CN102329882A

CN102329882A - Multiple rapid detection method, detection primer group and kit for three food-borne pathogenic bacteria

Info

Publication number: CN102329882A
Application number: CN201110314039A
Authority: CN
Inventors: 姜侃; 张东雷; 金燕飞; 汪新; 黄建锋; 陈小珍
Original assignee: ZHEJIANG INSTITUTE OF QUALITY INSPECTION AND TECHNICAL RESEARCH
Current assignee: ZHEJIANG INSTITUTE OF QUALITY INSPECTION AND TECHNICAL RESEARCH
Priority date: 2011-10-15
Filing date: 2011-10-15
Publication date: 2012-01-25

Abstract

The invention discloses a multiple rapid detection method, a detection primer group and a kit for three food-borne pathogenic bacteria. According to the invention, upstream and downstream primers of an external primer and upstream and downstream primers of an internal primer of a rapid detection primer group of salmonella correspondingly have sequences as shown in SEQNo.1, SEQNo.2, SEQNo.3 and SEQNo.4; upstream and downstream primers of an external primer and upstream and downstream primers of an internal primer of a rapid detection primer group of staphylococcus aureus have sequences as shown in SEQNo.5, SEQNo.6, SEQNo.7 and SEQNo.8; upstream and downstream primers of an external primer and upstream and downstream primers of an internal primer of a rapid detection primer group of Listeria monocytogenes have sequences as shown in SEQNo.9, SEQNo.10, SEQNo.11 and SEQNo.12. By using the method in the invention, the defect that the prior art can not be used for simultaneously judging whether multiple pathogenic bacteria exist in the same system at the same time is overcome, and whether the same detected sample contains one or more of salmonella, staphylococcus aureus and Listeria monocytogenes can be correctly detected.

Description

Three kinds of multiple fast detection methods of food-borne pathogens and detect primer sets and test kit

Technical field

The invention belongs to the microorganism detection field, relate to the triple method for quick of a kind of Salmonellas, streptococcus aureus and Listeria monocytogenes and detect primer sets and detection kit.

Background technology

Food safety is the significant problem that countries in the world are paid close attention to, and wherein food-borne pathogens is the one of the main reasons that influences food safety.Salmonellas (Salmonella spp) is the important pathogenic bacteria in public health field; According to statistics in the bacterial food poisoning that countries in the world take place; The normal row umber one of salmonellal food poisoning also is first cause with the Salmonellas in the bacterial food poisoning that China hinterland is taken place.Except that causing food poisoning, the bacterium of this genus can also cause diseases such as gastro-enteritis, typhoid fever and typhus fever.Streptococcus aureus (Staphylococcus aureus) is under the jurisdiction of Staphylococcus; It is modal pathogenic bacteria in the human suppurative infection; Simultaneously, Staphylococcus aureus enterotoxin is a worldwide health difficult problem, and etesian this type of poisoning of China is also very many.Listeria monocytogenes (Listeria monocytogenes) is a kind of food-borne pathogens with character such as heat-resisting (60 ℃), low temperature resistant (4 ℃), salt tolerant, anti-dryings, and 1986, WHO and FDA classified them one of as food four big pathogenic bacterium nineties in 20th century; 2000, classified as one of food-borne pathogens of emphasis monitoring by WHOization.

According to current national food safety supervisory system, Salmonellas, streptococcus aureus and Listeria monocytogenes all belong to the food-borne pathogens of paying close attention to.Conventional sense adopts traditional cultural method, and each pathogenic bacterium need use independently that detection method detects separately, and workload is big, and sense cycle is long, and receives multiple effects limit such as testing staff's specialty, experience, occurs erroneous judgement easily.Along with the progressively application of molecular biology method in food microorganisms detect, the detection efficiency of pathogenic bacterium also progressively promotes.Though but present regular-PCR method that adopts and fluorescence PCR method can improve detection efficiency, and plant and instrument is had relatively high expectations, testing staff's professional background and detectivity also there is certain requirement, therefore can't promote the use of on a large scale.

The LAMP method is a kind of novel constant temperature nucleic acid amplification technology; This technology is mainly utilized 6 specific regions on 4 kinds of different Auele Specific Primer identification target DNAs; And utilize a kind of active archaeal dna polymerase of strand displacement (BstDNA) that has; Rapid amplifying nucleic acid under constant temperature has guaranteed the high specific and the high-level efficiency that increase.Because the nucleic acid amplification mechanism that LAMP is unique, its product can demonstrate the stepped band of LAMP reflect typical by stem one cyclic DNA of multiple multiple target sequence and the mixture that Cauliflower shape DNA is formed on agarose gel electrophoresis; Simultaneously, when nucleic acid generates in a large number, pyrophosphate ion of from dNTPs, separating out and the Mg in the reaction system ²⁺In conjunction with, produce macroscopic amplified reaction by product-white magnesium pyrophosphate deposition; In addition, because amplified production generates in a large number, behind the adding optical dye (like Syber Green I, ethidium bromide, Gel Red etc.), can under uv lamp, observe obvious fluorescence.Therefore not only view mode is various for the LAMP amplification, and the result identifies easyly, is fit to very much high-throughout rapid detection.

In view of the LAMP technology has numerous advantages, be applied to the detection of pathogenic micro-organism gradually at present.Like mycobacterium, Enterobacter sakazakii, Salmonellas, streptococcus aureus etc.But existent method can only detect to a certain pathogenic bacterium in a system at present, when a plurality of pathogenic bacterium of needs rapid screening, needs respectively it to be detected, and still has big workload.Multiple constant temperature nucleic acid detection technique can under the same reaction conditions, realize the rapid screening of a plurality of purpose bacterium in same reaction system, have broad application prospects.Through the domestic and foreign literature inquiry, Shang Weijian has LAMP to be applied to the relevant report of Salmonellas, streptococcus aureus and the multiple rapid detection of Listeria monocytogenes.

Summary of the invention

The purpose of this invention is to provide a kind of three kinds of multiple fast detection methods of food-borne pathogens and detect primer sets and test kit, can only be in a system detect to a certain pathogenic bacterium and the defective that whether exists various pathogens to judge simultaneously in can not be simultaneously to same system thereby overcome present existent method.

For realizing above-mentioned purpose, the technical scheme that the present invention taked is:

The upstream primer of the outer primer of the rapid detection primer sets of Salmonellas of the present invention has the sequence shown in SEQ No.1, and the downstream primer of its outer primer has the sequence shown in SEQ No.2; The upstream primer of its inner primer has the sequence shown in SEQ No.3, and the downstream primer of its inner primer has the sequence shown in SEQ No.4.

The upstream primer of the outer primer of the rapid detection primer sets of streptococcus aureus of the present invention has the sequence shown in SEQ No.5, and the downstream primer of its outer primer has the sequence shown in SEQ No.6; The upstream primer of its inner primer has the sequence shown in SEQNo.7, and the downstream primer of its inner primer has the sequence shown in SEQ No.8.

The upstream primer of the outer primer of the rapid detection primer sets of Listeria monocytogenes of the present invention has the sequence shown in SEQ No.9, and the downstream primer of its outer primer has the sequence shown in SEQ No.10; The upstream primer of its inner primer has the sequence shown in SEQNo.11, and the downstream primer of its inner primer has the sequence shown in SEQ No.12.

The present invention uses the detection primer sets to the method that Salmonellas, streptococcus aureus and Listeria monocytogenes carry out multiple rapid detection to be: the detection primer sets of Salmonellas, the detection primer sets of streptococcus aureus and the detection primer sets of Listeria monocytogenes and the DNA of testing sample are carried out the LAMP reaction; Finish the back in reaction and reaction solution is carried out yin and yang attribute judge, with confirm whether to contain in the said sample in Salmonellas, streptococcus aureus, the Listeria monocytogenes wherein one or more; In the detection primer sets of said Salmonellas; The upstream primer of outer primer has the sequence shown in SEQ No.1; The downstream primer of outer primer has the sequence shown in SEQ No.2; The upstream primer of inner primer has the sequence shown in SEQ No.3, and the downstream primer of inner primer has the sequence shown in SEQ No.4; In the detection primer sets of said streptococcus aureus; The upstream primer of outer primer has the sequence shown in SEQ No.5; The downstream primer of outer primer has the sequence shown in SEQ No.6; The upstream primer of inner primer has the sequence shown in SEQ No.7, and the downstream primer of inner primer has the sequence shown in SEQ No.8; In the detection primer sets of said Listeria monocytogenes; The upstream primer of outer primer has the sequence shown in SEQNo.9; The downstream primer of outer primer has the sequence shown in SEQ No.10; The upstream primer of inner primer has the sequence shown in SEQ No.11, and the downstream primer of inner primer has the sequence shown in SEQ No.12.

Further, LAMP reaction according to the invention is undertaken by following first kind of scheme or second kind of scheme:

First kind of scheme: the temperature of reaction of said LAMP reaction is 59.5-62.5 ℃, and the reaction times is 70-110min;

Second kind of scheme: said LAMP reaction comprises following steps:

(1) reacts 70-110min down at 59.5-62.5 ℃ earlier;

(2) back is reacted 6-12min down at 80 ℃.

Further; The present invention is in the reaction solution that carries out said LAMP reaction; The upstream primer of the outer primer in the detection primer sets of said Salmonellas and the concentration of downstream primer are 0.001-0.003 μ M, and the upstream primer of inner primer and the concentration of downstream primer are 0.35-0.45 μ M; The upstream primer of the outer primer in the detection primer sets of said streptococcus aureus and the concentration of downstream primer are 0.001-0.003 μ M, and the upstream primer of inner primer and the concentration of downstream primer are 0.25-0.35 μ M; The upstream primer of the outer primer in the detection primer sets of said Listeria monocytogenes and the concentration of downstream primer are 0.001-0.003 μ M, and the upstream primer of inner primer and the concentration of downstream primer are 0.25-0.35 μ M; And carrying out also including trimethyl-glycine, the concentration that concentration is 0.6-0.7M in the reaction solution of said LAMP reaction is the MgSO of 4.4-5.2mM ₄, 10 * BstDNA polymerase buffer of 10% (volume), concentration be that BstDNA polysaccharase and the concentration of 0.4-0.6U/ μ L respectively is the acid of triphosphoric acid guanine deoxyribonucleoside, triphosphoric acid adenyl-deoxyribonucleotide, triphosphoric acid thymidylic acid and the triphosphoric acid deoxycytidylic acid of 0.6-1.0mM.

