CN103436602B - 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 technical field of molecular biological detection, 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 staphylococcus aureus gene and bacillus coli gene simultaneously.
Background technology
Bread is the staff of life, eats with An Weixian.Food-safety problem has become the global great security hidden trouble of puzzlement, and the food contamination particularly caused by pathogenic micro-organism and food origin disease 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 example, developed country has at least the crowd of 1/3rd to suffer from food origin disease every year, wherein about has less than 1,700,000 15 years old children to cause diarrhoea and dead because of food source property microbial contamination.Important pathogenic bacteria common in microbes 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.
Current China Food Hygiene Surveillance inspection department still rests on traditional Bacteria culturing, Serum Antibody Detection and biochemical character comparison level to the Pathogen test of food origin disease, identification of means, be generally detection time between 2-4 days, some even reaches 7 days, and biochemical test and Serologic test are unstable, detection sensitivity is lower, in addition, current detection means generally can only detect a kind of pathogenic micro-organism, and in most cases, the pollution of food is not that single bacterium infects, thus add the workload of qualification, reduce detection efficiency.
Along with the development of Protocols in Molecular Biology, people adopt round pcr to be applied to the diagnosis of bacterium.Such as China Patent Publication No. is mention a kind of method being detected Vibrio parahaemolyticus by means such as DNA extraction, pcr amplification, electrophoresis and gel imaging observations in the patent application of CN1526825.But round pcr is easily polluted, it needs special instrument and equipment in addition, high to the technical requirements of operator, thus limits it and applies 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 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, be easy to, in some infrastructure promotion and application, have application prospect very widely.LAMP technology is widely used in the detection detecting 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 all shows that the height of the method is special, highly sensitive.But 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 first Tyagi and Kramer in 1996 propose, at the annealing stage of pcr amplification, molecular beacon is combined with the target sequence of generation and sends fluorescence, in the extension stage, depart from target sequence and do not disturb amplification, along with the increase of cycle index, the amount of the molecular beacon be 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, and particularly it can carry out Real_time quantitative detection, even may be used for in-vivo analysis.
Due to change and the antibiotic abuse of ecotope, clinical symptom caused by a lot of pathogenic bacterium is not more and more true to type, usually needing to detect various bacteria could determine pathogenic former simultaneously, therefore, need badly set up a kind of can 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 staphylococcus aureus gene and bacillus coli gene simultaneously, staphylococcus aureus gene nuc and Escherichia coli O 157 can be detected fast and effectively: rfbE gene two kinds of food-borne pathogens of H7.
Dual molecular beacon-LAMP method detects the test kit of staphylococcus aureus gene and bacillus coli gene simultaneously, comprise LAMP reaction system, the primer sets of the rfbE gene containing detection staphylococcus aureus gene nuc and Escherichia coli O 157: H7 in described LAMP reaction system, this primer sets comprises a pair outer primer F3, B3 of nuc gene, a pair inner primer FIP, BIP and a molecular beacon probe; Also comprise a pair outer primer F3, B3 of rfbE gene, a pair 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 μM/L of outer primer F3, B3 of nuc gene and rfbE gene, each 0.4 μM/L of outer primer F3, B3 of inner primer FIP, BIP each 2.4 μMs/L, rfbE gene, each 2.4 μMs/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) trimethyl-glycine of the dNTP of 2.8mmol/l, 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.
The test kit adopting dual molecular beacon-LAMP method simultaneously to detect staphylococcus aureus gene and bacillus coli gene carries out the method detected, and comprises the following steps:
1) adopt LAMP reaction system, this reaction system comprises primer and the molecular beacon probe of staphylococcus aureus gene nuc, the primer of the rfbE gene of Escherichia coli O 157: H7 and molecular beacon probe;
2) testing sample process and template extraction
Get tested bacteria sample, after suspending with physiological saline, centrifugal, get precipitation, in precipitation, add sample pretreatment liquid, after mixing, after boiling 10min, cooling 10min, recentrifuge, gets supernatant, obtains the template DNA of testing sample;
3) LAMP isothermal duplication
The template DNA of testing sample is joined in LAMP reaction system, at 60 ~ 70 DEG C, reaction 50 ~ 70min;
4) color developing detection
Get the product after LAMP isothermal duplication, add nitrite ion, with positive control, directly change with visual inspection.
Step 2) described in sample pretreatment liquid be by the N,O-Diacetylmuramidase of 40 μ l20mg/mL, the Proteinase K of the SDS of 30 μ l10%, 10 μ l20mg/mL and the NaCl of 100 μ l5mol/L, fully after mixing, under 8000r/min, after centrifugal 10min, the supernatant liquor obtained.
Described N,O-Diacetylmuramidase first at 37 DEG C, is incubated 30min before use; Described Proteinase K first at 53 DEG C, is incubated 30min before use.
Step 2) described in 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, establishes the method that dual molecular beacon detects two kinds of food-borne pathogens simultaneously, has following advantage:
1, present method is highly sensitive, and sample, containing 20 ~ 100cfu/ml, can detect;
2, present method high specificity, with control group tens kinds of bacterium no cross reactions;
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 process.
Accompanying drawing explanation
Fig. 1 is LAMP of the present invention amplification electrophoretogram.
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, for streptococcus aureus and Escherichia coli O 157: H7, analyzes the detection method of dual molecular beacon-LAMP, thus realizes detecting two kinds of pathogenic bacterium fast and accurately simultaneously.
