CN101838708B

CN101838708B - Method for detecting canine influenza

Info

Publication number: CN101838708B
Application number: CN2010101401769A
Authority: CN
Inventors: 李守军; 师志海; 焦培荣; 远立国
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2010-03-30
Filing date: 2010-03-30
Publication date: 2012-07-18
Anticipated expiration: 2030-03-30
Also published as: CN101838708A

Abstract

The invention belongs to the field of biotechnology and particularly relates to a method for detecting canine influenza. In the invention, a PCR detection method is adopted, and a PCR reaction system comprises the following materials: primers having sequences SEQ ID No.1 and SEQ ID No.2 respectively, as well as dNTPs, Ex Taq, 10*Ex Taq buffer and cDNA templates. At the end of a PCR reaction, whether a target strip exists is detected by electrophoresis and ultra-violet analysis, so as to determine if a dog catches canine influenza. The detection method provided by the invention has high sensitivity and specificity, is simple, convenient and quick in operation, has low requirements on the quality of initial materials and is convenient to use in clinic.

Description

A kind of method that detects the dog influenza

Technical field

The invention belongs to biological technical field, be specifically related to a kind of method that detects the dog influenza.

Background technology

The dog influenza is caused that by various influenza A viruss this virus came to light in 2004 can cause the dog influenza.Dog does not have born immunological competence to this viroid; Therefore; This transmissible disease maybe be promptly propagated between only dog; Be the high disease of a kind of sickness rate, mainly show as various respiratory road symptoms such as a nasal discharge, sneeze, cough, in clinical manifestation, also obscure mutually with respiratory tract diseases such as canine distemper, parainfluenza easily.In China dog crowd, detect H3N2 hypotype canine influenza virus and successfully be separated to virus.Yet; All also there are not effective vaccine and medicine to prevent system both at home and abroad for the dog influenza at present; The main means of controlling this disease are through to dog early detection only, detect early and are with malicious positive dog, reduce and remove the H3N2 hypotype canine influenza virus among the dog crowd thereby treat.

Characteristics of incidence to epidemic virus; Whether confirm infective virus at short notice; Help to take measures rapidly to prevent that epidemic situation from spreading, thereby be reduced in the danger of human and animal's intraindividual variation, the outburst of for example crown SARS virus and H5 subtype avian influenza and H1N1 subtype influenza.Therefore, Rapid identification mikrobe cause of disease technology will brought into play very important effect in publilc health and social hygiene and agriculture benign development process.Serious infectious diseases PCR detection techniques such as H5 subtype avian influenza are set up in succession, and under the situation that China begins to occur, the rapid detection of H3N2 hypotype canine influenza virus becomes an important research project in H3N2 hypotype dog influenza.The PCR detection method is as a kind of susceptibility height, high specificity, easy and simple to handle quick; Be widely used in clinically; At present; All set up the PCR detection method for other kind animal influenza both at home and abroad, both at home and abroad how tame pets hospital has all begun to adopt this method to carry out the multiple disease of animal to detect and diagnose.But, do not see any at present yet about utilizing the PCR detection method to detect the report of the method for canine influenza virus.Opposite, the ELISA detection kit becomes present detection canine influenza virus a kind of means commonly used.Though the ELISA detection kit is also used in clinical, ELISA detects has certain false positive, and its price is very expensive.Another shortcoming that ELISA detects be that this detection kit all is batch detection, be not very favourable to this disease that is dispersed in outburst of H3N2 hypotype dog influenza, so on the basis of its expensive price, can increase suitable cost again.Thereby cause dog detection cost only too high, pets hospital and pet owners all are difficult to accept.Moreover, single dog only or very few several dog only carry out then causing when ELISA detects the serious waste of test kit.In addition, the influence factor of ELISA detecting operation is very many and complicated, also causes result's unreliability to a certain degree.In view of H3N2 hypotype dog influenza China begins to be dispersed under the situation of generation, need set up a kind of quick, easy H3N2CIV detection method, to promote and to promote the harmony of society to stablize healthy and rapid development, also have certain society public hygienics meaning simultaneously.

Summary of the invention

The objective of the invention is to be deficiency, a kind of method of economical and practical detection canine influenza virus is provided to prior art.

The present invention realizes above-mentioned purpose through following technical scheme:

A kind of method that detects the dog influenza adopts the PCR detection method.

At first, extract dog to be measured Nasopharyngeal swabs RNA only, further synthetic cDNA, subsequent use in-20 ℃ of preservation, one of RM that reacts as next step PCR.