A kind of quick detection kit of the present invention comprises the detection primer sets of detection primer sets, the Listeria monocytogenes of detection primer sets, the streptococcus aureus of Salmonellas, three kinds of positive reference substances, BstDNA polysaccharase, 10 * BstDNA polymerase buffer, MgSO ₄Solution, alkali solution of beet, the acid of triphosphoric acid guanine deoxyribonucleoside, triphosphoric acid adenyl-deoxyribonucleotide, triphosphoric acid thymidylic acid, triphosphoric acid deoxycytidylic acid and aseptic ultrapure water; The upstream primer of the outer primer in the detection primer sets of said Salmonellas has the sequence shown in SEQ No.1; The downstream primer of outer primer has the sequence shown in SEQ No.2; The upstream primer of inner primer has the sequence shown in SEQ No.3, and the downstream primer of inner primer has the sequence shown in SEQ No.4; The upstream primer of the outer primer in the detection primer sets of said streptococcus aureus has the sequence shown in SEQ No.5; The downstream primer of outer primer has the sequence shown in SEQ No.6; The upstream primer of inner primer has the sequence shown in SEQ No.7, and the downstream primer of inner primer has the sequence shown in SEQ No.8; The upstream primer of the outer primer in the detection primer sets of said Listeria monocytogenes has the sequence shown in SEQ No.9; The downstream primer of outer primer has the sequence shown in SEQ No.10; The upstream primer of inner primer has the sequence shown in SEQ No.11, and the downstream primer of inner primer has the sequence shown in SEQ No.12; Said three kinds of positive reference substances are respectively the genomic dna of the reference culture of Salmonellas, streptococcus aureus and Listeria monocytogenes.

Another kind of quick detection kit of the present invention comprises 2 * LAMP premixed liquid and three kinds of positive reference substances; Said three kinds of positive reference substances are respectively the genomic dna of the reference culture of Salmonellas, streptococcus aureus and Listeria monocytogenes, and the BstDNA polysaccharase, 2 * BstDNA polymerase buffer, the concentration that in said 2 * LAMP premixed liquid, include the detection primer sets of detection primer sets, the Listeria monocytogenes of detection primer sets, the streptococcus aureus of Salmonellas, trimethyl-glycine, concentration that concentration is 1.2-1.4M and be 0.8-1.2U/ μ L are the MgSO of 8.8-10.4mM ₄Solution, aseptic ultrapure water and concentration respectively are the acid of triphosphoric acid guanine deoxyribonucleoside, triphosphoric acid adenyl-deoxyribonucleotide, triphosphoric acid thymidylic acid and the triphosphoric acid deoxycytidylic acid of 1.2-2.0mM; The upstream primer of the outer primer in the detection primer sets of said Salmonellas has the sequence shown in SEQ No.1; The downstream primer of outer primer has the sequence shown in SEQ No.2; The upstream primer of inner primer has the sequence shown in SEQ No.3, and the downstream primer of inner primer has the sequence shown in SEQ No.4; The upstream primer of the outer primer in the detection primer sets of said streptococcus aureus has the sequence shown in SEQ No.5; The downstream primer of outer primer has the sequence shown in SEQ No.6; The upstream primer of inner primer has the sequence shown in SEQ No.7, and the downstream primer of inner primer has the sequence shown in SEQ No.8; The upstream primer of the outer primer in the detection primer sets of said Listeria monocytogenes has the sequence shown in SEQ No.9; The downstream primer of outer primer has the sequence shown in SEQ No.10; The upstream primer of inner primer has the sequence shown in SEQ No.11, and the downstream primer of inner primer has the sequence shown in SEQ No.12; The upstream primer of the outer primer in the detection primer sets of said Salmonellas and the concentration of downstream primer are 0.002-0.006 μ M, and the upstream primer of inner primer and the concentration of downstream primer are 0.7-0.9 μ M; The upstream primer of the outer primer in the detection primer sets of said streptococcus aureus and the concentration of downstream primer are 0.002-0.006 μ M, and the upstream primer of inner primer and the concentration of downstream primer are 0.5-0.7 μ M; The upstream primer of the outer primer in the detection primer sets of said Listeria monocytogenes and the concentration of downstream primer are 0.002-0.006 μ M, and the upstream primer of inner primer and the concentration of downstream primer are 0.5-0.7 μ M.

Compared with prior art, the invention has the beneficial effects as follows:

(1) the present invention is according to disclosed Salmonellas invasin protein A (invA) gene order, streptococcus aureus heat stable nuclease (nuc) gene order, listeria monocytogenes hemolysin albumen (hly) gene order, analyzes design and obtains triple LAMP detection primer sets of the present invention.Triple LAMP of the present invention detects the primer sets high specificity; As long as contain the genomic dna of these three kinds of pathogenic bacterium of Salmonellas, streptococcus aureus and Listeria monocytogenes in the DNA extraction liquid of the enrichment liquid of same institute sample article, can accurately detect simultaneously.

(2) utilize the rapid detection primer sets of Salmonellas of the present invention, streptococcus aureus and Listeria monocytogenes; Can be in same reaction system and under the same temperature of reaction condition; Realize simultaneously rapid detection to Salmonellas, streptococcus aureus and Listeria monocytogenes; As long as contain in Salmonellas, streptococcus aureus, the Listeria monocytogenes genomic dna one or more in the DNA extraction liquid of the enrichment liquid of same institute sample article; Can detect fast, accurate with judging; Can only be in a system detect and the defective that whether exists various pathogens to judge simultaneously in can not be simultaneously to same system thereby overcome prior art, improve detection efficiency greatly to a certain pathogenic bacterium.

(3) method for quick of the present invention is owing to utilize the rapid detection primer sets of Salmonellas of the present invention, streptococcus aureus and Listeria monocytogenes; And LAMP reaction system and reaction conditions be optimized; Thereby the genomic dna purpose fragment of can rapidly and efficiently increase Salmonellas, streptococcus aureus and Listeria monocytogenes; Whole LAMP amplification procedure can be accomplished in 70 minutes the soonest, and the amplified production yield is high, and is easy, accurate to the detection of above-mentioned three kinds of pathogenic bacterium.

(4) in method for quick of the present invention, the LAMP amplified reaction can be accomplished under steady temperature, and the temperature fluctuation range that allows big (± 1.5 ℃), and therefore less demanding to the plant and instrument of temperature control, water bath or plate heater all can meet the demands.

(5) method for quick of the present invention is highly sensitive, and the positive amplification of Salmonellas, streptococcus aureus and Listeria monocytogenes all only needs 1fg/ μ L purpose bacterium genomic dna.

(6) method for quick of the present invention to the decision method of detected result to breadboard physical condition require low, flexibility is stronger, and decision method is easy, can adopt and observe electrophoretic band, observe centrifugation or observe different modes result of determination such as fluorescence.

(7) compare with the plating method of routine; Method for quick of the present invention can shorten sense cycle greatly; Reduce and detect link; Thereby reduce because of detecting link too much or the probability of the erroneous judgement that causes of Personnel Skill Levels and empirical difference, make detected result more accurately and reliably, and the disposal that can be the food safety accident provides the important techniques support.

Embodiment

Below in conjunction with specific embodiment the present invention is done further elaboration, but the present invention is not done any qualification.

Embodiment 1: to the best Mg in the LAMP reaction system of the triple LAMP method for quick of the present invention ²⁺Confirming of concentration.

The concrete of present embodiment confirms that method is:

1. the extraction of sample DNA

11 are inoculated in nutrient broth respectively separately with Salmonellas and streptococcus aureus reference culture, and Listeria monocytogenes is inoculated in brain heart infusion meat soup, cultivate 18 hours for 36 ℃ ± 1 ℃.

Extract the DNA of bacteria in the above-mentioned cultured products respectively 1.2 use QIAGEN bacterial genomes DNA extraction test kit.

2.LAMP reaction

2.1 synthetic is used for the primer sets that Salmonellas, streptococcus aureus and Listeria monocytogenes detect respectively.Wherein, The upstream primer that is used for the outer primer of the primer sets that Salmonellas detects has the sequence shown in SEQ No.1; The downstream primer of outer primer has the sequence shown in SEQ No.2; The upstream primer of inner primer has the sequence shown in SEQ No.3, and the downstream primer of inner primer has the sequence shown in SEQ No.4; The upstream primer that is used for the outer primer of the primer sets that streptococcus aureus detects has the sequence shown in SEQ No.5; The downstream primer of outer primer has the sequence shown in SEQ No.6; The upstream primer of inner primer has the sequence shown in SEQ No.7, and the downstream primer of inner primer has the sequence shown in SEQ No.8; The upstream primer that is used for the outer primer of the primer sets that Listeria monocytogenes detects has the sequence shown in SEQ No.9; The downstream primer of outer primer has the sequence shown in SEQ No.10; The upstream primer of inner primer has the sequence shown in SEQNo.11, and the downstream primer of inner primer has the sequence shown in SEQ No.12.

