CN103436602A - Kit and method for simultaneous detection of Staphylococcus aureus gene and Escherichia coli gene by using dual molecular beacon-LAMP process - Google Patents
Kit and method for simultaneous detection of Staphylococcus aureus gene and Escherichia coli gene by using dual molecular beacon-LAMP process Download PDFInfo
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Abstract
The invention discloses a kit and a method for simultaneous detection of a Staphylococcus aureus gene and an Escherichia coli gene by using a dual molecular beacon-LAMP process, belonging to the technical field of molecular biological detection. The kit comprises an LAMP reaction system, wherein the LAMP reaction system comprises a primer group used for detecting the Staphylococcus aureus gene nuc and an rfbE gene of the Escherichia coli O157: H7, the primer group includes a pair of outer primers F3 and B3, a pair of inner primers FIP and BIP and a molecular beacon probe of the nuc gene and further includes a pair of outer primers F3 and B3, a pair of inner primers FIP and BIP and a molecular beacon probe of the rfbE gene. The method provided by the invention has the advantages of high sensitivity, strong specificity, avoidance of cross reaction with a dozen of bacteria of a control group, simple operation, realization of visual and clear observation of results and a fast detection speed.
Description
Technical field
The invention belongs to the molecular Biological Detection technical field, relate to a kind of method that dual molecular beacon-loop-mediated isothermal amplification technique detects two kinds of food-borne pathogens simultaneously, be specifically related to test kit and method that dual molecular beacon-LAMP method detects the rfbE gene of staphylococcus aureus gene nuc and Escherichia coli O 157: H7 simultaneously.
Background technology
Bread is the staff of life, and food is with An Weixian.Food contamination and food origin disease that food-safety problem has become due to puzzlement global a great security hidden trouble, particularly pathogenic micro-organism have become a worldwide public health problem.The World Health Organization (WHO) report claims the etesian food origin disease in the whole world billions of examples, developed country has at least 1/3rd crowd to suffer from food origin disease every year, wherein approximately has children below 1,700,000 15 years old to cause that because of the microbial contamination of food source property diarrhoea is dead.Important pathogenic bacteria common in microorganism property food poisoning is: streptococcus aureus, Vibrio parahemolyticus, bacillus cereus and Salmonellas, Clostridium botulinum, Liszt's monocyte hyperplasia bacterium, false unicellular bacterium and Escherichia coli O 157: H7 etc.
China's Food Hygiene Surveillance inspection department still rests on traditional Bacteria culturing, Serum Antibody Detection and biochemical character comparison level to cause of disease detection, the identification of means of food origin disease at present, be generally detection time between 2-4 days, some even reaches 7 days, and biochemical test and Serologic test unstable, detection sensitivity is lower, in addition, current detection means generally can only detect a kind of pathogenic micro-organism, and in most of situation, the pollution of food is not that single bacterium infects, thereby increased the workload of identifying, lowered detection efficiency.
Along with the development of Protocols in Molecular Biology, people adopt round pcr to be applied to the diagnosis of bacterium.Mention a kind of method that detects Vibrio parahaemolyticus by means such as DNA extraction, pcr amplification, electrophoresis and gel imaging observations in the patent application that is CN1526825 such as China Patent Publication No..Yet round pcr is easily polluted, it needs special instrument and equipment in addition, high to operator's technical requirements, thereby has limited it and applied widely.
Ring mediated isothermal amplification (LAMP) technology is a kind of new nucleic acid amplification method that the people such as Japanese Notomi T in 2000 develop, can be using opacity as identification of indicator, the white casse precipitation as long as detect by an unaided eye, just can identify and whether increase, and do not need the processes such as loaded down with trivial details electrophoresis and ultraviolet visualization, being easy to, in some mechanism of basic unit promotion and application, has application prospect very widely.The LAMP technology is widely used in the detection that detects many bacteriums such as EcoliO157:H7, streptococcus aureus, Enterobacter sakazakii, Vibrio parahemolyticus, enterococcus faecalis, Shigellae, artificial tuberculosis yersinia genus, blunt Fahrenheit bacterium (E.tarda), Salmonellas in food, and result has all demonstrated that the height of the method is special, highly sensitive.But the LAMP product is subject to Aerosol Pollution, causes false positive results.
