CN101168768A - Alexandrium constant temperature gene amplification fast detecting kit and method - Google Patents

Abstract

The invention discloses an Alexandrium constant temperature gene amplification rapid-test kit and the method thereof. The kit comprises primer mixed liquor, Bst DNA polymerase, reaction liquid, sample pretreatment liquid, visualization reagent, positive control and negative control, wherein the sequence of the primer comprises SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4. The kit is used to realize high specificity, rapidness, high sensitivity, and simple test through Alexandrium genome DNA extraction, constant temperature gene amplification reaction, and reaction product judgment.

Description

Alexandrium constant temperature gene amplification fast detecting kit and method

Technical field

The present invention relates to microorganism detection reagent and method, be specifically related to a kind of Alexandrium constant temperature gene amplification fast detecting kit and method.

Background technology

The part toxiferous algae kind of Alexandria Trentepohlia is the major cause that causes that harmful algal bloom takes place, the paralytic shellfish poisoning (PSP) (PST) that its metabolism produces is a kind of neural paralysis agent, this toxin can be enriched in shellfish or the fish body, by food chain, directly threatens human health.Therefore, need badly and carry out, improve detection sensitivity, thereby on the source, stop of the harm of algae toxin public health, water surrounding and natural resources to producing the detection of the harmful algae kind of PST toxin.The algae kind detection method of generally acknowledging is the morphological specificity of observing algae by optics or electron microscope; but; this detection means is subjected to the instrument restriction to be difficult to satisfy the demand of long term monitoring; and the phenotypic characteristic of algae usually can be subjected to the influence of environmental factors and culture condition, makes that similar or close algae kind is difficult to distinguish on form.Because the algae kind there are differences on gene level, makes molecular biology method become effective detection means.

At present, the method for PCR-based, fluorescence in situ hybridization method, restriction fragment length polymorphism analysis and immunological technique etc. have had report to be used for poisonous algae detection research.Though these research meanses have significantly improved detection sensitivity and specificity to a certain extent, need high-accuracy instrument in the testing process, this just makes detection means become complicated and expends.So, can not satisfy the needs of rapid detection on the spot.Except PCR method, also have other nucleic acid amplification methods, such as amplification of nucleic acid sequences method (NASBA), from sequence transfer printing (3SR) and strand displacement transfer printing (SDA).The detection level of these three kinds of methods all is no less than 10 copies, in the similar scope that just target nucleic acid can be increased in about 1 hour consuming time, however, they are not strong to the specific amplification of target sequence, also need detailed experimental implementation means to detect amplified production after the feasible amplification.The immunology detection technology is fast and convenient with low cost, but requires the monoclonal antibody of high quality high stability, otherwise because of accuracy is not enough, can only be the auxiliary detection means at present.So, the newest fruits of biotech development is applied to the pathogenic micro-organism detection, significant.

Dna circle mediated constant temperature nucleic acid amplification technology (loop-mediated isothermal amplification of DNA, LAMP) overcome the deficiency of gene amplification method in the past, can specificity under isothermal condition, carry out the amplification of nucleic acid efficiently, apace, has a lot of superior lifes, see document Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T.Loop-mediated isothermal amplification of DNA, Nucleic Acids Res.2000 Jun 15; 28 (12): E63.This method is normally carried out as follows: step 1, tested sample is carried out pre-treatment, the DNA of the tested sample of rapid extraction or RNA; The preparation of step 2, Auele Specific Primer: after determining the target gene of sample to be measured, obtain 2 pairs of Auele Specific Primers, each is a pair of for inner primer and outer primer; Step 3, carry out loop-mediated isothermal amplification technique (LAMP) reaction: will mix with the Bst archaeal dna polymerase through sample, primer, the reaction buffer of pre-treatment, be incubated 0.5 to 1.5 hour at 63～65 ℃ and carry out the endless chain replacement(metathesis)reaction; Step 4, analysis and judgement reaction product result.Though disclosed LAMP technology has high sensitivity, can be under isothermal condition amplification of nucleic acid efficiently, prior art also has weak point, mainly is: 1. its special primer requires the very harsh widespread use that limits this method; 2. the milky white precipitate thing judged result that only generates with reaction is accurately clear inadequately, also is difficult for the realization response miniaturization, limits it and further develops.Be necessary existing LAMP technology is improved in order to overcome these shortcomings.

Summary of the invention

The objective of the invention is to overcome above-mentioned technological deficiency, a kind of Alexandrium constant temperature gene amplification fast detecting kit and method are provided.

To achieve the object of the present invention, by the following technical solutions:

A kind of Alexandrium constant temperature gene amplification fast detecting kit of the present invention, its reagent comprises:

---the primer mixed solution: four primer concentrations are 10pmol/ μ l, primer sequence such as SEQ ID NO.1, SEQ ID NO.2, SEQ IDNO.3, SEQ ID NO.4;

---the Bst archaeal dna polymerase: concentration is 8U/ μ l;

---reaction solution: 10mM dNTP:10 * ThermoPol reaction buffer: 150mM MgSO ₄=8: 5: 2;

---sample pretreatment liquid: 20 mM Tris-HCl (pH 8.0), 2 mM EDTA, 1.2%Triton X-100;

---developer: fluorescence dye 1 * SYBR Green I.

