CN107058630A - A kind of multiple RT PCR detection methods of 3 kinds of genotype of bovine parainfluenza type-3 virus - Google Patents
A kind of multiple RT PCR detection methods of 3 kinds of genotype of bovine parainfluenza type-3 virus Download PDFInfo
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Abstract
According to the bovine parainfluenza type-3 virus delivered on Genbank（Bovine parainfluenza 3 virus, BPI3V）The HN genes of 3 kinds of genotype separation strains carry out sequence comparing analysis, with reference to the respective HN gene orders conserved regions design specific primer of 3 genotype separation strains NM09, TVMDL15, SD0835, the multiple RT PCR methods for 3 kinds of genotype that BPI3V is detected in same system are established.Method can detect that A type BPI3V length is 150bp, the specific fragment that Type B BPI3V length is 253bp and c-type BPI3V length is 342bp simultaneously, and the detection of minimum positive plasmid is respectively 0.89 × 104copies/µL、0.92×104copies/µL、1.53×104Copies/ μ L, with good stability and specificity.
Description
Technical field
The invention belongs to field of virus detection, and in particular to the detection method of viral infectious, more particularly to a kind of
The detection method and application field of 3 kinds of genotype of bovine parainfluenza type-3 virus.
Background technology
Bovine parainfluenza is a kind of acute, contagious disease as caused by bovine parainfluenza type-3 virus, using respiratory symptom as
Main, single viral infection symptoms are lighter, other virus and bacterias of mixed infection or can cause serious in the presence of stress factor
Clinical symptoms even result in the mortality of infected cattle.Bovine parainfluenza type-3 virus（The virus of Bovine parainfluenza 3,
BPI3V）, it is the sub-thread minus-stranded rna virus of paramyxovirus section Respirovirus, genome is by about more than 15000 nucleotides groups
Into, at least encode 6 kinds of structural proteins.It is divided into BPI3V Gene As type, BPI3V genes Type B and BPI3V gene Cs type 3 before the virales
Individual genotype.After bovine parainfluenza type-3 virus is separated first from nineteen fifty-nine by the Reisinger in the U.S. etc. from calf kidney,
Separated successively by many countries in the world.The domestic report first on BPI3V is to there is respiratory tract in damp grace of child in 2001 etc.
Doubtful bovine parainfluenza is found after the dead ox cut open inspection of symptom.It is in 2009, by Liu Peng to be split into BPI3V in China cows for the first time
Deng separation Inner Mongol separation strains (NM09) and BPI3V Gene A types, Chinese scholar Zhu are accredited as by Chinese scholar Wen etc. in 2012
Deng separating and identify BPI3V gene C types Shandong separation strains (SD0835).China is currently without being separated to BPI3V gene Type Bs.
Up to the present, many scholars establish the RT-PCR method for detection BPI3V both at home and abroad, but without 3 for BPI3V
Plant the RT-PCR detection method of hypotype.As both at home and abroad to the widely studied of BPI3V, more and more areas are found to have BPI3V
Prevalence, and the serosurvey in the multiple provinces of China displayed that bovine parainfluenza type-3 virus antibody positive rate was higher in recent years, its
Middle Inner Mongol seroprevalence is up to 84.31%（Huo Zhiyun, 2012）With 95.20%（Zhang Xinxin, 2016）.In view of the Inner Mongol is partly
Area's bovine parainfluenza serosurvey positive rate is high, but still insufficient to its Etiological.Multiple RT-PCR is operated with it simultaneously
Easy, quick advantage is usually used in clinical practice.HN gene of this research to establish BPI3V establishes detection as target sequence
BPI3V and the multiple RT-PCR detection method for distinguishing its 3 kinds of genotype.The detection side of effective practicality is provided for the sick inspection and quarantine
Method.
The content of the invention
According to the 3 of the BPI3V delivered on Genbank kinds of genotype separation strains NM09（Accession number：JQ063064)、
TVMDL15（Accession number：KJ647284)、SD0835（Accession number：HQ530153) respective HN genes carry out sequence comparing analysis,
3 pairs of specific primers are devised, the multiple RT-PCR detection method of detection BPI3V 3 kinds of genotype are established, so as to realize pair
BPI3V efficient, quick, accurate detection and genotyping.
It is an object of the invention to except the clinical detection that can be used for BPI3V by multiplex RT-PCR method, moreover it is possible to reflect simultaneously
Not Jian Ce BPI3V 3 kinds of genotype.
