CN105331746A - Duck flavivirus RT-LAMP detection kit - Google Patents

Abstract

The invention discloses a duck flavivirus RT-LAMP detection kit. The kit comprises RT-LAMP reaction liquid, primer mixed liquor, an enzyme mixture, visible fluorescent dye, DEPC water, a negative control and a positive control. A detection method comprises the steps that samples are collected, nucleic acid RNA is extracted, PCR amplification is conducted by means of the kit, and result detection is conducted after the reaction is completed. The duck flavivirus RT-LAMP detection kit has the advantages that rapidness is achieved, and only two hours are needed from sample treatment to result obtaining; the purpose of detecting duck flavivirus through one-step RT-LMP is achieved; sensitivity is achieved, and compared with a PCR method, the sensitivity is increased by 10-100 times; specificity is achieved, and a cross reaction with other common viruses of poultry does not occur; operation is easy, and good repeatability is achieved.

Description

A kind of duck flavivirus RT-LAMP detection reagent box

Technical field

The present invention relates to a kind of RT-LAMP test kit detecting duck flavivirus.

Background technology

Duck flavivirus has another name called duck tembusu virus (DuckTembusuvirus) and belongs to flaviviridae (Flaviviridae), Flavivirus (flavivirus).Jiangsu-zhejiang Shanghai Area one in 2010 is with and has been broken out a kind of disease being symptom with the decline of egg duck laying rate, growth retardation, loss of weight, appetite stimulator, Gao Re and Mortality, Causative virus has been separated in sick duck body, through gene order comparison, this virus is considered to belong to a new genotype, is the virus of the Flavivirus of TMUV group.

At present, duck flavivirus detects the separation of main dependovirus and molecular Biological Detection.Virus purification is the traditional detection method of duck flavivirus; Molecular biology for detection has RT-PCR, Nest RT-PCR, fluorescence quantitative RT-RCR etc., these method complex operations, consuming time longer.Loop-mediated isothermal amplification technique is that Japanese scholars Notomi is in a kind of gene isothermal amplification new technology of invention in 2000, be characterized in 6 zone design, the 3 pairs of special primers for target gene, utilize a kind of strand displacement archaeal dna polymerase to be incubated 30-60 minute in isothermal condition (about 63 DEG C), can nucleic acid amplification reaction be completed.Compared with Standard PCR, the method does not need the process such as electrophoresis and ultraviolet visualization, and sensitivity, specificity are all better than round pcr, and can realize on-the-spot high-throughput rapid detection without any need for special plant and instrument, and testing cost is low.The present invention intends utilizing LAMP technology, sets up a kind of sensitive special, the method for fast and convenient detection duck flavivirus.

Summary of the invention

The technical problem to be solved in the present invention is to provide a kind of simple to operate, test kit that result detects the RT-LAMP of duck flavivirus accurately, to overcome the deficiencies in the prior art.

For achieving the above object, adopt loop-mediated isothermal amplification technology, this technology is a kind of detection technique in nucleic acid level, has the features such as accuracy is good, highly sensitive, simple to operate in the present invention.

Test kit provided by the invention comprises following component:

1) RT-LAMP reaction solution: including concentration is 8mmol/LMgSO ₄, 5.6mmol/LdNTPMixture;

2) primer mixed solution; The primer sets detecting duck flavivirus is the ratio mixing of 5nM:5nM:40nM:40nM:20nM:20nM according to F3:B3:FIP:BIP:LF:LB;

Wherein involved detection primer sequence is:

Upstream inner primer FIP sequence:

5’-GCGGCATGTTTCAGCGACTGCATGGTTCCACGGAAGCG-3’,

Downstream inner primer BIP sequence:

5’-AACGCCCAAAAGTCCCGTCTACGGGGTTCACATTCGAGTGTG-3’.

Upstream outer primer F3 sequence:

5’-GCAGAAGGAAAACGTCCAGT-3’

Downstream outer primer B3 sequence:

5’-CCCATGTCAACCCCAGATC-3’,

Upper lantern primer LF sequence

5’-GGCTGAATAATTGTGGTAGGTGCT-3’,

Lower lantern primer LB sequence

5’-ACCGCTGAGATGGAGGATTATG-3’

3) enzyme mixture: the BstDNA polysaccharase of AMV ThermoScript II and 8000U/mL that concentration is 5U/ μ L mixes packing in 1:1 ratio;

4) visual fluorescence dyestuff.