2.2LAMP reaction system

2.2.1 the LAMP reaction system of Salmonellas

Use different Mg ²⁺Concentration is set up the LAMP reaction system of 7 groups of Salmonellass respectively, and is specific as follows:

The volume of each group reaction system is 25 μ L; Comprise respectively: the upstream primer and the downstream primer of the upstream primer of the outer primer that the upstream primer of the inner primer that the upstream primer of the outer primer that the upstream primer of the inner primer that the upstream primer that present embodiment 2.1 parts institute synthetic concentration is the outer primer that the Salmonellas of 0.002 μ M detects and the Salmonellas that downstream primer, concentration are 0.4 μ M detect and the streptococcus aureus that downstream primer, concentration are 0.002 μ M detect and the streptococcus aureus that downstream primer, concentration are 0.3 μ M detect and the Listeria monocytogenes that downstream primer, concentration are 0.002 μ M detect and the inner primer of the Listeria monocytogenes detection that downstream primer, concentration are 0.3 μ M; Concentration is the trimethyl-glycine of 0.65M; 10 * BstDNA polymerase buffer of 10% (volume); Concentration is the BstDNA polysaccharase of 0.5U/ μ L; Concentration respectively is the acid of triphosphoric acid guanine deoxyribonucleoside (dGTP), triphosphoric acid adenyl-deoxyribonucleotide (dATP), triphosphoric acid thymidylic acid (dTTP), the triphosphoric acid deoxycytidylic acid (dCTP) of 0.8mM, salmonella gene group dna profiling 1 μ L; And in above 7 groups of LAMP reaction systems, MgSO ₄Concentration is respectively: 3.6mM, 4.0mM, 4.4mM, 4.8mM, 5.2mM, 5.6mM, 6.0mM; More than the residual volume of each group reaction system replenish with aseptic ultrapure water.

2.2.2 the LAMP reaction system of streptococcus aureus

Use different Mg ²⁺Concentration is set up the LAMP reaction system of 7 groups of streptococcus aureuses respectively, and is specific as follows:

The volume of each group reaction system is 25 μ L; Comprise respectively: the upstream primer that present embodiment 2.1 parts institute synthetic concentration is the outer primer that the Salmonellas of 0.002 μ M detects and downstream primer, concentration are the upstream primer and the downstream primer of the inner primer of the upstream primer of the outer primer that detects of the upstream primer of the inner primer that detects of the said Salmonellas of 0.4 μ M and streptococcus aureus that downstream primer, concentration are 0.002 μ M and the streptococcus aureus detection that downstream primer, concentration are 0.3 μ M; Concentration is the upstream primer of the outer primer that the Listeria monocytogenes of 0.002 μ M detects and the upstream primer and the downstream primer of the inner primer of the Listeria monocytogenes detection that downstream primer, concentration are 0.3 μ M; Concentration is the trimethyl-glycine of 0.65M; 10 * BstDNA polymerase buffer of 10% (volume); Concentration is the BstDNA polysaccharase of 0.5U/ μ L; Concentration respectively is the acid of triphosphoric acid guanine deoxyribonucleoside (dGTP), triphosphoric acid adenyl-deoxyribonucleotide (dATP), triphosphoric acid thymidylic acid (dTTP), the triphosphoric acid deoxycytidylic acid (dCTP) of 0.8mM, staphylococcus aureus gene group dna profiling 1 μ L; And in above 7 groups of LAMP reaction systems, MgSO ₄Concentration adopt respectively: 3.6mM, 4.0mM, 4.4mM, 4.8mM, 5.2mM, 5.6mM, 6.0mM; More than the residual volume of each group reaction system replenish with aseptic ultrapure water.

2.2.3 the LAMP reaction system of Listeria monocytogenes

Use different Mg respectively ²⁺Concentration is set up 7 groups of streptococcus aureus LAMP reaction systems respectively, and is specific as follows:

The volume of each group reaction system is 25 μ L; Comprise respectively: the upstream primer and the downstream primer of the upstream primer of the outer primer that the upstream primer of the inner primer that the upstream primer that present embodiment 2.1 parts institute synthetic concentration is the outer primer that the said Salmonellas of 0.002 μ M detects and the Salmonellas that downstream primer, concentration are 0.4 μ M detect and the streptococcus aureus that downstream primer, concentration are 0.002 μ M detect and the inner primer of the streptococcus aureus detection that downstream primer, concentration are 0.3 μ M; Concentration is the upstream primer of the outer primer that the Listeria monocytogenes of 0.002 μ M detects and the upstream primer and the downstream primer of the inner primer of the Listeria monocytogenes detection that downstream primer, concentration are 0.3 μ M; Concentration is the trimethyl-glycine of 0.65M; 10 * BstDNA polymerase buffer of 10% (volume); Concentration is the BstDNA polysaccharase of 0.5U/ μ L; Concentration respectively is the acid of triphosphoric acid guanine deoxyribonucleoside (dGTP), triphosphoric acid adenyl-deoxyribonucleotide (dATP), triphosphoric acid thymidylic acid (dTTP), the triphosphoric acid deoxycytidylic acid (dCTP) of 0.8mM, Listeria monocytogenes genomic dna template 1 μ L; And in above 7 groups of LAMP reaction systems, MgSO ₄Concentration be respectively: 3.6mM, 4.0mM, 4.4mM, 4.8mM, 5.2mM, 5.6mM, 6.0mM; More than the residual volume of each reaction system replenish with aseptic ultrapure water.

2.3LAMP reaction conditions

Use thermostatical water bath, reaction conditions is: react 90min down at 61 ℃ earlier; The back is reacted 6min down at 80 ℃.

2.4 the result observes

The reaction solution of respectively organizing the LAMP reaction system of above Salmonellas, streptococcus aureus and Listeria monocytogenes is directly carried out agarose gel electrophoresis 30min respectively; Deposition condition is 0.5 * TBE, 2.0% sepharose, 150V voltage, automatically imaging observations under the gel imaging system.Wherein, during the Salmonellas amplification, work as Mg ²⁺Concentration demonstrates bright scalariform electrophoretic band when 4.0-5.6mM is interval; During the streptococcus aureus amplification, work as Mg ²⁺Concentration demonstrates bright scalariform electrophoretic band when 4.4-5.6mM is interval; During the Listeria monocytogenes amplification, work as Mg ²⁺Concentration demonstrates bright scalariform electrophoretic band when 4.0-5.2mM is interval.According to above electrophoresis observation result, take all factors into consideration the Mg when these three kinds of object bacteria of Salmonellas, streptococcus aureus and Listeria monocytogenes are carried out presenting bright scalariform electrophoretic band in the testing process ²⁺Concentration range is confirmed the best Mg in the LAMP reaction system of the present invention's three re-detection methods ²⁺Concentration is 4.4-5.2mM.

Embodiment 2: the confirming of the best LAMP temperature of reaction of the triple LAMP method for quick of the present invention.

The concrete of present embodiment confirms that step is following:

1. the extraction of sample DNA

1.1 Salmonellas and streptococcus aureus reference culture are inoculated in nutrient broth separately respectively, and Listeria monocytogenes is inoculated in brain heart infusion meat soup, cultivates 18 hours for 36 ℃ ± 1 ℃.

Extract the DNA of bacteria in the above cultured products respectively 1.2 use QIAGEN bacterial genomes DNA extraction test kit.

2.LAMP reaction

2.1 synthetic is used for the primer sets that Salmonellas, streptococcus aureus and Listeria monocytogenes detect respectively, each primer sets primer and sequence thereof are said consistent with 2.1 parts among the embodiment 1.

2.2LAMP reaction system

2.2.1 the LAMP reaction system of Salmonellas

Use different temperature of reaction and set up 6 groups of Salmonellas LAMP reaction systems respectively, specific as follows:

The volume of each group reaction system is 25 μ L; Comprise respectively: the upstream primer and the downstream primer of the upstream primer of the outer primer that the upstream primer of the inner primer that the upstream primer that present embodiment 2.1 parts institute synthetic concentration is the outer primer that the Salmonellas of 0.002 μ M detects and the Salmonellas that downstream primer, concentration are 0.4 μ M detect and the streptococcus aureus that downstream primer, concentration are 0.002 μ M detect and the inner primer of the streptococcus aureus detection that downstream primer, concentration are 0.3 μ M; Concentration is the upstream primer of the outer primer that the Listeria monocytogenes of 0.002 μ M detects and the upstream primer and the downstream primer of the inner primer of the Listeria monocytogenes detection that downstream primer, concentration are 0.3 μ M; Concentration is the trimethyl-glycine of 0.65M; 10 * BstDNA polymerase buffer of 10% (volume); Concentration is the BstDNA polysaccharase of 0.5U/ μ L; Concentration respectively is the acid of triphosphoric acid guanine deoxyribonucleoside (dGTP), triphosphoric acid adenyl-deoxyribonucleotide (dATP), triphosphoric acid thymidylic acid (dTTP), the triphosphoric acid deoxycytidylic acid (dCTP) of 0.8mM, and concentration is the MgSO of 4.8mM ₄, salmonella gene group dna profiling 1 μ L, more than the residual volume of each group reaction system replenish with aseptic ultrapure water.

2.2.2 the LAMP reaction system of streptococcus aureus

Use different temperature of reaction and set up the LAMP reaction system of 6 groups of streptococcus aureuses respectively, specific as follows:

Each group reaction system volume is 25 μ L; Comprise respectively: the upstream primer and the downstream primer of the upstream primer of the outer primer that the upstream primer of the inner primer that the upstream primer that present embodiment 2.1 parts institute synthetic concentration is the outer primer that the Salmonellas of 0.002 μ M detects and the Salmonellas that downstream primer, concentration are 0.4 μ M detect and the streptococcus aureus that downstream primer, concentration are 0.002 μ M detect and the inner primer of the streptococcus aureus detection that downstream primer, concentration are 0.3 μ M; Concentration is the upstream primer of the outer primer that the Listeria monocytogenes of 0.002 μ M detects and the upstream primer and the downstream primer of the inner primer of the Listeria monocytogenes detection that downstream primer, concentration are 0.3 μ M; Concentration is the trimethyl-glycine of 0.65M; 10 * BstDNA polymerase buffer of 10% (volume); Concentration is the BstDNA polysaccharase of 0.5U/ μ L; Concentration respectively is the acid of triphosphoric acid guanine deoxyribonucleoside (dGTP), triphosphoric acid adenyl-deoxyribonucleotide (dATP), triphosphoric acid thymidylic acid (dTTP), the triphosphoric acid deoxycytidylic acid (dCTP) of 0.8mM, and concentration is the MgSO of 4.8mM ₄, staphylococcus aureus gene group dna profiling 1 μ L, more than the residual volume of each group reaction system replenish with aseptic ultrapure water.