Bovine parainfluenza type-3 virus is detected it is an object of the invention to provide a kind of multiple RT-PCR（BPI3V）3 kinds of genotype
Primer, the primer is as shown in Table 1.
Bovine parainfluenza type-3 virus is detected it is an object of the invention to provide a kind of multiple RT-PCR（BPI3V）3 kinds of genotype
Kit, the kit include detection bovine parainfluenza type-3 virus（BPI3V）The primer of 3 kinds of genotype.
The present invention specifically provides a kind of multiple RT-PCR detection bovine parainfluenza type-3 virus（BPI3V）3 kinds of genotype detections
Method, is comprised the following steps that.
1. the design and synthesis of primer and probe：
According to bovine parainfluenza type-3 virus A type separation strains NM09, Type B separation strains TVMDL15, the c-type separation delivered on Genbank
Strain SD0835 respective HN Gene sequence comparisons analyses, the specific primer using the Software for Design of Oligo 6.0, primer transfers to treasured
Bioengineering (Dalian) Co., Ltd synthesizes, and amplification length is respectively：150bp、253bp、342bp.
2. the preparation of positive colony plasmid template
The μ L of venom 200 are taken, are extracted according to the specification of Takara viral DNAs/RNA extracts kits.A types BPI3V and Type B
BPI3V positive colonies plasmid is by precious bioengineering（Dalian）Co., Ltd synthesizes.Using c-type BPI3V cDNA, A type BPI3V and
Type B BPI3V positive colonies plasmid enters performing PCR reaction respectively, concurrently sets negative control（ddH2O）.Reaction uses 25 μ L body
System includes：The μ L of Mix 12.5, each 1.0 μ L of upstream and downstream primer, the μ L of DNA profiling 1.0 plus ddH2O to 25 μ L.Loop parameter is：95
DEG C min of pre-degeneration 5；95 DEG C of 20s, 50 DEG C~52 DEG C 30s, 72 DEG C of 40s, totally 35 circulations, last 72 DEG C of extensions 8min；Purpose
Fragment length is respectively 150bp, 253bp, 342bp,
C-type BPI3V cDNA PCR primer, which is reclaimed, to be connected after kits into pMD19-T carriers, conversion to DH5a large intestines
Angstrom uncommon bacterium, chooses the c-type positive colony plasmid identified and verified by Sequence analysis through double digestion and is used as its standard positive.
3. multiple RT-PCR reaction system and parameter：
Added in 50 μ L systems the 5 μ L of μ L, dNTP of high-fidelity enzyme 1 μ L, 10 × PCR Buffer 5, MgSO4 2 μ L,（BPI3Va、
BPI3Vb, BPI3Vc primer press 4:4:1 mixing）Each 1.5 μ L of upstream and downstream mix primer, the μ L of DNA profiling 3.0, ddH2O 31 μ L.
Loop parameter is：95 DEG C of min of pre-degeneration 5；95 DEG C of 20s, 51.5 DEG C of 30s, 72 DEG C of 40s, totally 35 circulations, last 72 DEG C of extensions
8min；Purpose fragment is 150bp+253bp+342bp.
In the present invention, numbering is JQ063064, KJ647284, HQ530153 BPI3V 3 kinds of genes in reference gene storehouse
Type separation strains NM09, TVMDL15, SD0835 HN gene orders design primer, and its complete genome sequence length is respectively
15474bp, 15456bp, 15474bp, those of ordinary skill in the art can be known by the channel of the public.
In the present invention, the C genotype cDNA using BPI3V amplifies the fragment that length is 342bp with c-type primer as template,
The fragment is cloned into pMD-19T carriers, obtains standard positive template.
In the present invention, the A types and Type B positive colony plasmid gene using BPI3V are expanded as template with A types and Type B primer
Increase the fragment for that length is 150bp and 253bp, confirm that synthetic standards positive template can use.
Meanwhile, the present invention fully takes into account, when BPI3V A types, Type B and the template of c-type three simultaneously expand when, can compete enzyme,
Primer, influence is produced on amplification, and research is optimized in the reaction condition and detection lower bound to coamplification system.Triple RT-PCR
System is then according to 4:1:1 proportions.Response parameter is：95℃5 min；95 DEG C of 20s, 51.5 DEG C of 30s, 72 DEG C of 40s, totally 35
Individual circulation；Last 72 DEG C of extensions 8min.
Further the present invention is to the specificity of infectious bovine rhinotrachetis virus detection method, sensitivity and may be repeated
Confirmation.
The amplification system provided with the present invention has expanded infectious bovine rhinotrachetis virus, Mycoplasma bovis, bovine viral respectively
Property diarrhea virus, PPR etc. nucleic acid samples, do not occur band, show that this method has preferable specificity.