The extraction of Nasopharyngeal swabs RNA can be adopted Invitrogen company's T RIzol LS Reagent test kit, and specific operation process is following:

Add the Trizol Ls Reagent of 750 μ L in the 250 μ L virus liquid, (15-30 ℃) shakes up 5min under the room temperature, adds 200 μ L chloroforms again, and (15-30 ℃) shakes up 1-2min under the room temperature; At 12000g, 4 ℃ of following centrifugal 10min draw the about 500 μ L of supernatant; Add 500 μ L Virahols, again at 12000g, 4 ℃ of following centrifugal 10min; After abandoning supernatant, add 70%DEPC ethanol 1000 μ L, at 7500g; Centrifugal 5min under 4 ℃ adds 20 μ LDEPC water after the thorough drying in super clean bench, preserve down for-20 ℃.

The PCR reaction system comprises following material: two sequences are respectively primer, dNTPs, Ex Taq, 10 * Ex Taq buffer, the cDNA template of SEQ ID NO:1, SEQ ID NO:2.The volume ratio of each material is following: sequence is respectively each 0.3 part of primer solution, 2 parts of dNTPs, 0.3 part of Ex Taq, the 10 * Ex Taq buffer (Mg of SEQ ID NO:1, SEQ ID NO:2 ²⁺Plus) 2.5 parts, 2 parts of cDNA templates, finally mend to 25 parts with water.Wherein the concentration of primer solution is 20pmol/ μ L, with the sterilization distilled water as solute; DNTPs solution is that various dNTP content are 2.5mM; The concentration of Ex Taq solution is 5U/ μ L.

The PCR reaction conditions is: 95 ℃ are reacted 5min down, carry out 1 circulation; Reaction 1min under 94 ℃, 53 ℃ are reacted 1min down, and 72 ℃ are reacted 2min down, carry out 30 circulations repeatedly; 72 ℃ are reacted 10min down; 4 ℃ are reacted 12-24h down.

After the PCR reaction finished, through electrophoresis and ultra-violet analysis, detection had or not the purpose band, thereby judged whether dog is only ill.And the contrast of purpose band be through with canine influenza virus RNA as the synthetic cDNA of masterplate after, carry out that said PCR reaction and electrophoresis and ultra-violet analysis confirm.Detailed process is: in 70 ℃ of water-baths, act on 5min add the random primer of 1 μ L at the viral RNA that 5 μ L extract after, then ice bath 10min.Add HPRI 0.5 μ L subsequently, AMV 1 μ L, 5 * AMV buffer, 4 μ L, dNTPs 4 μ L, R:O 1 μ L, DEPC water 3.5 μ L, effect is 1 hour in 42 ℃ of water-baths.Take out back ice bath 2-3min and carry out the PCR reaction.Primer is diluted to 20pmol/ μ L, and the volume that each component added in the 25 μ L systems is: each 0.3 μ L of upstream and downstream primer, dNTPs 2 μ L, Ex Taq 0.3 μ L, 10 * Ex Taq buffer (Mg ²⁺Plus) 2.5 μ L, cDNA template 2 μ L, moisturizing to 25 μ L, mixing.Carry out the PCR reaction by following condition: 95 ℃ of 5min, 1 circulation; 94 ℃ of 1min, 53 ℃ of 1min, 72 ℃ of 2min, 30 circulations; 72 ℃ of 10min; 4 ℃ of 12-24h.The judgement of reaction result: through 1.5% agarose gel electrophoresis, the positive detection product presents the purpose band, and feminine gender does not then have purpose shape band; Detected result is a decidable through uv analyzer, therefore is easy at clinical application.

The reaction principle of PCR is:

Just can separate into the dna molecular of two Dan Lian when (1) double chain DNA molecule heats under closing on the temperature of boiling point, archaeal dna polymerase is template with the single stranded DNA and utilizes the synthetic newborn DNA complementary strand of four kinds of dNTPs in the reaction mixture then.In addition, archaeal dna polymerase needs a bit of double-stranded DNA equally and starts the synthetic of new chain.

(2) all providing under the situation of one section Oligonucleolide primers for each bar chain, two single stranded DNAs all can be used as the template of synthetic newborn complementary strand.Because a pair of primer of in PCR reaction, being selected for use, be according to the principle design complimentary to one another of amplification section two ends sequence, so each bar nascent strand synthetic all be that annealed site from primer begins, and extend along opposite strand.

The primer that the present invention adopted is to get to the conservative region design of the gene order of carrying the dog influenza.Application of DNA STAR (Version 5.07) and Primer Premier (Version 5.0) genetic analysis software compare the M gene order of including many canine influenza virus strains in GenBank, and at relative conservative region design special primer.Selected conservative region size is about 1000bp.It is synthetic that primer after the design is served extra large handsome company.The present invention considers many-sided factor, after screening repeatedly, has adopted this two kinds of primers.Test of many times proves that this primer specificity is strong.

