faecalis, streptococcus suis 2-type, bacillus cereus) be inoculated in respectively in LB liquid nutrient medium, all at 37 ℃, under 200rpm condition, cultivate 24h, carry out DNA extraction, the extracting method of DNA carries out according to the working specification of precious biotech firm DNA extraction test kit.
(4) PCR reaction
(instrument is ABI7500 to pcr amplification reaction, band HRM module) cumulative volume is 25 μ L, amplification reaction system is: every 25 μ L PCR systems comprise 10 * buffer2.5 μ L, the Taq polymerase 0.5 μ L of concentration 5U/ μ L, the T aq antibody 0.5 μ L of concentration 1 μ g/ μ L, the dNTP2.5 μ L of 10mM, the MgCl of concentration 25mM
23 μ L, the BSA0.2 μ L that concentration is 20mg/ml, each 0.5 μ L of the upstream primer of concentration 10 μ M and downstream primer, the DNA masterplate 2 μ L of concentration 0.5 μ M, 1 μ L EvaGreen, spends nucleic acid water polishing to 25 μ L; PCR response procedures is: 95 ℃ of 10min, 95 ℃ of 10s, 40 circulations; 60 ℃ of 45s.PCR Sptting plate is distributed as: sample group, negative control group, positive controls.
(5) HRM analysis (program is: 95 ℃ of 15s, and 60 ℃ of 1min, 95 ℃ of 15s, 60 ℃ of 15s, melting speed is 0.2 ℃/s.Instrument uses ABI7500, band HRM module.
(6) experimental result
Result shows that specificity melting curve and fusing point peak all appear in Salmonellas, Listeria monocytogenes and streptococcus aureus sample, object bacteria Salmonellas, Listeria monocytogenes and T corresponding to streptococcus aureus difference
mvalue is 79.38 ± 0.138 ℃, the peak of 82.54 ± 0.149 ℃ and 77.36 ± 0.142 ℃.All there is not non-specific amplification in the bacterial strain of non-target bacterial classification, without specific melting curve and T
mvalue.Negative control and blank are all without amplified signal.Point out this HRM-PCR method to detect and there is high degree of specificity Salmonellas, Listeria monocytogenes and streptococcus aureus.
Embodiment 3
The detection of Salmonellas, Listeria monocytogenes and streptococcus aureus three gang mould versions
Choose above-mentioned three kinds of primer pairs, by the good HRM reaction system of above-mentioned optimization and response procedures, three kinds of object bacterium are detected simultaneously.
(1) sample
Utilize the HRM detection method that the present invention sets up to detect Salmonellas, Listeria monocytogenes, streptococcus aureus and other 10 strain negative control bacterial strains.Take distilled water as blank, three positive controls are set, with the positive masterplate of genomic dna of Salmonellas, Listeria monocytogenes, streptococcus aureus, using stoning sour water as blank group respectively.
(2) PCR primer sequence is in Table 1.
(3) sampling and DNA extraction
Salmonellas, Listeria monocytogenes, streptococcus aureus are inoculated in to mixed culture medium SSL simultaneously and (comprise 17 parts of Tryptoness, 3 parts of peptones, 2.5 parts of SODIUM PHOSPHATE, MONOBASIC, 2.5 parts of glucose, 1.0 parts of Vitamin C2s, 1000 parts of distilled water, 2.5 parts of Sodium.alpha.-ketopropionates, 5.0 parts, N.F,USP MANNITOL, 20.0 parts, sodium-chlor, 2.0 parts of lithium chlorides, 0.00015 part of potassium tellurite, 0.010 part of nalidixic acid, pH value 7.2-7.3.) in, 10 kinds of non-target bacterial classifications are inoculated in respectively in LB substratum, all at 37 ℃, under 200rpm condition, cultivate 24h, carry out DNA extraction, the extracting method of DNA carries out according to the working specification of precious biotech firm DNA extraction test kit.
(4) PCR reaction
The cumulative volume of pcr amplification reaction is 25 μ L, amplification reaction system is: every 25 μ L PCR systems comprise 10 * buffer2.5 μ L, the Taq polymerase 0.5 μ L of concentration 5U/ μ L, the T aq antibody 0.5 μ L of concentration 1 μ g/ μ L, the dNTP2.5 μ L of 10mM, the MgCl of concentration 25mM
23 μ L, each 0.5 μ L of the upstream primer of concentration 10 μ M and downstream primer, each 2 μ L of every kind of DNA masterplate of concentration 0.5 μ M, 1.25 μ L EvaGreen, spend nucleic acid water polishing to 25 μ L; PCR response procedures is: 95 ℃ of 10min, 95 ℃ of 10s, 40 circulations; 60 ℃ of 45s.PCR Sptting plate is distributed as: sample group, negative control group, positive controls.