Choose pMD19-T-HNa, pMD19-T-HNb, pMD19-T-HNc concentration be respectively 28ng/ μ L, 30ng/ μ L,
51ng/ μ L mixing positive plasmid carries out 10 times of doubling dilutions successively, and 9 gradients of dilution are respectively that template is expanded, knot
Fruit understands that the minimum positive plasmid detection that mix primer is expanded to hybrid template is respectively 0.89 × 104copies/µL、0.92×
104copies/µL、1.53×104Copies/ μ L, show that this method has good sensitivity.
Respectively with pMD19-T-HNa, pMD19-T-HNb, pMD19-T-HNc, pMD19-T-HNa+pMD19-T-HNb,
pMD19-T-HNb + pMD19-T-HNc、pMD19-T-HNa + pMD19-T-HNc、pMD19-T-HNa + pMD19-T-HNb
+ pMD19-T-HNc is template, carries out the amplification per 3 repetitions of class template, and amplified band is stable, shows that this method has good
Repeatability.
By implementing the specific content of the invention of the present invention, following beneficial effect can be reached.
The RT-PCR method that BPIV3 3 kinds of genotype are distinguished for identification is to be established first, be can be appreciated that by this method
The BPI3V information such as genotype is infected in livestock group, the research of viruses molecule epidemiology is further carried out with this and corresponding
Vaccine development.This method detection is quick and convenient, and the detection time of traditional isolation of virus at least will be in more than 7d, the invention
Detection time include sample pre-treatments to acquisition testing result within 3h.Detection sensitivity is high, and this method can detect 1pg's
Recombinant plasmid.Specificity is good, by infectious bovine rhinotrachetis virus, Mycoplasma bovis, bovine viral diarrhea virus, small ruminate
The nucleic acid samples such as epizootic disease detect that only BPIV3 positive templates amplify expected band.It is reproducible, by being mixed respectively to 7 kinds
Three repeatability detections are carried out with single template, band obtains uniformity result.This method can carry out qualitative detection and quickly divide
Type, method flow is simple, it is easy to operate, and can quickly grasp, as long as possessing molecular biology mechanism knowledge, without special training just
It can be rapidly completed.With the inventive method is to A types BPI3V viruses cDNA, c-type BPI3V virus cDNA and mixes its cDNA as mould
Plate is expanded to verify the accuracy of this method, while the clinical 33 parts of nose swabs collected and 12 parts of ox lungs are carried out with the system
Detection, obtains expected results.
This method can differentiate detection BPIV3 3 genotype simultaneously, have reality to the research of correlated virus inspection and quarantine technology
The reference on border.The genotype information of the BPI3V in livestock group is can be appreciated that by this method, while the milk raised to some areas
Ox carries out the research of viruses molecule epidemiology, Prevalence of diseases is understood with this, to prevent and controlling bovine parainfluenza to provide number
According to and technical support.This method can be widely applied for inspection and quarantining for import/export department, animal and veterinary department and cultivation unit, be
Effective prevention and control of epidemic disease are significant.
Brief description of the drawings
Fig. 1：Viral 3 kinds of hypotype substance PCR amplifications：Wherein 1 be negative control, 2,3,4 be BPI3Va, BPI3Vb,
BPI3Vc amplified fragments（342bp、150bp、253bp）, M is DL500marker.
Fig. 2：3 kinds of hypotype positive colony plasmid multiplex PCR results of virus：Wherein 1 be negative control, 2,3,4 be BPI3Va,
BPI3Vb, BPI3Vc amplified fragments（150bp、253bp、342bp）, 5,6,7 be BPI3Va+BPI3Vb, BPI3Vb+
BPI3Vc, BPI3Va+BPI3Vc amplified fragments（150bp+253bp、253bp+342bp、150bp+342bp）, 8 be BPI3Va
+ BPI3Vb+BPI3Vc amplified fragments（150bp+253bp+342bp）, M is DL500marker.
Fig. 3：Multiplex RT-PCR method sensitivity result of the test：Wherein 1 is negative control, and 2 ~ 10 be pMD19-T-HNa:
0.89×1010copies/µL~0.89×102copies/µL,pMD19-T-HNb:0.92×1010copies/µL~0.92×
102copies/µL,pMD19-T-HNc:1.53×1010copies/µL~1.53×102copies/µL;M is
DL500marker。
Fig. 4：Multiplex RT-PCR method specific test result：Wherein 1 is negative control, and 2 be positive control, 3~9 difference
For infectious bovine rhinotrachetis virus, goat capripoxvirus, sheep pox virus, bovine viral diarrhea virus, sheep of virus, small anti-
Hay epizootic disease virus, Mycoplasma bovis, M is DL500marker.