2.2.3 the LAMP reaction system of Listeria monocytogenes

Use different temperature of reaction and set up 6 groups of streptococcus aureus LAMP reaction systems respectively, specific as follows:

Each group reaction system volume is 25 μ L; Comprise respectively: the upstream primer and the downstream primer of the upstream primer of the outer primer that the upstream primer of the inner primer that the upstream primer that present embodiment 2.1 parts institute synthetic concentration is the outer primer that the said Salmonellas of 0.002 μ M detects and the Salmonellas that downstream primer, concentration are 0.4 μ M detect and the streptococcus aureus that downstream primer, concentration are 0.002 μ M detect and the inner primer of the streptococcus aureus detection that downstream primer, concentration are 0.3 μ M; Concentration is the upstream primer of the outer primer that the Listeria monocytogenes of 0.002 μ M detects and the upstream primer and the downstream primer of the inner primer of the Listeria monocytogenes detection that downstream primer, concentration are 0.3 μ M; Concentration is the trimethyl-glycine of 0.65M; 10 * BstDNA polymerase buffer of 10% (volume); Concentration is the BstDNA polysaccharase of 0.5U/ μ L; Concentration respectively is the acid of triphosphoric acid guanine deoxyribonucleoside (dGTP), triphosphoric acid adenyl-deoxyribonucleotide (dATP), triphosphoric acid thymidylic acid (dTTP), the triphosphoric acid deoxycytidylic acid (dCTP) of 0.8mM, and concentration is the MgSO of 4.8mM ₄, Listeria monocytogenes genomic dna template 1 μ L; More than the residual volume of each group reaction system replenish with aseptic ultrapure water.

2.3LAMP reaction conditions

Use thermostatical water bath; 6 groups of Salmonellas LAMP reaction systems, 6 groups of streptococcus aureus LAMP reaction systems, 6 groups of Listeria monocytogenes LAMP reaction systems described in 2.2 parts of present embodiment are reacted respectively under 6 kinds of differing tempss earlier separately; The temperature that is adopted is respectively: 58.5 ℃, 59.5 ℃, 61 ℃, 62.5 ℃, 63.5 ℃, 64.5 ℃, the reaction times is 90min; After more respectively at 80 ℃ of following reaction 6min.

2.4 the result observes

The reaction solution of respectively organizing the LAMP reaction system of above Salmonellas, streptococcus aureus and Listeria monocytogenes is directly carried out agarose gel electrophoresis 30min; Deposition condition is: 0.5 * TBE, 2.0% sepharose, 150V voltage, imaging observations under the gel imaging system automatically.Wherein, Salmonellas when amplification, when temperature of reaction at 59.5-63.5 ℃ when interval, present bright scalariform electrophoretic band; Streptococcus aureus when amplification, when temperature of reaction at 59.5-63.5 ℃ when interval, present bright scalariform electrophoretic band; Listeria monocytogenes when amplification, when temperature of reaction at 59.5-62.5 ℃ when interval, present bright scalariform electrophoretic band.According to above electrophoresis observation result; Take all factors into consideration the TR when these three kinds of object bacteria of Salmonellas, streptococcus aureus and Listeria monocytogenes are carried out presenting bright scalariform electrophoretic band in the testing process, confirm that the best LAMP temperature of reaction of the triple detection architecture of the present invention is 59.5-62.5 ℃.

Embodiment 3: the triple LAMP method for quick of the present invention are to the detection sensitivity of Salmonellas.

The concrete detection method of present embodiment is:

1. the extraction of sample DNA

1.1 the Salmonellas reference culture is inoculated in nutrient broth, cultivated 18 hours for 36 ℃ ± 1 ℃.

Extract DNA of bacteria in the above cultured products 1.2 use QIAGEN bacterial genomes DNA extraction test kit.

2.LAMP reaction

2.2LAMP reaction system

Set up 9 reaction tubess; The TV of reaction system is 25 μ L in each reaction tubes; Comprise respectively: the upstream primer and the downstream primer of the upstream primer of the outer primer that the upstream primer of the inner primer that the upstream primer that present embodiment 2.1 parts institute synthetic concentration is the outer primer that the Salmonellas of 0.002 μ M detects and the Salmonellas that downstream primer, concentration are 0.4 μ M detect and the streptococcus aureus that downstream primer, concentration are 0.002 μ M detect and the inner primer of the streptococcus aureus detection that downstream primer, concentration are 0.3 μ M; Concentration is the upstream primer of the outer primer that the Listeria monocytogenes of 0.002 μ M detects and the upstream primer and the downstream primer of the inner primer of the Listeria monocytogenes detection that downstream primer, concentration are 0.3 μ M; Concentration is the trimethyl-glycine of 0.65M; 10 * BstDNA polymerase buffer of 10% (volume); Concentration is the BstDNA polysaccharase of 0.5U/ μ L; Concentration respectively is the acid of triphosphoric acid guanine deoxyribonucleoside (dGTP), triphosphoric acid adenyl-deoxyribonucleotide (dATP), triphosphoric acid thymidylic acid (dTTP), the triphosphoric acid deoxycytidylic acid (dCTP) of 0.8mM, and concentration is the MgSO of 4.8mM ₄Each reaction tubes also need add salmonella gene group dna profiling liquid 1 μ L respectively; The concentration of used dna profiling liquid is respectively 10ng/ μ L, 1ng/ μ L, 100pg/ μ L, 10pg/ μ L, 1pg/ μ L, 100fg/ μ L, 10fg/ μ L, 1fg/ μ L, 0.1fg/ μ L in each reaction tubes, to test the detection sensitivity of this detection method to Salmonellas; More than the residual volume of each reaction system replenish with aseptic ultrapure water.

2.3LAMP reaction conditions

Use thermostatical water bath, elder generation reacts 90min down at 61 ℃, and the back is reacted 6min down at 80 ℃.

2.4 the result observes

Reaction solution in above each reaction tubes is directly carried out agarose gel electrophoresis 30min, and deposition condition is: 0.5 * TBE, 2.0% sepharose, 150V voltage, imaging observations under the gel imaging system automatically.

Through electrophoretic analysis to each reaction tubes reaction product; Find when the concentration of template DNA liquid is 10ng/ μ L, 1ng/ μ L, 100pg/ μ L, 10pg/ μ L, 1pg/ μ L, 100fg/ μ L, 10fg/ μ L; All can amplify comparatively bright scalariform electrophoretic band; And when the concentration of template DNA liquid is reduced to 1fg/ μ L, then can not amplify the scalariform electrophoretic band, explain that the inventive method is 10fg/ μ L to the detection sensitivity of Salmonellas DNA.

Embodiment 4: the triple LAMP method for quick of the present invention are to the detection sensitivity of streptococcus aureus.

The concrete detection method of present embodiment is:

1. the extraction of sample DNA

1.1 the streptococcus aureus reference culture is inoculated in nutrient broth, cultivated 18 hours for 36 ℃ ± 1 ℃.

2.LAMP reaction

2.2LAMP reaction system

Set up 9 reaction tubess; Each reaction tubes reaction system TV is 25 μ L; Comprise respectively: the upstream primer and the downstream primer of the upstream primer of the outer primer that the upstream primer of the inner primer that the upstream primer that present embodiment 2.1 parts institute synthetic concentration is the outer primer that the Salmonellas of 0.002 μ M detects and the Salmonellas that downstream primer, concentration are 0.4 μ M detect and the streptococcus aureus that downstream primer, concentration are 0.002 μ M detect and the inner primer of the streptococcus aureus detection that downstream primer, concentration are 0.3 μ M; The upstream primer that concentration is the outer primer that the Listeria monocytogenes of 0.002 μ M detects and downstream primer, concentration are the upstream primer and the downstream primer of inner primer of the said Listeria monocytogenes detection of 0.3 μ M; Concentration is the trimethyl-glycine of 0.65M; 10 * BstDNA polymerase buffer of 10% (volume); Concentration is the BstDNA polysaccharase of 0.5U/ μ L; Concentration respectively is the acid of triphosphoric acid guanine deoxyribonucleoside (dGTP), triphosphoric acid adenyl-deoxyribonucleotide (dATP), triphosphoric acid thymidylic acid (dTTP), the triphosphoric acid deoxycytidylic acid (dCTP) of 0.8mM, and concentration is the MgSO of 4.8mM ₄Each reaction tubes also need add staphylococcus aureus gene group dna profiling liquid 1 μ L; Used dna profiling liquid concentration is respectively 10ng/ μ L, 1ng/ μ L, 100pg/ μ L, 10pg/ μ L, 1pg/ μ L, 100fg/ μ L, 10fg/ μ L, 1fg/ μ L, 0.1fg/ μ L in each reaction tubes, to test the detection sensitivity of this detection method to streptococcus aureus; More than the residual volume of each reaction system replenish with aseptic ultrapure water.

2.3LAMP reaction conditions

2.4 the result observes

Through electrophoretic analysis to the reaction product in each reaction tubes; Find when the concentration of template DNA liquid is 10ng/ μ L, 1ng/ μ L, 100pg/ μ L, 10pg/ μ L, 1pg/ μ L, 100fg/ μ L, 10fg/ μ L; All can amplify comparatively bright scalariform electrophoretic band; And when the concentration of template DNA liquid is reduced to 1fg/ μ L, then can not amplify the scalariform electrophoretic band.Explain that present method is 10fg/ μ L to the detection sensitivity of streptococcus aureus DNA.

Embodiment 5: the triple LAMP method for quick of the present invention are to the detection sensitivity of Listeria monocytogenes.

The concrete detection method of present embodiment is:

1. the extraction of sample DNA

1.1 the Listeria monocytogenes reference culture is inoculated in brain heart infusion meat soup, cultivated 18 hours for 36 ℃ ± 1 ℃.