The toxin gene of food-borne pathogens announced according to GenBank or the conserved sequence of invasin gene, we select the heat stable nuclease Nuc gene of streptococcus aureus and the hemolysin gene rbfE of Escherichia coli O 157: H7, design LAMP primer and molecular beacon probe respectively, 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 of common bacterial food poisoning.
The method that dual molecular beacon-LAMP method detects food-borne pathogens mainly comprises the following steps:
1) according to the toxin gene of food-borne pathogens or the conserved sequence of invasin gene of Genbank announcement, primer and molecular beacon probe is designed respectively;
Heat stable nuclease gene nuc(Genbank:V01281 with streptococcus aureus) and Escherichia coli O 157: the rfbE gene (Genbank:AE005429) in H7 designs detection 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 process and template extraction
The sample scope of application comprises the samples such as food samples, ight soil and vomitus or needs to there is to bacterium the biological specimen detected.For ight soil or vomitus sample, according to the number of Specific amounts, suspend with 100-200 μ l physiological saline, 10000rpm abandons supernatant after centrifugal 2 minutes, then adds the pretreatment fluid of 80 μ l, and boiling water boiling is after 10 minutes, cool 10 minutes at once, centrifugal 2 minutes of 10000rpm, gets the template that 5 μ l supernatant liquors are used as amplification, and other samples judge whether to need to increase bacterium process according to actual needs.
3) LAMP amplification
LAMP reaction system amounts to 25 μ l and comprises:
Outer primer F3/B3 0.4 μM/L
Inner primer FIP/BIP 2.4 μMs/L
BstE 8U
dNTP 2.8mmol/l
Trimethyl-glycine 0.8mol/l
Buffer 2μl
Template 2 μ l
ddH2O 2μl
Temperature of reaction 65 DEG C of reaction times 60min
4) visual method directly observes colour-change, thus judges the situation of microbiological contamination in the situation that LAMP increases and sample.
According to distinct colors, LAMP amplified production can be judged, meanwhile, get 5 ~ 10 μ l products for electrophoresis, utilize electrophoresis to detect the result of LAMP amplification.
See 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 that the Auele Specific Primer with streptococcus aureus is designed has 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, present method high specificity, with control group tens kinds of bacterium no cross reactions, 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, whole testing process can be completed.
Claims (3)
1. dual molecular beacon-LAMP method detects the test kit of staphylococcus aureus gene and bacillus coli gene simultaneously, it is characterized in that, comprise LAMP reaction system, the primer sets of the rfbE gene containing detection staphylococcus aureus gene nuc and Escherichia coli O 157: H7 in described LAMP reaction system, this primer sets comprises a pair outer primer F3, B3 of nuc gene, a pair inner primer FIP, BIP and a molecular beacon probe; Also comprise a pair outer primer F3, B3 of rfbE gene, a pair 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 μM/L of outer primer F3, B3 of nuc gene and rfbE gene, each 0.4 μM/L of outer primer F3, B3 of inner primer FIP, BIP each 2.4 μMs/L, rfbE gene, each 2.4 μMs/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) trimethyl-glycine of the dNTP of 2.8mmol/l, 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.
2. the test kit adopting dual molecular beacon-LAMP method according to claim 1 simultaneously to detect staphylococcus aureus gene and bacillus coli gene carries out the method for non-diagnostic object detection, it is characterized in that, comprises the following steps:
1) adopt LAMP reaction system, this reaction system comprises primer and the molecular beacon probe of staphylococcus aureus gene nuc, the primer of the rfbE gene of Escherichia coli O 157: H7 and molecular beacon probe;
2) testing sample process and template extraction
Get tested bacteria sample, after suspending with physiological saline, centrifugal, get precipitation, in precipitation, add sample pretreatment liquid, after mixing, after boiling 10min, cooling 10min, recentrifuge, gets supernatant, obtains the template DNA of testing sample;
Described sample pretreatment liquid is by the N,O-Diacetylmuramidase of 40 μ l 20mg/mL, the SDS of 30 μ l 10%, the Proteinase K of 10 μ l 20mg/mL and the NaCl of 100 μ l 5mol/L, fully after mixing, under 8000r/min, after centrifugal 10min, and the supernatant liquor obtained;
Described N,O-Diacetylmuramidase first at 37 DEG C, is incubated 30min before use; Described Proteinase K first at 53 DEG C, is incubated 30min before use
3) LAMP isothermal duplication
The template DNA of testing sample is joined in LAMP reaction system, at 60 ~ 70 DEG C, reaction 50 ~ 70min;
4) color developing detection
Get the product after LAMP isothermal duplication, add nitrite ion, with positive control, directly change with visual inspection.
3. dual molecular beacon-LAMP method according to claim 2 detects the method for staphylococcus aureus gene and bacillus coli gene simultaneously, it is characterized in that: step 2) described in 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 |
| CN110878380B (en) * | 2019-12-23 | 2021-09-07 | 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 |
| CN114540516B (en) * | 2022-03-08 | 2023-06-20 | 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 |
| 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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| EP1770171A1 (en) * | 2005-09-29 | 2007-04-04 | Universität Zu Köln | DNA microarray for rapid identification of Candida albicans in blood cultures. |
| CN101487057B (en) * | 2009-01-23 | 2011-11-09 | 浙江省疾病预防控制中心 | Loop-mediated isothermal amplification fast detecting reagent kit for O157:H7 coliform |
| WO2011056933A1 (en) * | 2009-11-05 | 2011-05-12 | Becton, Dickinson And Company | Sequence-specific methods for homogenous, real-time detection of lamp products |
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