CN102703604B - Kit for rapidly detecting banana bunchy top virus by isothermal gene amplification and use method of kit - Google Patents

Abstract

The invention relates to a kit for rapidly detecting a banana bunchy top virus (BBTV) by isothermal gene amplification. Reagents of the kit include a primer mixed solution, a Bst DNA (deoxyribonucleic acid) polymerase of 8U/Mu l, a DNA extract, a reaction buffer solution, a nucleic acid dye, a positive control reagent and a negative control reagent, wherein the primer mixed solution consists of two pairs of primers; the DNA extract contains 100mM of Tris-HCL (pH is equal to 7.4), 1M of KCl, 10mM of EDTA and 2% (w/v) of PVPP; the reaction buffer solution contains 10mM of dNTP, a 10* ThermoPol reaction buffer solution, 150mM of MgSO4 and 5mM of betaine which are at the ratio of 8:5:2:10; the positive control reagent is a banana streak virus genome DNA; and the negative control reagent is 100mM of Tris-HCL (pH 8.0) and 50mM of EDTA. The invention also relates to a use method of the kit. By extraction of nucleic acid of the BBTV from a sample and isothermal gene amplification with the kit, the color of the reaction solution is finally judged through naked eyes, so that the high-specificity, rapid, high-sensitivity, simple and convenient molecular detection on the BBTV is achieved.

Description

A kind of abaca bunchy top virus constant temperature gene amplification fast detecting kit and using method thereof

Technical field

The present invention relates to the microorganism detection field, specifically, the present invention relates to a kind of abaca bunchy top virus constant temperature gene amplification fast detecting kit and using method thereof.

Background technology

Banana is the Musaceae banana, is the important cash crop of tropical and subtropical zone and food crop.(Banana bunchy top virus is that bananas such as restriction China Guangdong, Yunnan, Hainan are mainly planted one of important factor of regional industry sound development BBTV) to abaca bunchy top virus.The early stage morbidity of disease plant growth diseased plant obviously stunts, and blade is narrow and small, vertical, and the leaf margin yellow, the food value of leaf is crisp firmly, frangibility, master pulse and petiole performance are different in size, desultory dark striped (being commonly called as " playing blue veins ").The booting sequela all sprouts as blade, does not then show " bunchy top ", and blade is narrowless and small, yellow also, but the young leaflet tablet master pulse is existing " blue veins " still, and the bud of extraction is directly given birth to, and does not tie any of several broadleaf plants or knot any of several broadleaf plants performance extreme difference, and handle is elongated, crooked, fruit is little and lack, and the fruit end is carefully as finger, crisp, the British plain spirits of pulp.The cause of disease of banana bunchy top disease is peloid precursor virus (BBTV), is propagated in the persistence mode by banana any of several broadleaf plants arteries and veins aphid (Pentalonia nigronervosa).Banana bunchy top disease mainly is by suction bud and tissue cultured seedling band poison, and banana hands over arteries and veins aphid (Pentalonia nigroneryosa) to propagate.

Banana is mainly produced by asexual tissue culture batch production, abaca bunchy top virus can be along with the breeding of banana differentiation bud in the group training, if seedling (or any of several broadleaf plants head) carries abaca bunchy top virus and quarantines without strictness again, its cause of disease just may be propagated at faster speed with tissue cultured seedling, becomes an important primary source of infection of transmitted virus.Therefore, setting up quick, the stable detection method of a cover BBTV is the important assurance that nontoxic seedling is produced.

In recent years, there is a large amount of reports to confirm that the method success of PCR-based is for detection of abaca bunchy top virus, PCR method improves a lot than ordinary method on the operating time and aspect the specificity that detects and the sensitivity, but, PCR method must have accurate temperature cycle device, and its detection time also still long (2～3 hours), and reaction process is easy to the influence of contaminated thing, thus make this method can not satisfy the needs of actual detected.Therefore, need a kind of highly sensitive and willing method of exploitation, can replace the detection method of PCR to a certain extent.Therefore, the newest fruits of biotech development is applied to abaca bunchy top virus detects, 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, rapidly, has a lot of superiority, 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.2000Jun 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.

