CN102586455A

CN102586455A - Aflatoxin detection reagent kit based on loop-mediated isothermal gene amplification method

Info

Publication number: CN102586455A
Application number: CN2012100663513A
Authority: CN
Inventors: 万俊松
Original assignee: Individual
Current assignee: Individual
Priority date: 2012-03-14
Filing date: 2012-03-14
Publication date: 2012-07-18

Abstract

An aflatoxin detection reagent kit based on a loop-mediated isothermal gene amplification method is based on the basic principle of loop-mediated isothermal amplification (LAMP). The loop-mediated isothermal gene amplification method includes particular steps: preparing a culture medium of PDA (potato dextrose agar); cultivating strains; designing a loop-mediated isothermal gene amplification (LAMP) primer; extracting a fungus DNA (deoxyribose nucleic acid) template; and proportionally preparing, adding a PCR (polymerase chain reaction) tube and realizing water bath heat-insulation reaction, agarose gel electrophoresis and the like. The aflatoxin detection reagent kit is combined with a method (GB\T13092-2006) for detecting the total number of mold in feed, the concentration of selected fungus liquid is detected by an MPN (most probable number) method, and the aflatoxin detection reagent kit is prepared according to the method. When used for detecting aflatoxin fungi in the feed and food, the reagent kit is high in specificity and sensitivity, quick in detection and low in cost, can be used for detecting a plurality of samples at the same time, and is applicable to sanitation examination for primary-level feed and worthy of being widely popularized.

Description

A kind of flavacin detection kit based on ring matchmaker constant temperature gene amplification method

Technical field

The present invention relates to the gene test field, be specially a kind ofly, possess the flavacin detection kit based on ring matchmaker constant temperature gene amplification method of advantages such as speed is fast, high specificity based on ring matchmaker's constant temperature gene amplification method (LAMP) ultimate principle.

Background technology

Toxins, afla (Aflatoxin writes a Chinese character in simplified form AF) is a kind of mycotoxins, is mainly produced by Aspergillus flavus and Aspergillus parasiticus bacterium, and immunity system, mutagenesis, teratogenesis, carcinogenic that it influences body cause serious threat to animal body and HUMAN HEALTH.The up-to-date forage health standard of China all is strict with mould and toxin.The method that detects Toxins, afla at present has thin layer chromatography (TLC), HPLC (HPLC), enzyme-linked immunosorbent assay (ELISA) etc.But; These its treatment processs of method that detect mould all are confined to traditional method and fungi culture and biological polymerase chain reaction method (PCR); The former operates very trouble, and the time needs 4～7 days, the latter to instrument and reaction conditions than higher; Detect the also corresponding lifting of cost, be difficult to accomplish fairly large popularization.

Summary of the invention

The technical problem that the present invention solved is to provide a kind of flavacin detection kit based on ring matchmaker constant temperature gene amplification method, utilizes ring matchmaker's constant temperature gene amplification (LAMP) method to detect the Toxins, afla fungi, can be applicable to the sample detection of feed, food etc.

Ring matchmaker constant temperature gene amplification (Loop-mediated isothermal amplification is called for short LAMP) is a kind of new constant temperature gene amplification efficiently technology that Japanese scholar Notomi equals invention in 2000.This method is at 6 specific regions of constant temperature (60 ℃～65 ℃) through 4 primer B3, F3, BIP and FIP identifying purpose genes; Amplifying target genes fragment under the katalysis of Bst archaeal dna polymerase, reaction comprise two closely-related links of strand replacement reaction and ring mediation cyclic amplification.

The technical problem that the present invention solved adopts following technical scheme to realize:

A kind of flavacin detection kit based on ring matchmaker constant temperature gene amplification method is set up LAMP method rapid detection flavacin fungi based on the Ver-1 gene, and is applied to the detection of feed, food; Use AFB1 content of toxins in the ELISA method test sample, and comparative analysis ELISA and LAMP detected result, it specifically comprises following preparation process:

1. preparation PDA substratum: take by weighing yam 100 g that peeling is cleaned, put into 500 mL zero(ppm) water, with water boil to 100 ℃ and kept 30 minutes; Filter, add 10 g agar powders, supply water to 1000 mL; Adjust pH to 7.0, autoclaving after the packing, 4 ℃ of insulations are stored subsequent use;

2. cultured strain: flavus is inoculated on the solid potato culture medium (PDA) 28 ℃ of cultivations;

3. ring matchmaker's constant temperature gene amplification (LAMP) primer is set;

4. extract the fungal DNA template;

5. reaction reagents such as template, primer, dNTP and damping fluid are prepared in proportion and added the PCR pipe; With temperature condition following insulation 60 mins of reaction system as for 63 ℃; Thereafter at 3 minutes deactivation BstDNA of 80 ℃ of following heating baths polysaccharase, reaction terminating, product is electrophoresis in 2.0% sepharose;