Fig. 5：Multiplex RT-PCR method replica test result：Wherein 1~3 is BPI3Va, and 4~6 be BPI3Vc, and 7~9 are
BPI3Vb, 10~12 be BPI3Va+BPI3Vb, and 13~15 be BPI3Vb+BPI3Vc, and 16~18 be BPI3Va+BPI3Vc, 19
~21 be BPI3Va+BPI3Vb+BPI3Vc, and 22 be negative control, and M is DL500marker.
Fig. 6：Testing result of the multiplex RT-PCR method to viral nucleic acid：Wherein 1 is negative control, and 2 be positive control, 3
It is BPI3Vc for BPI3Va+BPI3Vc, 4；5 be BPI3Va, and M is DL500marker.
Embodiment
Embodiment one：The design of 3 kinds of genotype multiple RT-PCR detection method primer and probes of bovine parainfluenza type-3 virus with
Synthesis：
According to bovine parainfluenza type-3 virus A type separation strains NM09, Type B separation strains TVMDL15, the c-type separation delivered on Genbank
Strain SD0835 respective HN Gene sequence comparisons analyses, the specific primer using the Software for Design of Oligo 6.0, primer transfers to treasured
Bioengineering (Dalian) Co., Ltd synthesizes, and amplification length is respectively：150bp、253bp、342bp.Primer generation in the present invention
Number sequence is shown in Table 1, and numbering is JQ063064, KJ647284, HQ530153 BPI3V 3 kinds of genes wherein in reference gene storehouse
Type separation strains NM09, TVMDL15, SD0835 HN gene orders design primer, and its complete genome sequence length is respectively
15474bp, 15456bp, 15474bp, those of ordinary skill in the art can be known by the channel of the public.
The primer sequence of table 1 multiplex RT-PCR amplification, 3 kinds of BPI3V genotype genes
Embodiment two：The preparation of positive colony plasmid template
The μ L of venom 200 are taken, are extracted according to the specification of Takara viral DNAs/RNA extracts kits.A types BPI3V and Type B
BPI3V positive colonies plasmid is by precious bioengineering（Dalian）Co., Ltd synthesizes.Enter performing PCR using c-type BPI3V cDNA anti-
Should, concurrently set negative control（ddH2O）.Reaction is included using 25 μ L system：The μ L of Mix 12.5, each 1.0 μ of upstream and downstream primer
L, the μ L of DNA profiling 1.0 plus ddH2O to 25 μ L.Loop parameter is：95 DEG C of min of pre-degeneration 5；95 DEG C of 20s, 52 DEG C of 30s, 72 DEG C
40s, totally 35 circulations, last 72 DEG C of extensions 8min；Purpose fragment length is 342bp.PCR primer is reclaimed after kits
Connect into pMD19-T carriers, conversion to DH5a EHECs, choose and expand identification through PCR and tested by Sequence analysis
Card positive colony plasmid is used as its standard positive template.
1. the PCR amplification identifications of recombinant plasmid：3 genotype recombinant plasmids take 1 μ L to be template respectively, add Premix12.5
μ L, each 1 μ L of upstream and downstream primer (10pmoL), moisturizing enter performing PCR amplification to 25 μ L, and 2% agarose gel electrophoresis is detected, as a result
Referring to accompanying drawing 1.
2. the sequencing identification of recombinant plasmid：PCR is expanded to the bacterium solution incubated overnight for being accredited as positive plasmid, send company to survey
Sequence.Agarose gel electrophoresis is understood, carries out substance PCR reactions by template of BPI3V A types, Type B and c-type recombinant plasmid respectively,
150bp, 253bp and 342bp that size is consistent fragment are amplified, sequencing identification is correct, identified correct recombinant plasmid
As positive template.