Summary of the invention

The objective of the invention is to overcome above-mentioned technological deficiency, a kind of abaca bunchy top virus constant temperature gene amplification fast detecting kit is provided.

The present invention also aims to provide the using method of this abaca bunchy top virus constant temperature gene amplification fast detecting kit.

In order to realize purpose of the present invention, the invention provides a kind of abaca bunchy top virus constant temperature gene amplification fast detecting kit, its reagent comprises:

The primer mixed solution that is constituted by two pairs of primers, described two pairs of primers are: outer primer 1-F3, its nucleotide sequence is shown in SEQ ID NO:1 (5 '-TGATGCGGGATGAGTTTA-3 '), outer primer 2-B3, its nucleotide sequence is shown in SEQ ID NO:2 (5 '-CACGCATATCCTGTATGAC-3 '), inner primer 1-FIP, its nucleotide sequence is shown in SEQ ID NO:3 (5 '-CTTTCGTTTCTCAAGGTGTGCGTCGAGATGAAGAGAAGAAG-3 '), inner primer 2-BIP, its nucleotide sequence is shown in SEQ ID NO:4 (5 '-GGGAGCCAAGAAGAAGCAGGACCTTCGATTCTTGTATC-3 '), the concentration of described inner primer 1-FIP and described inner primer 2-BIP is 40pmol/ μ l, and the concentration of described outer primer 1-F3 and described outer primer 2-B3 is respectively 5pmol/ μ l;

Concentration is the BstDNA polysaccharase of 8U/ μ l;

DNA extraction liquid, described DNA extraction liquid comprises: 100mM Tris-HCl (pH7.4), 1M KC1,10mM EDTA and 2% (w/v) PVPP;

Reaction buffer, described reaction buffer comprises: 10mM dNTP, 10 * ThermoPol reaction buffer, 150mM MgSO ₄, 5mM trimethyl-glycine=8: 5: 2: 10;

Nucleic acid dye;

Positive control: bbtv dna;

Negative control: 100mM Tris-HCl (pH 8.0) and 50mM EDTA.

Preferably, described nucleic acid dye is 1000 * SYBR Green I.

As used herein, in reaction buffer, " reaction buffer comprises: 10mMdNTP, 10 * ThermoPol reaction buffer, 150mM MgSO ₄, 5mM trimethyl-glycine=8: 5: 2: 10 " refer to 10mM dNTP in the reaction buffer, 10 * ThermoPol reaction buffer, 150mM MgSO ₄The volume ratio of the aqueous solution of the aqueous solution, 5mM trimethyl-glycine be 8: 5: 2: 10.

The present invention also provides a kind of using method of abaca bunchy top virus constant temperature gene amplification fast detecting kit, and this method comprises the following steps:

1) the DNA nucleic acid extraction of test sample: add 30 μ lDNA extracting solutions in the 0.5mg detected sample, grind to form pasty state after, at 95 ℃ of 10min, under the 10000rpm centrifugal 2 minutes, get supernatant as detecting template DNA;

2) loop-mediated isothermal amplification: in 25 μ l PCR pipes, dispose reaction soln: the outer primer 1-F3 of 1 μ l, its nucleotide sequence is shown in SEQ ID NO:1; The outer primer 2-B3 of 1 μ l, its nucleotide sequence is shown in SEQ ID NO:2; The inner primer 1-FIP of 1 μ l, its nucleotide sequence is shown in SEQ ID NO:3; The inner primer 2-BIP of 1 μ l, its nucleotide sequence is shown in SEQ ID NO:4; 12.5 the reaction buffer of μ l; The Bst archaeal dna polymerase of 1 μ l; The template DNA of 2 μ l; Add water to 25 μ l then; When the positive control reaction is set, replace 1 with bbtv dna) in the detection template DNA that obtains, when the negative control reaction is set, replace 1 with 100mM Tris-HCl (pH 8.0) and 50mM EDTA) in the detection template DNA that obtains, will contain the PCR pipe of the reaction soln for preparing in 63 ℃ of isothermal reaction 90min;

3) adding the nucleic acid dye of 1 μ l analysis and judgement reaction product result: 2) in the gained reaction product, is orange as the reaction solution color, and the result is negative in expression, is green as the reaction solution color, and the result is positive in expression.