6. make template with reference to strain DNA extraction liquid, adopt 25 μ L reaction systems.Change temperature of reaction, reaction times, Mg respectively ²⁺Concentration, the concentration of dNTP, the concentration of Betaine, carry out the LAMP reaction respectively, judge LAMP result with agarose gel electrophoresis, to select best amplification condition;

7. with LAMP detection method amplification flavus, gel electrophoresis imaging inspection amplified production is judged the specificity of this test;

8. the template of above preparation is made the 10n doubling dilution.Get each extent of dilution 1 μ L respectively and carry out the LAMP amplification.In conjunction with the measuring method of total number of molds in the feed (T13092-2006), measure the bacterial concentration of institute's picking with the MPN method;

9. set up flavacin fungi detection kit according to the method described above;

In the present invention, gel is used ethidium bromide staining, observes in order to make things convenient for ultraviolet (uv) transmission.

In the present invention, said flavacin fungi detection kit can be used SYBR Green I determining method and gel electrophoresis imaging method judgement sample detected result.

Beneficial effect: the present invention than traditional P CR technology sooner, more accurate.Simultaneously, the LAMP method adopts 6 specific sites of 4 primer identification, has guaranteed this method high degree of specificity, and simultaneously, the present invention also possesses following advantage:

1. the present invention makes full use of the peculiar advantage of LAMP on the method for detected result, when having used traditional gel imaging to detect, also utilizes SYBR Green I to implement visual detection.SYBR Green I implements visual detection and has avoided the pollution that EB brings in the actually operating, and the speed that detects was practiced thrift more than 40 minutes;

2. the present invention detects the plain fungi stability of forage poisoning height, favorable repeatability.Detected 180 feed samples with this method, the positive rate and the ELISA method goodness of fit are high;

3. the LAMP method detects in the feed Toxins, afla fungi to detect cost not high among the present invention, and each sample only needs about 3.0 yuan.The used Bst enzyme of LAMP method price is more expensive than traditional T aq enzyme, but in this experiment to after 5 times of dilutions of this enzyme, on the one hand test-results is stable, cost obviously reduces on the other hand, on average the Bst enzyme of each sample only needs 0.30 yuan;

4. required for the present inventionly want instrument fairly simple, key instrument is not as long as the ortho-water bath needs the PCR appearance, and suitable substrate is promoted;

5. test sample speed of the present invention is very fast, and proliferation time needed only 60 minutes, and detected result is judged only needs several minutes.

Description of drawings

The primer that Fig. 1: LAMP is used and be used for the part A spergillus flavus ver-1 gene order of outside primer (FIP and BIP) and inner primer (F3 and B3) design.

Fig. 2: three kinds of differences are split the LAMP reaction result of bacterium method.

Fig. 3: difference is split the LAMP reaction result of bacterium time.

Fig. 4: different dNTPs concentration are to the influence of LAMP reaction.

Fig. 5: the AMP method detects feed and produces Toxins, afla fungi operation steps.

Fig. 6: SYBR Green I determining method detected result.

Fig. 7: gel electrophoresis imaging method judgement sample detected result.

Embodiment

For technique means, creation characteristic that the present invention is realized, reach purpose and effect and be easy to understand and understand, below in conjunction with concrete diagram, further set forth the present invention.

Embodiment 1:

Make amplification template based on Aspergillus flavus ver-1 gene gene order, utilize software and range estimation to overlap Auele Specific Primer F3, B3, FIP and BIP in sequence 25bp～221bp place design one.Each primer sequence.Concrete outcome is seen Fig. 1, and in Fig. 1, F3 and B3 are outer primer, also claim short primer, and FIP and BIP are inner primer, also claim long primer.Wherein BIP is formed by connecting B2 and BIC, and FIP is formed by connecting F2 and FIC.Article four, 6 sites of primer identifying purpose gene.

Embodiment 2:

Adopting liquid nitrogen extraction method, microwave thermal to shake the different bacterium method of splitting of three kinds of method and simple and easy extraction methods can successful lysing cell; Comparatively speaking simple and easy extraction method is simple; And effect stability adopts three kinds of method ruptured cells for this test and makes template LAMP amplification electrophoresis result such as Fig. 2, in Fig. 2; The LAMP reaction result that splits the bacterium method of 1 expression liquid nitrogen extraction method; The 2 expression microwave thermal methods of shaking are split the LAMP reaction result of bacterium method, the LAMP reaction result of the reaction method that 3 expressions are traditional, the no template contrast of 4 expressions.

Embodiment 3:

Fungi is 100 ℃ of difference water-bath 1 min, 2 min, 3 min, 4 min, 5 min, 6 min and 7 min in lysate, behind centrifugal 1 min of 10000 r/min, get supernatant, as the template of LAMP detection.Revision test shows, splits the bacterium effect stability in 5 minutes.Concrete outcome is seen Fig. 3, in the drawings, 1 ~ 7 reaction and display when representing 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes respectively, the no template of 8 expressions contrasts.