Molecular beacons technology is a kind of molecular probe with neck ring configuration based on nucleic acid base pair principle and fluorescence resonance energy principle design that at first Tyagi in 1996 and Kramer propose, annealing stage at pcr amplification, molecular beacon is combined and is sent fluorescence with the target sequence of generation, in the extension stage, break away from target sequence and do not disturb amplification, along with the increase of cycle index, the amount of the molecular beacon of being combined with template also increases, and final fluorescence intensity is directly proportional to the template amount of amplification.This technology has high specificity, and easy and simple to handle, highly sensitive, particularly it can carry out the real-time quantitative detection, even can be for in-vivo analysis.
Variation and antibiotic abuse due to ecotope, a lot of caused clinical symptom of pathogenic bacterium more and more are not true to type, usually needing to detect various bacteria could determine pathogenic former simultaneously, therefore, need badly set up a kind of can be fast, high specific and susceptibility, detect the diagnostic techniques of multiple pathogenic microorganisms simultaneously.
Summary of the invention
The object of the present invention is to provide a kind of dual molecular beacon-LAMP method to detect test kit and the method for the rfbE gene of staphylococcus aureus gene nuc and Escherichia coli O 157: H7 simultaneously, can detect fast and effectively two kinds of food-borne pathogens of rfbE gene of staphylococcus aureus gene nuc and Escherichia coli O 157: H7.
Dual molecular beacon-LAMP method detects the test kit of the rfbE gene of staphylococcus aureus gene nuc and Escherichia coli O 157: H7 simultaneously, comprise the LAMP reaction system, the primer sets that contains the rfbE gene that detects staphylococcus aureus gene nuc and Escherichia coli O 157: H7 in described LAMP reaction system, a pair of outer primer F3, B3 that this primer sets comprises the nuc gene, a pair of inner primer FIP, BIP and a molecular beacon probe; The a pair of outer primer F3, the B3 that also comprise the rfbE gene, a pair of inner primer FIP, BIP and a molecular beacon probe;
Wherein, the primer sets of described nuc gene is:
The nucleotide sequence of outer primer F3 is as shown in SEQ.ID.NO.1;
The nucleotide sequence of outer primer B3 is as shown in SEQ.ID.NO.2;
The nucleotide sequence of inner primer FIP is as shown in SEQ.ID.NO.3;
The nucleotide sequence of inner primer BIP is as shown in SEQ.ID.NO.4;
The nucleotide sequence of molecular beacon probe is as shown in SEQ.ID.NO.5;
The primer sets of described rfbE gene is:
The nucleotide sequence of outer primer F3 is as shown in SEQ.ID.NO.6;
The nucleotide sequence of outer primer B3 is as shown in SEQ.ID.NO.7;
The nucleotide sequence of inner primer FIP is as shown in SEQ.ID.NO.8;
The nucleotide sequence of inner primer BIP is as shown in SEQ.ID.NO.9;
The nucleotide sequence of molecular beacon probe is as shown in SEQ.ID.NO.10;
The cumulative volume of described LAMP reaction system is 25 μ l, comprises following component:
1) the testing sample template DNA of 2 μ l;
2) each 0.4 μ M/L of outer primer F3, the B3 of nuc gene and rfbE gene, each 2.4 μ M/L of inner primer FIP, BIP, each 0.4 μ M/L of outer primer F3, the B3 of rfbE gene, each 2.4 μ M/L of inner primer FIP, BIP;
3) archaeal dna polymerase: every μ l is containing the Bst archaeal dna polymerase of 8 activity units;
4) dNTP of 2.8mmol/l, the trimethyl-glycine of 0.8mol/l, the Buffer of 2 μ l, the ddH of 2 μ l
2o.