Preferably, described nucleic acid dye is 1000 * SYBR Green I.

Abaca bunchy top virus constant temperature gene amplification fast detecting kit of the present invention has following beneficial effect:

1, high specific: the present invention according to banana bunchy top disease virus gene conserved regions design four Auele Specific Primers, primer sequence is SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.Use above-mentioned four primers, whether the existence that just can judge target substance according to whether increasing, and positive rate is greater than 95%, and false positive rate is less than 3%;

2, need not the high purity template DNA: DNA extraction method does not simply fast need whizzer, liquid nitrogen, phenol, chloroform instrument and reagent;

3, realizing that sample is on-the-spot detects: in the open air or the scene, field, directly the streak virus in the sample is carried out on-the-spot gene amplification and detect;

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

5, highly sensitive: can detect the target of individual cells in complex system, the lowest detection limit reaches 2pg DNA, and the recall rate of sample reaches more than 95%;

6, evaluation is easy: by visual inspection colour-change result of determination, need not other any analytical procedures such as electrophoresis.

Embodiment

Following examples are used for explanation the present invention, but are not used for limiting the scope of the invention.

Embodiment 1

(Banana bunchy top virus BBTV) can be available from grinding territory (Shanghai) chemical reagent company limited for abaca bunchy top virus.Press following formulation abaca bunchy top virus isothermal gene amplification fast detecting kit:

The primer mixed solution that is constituted by two pairs of primers, described two pairs of primers are: outer primer 1-F3, its nucleotide sequence is shown in SEQ ID NO:1, outer primer 2-B3, its nucleotide sequence is shown in SEQ ID NO:2, inner primer 1-FIP, its nucleotide sequence is shown in SEQ ID NO:3, inner primer 2-BIP, its nucleotide sequence is shown in SEQ ID NO:4, the concentration of described inner primer 1-FIP and described inner primer 2-BIP is 40pmol/ μ l, and the concentration of described outer primer 1-F3 and described outer primer 2-B3 is respectively 5pmol/ μ l;

Concentration is the Bst archaeal dna polymerase of 8U/ μ l;

DNA extraction liquid, described DNA extraction liquid comprises: 100mM Tris-HCl (pH7.4), 1M KC1,10mM EDTA and 2%PVPP;

Reaction buffer, described reaction buffer comprises: 10mM dNTP (available from sigma), 10 * ThermoPol reaction buffer (available from NEB), 150mM MgSO ₄(available from sigma), 5mM trimethyl-glycine (available from sigma)=8: 5: 2: 10;

Nucleic acid dye 1000 * SYBR Green I;

Positive control: bbtv dna;

Negative control: 100mM Tris-HCl (pH 8.0) and 50mM EDTA.

Use the mentioned reagent box by the following method abaca bunchy top virus to be detected:

1) the DNA nucleic acid extraction of test sample: add 30 μ l DNA extraction liquid in the 0.5mg detected sample (plant is got middle arteries and veins, and seedling is got bulb), grind to form pasty state after, at 95 ℃ of 10min, under the 10000rpm centrifugal 2 minutes, get supernatant as detecting template DNA;

2) loop-mediated isothermal amplification: in 25 μ l PCR pipes, dispose reaction soln: the outer primer 1-F3 of 1 μ l, its nucleotide sequence is shown in SEQ ID NO:1; The outer primer 2-B3 of 1 μ l, its nucleotide sequence is shown in SEQ ID NO:2; The inner primer 1-FIP of 1 μ l, its nucleotide sequence is shown in SEQ ID NO:3; The inner primer 2-BIP of 1 μ l, its nucleotide sequence is shown in SEQ ID NO:4; 12.5 the reaction buffer of μ l; The Bst archaeal dna polymerase of 1 μ l; The template DNA of 2 μ l; Add water to 25 μ l then; When the positive control reaction is set, replace 1 with bbtv dna) in the detection template DNA that obtains, when the negative control reaction is set, replace 1 with 100mM Tris-HCl (pH 8.0) and 50mM EDTA) in the detection template DNA that obtains, will contain the PCR pipe of the reaction soln for preparing in 63 ℃ of isothermal reaction 90min;