5) DEPC water: include the coke diethyl phthalate that concentration is 1 ‰.

6) negative control: ddH2O.

7) positive control: utilize the pseudovirion that duck flavivirus E is gene constructed.

Detection method concrete steps provided by the invention are:

After gathering avian sample, the conventional kit of the positive control in sample and this test kit is extracted nucleic acid RNA, from mentioned reagent box, takes out RT-LAMP reaction solution, enzyme mixture, primer mixed solution, after DEPC water at room temperature melts, the centrifugal 5s of 2000r/min; Often pipe test reaction system adds: RT-LAMP reaction solution, 7.5 μ L; Primer mixed solution, 6 μ L; Enzyme mixture, 2 μ L; Visual fluorescence dyestuff, 1 μ L; DEPC water, 3.5 μ L; Add RNA solution and each 5 μ L of negative control of positive control and the sample prepared after processing again respectively, cover tightly pipe lid, in the centrifugal 5s of 4000r/min; PCR pipe is sequenced, puts into 63 DEG C of water-bath water-baths 1 hour or put into PCR instrument and run 63 DEG C of insulations 1 hour.

Carry out result detection after reaction terminates, specifically comprise three kinds:

(1) Visual detection methods of RT-LAMP reaction:

Negative control presents shallow safran, and positive control is fluorescent green, and sample carries out judging the negative positive according to the color of liquid in pipe after reaction.

(2) turbidity detection method of RT-LAMP reaction

After reaction terminates, all PCR pipe are carried out centrifugal 10000rpm, 1min, at the bottom of observation tube, without precipitation at the bottom of negative control pipe, positive control adularescent precipitates, and sample carries out judgement yin and yang attribute according to or without white precipitate.

(3) gel electrophoresis detection method of RT-LAMP reaction

With 1.5% agarose gel electrophoresis containing ethidium bromide (EB).Nucleic acid belt is observed under ultraviolet transilluminator.After the gel electrophoresis of positive control amplified production, as seen from the conditions of streaking of loading wells and the band of a lot of different amplification length, negative control without band, sample according to band after electrophoresis with or without carrying out judgement yin and yang attribute.

Effective principle: positive control and negative control are set up respectively.

Beneficial effect of the present invention is:

1) quick: only to need 2 hours from sample preparation to going out result; 2) in the present invention, filter out the combination of best primer concentration, overcome the deficiency that two-step approach detects duck flavivirus, achieve the object that single stage method RT-LAMP detects duck flavivirus.3) sensitive: to use more highly sensitive than traditional PCR method 10 ~ 100 times of the method; 4) special: cross reaction not to occur with other viruses of common bird, the specificity of the method, higher than traditional PCR method, has stopped the false positive results that non-specific amplification causes greatly; 5) the invention provides the reagent of good quality and the schedule of operation of optimization, therefore simple to operate, there is good reproducibility.

Accompanying drawing explanation

Fig. 1 .RT-LAMPFD dyestuff result.1. negative control; 2. positive control.

Fig. 2 .RT-LAMP agarose gel electrophoresis result.1. positive control; 2. negative control; M.DNA molecular criteria DL2000.

Fig. 3 .RT-LAMP compares with RT-PCR sensitivity.A.FD dyestuff result; B.RT-LAMP agarose gel electrophoresis result; C.RT-PCR agarose gel electrophoresis result; M.DNA molecular mass standard DL2000; The viral RNA (10 of 1-9.10 times of gradient dilution ¹-10 ⁹) 10. negative control.

The specific test result of Fig. 4 .RT-LAMP detection method.A. agarose gel electrophoresis result; B.FD dyestuff result M.DNA molecular criteria DL2000

1. duck flavivirus; 2. H 5 N 1 avian influenza hypotype; 3. duck enteritis virus; 4, virulent duck enteritis virus.

Embodiment

embodiment 1. duck flavivirus RT-LAMP detection reagent box

The composition of test kit

1, RT-LAMP reaction solution: including concentration is 8mmol/LMgSO ₄, 5.6mmol/LdNTPMixture;

2, primer mixed solution; The primer sets detecting duck flavivirus is the ratio mixing of 5nM:5nM:40nM:40nM:20nM:20nM according to F3:B3:FIP:BIP:LF:LB;

Wherein involved detection primer sequence is:

Upstream inner primer FIP sequence:

5’-GCGGCATGTTTCAGCGACTGCATGGTTCCACGGAAGCG-3’,

Downstream inner primer BIP sequence:

5’-AACGCCCAAAAGTCCCGTCTACGGGGTTCACATTCGAGTGTG-3’.