5 ' end of the nucleotide sequence of the molecular beacon probe of described Nuc gene is connected with fluorescence dye Rox, and 3 ' end is connected with quenching of fluorescence group DABCYL.
5 ' end of the nucleotide sequence of the molecular beacon probe of described rfbE gene is connected with fluorescence dye FAM, and 3 ' end is connected with quenching of fluorescence group DABCYL.
Dual molecular beacon-LAMP method detects the method that the test kit of the rfbE gene of staphylococcus aureus gene nuc and Escherichia coli O 157: H7 is detected simultaneously, comprises the following steps:
1) adopt the LAMP reaction system, this reaction system comprises primer and the molecular beacon probe of staphylococcus aureus gene nuc, primer and the molecular beacon probe of the rfbE gene of Escherichia coli O 157: H7;
2) testing sample is processed and template extraction
Get the tested bacteria sample, after suspending with physiological saline, centrifugal, get precipitation, add sample pretreatment liquid in precipitation, after mixing, after boiling 10min, cooling 10min, recentrifuge, get supernatant, obtains the template DNA of testing sample;
3) LAMP isothermal duplication
The template DNA of testing sample is joined in the LAMP reaction system, under 60～70 ℃, react 50～70min;
4) color developing detection
Get the product after the LAMP isothermal duplication, add nitrite ion, with positive control, directly change with visual inspection.
Step 2) described sample pretreatment liquid is by the N,O-Diacetylmuramidase of 40 μ l20mg/mL, the SDS of 30 μ l10%, and the NaCl of the Proteinase K of 10 μ l20mg/mL and 100 μ l5mol/L, after fully mixing, under 8000r/min, after centrifugal 10min, the supernatant liquor obtained.
Described N,O-Diacetylmuramidase is first under 37 ℃ before use, insulation 30min; Described Proteinase K is first under 53 ℃ before use, insulation 30min.
Step 2) described centrifugal be under 8000～12000r/min, centrifugal 1～3min.
Compared with prior art, the present invention has following useful technique effect:
The present invention, on the basis of LAMP amplification technique, has set up the method that dual molecular beacon detects two kinds of food-borne pathogens simultaneously, and following advantage is arranged:
1, present method is highly sensitive, and sample, containing 20～100cfu/ml, can detect;
2, present method high specificity, with tens kinds of bacterium no cross reactions of control group;
3, simple to operate, result is observed simple and clear;
4, detection speed is fast, connects sample pre-treatments and amplified reaction, only needs 2 hours, can complete whole testing processes.
The accompanying drawing explanation
Fig. 1 is LAMP amplification electrophoretogram of the present invention.
Embodiment
Below in conjunction with concrete the drawings and specific embodiments, the present invention is described in further detail, and the explanation of the invention is not limited.
The present invention be take streptococcus aureus and Escherichia coli O 157: H7 as example, analyzes the detection method of dual molecular beacon-LAMP, thereby realizes detecting fast and accurately two kinds of pathogenic bacterium simultaneously.
The toxin gene of the food-borne pathogens of announcing according to GenBank or the conserved sequence of invasin gene, we select the hemolysin gene rbfE of heat stable nuclease Nuc gene and the Escherichia coli O 157 of streptococcus aureus: H7, design respectively LAMP primer and molecular beacon probe, with different fluorescent agent marks, set up dual molecular beacon-LAMP method, to detect the reaction system of these two kinds of food-borne pathogens, and this detection system is applied to the quick diagnosis that common bacterial is poisoned by food.