Embodiment 4:

With 0.2 mmol/L, 0.4 mmol/L, 0.6 mmol/L, 0.8 mmol/L, 1 mmol/L, 1.2 mmol/L, 1.4 mmol/several different dNTPs concentration such as L are carried out the LAMP amplification.In order to detect the influence of different dNTPs concentration to LAMP reaction, the result shows does not have specific band when contain dNTPs concentration less than 6 mM in the reaction system, during 6 mM appearance than filaments of sun band, when concentration band when 10 mM are above very obvious.Concrete outcome is seen Fig. 4, and wherein 1 ~ 7 representes 0.2 mmol/L, 0.4 mmol/L, 0.6 mmol/L successively respectively; 0.8 mmol/L, 1 mmol/L, 1.2 mmol/L; 1.4 the dNTPs concentration of mmol/L is to the influence of LAMP, wherein, 8 represent no template control group.

Embodiment 5:

Through TE, LAMP detects the working method of producing the Toxins, afla fungi in feed, the food and finally is able to confirm, and attempts being applied to reality.This method comprises three big steps altogether: 1. sample preparation; 2. LAMP amplification; 3. detected result is judged.Specifically see Fig. 5.

Embodiment 6:

SYBR Green I determining method: in the LAMP product, add SYBR Green I, observations under naked eyes, the positive amplified production is green, and is bright, and negative sample does not have amplified production, color is a safran, dimness.Concrete outcome is seen Fig. 6, and wherein the left side is negative, and the right is positive.

Embodiment 7:

Gel electrophoresis imaging method judgement sample detected result: the sepharose with 2%, electrophoresis is 45 minutes under EB 2%, the 80 V voltage, observes with gel imaging system imaging back, and the positive of specific band arranged among the result, otherwise negative.Concrete outcome is seen Fig. 7.

Random sampling totally 180 parts of feed samples from the Hunan, wherein feed factory is 90 parts, and 90 parts of plants have 200 of the food samples that gather in each market from the Hunan in addition.Respectively these are carried out ELISA and detect AFB1 and its Toxins, afla fungi of LAMP method detection, and the result is compared analysis.Comprehensive LAMP method and ELISA method detected result, two kinds of detected results are male in 180 samples has 29 parts, and what two kinds of detected results were all negative has 136, and it is identical promptly to have two kinds of test result of 165 samples, and identical rate is 91.67%.

More than show and described ultimate principle of the present invention and principal character and advantage of the present invention.The technician of the industry should understand; The present invention is not restricted to the described embodiments; That describes in the foregoing description and the specification sheets just explains principle of the present invention; Under the prerequisite that does not break away from spirit and scope of the invention, the present invention also has various changes and modifications, and these variations and improvement all fall in the scope of the invention that requires protection.The present invention requires protection domain to be defined by appending claims and equivalent thereof.

Claims

1. the flavacin detection kit based on ring matchmaker constant temperature gene amplification method is characterized in that, specifically comprises following preparation process:

1) preparation PDA substratum: take by weighing yam 100 g that peeling is cleaned, put into 500 mL zero(ppm) water, with water boil to 100 ℃ and kept 30 minutes; Filter, add 10 g agar powders, supply water to 1000 mL; Adjust pH to 7.0, autoclaving after the packing, 4 ℃ of insulations are stored subsequent use;

2) cultured strain: flavus is inoculated on the solid potato culture medium (PDA) 28 ℃ of cultivations;

3) ring matchmaker's constant temperature gene amplification (LAMP) primer is set;

4) extract the fungal DNA template;

5) reaction reagents such as template, primer, dNTP and damping fluid are prepared adding PCR pipe in proportion; With temperature condition following insulation 60 mins of reaction system as for 63 ℃; Thereafter at 3 minutes deactivation BstDNA of 80 ℃ of following heating baths polysaccharase, reaction terminating, product is electrophoresis in 2.0% sepharose;

6) make template with reference to strain DNA extraction liquid, adopt 25 μ L reaction systems, change temperature of reaction, reaction times, Mg respectively ²⁺Concentration, the concentration of dNTP, the concentration of Betaine, carry out the LAMP reaction respectively, judge LAMP result with agarose gel electrophoresis, to select best amplification condition;

7) with LAMP detection method amplification flavus, gel electrophoresis imaging inspection amplified production is judged the specificity of this test;

8) template of above preparation is made the 10n doubling dilution, gets each extent of dilution 1 μ L respectively and carries out the LAMP amplification, combine total number of molds in the feed measuring method (T13092-2006), with the bacterial concentration of MPN method mensuration institute picking;

9) set up flavacin fungi detection kit according to the method described above.

2. a kind of flavacin detection kit based on ring matchmaker constant temperature gene amplification method according to claim 1 is characterized in that said gel is used ethidium bromide staining, observes in order to make things convenient for ultraviolet (uv) transmission.

3. a kind of flavacin detection kit according to claim 1 based on ring matchmaker constant temperature gene amplification method; It is characterized in that said flavacin fungi detection kit can be used SYBR Green I determining method and gel electrophoresis imaging method judgement sample detected result.