(6) positive control is the Alexandrium mimutum Halim by using genomic dna, and negative control is not for containing the reaction mixture of template.

The method that detects Alexandrium with above-mentioned test kit may further comprise the steps:

(1) Alexandrium extracting genome DNA process: the enrichment liquid of getting incubated overnight is in centrifuge tube, and centrifugal 2 minutes of 1000rpm removes supernatant; Add sample pretreatment liquid in centrifuge tube, mix with precipitation gained thalline; Boil in the boiling water after 10 minutes and placed cooled on ice immediately 10 minutes; Centrifugal 2 minutes of 10000-12000rpm, supernatant promptly can be used as stand-by template DNA; Wherein the amount ratio of enrichment liquid and sample pretreatment liquid is 5: 8;

(2) constant temperature gene amplification reaction: prepare reaction mixture at the 200ulPCR pipe: primer mixture 1.5 μ l, reaction solution 5.5 μ l, Bst archaeal dna polymerase 0.5 μ l, template DNA 2 μ l add water to 11.5 μ l; When the positive control reaction is set, substitute Alexandrium genomic dna to be checked, when the negative control reaction is set, substitute Alexandrium genomic dna to be checked with the reaction mixture that does not contain template with the Alexandrium mimutum Halim by using genomic dna; To prepare the PCR pipe of reaction mixture in 65 ℃ of reactions 1.5 hours;

(3) analysis and judgement reaction product result: obtain adding 2.5 μ l fluorescence dye SYBR Green I in the product in (2), mixing leaves standstill 5min; If shows green is then positive, orange then negative.

A kind of Alexandrium constant temperature gene amplification fast detecting kit of the present invention and method have following beneficial effect: high specific: the present invention has designed four Auele Specific Primers according to the Alexandrium rDNA: primer sequence such as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ IDN are O.4;

Use above-mentioned four primers, whether the existence that just can judge target substance according to whether increasing, and positive rate can reach greater than 99.9%, and false positive rate is less than 0.1%;

2, fast efficient amplification: amplification can be finished less than 1 hour, and the productive rate height;

3, highly sensitive: be used for the detection of pathogenic micro-organism and the diagnosis of transmissible disease, the bacterium microbe lowest detection limit is reached 10CFU/ml, the recall rate of sample reaches 99%;

4, evaluation is easy: identify by visual inspection, need not other any analytical procedures such as electrophoresis.

Embodiment

Press following formulation Alexandrium constant temperature gene amplification fast detecting kit:

---the primer mixed solution: four primer concentrations are 10pmol/ μ l, primer sequence such as SEQ ID NO.1, SEQ ID NO.2, SEQ IDNO.3, SEQ ID NO.4;

---the Bst archaeal dna polymerase: concentration is 8U/ μ l;

---reaction solution: 80 μ l 10mM dNTP, 50 μ l, 10 * ThermoPol reaction buffer, 20 μ l 150mM MgSO ₄

---sample pretreatment liquid: 20 mM Tris-HCl (pH 8.0), 2 mM EDTA, 1.2%TritonX-100.

---developer: fluorescence dye 1 * SYBR Green I.

---positive control is the Alexandrium mimutum Halim by using genomic dna, and negative control is not for containing the reaction mixture of template.

By the following method Alexandrium is detected with above-mentioned test kit:

1, the simple and easy leaching process of Alexandrium genomic dna:

(1) enrichment liquid of getting 50 μ l incubated overnight is in centrifuge tube, and centrifugal 2 minutes of 1000rpm removes supernatant;

(2) add 80 μ l sample pretreatment liquid in centrifuge tube, mix with precipitation gained thalline;

(3) boil in the boiling water after 10 minutes and placed cooled on ice immediately 10 minutes;

(4) 10000rpm is centrifugal 2 minutes, and supernatant promptly can be used as stand-by template DNA.

2, loop-mediated isothermal amplification (LAMP) reaction:

(1) in 200ulPCR pipe preparation reaction system: primer mixture 1.5 μ l, reaction solution 5.75 μ l, Bst archaeal dna polymerase 0.5 μ l, template DNA 2 μ l add water to 11.5 μ l; When the positive control reaction is set, substitute Alexandrium genomic dna to be checked, when the negative control reaction is set, substitute Alexandrium genomic dna to be checked with the reaction mixture that does not contain template with the Alexandrium mimutum Halim by using genomic dna;

(2) will prepare the PCR pipe of reaction mixture in 65 ℃ of reactions 1.5 hours.

3, analysis and judgement reaction product result:

Add 2.5 μ l fluorescence dyes (SYBR Green I) in reaction product, mixing leaves standstill 5min.If shows green is then positive, orange then negative.Negative control is orange, and negative control is green.