Upstream outer primer F3 sequence:

5’-GCAGAAGGAAAACGTCCAGT-3’

Downstream outer primer B3 sequence:

5’-CCCATGTCAACCCCAGATC-3’,

Upper lantern primer LF sequence

5’-GGCTGAATAATTGTGGTAGGTGCT-3’,

Lower lantern primer LB sequence

5’-ACCGCTGAGATGGAGGATTATG-3’

1) concentration optimization of primer and probe: by outer primer: inner primer: ring primer combines according to 1: 1: 1,1: 2: 1,1: 2: 2,1: 4: 2,1: 4: 4,1: 6: 4,1: 8: 4 respectively.Other conditions are optimized according to listed by table 1

Table 1RT-LAMP reaction system

Component	Final concentration
		RT-LAMP reaction solution	1×
ThermoScript II AMV	5U
		Bst archaeal dna polymerase	8U
Visual fluorescence dyestuff	1μL
		Template ribonucleic acid	5μL
Mend DEPC water extremely	25μL

Adopt said ratio to increase to extracted nucleic acid, 63 DEG C of water-baths 1 hour, often organize and all adopt multiple pipe to test.

2) optimum result: find from repeatedly revision test, outer primer after optimizing: inner primer: the optimum proportion of ring primer is 1:8:4, and wherein outer primer concentration is 5nM, and inner primer concentration is 40nM, and ring primer concentration is 20nM.

3, enzyme mixture: the BstDNA polysaccharase of AMV ThermoScript II and 8U/ μ L that concentration is 5U/ μ L mixes packing in 1:1 ratio;

4, visual fluorescence dyestuff.

5, DEPC water: include the coke diethyl phthalate that concentration is 1 ‰.

6, negative control: ddH2O.

7, positive control: utilize the pseudovirion that duck flavivirus E is gene constructed.

the method of embodiment 2. detection duck flavivirus of the present invention

One, sample preparation

Sample extracts according to the specification sheets of QIAgenRNeasyMiniKit, or adopts the RNA of equivalence to extract reagent.The RNA extracted is in-80 DEG C of preservations, for subsequent use.

Two, RT-PCR amplified reaction

(1) amplifing reagent prepares: take out after RT-LAMP reaction solution, enzyme mixture, primer mixed solution and visual fluorescence dyestuff at room temperature melt from test kit, the centrifugal 5s of 2000r/min.If required PCR pipe number is n (n=sample number+1 pipe negative control+1 pipe positive control).Each test reaction system configurations is as following table 2:

Table 2. configuration scheme

Reagent	Single reaction consumption
		RT-LAMP reaction solution	7.5 μL
Primer mixed solution	6 μL
		Enzyme mixture	2 μL
Visual fluorescence dyestuff	1 μL
		DEPC water	4.5 μL

Calculate the usage quantity of each reagent, add in a proper volume EP pipe, fully mix, each packing 20 μ L in each PCR pipe.

(2) sample after process is added

In the PCR pipe of each setting, add each 5 μ L of the RNA solution prepared in above-mentioned sample handling procedure respectively, cover tightly pipe lid, in the centrifugal 5s of 4000r/min.PCR pipe is sequenced, puts into water-bath or PCR instrument.

(3) RT-LAMP reaction

Condition is: hatch 1 hour for 63 DEG C.

Three, result judges

(1) quality control standard

1, Visual detection methods (Fig. 1)

Negative control is visual in lightcoral.

Positive control is visual in fluorescent green.

2, turbidity detection method (Fig. 1)

After negative control is centrifugal, without precipitation at the bottom of pipe.

After positive control is centrifugal, adularescent precipitation at the bottom of pipe.

3, gel electrophoresis detection method (Fig. 2)

Negative control electrophoresis is without band

Positive control electrophoresis is as seen from the conditions of streaking of loading wells and the band of a lot of different amplification length.

(2) result describes and judges

Under the prerequisite that yin and yang attribute contrast is set up, a kind of detection method is selected to judge.

1, Visual detection methods

Visual is negative in lightcoral, and visual is positive in fluorescent green.

2, turbidity detection method

After centrifugal, without precipitation at the bottom of pipe is negative, and at the bottom of pipe, adularescent is precipitated as the positive.