Extract the DNA of bacteria in the above cultured products 1.2 use QIAGEN bacterial genomes DNA extraction test kit.

2.LAMP reaction

2.2LAMP reaction system

Set up 9 reaction tubess; The reaction system TV of each reaction tubes is 25 μ L; Comprise respectively: the upstream primer and the downstream primer of the upstream primer of the outer primer that the upstream primer of the inner primer that the upstream primer that present embodiment 2.1 parts institute synthetic concentration is the outer primer that the said Salmonellas of 0.002 μ M detects and the Salmonellas that downstream primer, concentration are 0.4 μ M detect and the streptococcus aureus that downstream primer, concentration are 0.002 μ M detect and the inner primer of the streptococcus aureus detection that downstream primer, concentration are 0.3 μ M; Concentration is the upstream primer of the outer primer that the Listeria monocytogenes of 0.002 μ M detects and the upstream primer and the downstream primer of the inner primer of the Listeria monocytogenes detection that downstream primer, concentration are 0.3 μ M; Concentration is the trimethyl-glycine of 0.65M; 10 * BstDNA polymerase buffer of 10% (volume); Concentration is the BstDNA polysaccharase of 0.5U/ μ L; Concentration respectively is the acid of triphosphoric acid guanine deoxyribonucleoside (dGTP), triphosphoric acid adenyl-deoxyribonucleotide (dATP), triphosphoric acid thymidylic acid (dTTP), the triphosphoric acid deoxycytidylic acid (dCTP) of 0.8mM, and concentration is the MgSO of 4.8mM ₄In addition; Each reaction tubes also need add Listeria monocytogenes genomic dna template liquid 1 μ L; The concentration of used dna profiling liquid is respectively 10ng/ μ L, 1ng/ μ L, 100pg/ μ L, 10pg/ μ L, 1pg/ μ L, 100fg/ μ L, 10fg/ μ L, 1fg/ μ L, 0.1fg/ μ L in each reaction tubes, with the detection sensitivity of test detection method of the present invention to Listeria monocytogenes; More than the residual volume of reaction system of each reaction tubes replenish with aseptic ultrapure water.

2.3LAMP reaction conditions

Use thermostatical water bath, reaction conditions is: elder generation reacts 90min down at 61 ℃, and the back is reacted down at 80 ℃ and is 6min.

2.4 the result observes

The reaction solution of above each reaction tubes is directly carried out agarose gel electrophoresis 30min, and deposition condition is: 0.5 * TBE, 2.0% sepharose, 150V voltage, imaging observations under the gel imaging system automatically.

Through electrophoretic analysis to the reaction product of each reaction tubes; Find when the concentration of template DNA liquid is 10ng/ μ L, 1ng/ μ L, 100pg/ μ L, 10pg/ μ L, 1pg/ μ L, 100fg/ μ L, 10fg/ μ L; All can amplify comparatively bright scalariform electrophoretic band; And when the concentration of template DNA liquid is reduced to 1fg/ μ L, then can not amplify the scalariform electrophoretic band, explain that the inventive method is 10fg/ μ L to the detection sensitivity of Listeria monocytogenes DNA.

Embodiment 6: specificity, the analysis of the accuracy of the triple LAMP method for quick of the present invention

The concrete detection method of present embodiment is:

1. the extraction of sample DNA

1.1 Salmonellas and streptococcus aureus reference culture are inoculated in nutrient broth respectively, and Listeria monocytogenes is inoculated in brain heart infusion meat soup, cultivates 18 hours for 36 ℃ ± 1 ℃.

Extract DNA of bacteria in the above cultured products respectively 1.2 use QIAGEN bacterial genomes DNA extraction test kit.

1.3 three groups of DNA extraction liquid are pressed the equal-volume mixed, detect template as final LAMP.

2.LAMP reaction

2.1 synthetic is used for the primer sets that Salmonellas, streptococcus aureus and Listeria monocytogenes detect respectively, each primer sets primer and sequence thereof are said consistent with 2.1 parts among the embodiment 1.Be used for the primer that triple LAMP products to Salmonellas, streptococcus aureus and Listeria monocytogenes carry out the PCR order-checking and see table 1 for details.

The triple LAMP product of table 1. Salmonellas, streptococcus aureus and Listeria monocytogenes sequencing primer

2.2LAMP reaction system

The reaction system TV is 100 μ L; Comprise: the upstream primer and the downstream primer of the upstream primer of the outer primer that the upstream primer of the inner primer that the upstream primer that present embodiment 2.1 parts institute synthetic concentration is the outer primer that the Salmonellas of 0.002 μ M detects and the Salmonellas that downstream primer, concentration are 0.4 μ M detect and the streptococcus aureus that downstream primer, concentration are 0.002 μ M detect and the inner primer of the streptococcus aureus detection that downstream primer, concentration are 0.3 μ M; Concentration is the upstream primer of the outer primer that the Listeria monocytogenes of 0.002 μ M detects and the upstream primer and the downstream primer of the inner primer of the Listeria monocytogenes detection that downstream primer, concentration are 0.3 μ M; Concentration is the trimethyl-glycine of 0.65M; 10 * BstDNA polymerase buffer of 10% (volume); Concentration is the BstDNA polysaccharase of 0.5U/ μ L; Concentration respectively is the acid of triphosphoric acid guanine deoxyribonucleoside (dGTP), triphosphoric acid adenyl-deoxyribonucleotide (dATP), triphosphoric acid thymidylic acid (dTTP), the triphosphoric acid deoxycytidylic acid (dCTP) of 0.8mM, and concentration is the MgSO of 4.8mM ₄, hybrid dna template 12 μ L, the residual volume of above reaction system replenishes with aseptic ultrapure water.

2.3LAMP reaction conditions

3. product reclaims sequence verification

3.1 above reaction solution is directly carried out agarose gel electrophoresis 40min; Deposition condition is: 0.5 * TBE, 2.0% sepharose, 150V voltage; Molecular weight is reclaimed in the product rubber tapping in 100bp-1000bp zone; Use QIAGEN sepharose purification kit and carry out purifying, as the pcr amplification template.

3.2 respectively the product that is reclaimed is carried out sequencing analysis; Wherein measured ordered sequence and the Salmonellas invA gene order of sequencing primer invA-F of the Salmonellas shown in the application table 1 (having sequence shown in SEQ No.13) and invA-R (having sequence shown in SEQ No.14) coincide; Ordered sequence that streptococcus aureus sequencing primer nuc-F (having sequence shown in SEQ No.15) shown in the application table 1 and nuc-R (having sequence shown in SEQ No.16) are measured and streptococcus aureus nuc gene order are coincide, and ordered sequence that Listeria monocytogenes sequencing primer hly-F (having sequence shown in SEQ No.17) shown in the application table 1 and hly-R (having sequence shown in SEQ No.18) are measured and Listeria monocytogenes hly gene order are coincide.

4. conclusion

Through the sequencing analysis of above LAMP product to present embodiment, can verify that detection method of the present invention is strong to Salmonellas, streptococcus aureus and Listeria monocytogenes detection specificity, the product sequence that is increased is coincide with expection.Embodiment 7: to triple LAMP rapid detection of Salmonellas, streptococcus aureus, Listeria monocytogenes.

Present embodiment adopts milk powder as test sample, and concrete detection method is:

1. the extraction of sample DNA

Increase in the bacterial context soup 1.1 take by weighing 25g milk powder sample to 225mL buffered peptone water nutrient solution, 225mL 7.5% sodium-chlor meat soup and 225mL listeria bacteria respectively, cultivated 18 hours for 36 ℃ ± 1 ℃.

Extract the DNA of bacteria in above three kinds of cultured products respectively 1.2 use QIAGEN bacterial genomes DNA extraction test kit, obtain three groups of DNA extraction liquid.

1.3 above-mentioned three groups of DNA extraction liquid are pressed the equal-volume mixed, as final detection template.

2.LAMP reaction

2.2LAMP reaction system

Set up 2 parallel reactor pipes; The reaction system TV of each parallel reactor pipe is 25 μ L; Comprise respectively: present embodiment 2.1 parts institute synthetic concentration is the upstream primer and the downstream primer of the inner primer of the upstream primer of the outer primer that detects of the upstream primer of the inner primer that detects of upstream primer and the Salmonellas that downstream primer, concentration are 0.35 μ M of the outer primer that detects of the Salmonellas of 0.001 μ M and streptococcus aureus that downstream primer, concentration are 0.001 μ M and the streptococcus aureus detection that downstream primer, concentration are 0.25 μ M; Concentration is the upstream primer of the outer primer that the Listeria monocytogenes of 0.001 μ M detects and the upstream primer and the downstream primer of the inner primer of the Listeria monocytogenes detection that downstream primer, concentration are 0.25 μ M; Concentration is the trimethyl-glycine of 0.6M; 10 * BstDNA polymerase buffer of 10% (volume); Concentration is the BstDNA polysaccharase of 0.4U/ μ L; Concentration respectively is the acid of triphosphoric acid guanine deoxyribonucleoside (dGTP), triphosphoric acid adenyl-deoxyribonucleotide (dATP), triphosphoric acid thymidylic acid (dTTP), the triphosphoric acid deoxycytidylic acid (dCTP) of 0.6mM, and concentration is the MgSO of 4.4mM ₄, the residual volume of the reaction system in the hybrid dna template 3 μ L, above parallel reactor pipe replenishes with aseptic ultrapure water.

2.3LAMP reaction conditions

Use thermostatical water bath, elder generation reacts 110min down at 59.5 ℃, and the back is reacted 6min down at 80 ℃.

2.4 the result observes

With the electrophoresis 30min under 0.5 * TBE, 2.0% sepharose, 150V voltage conditions of the reaction solution in above two parallel reactor pipes; In gel imaging system, observe then; Find all to appear bright stepped purpose band, explain exist in the sample of present embodiment in Salmonellas, streptococcus aureus and the Listeria monocytogenes wherein one or more.