(5) HRM analyzes, and program is: 95 ℃ of 15s, and 60 ℃ of 1min, 95 ℃ of 15s, 60 ℃ of 15s, melting speed is 0.2 ℃/s.Instrument is QIAGEN Luo De gene 6000 type detection systems (German QIAGEN company), band HRM module.
(6) experimental result
Result shows that specificity melting curve and fusing point peak appear in three gang mould versions, object bacteria Salmonellas, Listeria monocytogenes and T corresponding to streptococcus aureus difference
mvalue is 79.38 ± 0.138 ℃, the peak of 82.54 ± 0.149 ℃ and 77.36 ± 0.142 ℃, as shown in Figure 4.All there is not non-specific amplification in the bacterial strain of non-target bacterial classification, without specific melting curve and T
mvalue.Negative control and blank are without amplified signal.Point out this HRM-PCR method to detect simultaneously and there is high degree of specificity Salmonellas, Listeria monocytogenes and streptococcus aureus.
Embodiment 4
The detection of artificial contaminated bacteria samples Salmonellas, Listeria monocytogenes and streptococcus aureus chicken sample
(1) preparation of artificial contaminated bacteria samples sample
Milk, chicken, beef, pork, egg sample from market purchasing, adopt GB4789-2008 proof not have after Salmonellas, Listeria monocytogenes and streptococcus aureus, three kinds of object bacteria bacterium liquid of every 25g sample inoculation, make the final concentration of three kinds of object bacteria in sample all be controlled at 5,10, the homogenate of 20CFU/25g food, bacteria concentration is determined by traditional colony counting method.Postvaccinal sample mixes with 225mL mixed culture medium (SSL) after being placed in 20 ℃ of cultivation 1h, homogenate 10s.Nonvaccinated sample is as negative control, and the object bacteria masterplate of mentioning in embodiment 1 is as positive control.Mixing sample is cultivated 24h centrifugal 5min under 1,000 * g condition at 37 ℃, with filter bag (Tekmar, Cincinnati, Ohio) filter out coarse swill, then under 8,000 * g condition centrifugal 5min.
(2) PCR primer sequence is in Table 1.
(3) extraction of masterplate DNA
Get 500 μ L supernatant liquors for DNA extraction.The working specification of the DNA extraction test kit that the extracting method of DNA is produced according to precious biotech firm carries out.
(4) PCR reaction: (instrument is ABI7500 to pcr amplification reaction, band HRM module) cumulative volume is 25 μ L, amplification reaction system is: every 25 μ L PCR systems comprise 10 * buffer2.5 μ L, the Taq polymerase 0.5 μ L of concentration 5U/ μ L, the T aq antibody 0.5 μ L of concentration 1 μ g/ μ L, the dNTP2.5 μ L of 10mM, the MgCl of concentration 25mM
23 μ L, each 0.5 μ L of the upstream primer of concentration 10 μ M and downstream primer, each 2 μ L of every kind of DNA masterplate of concentration 0.5 μ M, 1 μ L EvaGreen, spends nucleic acid water polishing to 25 μ L; PCR response procedures is: 95 ℃ of 10min, 95 ℃ of 10s, 40 circulations; 60 ℃ of 45s.PCR Sptting plate is distributed as: sample group, negative control group, positive controls.
(5) HRM analyzes (instrument is ABI7500, band HRM module), and program is: 95 ℃ of 15s, and 60 ℃ of 1min, 95 ℃ of 15s, 60 ℃ of 15s, melting speed is 0.2 ℃/s.
(6) experimental result
The T that table 4 artificial contaminated bacteria samples sample HRM detects
mvalue
HRM detection method detection sensitivity of the present invention can reach the homogenate of 5CFU/25g food, points out above-mentioned primer pair to have good sensitivity.Negative control is without amplification curve.