The method that dual molecular beacon-LAMP method detects food-borne pathogens mainly comprises the following steps:
The toxin gene of the food-borne pathogens of 1) announcing according to Genbank or the conserved sequence of invasin gene, design respectively primer and molecular beacon probe;
Heat stable nuclease gene nuc(Genbank:V01281 with streptococcus aureus) and Escherichia coli O 157: the rfbE gene (Genbank:AE005429) in H7 designs and detects with primer and corresponding molecular beacon.
Primer and the probe of described molecular beacon are respectively:
The LAMP amplimer of Nuc gene:
F3：TGCAAAGAAAATTGAAGTCGA
B3:GGTTGTCTTCGCTCCAAAT
FIP:
CGTTTACCATTTTTCCATCAGCATATTTGACAAAGGTCAAAGAACT
BIP:
TCAAGGCTTGGCTAAAGTTGCTTATTCGCTTGTGCTTCACTT
The probe of Nuc gene:
Rox-5’-CGATGCAGTCTAAGTAGCTCAGCAAATGCATCG-3’-DABCYL
The LAMP amplimer of rfbE gene:
F3：AACAGTCTTGTTGTACAAGTCCA
B3：CGTGCTTTGATATTTTTCCG
FIP:
CTCTCTTTCCTCTGCGGTCCGATGTTTTTCACACTTATTGGAT
BIP:
TAAGGAATCACCTTGCAGATAAACTAGTACATTGGCATCGTGT
The probe of rfbE gene:
5’-FAM-CGGCCAAGGATTAGCTGTACATAGGCCG-DABCYL-3’
2) testing sample is processed and template extraction
The sample scope of application comprises that the samples such as food samples, ight soil and vomitus maybe need bacterium is existed to the biological specimen detected.Take ight soil or vomitus sample is example, number according to concrete amount, with 100-200 μ l physiological saline, suspend, 10000rpm abandons supernatant after centrifugal 2 minutes, then adds the pretreatment fluid of 80 μ l, and boiling water boiling is after 10 minutes, cooling 10 minutes at once, centrifugal 2 minutes of 10000rpm, get the template of 5 μ l supernatant liquors as amplification, and other samples judge whether to increase the bacterium process according to actual needs.
3) LAMP amplification
The LAMP reaction system amounts to 25 μ l and comprises:
Outer primer F3/B30.4 μ M/L
Inner primer FIP/BIP2.4 μ M/L
BstE 8U
dNTP2.8mmol/l
Trimethyl-glycine 0.8mol/l
Buffer2μl
Template 2 μ l
ddH2O2μl
65 ℃ of reaction times 60min of temperature of reaction
4) visual method is directly observed colour-change, thus the situation of microbiological contamination in the situation of judgement LAMP amplification and sample.
Can be according to distinct colors, judgement LAMP amplified production, simultaneously, get 5～10 μ l products for electrophoresis, utilizes electrophoresis to detect the result of LAMP amplification.
Referring to Fig. 1, result shows, band 4, band 5 all have obvious scalariform band, and other blank and intestinal bacteria control group are all without obvious scalariform band, as can be seen here, only have streptococcus aureus to show positive, and remaining blank and control group all show feminine gender, also illustrate with the Auele Specific Primer of streptococcus aureus design and have good specificity.
In addition, the inventive method adopts tens kinds of bacteriums to do control experiment, bacterium is as follows: streptococcus aureus (ATCC6538), streptococcus aureus (ATCC25923), streptococcus aureus (GIM1.55) intestinal bacteria (ATCC8739), colon bacillus (ATCC25922), produce enterotoxin colon bacillus (ATCC35401) Listeria monocytogenes (GIM1.229), Salmonella typhimurium (CMCC50115), shigella dysenteriae (CMCC51252), Salmonella choleraesuls (ATCC13312), shigella flexneri (CMCC51572), Song Shi Shigellae (CMCC51592) and Vibrio parahemolyticus (ATCC17802), equal no cross reaction.
In sum, present method is highly sensitive, and present method high specificity, with tens kinds of bacterium no cross reactions of control group, simple to operate, result is observed simple and clear, and detection speed is fast, connect sample pre-treatments and amplified reaction, only need 2 hours, can complete whole testing processes.