2.5 result verification

Adopt GB 4789-2010 " food microbiological analysis of food safety national standard " that the sample of present embodiment is detected; Detect Salmonellas, streptococcus aureus and Listeria monocytogenes, consistent with the described LAMP detected result of present embodiment 2.4 parts.

Embodiment 8: the triple LAMP rapid detection of Salmonellas, streptococcus aureus and Listeria monocytogenes

1. the extraction of sample DNA

11 take by weighing 25g milk powder sample to 225mL buffered peptone water nutrient solution, 225mL 7.5% sodium-chlor meat soup and 225mL listeria bacteria respectively increases in the bacterial context soup, cultivates 18 hours for 36 ℃ ± 1 ℃.

1.3 three groups of DNA extraction liquid that will extract are pressed the equal-volume mixed, as final detection template.

2.LAMP reaction

2.2LAMP reaction system

Set up 2 parallel reactor pipes; The reaction system TV of each reaction tubes is 25 μ L; Comprise respectively: present embodiment 2.1 parts institute synthetic concentration is the upstream primer and the downstream primer of the inner primer of the upstream primer of the outer primer that detects of the upstream primer of the inner primer that detects of upstream primer and the Salmonellas that downstream primer, concentration are 0.4 μ M of the outer primer that detects of the Salmonellas of 0.002 μ M and streptococcus aureus that downstream primer, concentration are 0.002 μ M and the streptococcus aureus detection that downstream primer, concentration are 0.3 μ M; Concentration is the upstream primer of the outer primer that the Listeria monocytogenes of 0.002 μ M detects and the upstream primer and the downstream primer of the inner primer of the Listeria monocytogenes detection that downstream primer, concentration are 0.3 μ M; Concentration is the trimethyl-glycine of 0.65M; 10 * BstDNA polymerase buffer of 10% (volume); Concentration is the BstDNA polysaccharase of 0.5U/ μ L; Concentration respectively is the acid of triphosphoric acid guanine deoxyribonucleoside (dGTP), triphosphoric acid adenyl-deoxyribonucleotide (dATP), triphosphoric acid thymidylic acid (dTTP), the triphosphoric acid deoxycytidylic acid (dCTP) of 0.8mM, and concentration is the MgSO of 4.8mM ₄, hybrid dna template 3 μ L, the residual volume of above reaction system replenishes with aseptic ultrapure water.

2.3LAMP reaction conditions

Use thermostatical water bath, reaction conditions is: elder generation reacts 90min down at 61 ℃, and the back is reacted 9min down at 80 ℃.

2.4 the result observes

In the reaction solution of above each reaction tubes, add fluorescent color-developing agent (dyestuff SYBR Green I) back observations under uv lamp respectively; Two parallel reactor Guan Jun present obvious fluorescence, explain contain in institute's sample article in Salmonellas, streptococcus aureus and the Listeria monocytogenes wherein one or more.

2.5 result verification

Adopt GB 4789-2010 " food microbiological analysis of food safety national standard " that the present embodiment sample is detected, detect Salmonellas, streptococcus aureus and Listeria monocytogenes, consistent with the detected result of present embodiment 2.4 parts.

Embodiment 9: the triple LAMP rapid detection of Salmonellas, streptococcus aureus and Listeria monocytogenes

1. the extraction of sample DNA

Extract DNA of bacteria in above three kinds of cultured products respectively 1.2 use QIAGEN bacterial genomes DNA extraction test kit, obtain three groups of DNA extraction liquid.

1.3 with three groups of DNA extraction liquid that extracted by the equal-volume mixed, as final detection template.

2.LAMP reaction

2.2LAMP reaction system

Set up 2 parallel reactor pipes; The reaction system TV of each reaction tubes is 25 μ L; Comprise respectively: present embodiment 2.1 parts institute synthetic concentration is the upstream primer and the downstream primer of the inner primer of the upstream primer of the outer primer that detects of the upstream primer of the inner primer that detects of upstream primer and the Salmonellas that downstream primer, concentration are 0.45 μ M of the outer primer that detects of the Salmonellas of 0.003 μ M and streptococcus aureus that downstream primer, concentration are 0.003 μ M and the streptococcus aureus detection that downstream primer, concentration are 0.35 μ M; Concentration is the upstream primer of the outer primer that the Listeria monocytogenes of 0.003 μ M detects and the upstream primer and the downstream primer of the inner primer of the Listeria monocytogenes detection that downstream primer, concentration are 0.35 μ M; Concentration is the trimethyl-glycine of 0.7M; 10 * BstDNA polymerase buffer of 10% (volume); Concentration is the BstDNA polysaccharase of 0.6U/ μ L; Concentration respectively is the acid of triphosphoric acid guanine deoxyribonucleoside (dGTP), triphosphoric acid adenyl-deoxyribonucleotide (dATP), triphosphoric acid thymidylic acid (dTTP), the triphosphoric acid deoxycytidylic acid (dCTP) of 1.0mM, and concentration is the MgSO of 5.2mM ₄, hybrid dna template 3 μ L, more than the residual volume of reaction system in each reaction tubes replenish with aseptic ultrapure water.

2.3LAMP reaction conditions

Use thermostatical water bath, reaction conditions is: elder generation reacts 70min down at 62.5 ℃, and the back is reacted 12min down at 80 ℃.

2.4 the result observes

With above each reaction solution observations behind centrifugal 5min under the 6000rpm condition.Two parallel reactor pipes bottom presents obvious sediment, explain contain in institute's sample article in Salmonellas, streptococcus aureus and the Listeria monocytogenes wherein one or more.

2.5 result verification

Adopt GB 4789-2010 " food microbiological analysis of food safety national standard " that the present embodiment sample is detected, detect Salmonellas, streptococcus aureus and Listeria monocytogenes, consistent with the detected result of present embodiment 2.4 parts.Embodiment 10: Salmonellas, streptococcus aureus, the triple LAMP rapid detection of Listeria monocytogenes

1. the extraction of sample DNA

2.LAMP reaction

2.2, the LAMP reaction system

Set up 2 parallel reactor pipes; The reaction system TV of each reaction tubes is 25 μ L; Comprise respectively: present embodiment 2.1 parts institute synthetic concentration is the upstream primer and the downstream primer of the inner primer of the upstream primer of the outer primer that detects of the upstream primer of the inner primer that detects of upstream primer and the Salmonellas that downstream primer, concentration are 0.35 μ M of the outer primer that detects of the Salmonellas of 0.001 μ M and streptococcus aureus that downstream primer, concentration are 0.001 μ M and the streptococcus aureus detection that downstream primer, concentration are 0.25 μ M; Concentration is the upstream primer of the outer primer that the Listeria monocytogenes of 0.001 μ M detects and the upstream primer and the downstream primer of the inner primer of the Listeria monocytogenes detection that downstream primer, concentration are 0.25 μ M; Concentration is the trimethyl-glycine of 0.6M; 10 * BstDNA polymerase buffer of 10% (volume); Concentration is the BstDNA polysaccharase of 0.4U/ μ L; Concentration respectively is the acid of triphosphoric acid guanine deoxyribonucleoside (dGTP), triphosphoric acid adenyl-deoxyribonucleotide (dATP), triphosphoric acid thymidylic acid (dTTP), the triphosphoric acid deoxycytidylic acid (dCTP) of 0.6mM, and concentration is the MgSO of 4.4mM ₄, hybrid dna template 3 μ L, more than the residual volume of reaction system in each reaction tubes replenish with aseptic ultrapure water.

2.3 LAMP reaction conditions

Use thermostatical water bath, react 110min down at 59.5 ℃.

2.4 the result observes

With reaction solution centrifugal 5min under the 6000rpm condition, sample hose bottom presents deposition, explain exist in the present embodiment sample in Salmonellas, streptococcus aureus and the Listeria monocytogenes wherein one or more.

2.5 result verification

Adopt GB 4789-2010 " food microbiological analysis of food safety national standard " that the present embodiment sample is detected, detect Salmonellas, streptococcus aureus and Listeria monocytogenes, consistent with the LAMP detected result of present embodiment 2.4 parts.

Embodiment 11: Salmonellas, streptococcus aureus, the triple LAMP rapid detection of Listeria monocytogenes

1. the extraction of sample DNA

2.LAMP reaction

2.2LAMP reaction system

Set up 2 parallel reactor pipes; The reaction system TV of each reaction tubes is 25 μ L; Comprise respectively: present embodiment 2.1 parts institute synthetic concentration is the upstream primer and the downstream primer of the inner primer of the upstream primer of the outer primer that detects of the upstream primer of the inner primer that detects of upstream primer and the Salmonellas that downstream primer, concentration are 0.4 μ M of the outer primer that detects of the Salmonellas of 0.002 μ M and streptococcus aureus that downstream primer, concentration are 0.002 μ M and the streptococcus aureus detection that downstream primer, concentration are 0.3 μ M; Concentration is the upstream primer of the outer primer that the Listeria monocytogenes of 0.002 μ M detects and the upstream primer and the downstream primer of the inner primer of the Listeria monocytogenes detection that downstream primer, concentration are 0.3 μ M; Concentration is the trimethyl-glycine of 0.65M; 10 * BstDNA polymerase buffer of 10% (volume); Concentration is the BstDNA polysaccharase of 0.5U/ μ L; Concentration respectively is the acid of triphosphoric acid guanine deoxyribonucleoside (dGTP), triphosphoric acid adenyl-deoxyribonucleotide (dATP), triphosphoric acid thymidylic acid (dTTP), the triphosphoric acid deoxycytidylic acid (dCTP) of 0.8mM, and concentration is the MgS0 of 4.8mM ₄, hybrid dna template 3 μ L, more than the residual volume of reaction system in each reaction tubes replenish with aseptic ultrapure water.

2.3 LAMP reaction conditions

Use thermostatical water bath, react 90min down at 61 ℃.

2.4 the result observes

With the centrifugal 5min under the 6000rpm condition respectively of the reaction solution in each reaction tubes; The bottom of each reaction tubes all presents deposition, explain exist in the reaction solution in each reaction tubes of present embodiment in Salmonellas, streptococcus aureus and the Listeria monocytogenes wherein one or more.