In addition, each component concentrations proportioning is through drawing after repeatedly the experiment adjustment is optimized, can reaching good reaction result in the PCR reaction system of the present invention.

Compared with prior art, the present invention has following beneficial effect:

1. detection method susceptibility provided by the invention is high.The generation of PCR product increases with exponential manner, can with the target DNA of denier more than millions of times the DNA of enough check and analysis amounts that increases.This is the topmost characteristics of round pcr, and it can be analyzed micro-samples such as single copy gene, individual cells.

2. detection method high specificity provided by the invention.Since extracting heat-resisting TaqDNA polysaccharase, when thermally denature is handled by digestion, needn't in each cyclic amplification, add new enzyme again, and under comparatively high temps successive reaction, significantly improved the specificity of PCR reaction product.

3. detection method of the present invention is easy and simple to handle, quick.Only need to press finite concentration to reaction material and mix, reaction is just undertaken by the program of being imported.Whole PCR operating process to product analysis, can be accomplished all experiments from sample disposal, pcr amplification in 4h.

4. detection method of the present invention is low to the parent material specification of quality, is convenient to application clinically.

Description of drawings

Fig. 1 is the result of the inventive method detection case dog sample, and wherein M representes DNA Marker (DL2000), 1 expression positive control, 2 expression test sample, 3 expression negative controls.

Fig. 2 is the result of the inventive method detection healthy dogs sample, and wherein M representes DNA Marker (DL2000), 1 expression positive control, 2 expression test sample.

Embodiment

Below further specify content of the present invention through embodiment.

Embodiment 1

Get 1 part of the Nasopharyngeal swabs of clinical case dog, put into the centrifuge tube that 750 μ L saline water are housed, repeatedly extruding for several times, supernatant carries out RNA as viral liquid and extracts.

The concrete operations step is following:

The viral RNA of 5 μ L said extracted acts on 5min after adding the random primer of 1 μ L in 70 ℃ of water-baths, then ice bath 10min.Add HPRI 0.5 μ L subsequently, AMV 1 μ L, 5 * AMV buffer4 μ L, dNTPs 4 μ L, R:O 1 μ L, DEPC water 3.5 μ L, effect is 1 hour in 42 ℃ of water-baths.Take out back ice bath 2-3min and carry out the PCR reaction.

25 μ L systems are adopted in the PCR reaction.Testing sample and positive control and negative control carry out for three groups simultaneously.Wherein positive control is the H3N2 hypotype dog influenza RNA synthetic cDNA of institute; Negative control is the sterilization distilled water.The volume that each component added in three groups is: sequence is respectively each 0.3 μ L of primer of SEQ ID NO:1, SEQ IDNO:2; DNTPs 2 μ L; Ex Taq 0.3 μ L, 10 * Ex Taq buffer, 2.5 μ L, cDNA template 2 μ L (negative control does not add the cDNA template); Moisturizing to 25 μ L, mixing.Carry out the PCR reaction by following condition: 95 ℃ of 5min, 1 circulation; 94 ℃ of 1min, 53 ℃ of 1min, 72 ℃ of 2min, 30 circulations; 72 ℃ of 10min; 4 ℃ of 12-24h perhaps directly carry out agarose gel electrophoresis and do result's judgement.

Above-mentioned experimental group and DNA Marker (DL 2000) are through 1.5% agarose gel electrophoresis, and the result is as shown in Figure 1.

Can be known by the result: the purpose band appears in positive; Negative control does not then have purpose shape band; Testing sample has purpose shape band, is judged as the positive.

Embodiment 2

Like embodiment 1, but its sample is the Nasopharyngeal swabs of healthy dogs.

The result is as shown in Figure 2: the purpose band appears in positive; Negative control does not then have purpose shape band; Testing sample does not have purpose shape band, is judged as feminine gender.

A kind of method sequence table that detects the dog influenza

SEQUENCE?LISTING

< 110>Agricultural University Of South China

< 120>a kind of method that detects the dog influenza

<130>

<160>2

<170>PatentIn?version?3.2

<210>1

<211>23

<212>DNA

< 213>artificial sequence

<400>1

agcaaaagca?ggtagatrtt?kaa 23

<210>2

<211>26

<212>DNA

< 213>artificial sequence

<400>2

agtagaaaca?aggtagtttt?ttactc 26

Claims

1. a primer that detects the dog influenza is characterized in that sequence is SEQ ID NO:1, SEQ IDNO:2.