Claims (7)
1. dual molecular beacon-LAMP method detects the test kit of the rfbE gene of staphylococcus aureus gene nuc and Escherichia coli O 157: H7 simultaneously, it is characterized in that, comprise the LAMP reaction system, the primer sets that contains the rfbE gene that detects staphylococcus aureus gene nuc and Escherichia coli O 157: H7 in described LAMP reaction system, a pair of outer primer F3, B3 that this primer sets comprises the nuc gene, a pair of inner primer FIP, BIP and a molecular beacon probe; The a pair of outer primer F3, the B3 that also comprise the rfbE gene, a pair of inner primer FIP, BIP and a molecular beacon probe;
Wherein, the primer sets of described nuc gene is:
The nucleotide sequence of outer primer F3 is as shown in SEQ.ID.NO.1;
The nucleotide sequence of outer primer B3 is as shown in SEQ.ID.NO.2;
The nucleotide sequence of inner primer FIP is as shown in SEQ.ID.NO.3;
The nucleotide sequence of inner primer BIP is as shown in SEQ.ID.NO.4;
The nucleotide sequence of molecular beacon probe is as shown in SEQ.ID.NO.5;
The primer sets of described rfbE gene is:
The nucleotide sequence of outer primer F3 is as shown in SEQ.ID.NO.6;
The nucleotide sequence of outer primer B3 is as shown in SEQ.ID.NO.7;
The nucleotide sequence of inner primer FIP is as shown in SEQ.ID.NO.8;
The nucleotide sequence of inner primer BIP is as shown in SEQ.ID.NO.9;
The nucleotide sequence of molecular beacon probe is as shown in SEQ.ID.NO.10;
The cumulative volume of described LAMP reaction system is 25 μ l, comprises following component:
1) the testing sample template DNA of 2 μ l;
2) each 0.4 μ M/L of outer primer F3, the B3 of nuc gene and rfbE gene, each 2.4 μ M/L of inner primer FIP, BIP, each 0.4 μ M/L of outer primer F3, the B3 of rfbE gene, each 2.4 μ M/L of inner primer FIP, BIP;
3) archaeal dna polymerase: every μ l is containing the Bst archaeal dna polymerase of 8 activity units;
4) dNTP of 2.8mmol/l, the trimethyl-glycine of 0.8mol/l, the Buffer of 2 μ l, the ddH of 2 μ l
2o.
2. dual molecular beacon according to claim 1-LAMP method detects the test kit of the rfbE gene of staphylococcus aureus gene nuc and Escherichia coli O 157: H7 simultaneously, it is characterized in that, 5 ' end of the nucleotide sequence of the molecular beacon probe of described Nuc gene is connected with fluorescence dye Rox, and 3 ' end is connected with quenching of fluorescence group DABCYL.
3. dual molecular beacon according to claim 1-LAMP method detects the test kit of the rfbE gene of staphylococcus aureus gene nuc and Escherichia coli O 157: H7 simultaneously, it is characterized in that, 5 ' end of the nucleotide sequence of the molecular beacon probe of described rfbE gene is connected with fluorescence dye FAM, and 3 ' end is connected with quenching of fluorescence group DABCYL.
4. the method that the test kit that adopts dual molecular beacon claimed in claim 1-LAMP method simultaneously to detect the rfbE gene of staphylococcus aureus gene nuc and Escherichia coli O 157: H7 is detected, is characterized in that, comprises the following steps:
1) adopt the LAMP reaction system, this precursor reactant system comprises primer and the molecular beacon probe of staphylococcus aureus gene nuc, primer and the molecular beacon probe of the rfbE gene of Escherichia coli O 157: H7;
2) testing sample is processed and template extraction
Get the tested bacteria sample, after suspending with physiological saline, centrifugal, get precipitation, add sample pretreatment liquid in precipitation, after mixing, after boiling 10min, cooling 10min, recentrifuge, get supernatant, obtains the template DNA of testing sample;
3) LAMP isothermal duplication
The template DNA of testing sample is joined in the LAMP reaction system, under 60～70 ℃, react 50～70min;
4) color developing detection
Get the product after the LAMP isothermal duplication, add nitrite ion, with positive control, directly change with visual inspection.