2.5 result verification

Adopt GB 4789-2010 " food microbiological analysis of food safety national standard " that the present embodiment sample is detected, detect Salmonellas, streptococcus aureus and Listeria monocytogenes, consistent with the LAMP detected result.

Embodiment 12: triple LAMP rapid detection of Salmonellas, streptococcus aureus, Listeria monocytogenes

1. the extraction of sample DNA

2.LAMP reaction

2.1 synthetic is used for the primer sets that Salmonellas, streptococcus aureus and Listeria monocytogenes detect respectively, 2.1 parts are said consistent among each primer sets primer and sequence thereof and the embodiment 1.

2.2LAMP reaction system

Set up 2 parallel reactor pipes; The reaction system TV of each reaction tubes is 25 μ L; Comprise respectively: present embodiment 2.1 parts institute synthetic concentration is the upstream primer and the downstream primer of the inner primer of the upstream primer of the outer primer that detects of the upstream primer of the inner primer that detects of upstream primer and the Salmonellas that downstream primer, concentration are 0.45 μ M of the outer primer that detects of the Salmonellas of 0.003 μ M and streptococcus aureus that downstream primer, concentration are 0.003 μ M and the streptococcus aureus detection that downstream primer, concentration are 0.35 μ M; Concentration is the upstream primer of the outer primer that the Listeria monocytogenes of 0.003 μ M detects and the upstream primer and the downstream primer of the inner primer of the Listeria monocytogenes detection that downstream primer, concentration are 0.35 μ M; Concentration is the trimethyl-glycine of 0.7M; 10 * BstDNA polymerase buffer of 10% (volume); Concentration is the BstDNA polysaccharase of 0.6U/ μ L; Concentration respectively is the acid of triphosphoric acid guanine deoxyribonucleoside (dGTP), triphosphoric acid adenyl-deoxyribonucleotide (dATP), triphosphoric acid thymidylic acid (dTTP), the triphosphoric acid deoxycytidylic acid (dCTP) of 1.0mM, and concentration is the MgSO of 5.2mM ₄, hybrid dna template 3 μ L, more than the residual volume of reaction system in each parallel reactor pipe replenish with aseptic ultrapure water.

2.3LAMP reaction conditions

Use thermostatical water bath, react 70min down at 62.5 ℃.

2.4 the result observes

2.5 result verification

Embodiment 13: Salmonellas, streptococcus aureus, the triple LAMP quick detection kit of Listeria monocytogenes

This test kit is made up of following reagent:

1. the detection primer of Salmonellas, streptococcus aureus, Listeria monocytogenes LAMP comprises:

(1) is used for the upstream primer of the outer primer that Salmonellas LAMP detects, has the nucleotide sequence shown in SEQ No.1; Be used for the downstream primer of the outer primer of Salmonellas LAMP detection, have the nucleotide sequence shown in SEQ No.2, be used for the upstream primer of the inner primer of Salmonellas LAMP detection, have the nucleotide sequence shown in SEQ No.3; Be used for the downstream primer of the inner primer of Salmonellas LAMP detection, have the nucleotide sequence shown in SEQ No.4.

(2) be used for the upstream primer of the outer primer that streptococcus aureus LAMP detects, have the nucleotide sequence shown in SEQ No.5; Be used for the downstream primer of the outer primer of streptococcus aureus LAMP detection, have the nucleotide sequence shown in SEQ No.6; Be used for the upstream primer of the inner primer of streptococcus aureus LAMP detection, have the nucleotide sequence shown in SEQ No.7; Be used for the downstream primer of the inner primer of streptococcus aureus LAMP detection, have the nucleotide sequence shown in SEQ No.8.

(3) be used for the upstream primer of the outer primer that Listeria monocytogenes LAMP detects, have the nucleotide sequence shown in SEQ No.9; Be used for the downstream primer of the outer primer of Listeria monocytogenes LAMP detection, have the nucleotide sequence shown in SEQ No.10; Be used for the upstream primer of the inner primer of Listeria monocytogenes LAMP detection, have the nucleotide sequence shown in SEQ No.11; Be used for the downstream primer of the inner primer of Listeria monocytogenes LAMP detection, have the nucleotide sequence shown in SEQ No.12.

2.BstDNA polysaccharase;

3. positive reference substance: the genome of the reference culture of Salmonellas; The genomic dna of the reference culture of streptococcus aureus; The genomic dna of the reference culture of Listeria monocytogenes.

4. blank article: aseptic ultrapure water;

5.10 * BstDNA polymerase buffer;

6.MgSO ₄Solution;

7. alkali solution of beet;

8. the mol ratio of the mixing solutions of triphosphoric acid guanine deoxyribonucleoside acid, triphosphoric acid adenyl-deoxyribonucleotide, triphosphoric acid thymidylic acid and triphosphoric acid deoxycytidylic acid and the acid of triphosphoric acid guanine deoxyribonucleoside, triphosphoric acid adenyl-deoxyribonucleotide, triphosphoric acid thymidylic acid and triphosphoric acid deoxycytidylic acid is 1: 1: 1: 1

9. developer: SYBR Green I.

Embodiment 14: triple LAMP quick detection kit of Salmonellas, streptococcus aureus, Listeria monocytogenes

This test kit is made up of following reagent:

1. Salmonellas, streptococcus aureus Listeria monocytogenes LAMP detect primer, and primer sets that comprises and sequence thereof are said consistent with part 1 among the embodiment 13.

2.BstDNA polysaccharase;

4. blank article: aseptic ultrapure water;

5.10 * BstDNA polymerase buffer;

6.MgSO ₄Solution;

7. alkali solution of beet.

8. the mol ratio of the mixing solutions of triphosphoric acid guanine deoxyribonucleoside acid, triphosphoric acid adenyl-deoxyribonucleotide, triphosphoric acid thymidylic acid and triphosphoric acid deoxycytidylic acid and the acid of triphosphoric acid guanine deoxyribonucleoside, triphosphoric acid adenyl-deoxyribonucleotide, triphosphoric acid thymidylic acid and triphosphoric acid deoxycytidylic acid is 1: 1: 1: 1.Embodiment 15: Salmonellas, streptococcus aureus, the triple LAMP quick detection kit of Listeria monocytogenes

The present embodiment test kit is made up of following reagent:

1. the triple LAMP of Salmonellas, streptococcus aureus and Listeria monocytogenes detect 2 * premixed liquid, and concrete composition and concentration that this LAMP detects 2 * premixed liquid are:

Concentration is the upstream primer and the downstream primer of the inner primer of the upstream primer of the outer primer that detects of the upstream primer of the inner primer that detects of upstream primer and the Salmonellas that downstream primer, concentration are 0.7 μ M of the outer primer that detects of the Salmonellas of 0.002 μ M and streptococcus aureus that downstream primer, concentration are 0.002 μ M and the streptococcus aureus detection that downstream primer, concentration are 0.5 μ M; Concentration is the upstream primer of the outer primer that the Listeria monocytogenes of 0.002 μ M detects and the upstream primer and the downstream primer of the inner primer of the Listeria monocytogenes detection that downstream primer, concentration are 0.5 μ M; Concentration is the trimethyl-glycine of 1.2M; 2 * BstDNA polymerase buffer; Concentration is the BstDNA polysaccharase of 0.8U/ μ L; Concentration respectively is the acid of triphosphoric acid guanine deoxyribonucleoside (dGTP), triphosphoric acid adenyl-deoxyribonucleotide (dATP), triphosphoric acid thymidylic acid (dTTP), the triphosphoric acid deoxycytidylic acid (dCTP) of 1.2mM, and concentration is the MgSO of 8.8mM ₄Wherein said each primer sets and sequence thereof are said consistent with part 1 among the embodiment 13.

2. positive reference substance: salmonella gene group DNA; Staphylococcus aureus gene group DNA; Listeria monocytogenes bacterium genomic dna.

3. blank article: aseptic ultrapure water;

4. developer: SYBR Green I.

Embodiment 16: Salmonellas, streptococcus aureus, the triple LAMP quick detection kit of Listeria monocytogenes

The present embodiment test kit is made up of following reagent:

1. the triple LAMP of Salmonellas, streptococcus aureus and Listeria monocytogenes detect 2 * premixed liquid, and it is specifically formed and concentration is:

Concentration is the upstream primer and the downstream primer of the inner primer of the upstream primer of the outer primer that detects of the upstream primer of the inner primer that detects of upstream primer and the Salmonellas that downstream primer, concentration are 0.8 μ M of the outer primer that detects of the Salmonellas of 0.004 μ M and streptococcus aureus that downstream primer, concentration are 0.004 μ M and the streptococcus aureus detection that downstream primer, concentration are 0.6 μ M; Concentration is the upstream primer of the outer primer that the Listeria monocytogenes of 0.004 μ M detects and the upstream primer and the downstream primer of the inner primer of the Listeria monocytogenes detection that downstream primer, concentration are 0.6 μ M; Concentration is the trimethyl-glycine of 1.3M; 2 * BstDNA polymerase buffer; Concentration is the BstDNA polysaccharase of 1.0U/ μ L; Concentration respectively is the acid of triphosphoric acid guanine deoxyribonucleoside (dGTP), triphosphoric acid adenyl-deoxyribonucleotide (dATP), triphosphoric acid thymidylic acid (dTTP), the triphosphoric acid deoxycytidylic acid (dCTP) of 1.6mM, and concentration is the MgSO of 9.6mM ₄Wherein above-mentioned each primer sets and sequence thereof are said consistent with part 1 among the embodiment 13.

3. blank article: aseptic ultrapure water;

Embodiment 17: Salmonellas, streptococcus aureus, the triple LAMP quick detection kit of Listeria monocytogenes.