3, gel electrophoresis detection method

After electrophoresis, without band is negative, and as seen from the conditions of streaking of loading wells and a lot of different amplification length band be the positive.

embodiment 3, RT-LAMP method of the present invention detect specific test and the susceptibility test of duck flavivirus

The RT-LAMP detection method of foundation is detected different dilution duck flavivirus duck embryo allantoic liquid and compares with RT-PCR method, the sensitivity of assessment the method; Common avian viral is detected, to assess the specificity of the method by RT-LAMP detection method.

One, material

1. duck flavivirus RT-LAMP detection reagent box (embodiment 1)

2. bird flue virus H 5 N 1 subtype (AIV), duck enteritis virus (DEV), virulent duck enteritis virus (DVH) etc. are preserved by Dong Jian section of Shangdong Entry-Exit Inspection And Quarantine Bureau.

3SPF duck embryo: purchased from poultry institute of academy of agricultural sciences of Shandong Province

Two, the method for duck flavivirus specific test and susceptibility test is detected

1. susceptibility test

The infection allantoic fluid of duck flavivirus is done 10 ¹, 10 ², 10 ³, 10 ⁴, 10 ⁵, 10 ⁶, 10 ⁷, 10 ⁸, 10 ⁹doubly dilution, extract RNA, be divided into two parts, portion does and is RT-LAMP with test kit, and portion does conventional RT-PCR.

2. specific test

The RT-LAMP detection method utilizing this research to set up, detects AIV, DEV, DVH that all duck flavivirus and laboratory are preserved, determines the specificity of RT-LAMP method.

Three, result

1. use the viral allantoic fluid RNA of method to 10 times of gradient dilutions of RT-LAMP detection method and the RT-PCR optimized to detect, result shows, and the remolding sensitivity RT-PCR method of RT-LAMP method is high 10 times, sees Fig. 3 simultaneously.

2. for verifying the specificity of the RT-LAMP detection method set up, be that template detects with the nucleic acid of duck flavivirus, H 5 N 1 avian influenza hypotype, duck enteritis virus (DEV), virulent duck enteritis virus (DVH) respectively, result shows only duck flavivirus and can amplify band and see green fluorescence, the specificity good (Fig. 4) of method.

Nucleotides sequence list

<110> Inspection and Quarantine Technology Center, Shandong Inspection and Quarantine

<120> duck flavivirus RT-LAMP detection reagent box

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<170>PatentInversion3.5

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GCGGCATGTTTCAGCGACTGCATGGTTCCACGGAAGCG38

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AACGCCCAAAAGTCCCGTCTACGGGGTTCACATTCGAGTGTG42

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GCAGAAGGAAAACGTCCAGT20

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CCCATGTCAACCCCAGATC19

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GGCTGAATAATTGTGGTAGGTGCT24

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ACCGCTGAGATGGAGGATTATG22

Claims (1)

1. a duck flavivirus RT-LAMP detection reagent box, is characterized in that, this test kit comprises following component:

RT-LAMP reaction solution: including concentration is 8mmol/LMgSO ₄, 5.6mmol/LdNTPMixture;

2) primer mixed solution; The primer sets detecting duck flavivirus is the ratio mixing of 5nM:5nM:40nM:40nM:20nM:20nM according to F3:B3:FIP:BIP:LF:LB;

Wherein involved detection primer sequence is:

Upstream inner primer FIP sequence:

5’-GCGGCATGTTTCAGCGACTGCATGGTTCCACGGAAGCG-3’；

Downstream inner primer BIP sequence:

5’-AACGCCCAAAAGTCCCGTCTACGGGGTTCACATTCGAGTGTG-3’；

Upstream outer primer F3 sequence:

5’-GCAGAAGGAAAACGTCCAGT-3’；

Downstream outer primer B3 sequence:

5’-CCCATGTCAACCCCAGATC-3’；

Upper lantern primer LF sequence:

5’-GGCTGAATAATTGTGGTAGGTGCT-3’；

Lower lantern primer LB sequence:

5’-ACCGCTGAGATGGAGGATTATG-3’；

3) enzyme mixture: the BstDNA polysaccharase of AMV ThermoScript II and 8000U/mL that concentration is 5U/ μ L mixes packing in 1:1 ratio;

4) visual fluorescence dyestuff;

5) DEPC water: include the coke diethyl phthalate that concentration is 1 ‰;

6) negative control: ddH2O;

7) positive control: utilize the pseudovirion that duck flavivirus E is gene constructed.