Embodiment 5
The detection of nature microbiological contamination Salmonellas, Listeria monocytogenes and streptococcus aureus pork sample
(1) preparation of natural microbiological contamination pork sample
Directly select from 120 parts of fresh pork samples of market purchasing and detect, the homogenate of 25g sample is mixed with 200mL mixed culture medium (SSL).Mixing sample is cultivated 24h at 37 ℃, centrifugal 5min under 1,000 * g condition, rear with filter bag (Tekmar, Cincinnati, Ohio) filter out coarse swill, then under 8,000 * g condition centrifugal 5min.
(2) PCR primer sequence is in Table 1.
(3) extraction of masterplate DNA
Get 500 μ L supernatant liquors for DNA extraction.The extracting method of DNA carries out according to the working specification of Dneasy Tissue kit.
(4) PCR reaction: the cumulative volume of pcr amplification reaction is 25 μ L, amplification reaction system is: 2.5 μ L10 * buffer, 0.5 μ L Taq polymerase (5U/ μ L), 0.5 μ L Taq antibody (1 μ g/ μ L), dNTP (10mM), 3 μ L MgCl
2(25mM), each 0.5 μ L of the every pair of upstream primer and downstream primer (10 μ M), each 2 μ L of every kind of DNA masterplate (0.5 μ M), 1 μ L EvaGreen, spends nucleic acid water polishing to 25 μ L; PCR response procedures is: 95 ℃ of 10min; 95 ℃ of 10s, 60 ℃ of 45s, 40 circulations.PCR Sptting plate is distributed as: sample group, negative control group, positive controls.
(5) HRM analyzes, and program is: 95 ℃ of 15s, and 60 ℃ of 1min, 95 ℃ of 15s, 60 ℃ of 15s, melting speed is 0.2 ℃/s.Instrument uses QIAGEN Luo De gene 6000 type detection systems (German Qiagen company).
(6) every part of sample standard deviation detects relatively by National Standard Method GB4789-2008 and sequencing simultaneously.
Sequencing adopts object fragment is cloned into the method for pUC-19T plasmid (precious biotech firm).PCR primer, condition and program are identical with HRM method.
(7) experimental result
By two kinds of methods, 120 parts of commercially available pork samples are detected, for Salmonellas, Listeria monocytogenes and streptococcus aureus, the positive rate of HRM method all with National Standard Method difference to some extent, detected result confirms that HRM method can make Salmonellas positive rate be increased to 25% from 24.17% of National Standard Method; Listeria monocytogenes is consistent with National Standard Method with streptococcus aureus positive rate.This checkout discrepancy may be to compare National Standard Method and have higher sensitivity due to this method, illustrates that HRM method has extensive and good suitability.
The detected result of the natural microbiological contamination sample of table 5
"+" representative detects positive, and "-" representative detects negative.
Claims (8)
1. detect a test kit for Salmonellas, Listeria monocytogenes and streptococcus aureus simultaneously, it is characterized in that, comprise the upstream and downstream primer of above-mentioned three kinds of bacterium, specific as follows:
2. test kit according to claim 1, is characterized in that, also comprises 10 * buffer, MgCl
2, dNTP, BSA, 20 * Evagreen, Taq enzyme, Taq enzyme antibody and H
2o.
3. test kit according to claim 1 and 2, is characterized in that, this test kit is the test kit of 25 μ L reaction systems, and each component concentration is as follows:
And the H of enough polishing reaction system to 25 μ L
2o.
4. detect a method for Salmonellas, Listeria monocytogenes and streptococcus aureus simultaneously, it is characterized in that, comprise the steps:
(1) testing sample is cultivated in the mixed culture medium of Salmonellas, Listeria monocytogenes and streptococcus aureus, choose respectively invA, hly, Sa442 is the goal gene of Salmonellas (Salmonella), Listeria monocytogenes (Listeriamonocytogenes) and streptococcus aureus (Staphylococcus aureus), design and invA, the supporting upstream and downstream primer of hly, Sa442, carry out DNA extraction to the product after above-mentioned mixed culture; Wherein, primer sequence is as follows:
(2) adopt pcr amplification primer to carry out pcr amplification to the mixing masterplate DNA of said extracted; Pcr amplification product is carried out to high resolving power liquation, and program is: 95 ℃ of 15s, and 60 ℃ of 1min, 95 ℃ of 15s, 60 ℃ of 15s, melting speed is 0.2 ℃/s;
(3) by high resolving power melting curve method, testing sample is identified, used T
mvalue is as interpretation standard, as the T recording
mvalue is 79.38 ± 0.5 ℃, and when 82.54 ± 0.5 ℃ and 77.36 ± 0.5 ℃, corresponding bacterial strain is respectively Salmonellas, Listeria monocytogenes and streptococcus aureus.