The present embodiment test kit is made up of following reagent:

Concentration is the upstream primer and the downstream primer of the inner primer of the upstream primer of the outer primer that detects of the upstream primer of the inner primer that detects of upstream primer and the Salmonellas that downstream primer, concentration are 0.9 μ M of the outer primer that detects of the Salmonellas of 0.006 μ M and streptococcus aureus that downstream primer, concentration are 0.006 μ M and the streptococcus aureus detection that downstream primer, concentration are 0.7 μ M; Concentration is the upstream primer of the outer primer that the Listeria monocytogenes of 0.006 μ M detects and the upstream primer and the downstream primer of the inner primer of the Listeria monocytogenes detection that downstream primer, concentration are 0.7 μ M; Concentration is the trimethyl-glycine of 1.4M; 2 * BstDNA polymerase buffer; Concentration is the BstDNA polysaccharase of 1.2U/ μ L; Concentration respectively is the acid of triphosphoric acid guanine deoxyribonucleoside (dGTP), triphosphoric acid adenyl-deoxyribonucleotide (dATP), triphosphoric acid thymidylic acid (dTTP), the triphosphoric acid deoxycytidylic acid (dCTP) of 2.0mM, and concentration is the MgSO of 10.4mM ₄Wherein said each primer sets and sequence thereof are said consistent with part 1 among the embodiment 13.

3. blank article: aseptic ultrapure water.

< 110>Zhejiang Province Quality Technology Supervision Detection Research Institute

< 120>three kinds of multiple fast detection methods of food-borne pathogens and detect primer sets and test kit

<160> 18

<170> PatentIn?version?3.1

<210> 1

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gcgcggcatc?cgcatcaata?tctggatggt?atgcccgg 38

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Claims

1. the rapid detection primer sets of a Salmonellas, it is characterized in that: the upstream primer of its outer primer has the sequence shown in SEQ No.1, and the downstream primer of its outer primer has the sequence shown in SEQ No.2; The upstream primer of its inner primer has the sequence shown in SEQ No.3, and the downstream primer of its inner primer has the sequence shown in SEQ No.4.

2. the rapid detection primer sets of a streptococcus aureus, it is characterized in that: the upstream primer of its outer primer has the sequence shown in SEQ No.5, and the downstream primer of its outer primer has the sequence shown in SEQ No.6; The upstream primer of its inner primer has the sequence shown in SEQ No.7, and the downstream primer of its inner primer has the sequence shown in SEQ No.8.

3. the rapid detection primer sets of a Listeria monocytogenes, it is characterized in that: the upstream primer of its outer primer has the sequence shown in SEQ No.9, and the downstream primer of its outer primer has the sequence shown in SEQ No.10; The upstream primer of its inner primer has the sequence shown in SEQ No.11, and the downstream primer of its inner primer has the sequence shown in SEQ No.12.

4. one kind is used primer sets is carried out multiple rapid detection to Salmonellas, streptococcus aureus and Listeria monocytogenes the method that detects; It is characterized in that: the detection primer sets of Salmonellas, the detection primer sets of streptococcus aureus and the detection primer sets of Listeria monocytogenes and the DNA of testing sample are carried out the LAMP reaction; Finish the back in reaction and reaction solution is carried out yin and yang attribute judge, with confirm whether to contain in the said sample in Salmonellas, streptococcus aureus, the Listeria monocytogenes wherein one or more; In the detection primer sets of said Salmonellas; The upstream primer of outer primer has the sequence shown in SEQ No.1; The downstream primer of outer primer has the sequence shown in SEQ No.2; The upstream primer of inner primer has the sequence shown in SEQ No.3, and the downstream primer of inner primer has the sequence shown in SEQ No.4; In the detection primer sets of said streptococcus aureus; The upstream primer of outer primer has the sequence shown in SEQ No.5; The downstream primer of outer primer has the sequence shown in SEQ No.6; The upstream primer of inner primer has the sequence shown in SEQ No.7, and the downstream primer of inner primer has the sequence shown in SEQ No.8; In the detection primer sets of said Listeria monocytogenes; The upstream primer of outer primer has the sequence shown in SEQ No.9; The downstream primer of outer primer has the sequence shown in SEQ No.10; The upstream primer of inner primer has the sequence shown in SEQ No.11, and the downstream primer of inner primer has the sequence shown in SEQ No.12.

5. method according to claim 4 is characterized in that: said LAMP reaction is undertaken by following first kind of scheme or second kind of scheme:

Second kind of scheme: said LAMP reaction comprises following steps:

(1) reacts 70-110min down at 59.5-62.5 ℃ earlier;

(2) back is reacted 6-12min down at 80 ℃.

6. according to claim 4 or 5 described methods; It is characterized in that: in the reaction solution that carries out said LAMP reaction; The upstream primer of the outer primer in the detection primer sets of said Salmonellas and the concentration of downstream primer are 0.001-0.003 μ M, and the upstream primer of inner primer and the concentration of downstream primer are 0.35-0.45 μ M; The upstream primer of the outer primer in the detection primer sets of said streptococcus aureus and the concentration of downstream primer are 0.001-0.003 μ M, and the upstream primer of inner primer and the concentration of downstream primer are 0.25-0.35 μ M; The upstream primer of the outer primer in the detection primer sets of said Listeria monocytogenes and the concentration of downstream primer are 0.001-0.003 μ M, and the upstream primer of inner primer and the concentration of downstream primer are 0.25-0.35 μ M; And carrying out also including trimethyl-glycine, the concentration that concentration is 0.6-0.7 M in the reaction solution of said LAMP reaction is the MgSO of 4.4-5.2mM ₄, 10 % (volume) 10 * BstDna polymerase buffer liquid, concentration are 0.4-0.6U/ μ L's BstArchaeal dna polymerase and concentration respectively are the acid of triphosphoric acid guanine deoxyribonucleoside, triphosphoric acid adenyl-deoxyribonucleotide, triphosphoric acid thymidylic acid and the triphosphoric acid deoxycytidylic acid of 0.6-1.0mM.

7. quick detection kit is characterized in that: comprise the detection primer sets of detection primer sets, the Listeria monocytogenes of detection primer sets, the streptococcus aureus of Salmonellas, three kinds of positive reference substances, BstArchaeal dna polymerase, 10 * BstDna polymerase buffer liquid, MgSO ₄Solution, alkali solution of beet, the acid of triphosphoric acid guanine deoxyribonucleoside, triphosphoric acid adenyl-deoxyribonucleotide, triphosphoric acid thymidylic acid, triphosphoric acid deoxycytidylic acid and aseptic ultrapure water; The upstream primer of the outer primer in the detection primer sets of said Salmonellas has the sequence shown in SEQ No.1; The downstream primer of outer primer has the sequence shown in SEQ No.2; The upstream primer of inner primer has the sequence shown in SEQ No.3, and the downstream primer of inner primer has the sequence shown in SEQ No.4; The upstream primer of the outer primer in the detection primer sets of said streptococcus aureus has the sequence shown in SEQ No.5; The downstream primer of outer primer has the sequence shown in SEQ No.6; The upstream primer of inner primer has the sequence shown in SEQ No.7, and the downstream primer of inner primer has the sequence shown in SEQ No.8; The upstream primer of the outer primer in the detection primer sets of said Listeria monocytogenes has the sequence shown in SEQ No.9; The downstream primer of outer primer has the sequence shown in SEQ No.10; The upstream primer of inner primer has the sequence shown in SEQ No.11, and the downstream primer of inner primer has the sequence shown in SEQ No.12; Said three kinds of positive reference substances are respectively the genomic dna of the reference culture of Salmonellas, streptococcus aureus and Listeria monocytogenes.

8. quick detection kit; It is characterized in that: comprise 2 * LAMP premixed liquid and three kinds of positive reference substances; Said three kinds of positive reference substances are respectively the genomic dna of the reference culture of Salmonellas, streptococcus aureus and Listeria monocytogenes, and in said 2 * LAMP premixed liquid, including the detection primer sets of Salmonellas, the detection primer sets of streptococcus aureus, the detection primer sets of Listeria monocytogenes, trimethyl-glycine, the concentration that concentration is 1.2-1.4 M is 0.8-1.2U/ μ L's BstArchaeal dna polymerase, 2 * BstDna polymerase buffer liquid, concentration are the MgSO of 8.8-10.4mM ₄Solution, aseptic ultrapure water and concentration respectively are the acid of triphosphoric acid guanine deoxyribonucleoside, triphosphoric acid adenyl-deoxyribonucleotide, triphosphoric acid thymidylic acid and the triphosphoric acid deoxycytidylic acid of 1.2-2.0mM; The upstream primer of the outer primer in the detection primer sets of said Salmonellas has the sequence shown in SEQ No.1; The downstream primer of outer primer has the sequence shown in SEQ No.2; The upstream primer of inner primer has the sequence shown in SEQ No.3, and the downstream primer of inner primer has the sequence shown in SEQ No.4; The upstream primer of the outer primer in the detection primer sets of said streptococcus aureus has the sequence shown in SEQ No.5; The downstream primer of outer primer has the sequence shown in SEQ No.6; The upstream primer of inner primer has the sequence shown in SEQ No.7, and the downstream primer of inner primer has the sequence shown in SEQ No.8; The upstream primer of the outer primer in the detection primer sets of said Listeria monocytogenes has the sequence shown in SEQ No.9; The downstream primer of outer primer has the sequence shown in SEQ No.10; The upstream primer of inner primer has the sequence shown in SEQ No.11, and the downstream primer of inner primer has the sequence shown in SEQ No.12; The upstream primer of the outer primer in the detection primer sets of said Salmonellas and the concentration of downstream primer are 0.002-0.006 μ M, and the upstream primer of inner primer and the concentration of downstream primer are 0.7-0.9 μ M; The upstream primer of the outer primer in the detection primer sets of said streptococcus aureus and the concentration of downstream primer are 0.002-0.006 μ M, and the upstream primer of inner primer and the concentration of downstream primer are 0.5-0.7 μ M; The upstream primer of the outer primer in the detection primer sets of said Listeria monocytogenes and the concentration of downstream primer are 0.002-0.006 μ M, and the upstream primer of inner primer and the concentration of downstream primer are 0.5-0.7 μ M.