The primer sequence table

SEQUENCE?LISTING

＜110〉South China Science ﹠ Engineering University

Beautiful, the king

Of heap of stone, stone

Beautiful jade, Lee

＜120〉Alexandrium constant temperature gene amplification fast detecting kit and method

<130>Notomi?T，Okayama?H，Masubuchi?H，Yonekawa?T，Watanabe?K，Amino

N，Hase?T.Loop-mediated?isothermal?amplification?of?DNA，Nucleic

Acids?Res.2000?Jun?15；28(12)：E63.

<160>1

<170>PatentIn?version?3.4

<210>1

<211>18

<212>DNA

<213>Alexandrium

<400>1

tgcagggatc?agtcgtac 18

SEQUENCE?LISTING

＜110〉South China Science ﹠ Engineering University

Beautiful, the king

Of heap of stone, stone

Beautiful jade, Lee

＜120〉Alexandrium constant temperature gene amplification fast detecting kit and method

<130>Notomi?T，Okayama?H，Masubuchi?H，Yonekawa?T，Watanabe?K，Amino

N，Hase?T.Loop-mediated?isothermal?amplification?of?DNA，Nucleic

Acids?Res.2000?Jun?15；28(12)：E63.

<160>1

<170>PatentIn?version?3.4

<210>1

<211>20

<212>DNA

<213>Alexandrium

<400>1

agatcatcca?gtgttgtacg 20

SEQUENCE?LISTING

＜110〉South China Science ﹠ Engineering University

Beautiful, the king

Of heap of stone, stone

Beautiful jade, Lee

＜120〉the Alexandrium constant temperature gene amplification rapid detection is tried sharp box and method

<130>Notomi?T，Okayama?H，Masubuchi?H，Yonekawa?T，Watanabe?K，Amino

N，Hase?T.Loop-mediated?isothermal?amplification?of?DNA，Nucleic

Acids?Res.2000?Jun?15；28(12)：E63.

<160>1

<170>PatentIn?version?3.4

<210>1

<211>46

<212>DNA

<213>Alexandrium

<400>1

gtcagtgagg?ttccactatg?cgttttaatc?gccattcgtt?gactac 46

SEQUENCE?LISTING

＜110〉South China Science ﹠ Engineering University

Beautiful, the king

Of heap of stone, stone

Beautiful jade, Lee

＜120〉Alexandrium constant temperature gene amplification fast detecting kit and method

<130>Notomi?T，Okayama?H，Masubuchi?H，Yonekawa?T，Watanabe?K，Amino

N，Hase?T.Loop-mediated?isothermal?amplification?of?DNA，Nucleic

Acids?Res.2000?Jun?15；28(12)：E63.

<160>1

<170>PatertIn?version?3.4

<210>1

<211>42

<212>DNA

<213>Alexandrium

<400>1

tctgtggcaa?gagcgatggtt?ttttccctct?gtatttgccg?aa 42

Claims (2)

1. an Alexandrium constant temperature gene amplification fast detecting kit is characterized in that, its reagent comprises:

---the primer mixed solution: four primer concentrations are 10pmol/ μ l, primer sequence such as SEQ ID NO.1, SEQ ID NO.2, SEQ IDNO.3, SEQ ID NO.4;

---the Bst archaeal dna polymerase: concentration is 8U/ μ l;

---react night: 10mM dNTP:10 * ThermoPol reaction buffer: 150mM MgSO ₄=8: 5: 2;

---sample pretreatment liquid: 20 mM Tris-HCl (pH 8.0), 2 mM EDTA, 1.2%Triton X-100;

---developer: fluorescence dye 1 * SYBR Green I;

---positive control is the Alexandrium mimutum Halim by using genomic dna, and negative control is not for containing the reaction mixture of template.

2. the method with the described test kit detection of claim 1 Alexandrium is characterized in that, may further comprise the steps:

(1) Alexandrium extracting genome DNA process to be checked: the enrichment liquid of getting incubated overnight is in centrifuge tube, and centrifugal 2 minutes of 1000rpm removes supernatant; Add sample pretreatment liquid in centrifuge tube, mix with precipitation gained thalline; Boil in the boiling water after 10 minutes and placed cooled on ice immediately 10 minutes; Centrifugal 2 minutes of 10000-12000rpm, supernatant promptly can be used as Alexandrium DNA to be checked; Wherein the amount ratio of enrichment liquid and sample pretreatment liquid is 5: 8;

(2) constant temperature gene amplification reaction: prepare reaction mixture at the 200ulPCR pipe: primer mixture 1.5 μ l, reaction solution 5.75 μ l, Bst archaeal dna polymerase 0.5 μ l, Alexandrium genomic dna 2 μ l to be checked add water to 11.5 μ l; When the positive control reaction is set, substitute Alexandrium genomic dna to be checked, when the negative control reaction is set, substitute Alexandrium genomic dna to be checked with the reaction mixture that does not contain template with the Alexandrium mimutum Halim by using genomic dna; To prepare the PCR pipe of reaction mixture in 65 ℃ of reactions 1.5 hours;

(3) analysis and judgement reaction product result: obtain adding 2.5 μ l fluorescence dye SYBR Green I in the product in (2), mixing leaves standstill 5min; If shows green is then positive, orange then negative.