5. method according to claim 4, it is characterized in that, the reaction system of described pcr amplification is: every 25 μ L PCR systems comprise 10 * buffer2.5 μ L, the Taq enzyme 0.5 μ L of concentration 5U/ μ L, the T aq antibody 0.5 μ L of concentration 1 μ g/ μ L, the dNTP2.5 μ L of 10mM, the MgCl of concentration 25mM
23 μ L, the BSA0.2 μ L that concentration is 20mg/ml, each 0.5 μ L of the upstream primer of concentration 10 μ M and downstream primer, mixes masterplate DNA2 μ L, and 1 μ L fluorescence dye EvaGreen, spends nucleic acid water polishing to 25 μ L.
6. method according to claim 5, is characterized in that, described PCR response procedures is: 95 ℃ of 10min; 95 ℃ of 10s, 60 ℃ of 45s, 40 circulations.
7. according to the method described in claim 4 or 5 or 6, it is characterized in that, described mixed culture medium by weight, comprise 0.010 part of 17 parts of Tryptoness, 3 parts of peptones, 2.5 parts of SODIUM PHOSPHATE, MONOBASIC, 2.5 parts of glucose, 1.0 parts of Vitamin C2s, 1000 parts of distilled water, 2.5 parts of Sodium.alpha.-ketopropionates, 5.0 parts, N.F,USP MANNITOL, 20.0 parts, sodium-chlor, 2.0 parts of lithium chlorides, 0.00015 part of potassium tellurite and nalidixic acid, pH value 7.2-7.3.
8. method according to claim 7, is characterized in that, described culture condition is: 35-38 ℃, 100-300rpm are cultivated 12-36h.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107119029A (en) * | 2017-05-09 | 2017-09-01 | 广州海力特生物科技有限公司 | A kind of preparation method of hot start Taq polymerase |
| CN110172503A (en) * | 2019-05-27 | 2019-08-27 | 郑州轻工业学院 | A kind of food-borne pathogens Listeria monocytogenes PCDR method detection reagent |
| CN111560448A (en) * | 2020-04-21 | 2020-08-21 | 江苏省家禽科学研究所 | Method for detecting 4 common food-borne pathogenic bacteria in poultry by high-resolution dissolution curve technology |
| CN112831603A (en) * | 2021-01-27 | 2021-05-25 | 河南省疾病预防控制中心 | Kit and method for detecting food-borne pathogenic bacteria based on multiple PCR (polymerase chain reaction) technology |
| CN114231647A (en) * | 2021-11-23 | 2022-03-25 | 广西壮族自治区食品药品检验所 | Method for detecting food-borne pathogenic bacteria staphylococcus aureus by using rolling circle isothermal amplification technology based on visualization method and application thereof |
-
2014
- 2014-07-03 CN CN201410317876.9A patent/CN104152546A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| MUSHUXI: "高分辨溶解曲线分析技术", 《小木虫》 * |
| 刘园园: "沙门氏菌、金黄色葡萄球菌及单增李斯特菌的同步快速检测方法的研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
| 姜侃等: "三重LAMP法检测食品中沙门氏菌、单增李斯特菌和金黄色葡萄球菌", 《食品科学》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107119029A (en) * | 2017-05-09 | 2017-09-01 | 广州海力特生物科技有限公司 | A kind of preparation method of hot start Taq polymerase |
| CN110172503A (en) * | 2019-05-27 | 2019-08-27 | 郑州轻工业学院 | A kind of food-borne pathogens Listeria monocytogenes PCDR method detection reagent |
| CN111560448A (en) * | 2020-04-21 | 2020-08-21 | 江苏省家禽科学研究所 | Method for detecting 4 common food-borne pathogenic bacteria in poultry by high-resolution dissolution curve technology |
| CN112831603A (en) * | 2021-01-27 | 2021-05-25 | 河南省疾病预防控制中心 | Kit and method for detecting food-borne pathogenic bacteria based on multiple PCR (polymerase chain reaction) technology |
| CN114231647A (en) * | 2021-11-23 | 2022-03-25 | 广西壮族自治区食品药品检验所 | Method for detecting food-borne pathogenic bacteria staphylococcus aureus by using rolling circle isothermal amplification technology based on visualization method and application thereof |
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