5. dual molecular beacon according to claim 4-LAMP method detects the detection method of the rfbE gene of staphylococcus aureus gene nuc and Escherichia coli O 157: H7 simultaneously, it is characterized in that: step 2) described sample pretreatment liquid is by the N,O-Diacetylmuramidase of 40 μ l20mg/mL, the SDS of 30 μ l10%, the NaCl of the Proteinase K of 10 μ l20mg/mL and 100 μ l5mol/L, after fully mixing, under 8000r/min, after centrifugal 10min, the supernatant liquor obtained.
6. dual molecular beacon according to claim 5-LAMP method detects the detection method of the rfbE gene of staphylococcus aureus gene nuc and Escherichia coli O 157: H7 simultaneously, it is characterized in that: described N,O-Diacetylmuramidase is first under 37 ℃ before use, insulation 30min; Described Proteinase K is first under 53 ℃ before use, insulation 30min.
7. dual molecular beacon according to claim 4-LAMP method detects the detection method of the rfbE gene of staphylococcus aureus gene nuc and Escherichia coli O 157: H7 simultaneously, it is characterized in that: step 2) described centrifugal be under 8000～12000r/min, centrifugal 1～3min.
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| CN105950778A (en) * | 2016-07-20 | 2016-09-21 | 中国疾病预防控制中心传染病预防控制所 | Nucleotide sequence for detecting enterococcus faecalis and staphylococcus aureus with MERT-LAMP technology |
| CN106434917A (en) * | 2016-09-24 | 2017-02-22 | 中华人民共和国广州机场出入境检验检疫局 | LAMP primer group and detection kit for staphylococcus aureus and use method of detection kit |
| CN108277289A (en) * | 2017-12-29 | 2018-07-13 | 广东环凯生物科技有限公司 | Escherichia coli O157:The dry powdered LAMP quick detection kits of H7 |
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| CN105950778A (en) * | 2016-07-20 | 2016-09-21 | 中国疾病预防控制中心传染病预防控制所 | Nucleotide sequence for detecting enterococcus faecalis and staphylococcus aureus with MERT-LAMP technology |
| CN106434917A (en) * | 2016-09-24 | 2017-02-22 | 中华人民共和国广州机场出入境检验检疫局 | LAMP primer group and detection kit for staphylococcus aureus and use method of detection kit |
| CN108277289A (en) * | 2017-12-29 | 2018-07-13 | 广东环凯生物科技有限公司 | Escherichia coli O157:The dry powdered LAMP quick detection kits of H7 |
| CN110878380A (en) * | 2019-12-23 | 2020-03-13 | 广西壮族自治区兽医研究所 | Primer composition, kit and method for detecting vesicular stomatitis virus Indiana type and new Jersey type |
| CN110878381A (en) * | 2019-12-23 | 2020-03-13 | 广西壮族自治区兽医研究所 | Primer composition, kit and method for detecting mycoplasma bovis and infectious bovine rhinotracheitis virus |
| CN114540516A (en) * | 2022-03-08 | 2022-05-27 | 河南中检食安生物科技有限公司 | LAMP double-strand detection probe, kit and detection method for staphylococcus aureus |
| CN116694804A (en) * | 2023-06-16 | 2023-09-05 | 浙江大学 | LAMP primer probe group, kit and detection method for detecting fusarium graminearum |
| CN116694804B (en) * | 2023-06-16 | 2024-02-02 | 浙江大学 | LAMP primer probe group, kit and detection method for detecting fusarium graminearum |
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