Embodiment three：The foundation of multiplex RT-PCR amplification system
Using single template and hybrid template, the component of wherein hybrid template is isoconcentration mixing, concurrently sets negative control
（ddH2O）.Reaction uses 50 μ L system, including：1 μ L, 10 × PCR Buffer of high-fidelity enzyme 5 μ L, dNTP 5 μ L, MgSO4
2µL、（BPI3Va, BPI3Vb, BPI3Vc primer press 4:4:1 mixing）Each 1.5 μ L of upstream and downstream mix primer, the μ of DNA profiling 3.0
L、ddH2O 31µL.Reaction condition is：95 DEG C of min of pre-degeneration 5；95 DEG C of 20s, 51.5 DEG C of 30s, 72 DEG C of 40s, totally 35 circulations,
Last 72 DEG C of extensions 8min.As a result show, each self-template of primer pair has preferable specificity, and Gel electrophoresis results are referring to accompanying drawing
2。
Example IV：Sensitivity experiment, specific test and replica test
It is respectively 28ng/ μ L, 30ng/ μ L, 51ng/ μ L to choose pMD19-T-HNa, pMD19-T-HNb, pMD19-T-HNc concentration
Mixing positive plasmid carry out 10 times of doubling dilutions successively, 9 gradients of dilution are respectively that template is expanded, and take the mark of preparation
Zhunyang property mixes plasmid as positive control, while setting up negative control.2% agarose coagulates electrophoresis detection.Reaction result is by accompanying drawing
3 understand that the sensitivity that mix primer is expanded to hybrid template is higher, to pMD19-T-HNa, pMD19-T-HNb, pMD19-
T-HNc minimum positive plasmid detection is respectively 0.89 × 104copies/µL、0.92×104copies/µL、1.53×
104copies/µL.Specific test is with infectious bovine rhinotrachetis virus, Mycoplasma bovis, bovine viral diarrhea virus, small anti-
The grade nucleic acid samples of hay epizootic disease enter performing PCR reaction by the reaction system and response parameter after optimization.Simultaneously set up negative control and
Positive control, 2% agarose coagulates electrophoresis detection.Reaction result by accompanying drawing 4 understand except positive control occur 3 purpose bands in addition to, its
It is remaining not occur band including negative control, show that this law specificity is high.Respectively with pMD19-T-HNa, pMD19-T-HNb,
pMD19-T-HNc、pMD19-T-HNa + pMD19-T-HNb、pMD19-T-HNb + pMD19-T-HNc、pMD19-T-HNa +
PMD19-T-HNc, pMD19-T-HNa+pMD19-T-HNb+pMD19-T-HNc are template, are carried out per 3 repetitions of class template
Amplification, while setting up negative control and positive control, 2% agarose coagulates electrophoresis detection.Reaction result is understood to expand by accompanying drawing 5
Band is stable, shows that this method has good repeatability.
Embodiment five：Testing result of the multiplex RT-PCR method to viral nucleic acid and clinical sample
Using built multiplex RT-PCR method, with A types BPI3V viruses cDNA, c-type BPI3V virus cDNA and mix its cDNA and make
Amplify 150bp, 342bp, 150bp+342bp purpose fragment respectively for template, it was confirmed that the accuracy of this method.As a result join
See accompanying drawing 6.33 parts of nose swab samples of the clinic detected using this law and 11 parts of ox lung samples are feminine gender.
Above example further illustrates present disclosure, but should not be construed as limiting the invention.Without departing substantially from this
In the case of spirit and essence, the modifications or substitutions done to the inventive method, step or condition belong to the present invention's
Category.
Claims (2)
1. a kind of multiple RT-PCR detects bovine parainfluenza type-3 virus（BPI3V）The method of 3 kinds of genotype, including detection ox sidestream
Feel 3 types virus（BPI3V）The primer of 3 kinds of genotype.
2. described in 3 kinds of genotype primer be used for 3 kinds of genotype of clinical detection bovine parainfluenza type-3 virus.
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Cited By (2)
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| CN107988431A (en) * | 2017-12-13 | 2018-05-04 | 广西壮族自治区兽医研究所 | A kind of RT-PCR amplimers of quick detection 3 type of bovine parainfluenza virus and its application |
| CN108315491A (en) * | 2018-04-20 | 2018-07-24 | 山东师范大学 | RPA- Sidestream chromatographies detection primer, probe and the kit of 3 type of bovine parainfluenza virus |
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| CN103627816A (en) * | 2013-08-26 | 2014-03-12 | 中国农业科学院兰州兽医研究所 | Multiplex RT-PCR detection kit for porcine reproductive and respiratory syndrome virus and application thereof |
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| CN107988431A (en) * | 2017-12-13 | 2018-05-04 | 广西壮族自治区兽医研究所 | A kind of RT-PCR amplimers of quick detection 3 type of bovine parainfluenza virus and its application |
| CN108315491A (en) * | 2018-04-20 | 2018-07-24 | 山东师范大学 | RPA- Sidestream chromatographies detection primer, probe and the kit of 3 type of bovine parainfluenza virus |
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