CN101899534A - Kit and method for detecting goose parvovirus and Muscovy duck parvovirus - Google Patents

Abstract

The invention relates to a kit for quickly identifying and diagnosing goose parvovirus (GPV) and Muscovy duck parvovirus (MDPV) in an infected Muscovy duck group by using secondary polymerase chain reaction (PCR) technology and a detection method. The kit contains Taq DNA polymerase (5U/mul L), 10*PCR buffer, Mg<2+> (10mmol/L), dNTP (10mmol/L) and two pairs of specific primers. The virus type of the infected Muscovy duck group can be identified by performing extraction and the PCR on DNA in a texture sample, so that the defect that two viruses cannot be quickly identified in the prior art is overcome. The kit has the characteristics of strong specificity, high sensitivity, quickness and the like, and is suitable for clinical quick identification and diagnosis.

Description

Be used to detect the test kit and the detection method thereof of gosling plague virus and kind duck parvovirus

Technical field

The invention belongs to biological technical field, relate to the detection kit of a kind of quick discriminating gosling plague virus and kind duck parvovirus, and relate to a kind of method of using this kind test kit to differentiate gosling plague virus and kind duck parvovirus fast.

Background technology

Gosling plague (Gosling Plague GP) claims the goose parvovirus disease again, find in Yangzhou by fixed 1 years of Chinese scholar side the earliest, and in 1961 with the goose embryo separate this disease virus (Goose Parvovirus, GPV).This disease mainly betides young goose and young kind duck in 1 monthly age, is a kind of height contagious disease fast, that mortality ratio is high of propagating.The young goose M ﹠ M of 10 ages in days can reach 100%, and the lethality rate in the susceptible group is up to 70%.This disease all has in various degree popular all over the world, extensively betide states such as China, European various countries, Israel, Japan, is one of principal disease of supporting the goose industry of harm at present.

Kind duck parvovirus (Muscovy Duck Parvovirus, MDPV) disease, China's report in 1985, France's report in 1989.3 ages in week of main infringement are with interior young bird kind duck (three weeks are sick), and the M ﹠ M height is with enteritis, diarrhoea, weak foot and the acute and subacute infectious disease of breathing and examining feature for facing.

The cause of disease goose parvovirus of two kinds of diseases and a kind duck parvovirus all belong to Parvoviridae, parvovirus belongs to.Virus is lattice arrangement under Electronic Speculum, solid and hollow two kinds of particles is arranged, no cyst membrane, diameter 20～24nm; The nucleic acid single stranded DNA, the 5.1kbp size.

Because two kinds of viruses all can infect young kind duck, and symptom is similar with pathological change, can both cause death.Thereby, how two kinds of cause of diseases are differentiated fast to diagnosing the illness it is an importance.

Summary of the invention

Purpose of the present invention just is to develop a kind of detection kit and detection method thereof of differentiating goose parvovirus and kind duck parvovirus.

Technical scheme of the present invention is: a kind of test kit that is used to detect gosling plague virus and kind duck parvovirus comprises following composition:

10 * PCR buffer, Mg2+ (10mmol/L), dNTP (10mmol/L), Taq archaeal dna polymerase (5U/ μ L), 2 pairs of Auele Specific Primers.Wherein:

First pair of primer only is that the gosling plague viral genome is special, and a kind duck parvovirus genome is non-complementary:

F1：5’-gcagg?aacaa?ttacc?ag-3’

R1：5’-acc?acctc?ccgca?ctgac-3’

Second pair of primer is two kinds of fragments that aquatic bird parvovirus genome all has:

F2：5’-cggaa?ggcac?catgc?cgccg-3’

R2：5’-cagaa?tttac?agatt?ttgag-3’

Another technical scheme of the present invention is:

Use the mentioned reagent box to detect the method for gosling plague virus and kind duck parvovirus, its step comprises:

(1) from sample to be tested, extracts viral genome: adopt the DNA extraction test kit to carry out;

(2) carry out PCR simultaneously with two pairs of primers, system is as follows:

10×pCR?buffer 5μL

Mg2+10mmol/L 3μL

dNTP?10mmol/L 1μL

Taq archaeal dna polymerase 5U/ μ L 1 μ L

Primers F 11 μ L

Primer R1 1 μ L

Primers F 21 μ L

Primer R2 1 μ L

Amplification condition is: 95 ℃ of sex change 5 minutes, 94 ℃ 1 minute, 50 ℃ 1 minute, 72 ℃ 1 minute, 30 circulations, 72 ℃ were extended 10 minutes.

(3) electrophoresis is identified: get 10 microlitre PCR products and carry out electrophoresis with 1% sepharose, if two bands that size is about 774bp and 1145bp size appear in electrophoresis result simultaneously, then be judged to be gosling plague virus and infect; If the band that size is about the 1145bp size only appears in electrophoresis result, then be judged to be a kind duck parvovirus infections; If electrophoresis result does not amplify band, then judging does not have this two kinds of virus infectiones.

The present invention compared with prior art, its advantage and positively effect show: the present invention has set up the detection kit and the detection method thereof of quick discriminating gosling plague virus and kind duck parvovirus, this test kit according to the design of aquatic bird parvovirus gene order characteristics two pairs of primers, the a pair of distinctive gene fragment of goose parvovirus that can only increase, another is to the two kinds of viral common gene fragments that can increase.The differential diagnosis technology that the present invention sets up, high specificity has very high sensitivity, can be used for the differential diagnosis of gosling plague virus and kind duck parvovirus.

The invention solves and can't differentiate gosling plague virus and kind duck parvovirus defective in the prior art fast, differential diagnosis kit that provides and detection method thereof, fast, accuracy is high, susceptibility good, can be applicable to the veterinary clinic field.