3) adding nucleic acid dye 1000 * SYBR Green I of 1 μ l analysis and judgement reaction product result: 2) in the gained reaction product, is orange as the reaction solution color, and the result is negative in expression, is green as the reaction solution color, and the result is positive in expression.

Claims (4)

1. an abaca bunchy top virus constant temperature gene amplification fast detecting kit is characterized in that, its reagent comprises:

The primer mixed solution that is constituted by two pairs of primers, described two pairs of primers are: outer primer 1-F3, its nucleotide sequence is shown in SEQ ID NO:l, outer primer 2-B3, its nucleotide sequence is shown in SEQ ID NO:2, inner primer 1-FIP, its nucleotide sequence is shown in SEQ ID NO:3, inner primer 2-BIP, its nucleotide sequence is shown in SEQ ID NO:4, the concentration of described inner primer 1-FIP and described inner primer 2-BIP is 40pmol/ μ l, and the concentration of described outer primer 1-F3 and described outer primer 2-B3 is respectively 5pmol/ μ l;

Concentration is the Bst archaeal dna polymerase of 8U/ μ l;

DNA extraction liquid, described DNA extraction liquid comprises: 100mM Tris-HCl pH7.4,1M KC1,10mM EDTA and 2%(w/v) PVPP;

Reaction buffer, described reaction buffer comprises: 10mM dNTP, 10 * ThermoPol reaction buffer, 150mM MgSO ₄, 5mM trimethyl-glycine=8:5:2:10;

Nucleic acid dye;

Positive control: bbtv dna;

Negative control: 100mM Tris-HCl pH8.0 and 50mM EDTA.

2. abaca bunchy top virus constant temperature gene amplification fast detecting kit according to claim 1 is characterized in that, described nucleic acid dye is 1000 * SYBR Green I.

3. the using method of an abaca bunchy top virus constant temperature gene amplification fast detecting kit, this method comprises the following steps:

1) the DNA nucleic acid extraction of test sample: add 30 μ l DNA extraction liquid in the 0.5mg detected sample, grind to form pasty state after, at 95 ℃ of 10min, under the 10000rpm centrifugal 2 minutes, get supernatant as detecting template DNA;

2) loop-mediated isothermal amplification:

In 25 μ l PCR pipes, dispose reaction soln: the outer primer 1-F3 of 1 μ l, its nucleotide sequence is shown in SEQ ID NO:l; The outer primer 2-B3 of 1 μ l, its nucleotide sequence is shown in SEQ ID NO:2; The inner primer 1-FIP of 1 μ l, its nucleotide sequence is shown in SEQ ID NO:3; The inner primer 2-BIP of 1 μ l, its nucleotide sequence is shown in SEQ ID NO:4; 12.5 the reaction buffer of μ l; The Bst archaeal dna polymerase of 1 μ l; The template DNA of 2 μ l;

Add water to 25 μ l then;

When the positive control reaction is set, replace 1 with bbtv dna) in the detection template DNA that obtains, when the negative control reaction is set, replace 1 with 100mM Tris-HCl pH8.0 and 50mM EDTA) in the detection template DNA that obtains, will contain the PCR pipe of the reaction soln for preparing in 63 ℃ of isothermal reaction 90min;

3) adding the nucleic acid dye of 1 μ l analysis and judgement reaction product result: 2) in the gained reaction product, is orange as the reaction solution color, and the result is negative in expression, is green as the reaction solution color, and the result is positive in expression.

4. the using method of abaca bunchy top virus constant temperature gene amplification fast detecting kit according to claim 3 is characterized in that, described nucleic acid dye is 1000 * SYBR Green I.