Description of drawings

Fig. 1 is that double PCR identifies GPV and MDPV electrophorogram, and wherein M is DL2000 standard molecular weight Marker, the positive GPV contrast of the 1st swimming lane, the positive MDPV contrast of the 2nd swimming lane, the negative contrast of the 3rd swimming lane, the 4th, 5 swimming lanes are that GPV infects pathological material of disease, and the 6th swimming lane is that MDPV infects pathological material of disease.

Embodiment

Following example further specifies the present invention, but should not be used as limitation of the present invention.

Embodiment 1: make the detection kit of differentiating gosling plague virus and kind duck parvovirus fast by following prescription:

10 * PCR buffer, Mg2+ (10mmol/L), dNTP (10mmol/L), Taq archaeal dna polymerase (5U/ μ L), 2 pairs of Auele Specific Primers (primer is synthetic by Invitrogen company).Wherein:

First pair of primer only is that the gosling plague viral genome is special, and a kind duck parvovirus genome is non-complementary:

F1：5’-gcagg?aacaa?ttacc?ag-3’(SEQ?ID?NO.1)

R1：5’-acc?acctc?ccgca?ctgac-3’(SEQ?ID?NO.2)

Second pair of primer is two kinds of fragments that aquatic bird parvovirus genome all has:

F2：5’-cggaa?ggcac?catgc?cgccg-3’(SEQ?ID?NO.3)

R2：5’-cagaa?tttac?agatt?ttgag-3’(SEQ?ID?NO.4)

Embodiment 2: utilize the detection kit of differentiating gosling plague virus and kind duck parvovirus fast to detect parvovirus infections situation among kind duck group

(1) reaches and extract viral genome infected tissue's (negative control) from pathological material of disease (2 parts of gosling plague virus infection pathological material of diseases, 1 part of kind of duck parvovirus infections pathological material of disease): adopt Quiagen company DNA extraction test kit to carry out;

(2) carry out PCR simultaneously with two pairs of primers, GPV is got in setting simultaneously and MDPV nucleic acid is done positive template contrast, and system is as follows:

10×PCR?buffer 5μL

Mg2+10mmol/L 3μL

dNTP?10mmol/L 1μL

Taq archaeal dna polymerase 5U/ μ L 1 μ L

Primers F 11 μ L

Primer R1 1 μ L

Primers F 21 μ L

Primer R2 1 μ L

Amplification condition is: 95 ℃ of sex change 5 minutes, 94 ℃ 1 minute, 50 ℃ 1 minute, 72 ℃ 1 minute, 30 circulations, 72 ℃ were extended 10 minutes.

(3) electrophoresis is identified: as shown in Figure 1, gosling plague virus infects pathological material of disease (the 4th, 5 swimming lane) and occurs two bands that size is about 774bp and 1145bp size simultaneously; The band that size is about the 1145bp size only appears in kind duck parvovirus infections pathological material of disease (the 6th swimming lane); And negative control (negative control) does not amplify band.

SEQUENCE?LISTING

＜110〉Yangzhou University

＜120〉be used to detect the test kit and the detection method thereof of gosling plague virus and kind duck parvovirus

<130>

<160>4

<170>PatentIn?version?3.3

 

<210>1

<211>17

<212>DNA

＜213〉artificial sequence

 

<400>1

gcaggaacaa?ttaccag 17

 

<210>2

<211>18

<212>DNA

＜213〉artificial sequence

 

<400>2

accacctccc?gcactgac 18

 

<210>3

<211>20

<212>DNA

＜213〉artificial sequence

<400>3

cggaaggcac?catgccgccg 20

 

<210>4

<211>20

<212>DNA

＜213〉artificial sequence

 

<400>4

cagaatttac?agattttgag 20

Claims (2)

1. test kit that is used to detect gosling plague virus and kind duck parvovirus comprises following composition:

10 * PCR buffer, Mg2+10mmol/L, dNTP 10mmol/L, Taq archaeal dna polymerase 5U/ μ L, 2 pairs of Auele Specific Primers, wherein:

First pair of primer is:

F1：5’-gcagg?aacaa?ttacc?ag-3’

R1：5’-acc?acctc?ccgca?ctgac-3’

Second pair of primer is:

F2：5’-cggaa?ggcac?catgc?cgccg-3’

R2：5’-cagaa?tttac?agatt?ttgag-3’。

2. the using method of the described detection kit of claim 1, its step comprises:

(1) from sample to be tested, extracts viral genome: adopt the DNA extraction test kit to carry out;

(2) carry out PCR simultaneously with two pairs of primers, system is as follows:

10×PCR?buffer 5μL

Mg2+10mmol/L 3μL

dNTP?10mmol/L 1μL

Taq archaeal dna polymerase 5U/ μ L 1 μ L

Primers F 11 μ L

Primer R1 1 μ L

Primers F 21 μ L

Primer R2 1 μ L

Amplification condition is: 95 ℃ of sex change 5 minutes, 94 ℃ 1 minute, 50 ℃ 1 minute, 72 ℃ 1 minute, 30 circulations, 72 ℃ were extended 10 minutes.

(3) electrophoresis is identified: get 10 microlitre PCR products and carry out electrophoresis with 1% sepharose, if two bands that size is about 774bp and 1145bp size appear in electrophoresis result simultaneously, then be judged to be gosling plague virus and infect; If the band that size is about the 1145bp size only appears in electrophoresis result, then be judged to be a kind duck parvovirus infections; If electrophoresis result does not amplify band, then judging does not have this two kinds of virus infectiones.

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