CN103343170B - RT-PCR (reverse transcription-polymerase chain reaction) detection kit for bovine viral diarrhea viruses - Google Patents
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Abstract
The invention relates to an RT-PCR (reverse transcription-polymerase chain reaction) detection kit for the BVDV (bovine viral diarrhea virus). A pair of primers capable of specifically amplifying the BVDV is designed according to the BVDV sequence and the CSFV (classical swine fever virus) sequence in the GenBank, the target fragment is amplified to have a length of 470bp, and the RT-PCR liquids and the RT-PCR conditions are optimized, so that the RT-PCR detection kit for the BVDV is invented. The RT-PCR detection kit for the BVDV has good specificity and high sensitivity and is easy to operate. Particularly, the RT-PCR detection kit for the BVDV can be used for specifically amplifying all the BVDVes without causing the cross reaction with other pathogens, can achieve the detection limit of 10<1.5>TCID50/0.1ml and adopts the premixed reaction liquids, so that the operation steps can be reduced, the reaction time can be shortened and the pollution risk can be reduced.
Description
Art
Bovine viral diarrhea virus RT-PCR detection kit involved in the present invention, belongs to veterinary biologics field.
Technical background
Bovine viral diarrhoea/mucosal disease is the transmissible disease caused by bovine viral diarrhea virus (Bovine Viral Diarrhea Virus, BVDV).BVDV worldwide extensively distributes, and bring huge financial loss to whole world breast, beef cattle, the animals such as goat, sheep, pig, deer and wallaby are all host and the contagium of BVDV.All may there is BVDV as bovine serum, frozen embryo, these products of ox seminal fluid in the biological products in a lot of ox source, cause the propagation of BVDV.In addition, all need in a lot of pig vaccine production process to use ox source starting material, as bovine serum, pancreatin, bull testis cell etc.If these ox source materials are polluted by BVDV, then the pollution of vaccine be can cause, the clinical symptom and the pathological change that occur being similar to swine fever with the pig of this vaccine immunity caused.
At present, detect BVDV method and mainly contain virus purification, immunofluorescent test, enzyme linked immunosorbent assay, serum neutralization test and electron microscopic examination etc., these method time and effort consumings, susceptibility is low, can not meet the requirement of clinical detection.In addition, BVDV strain and Pestivirus suis (the Classical Swine Fever Virus belonged to together, CSFV) nucleotide homology about 60%, amino acid identity about 85%, there is cross reaction in serology, be difficult to carry out discriminating diagnosis and detection to BVDV and CSFV with the Methods of Detection of Pathogens of routine.At present, in China's live vaccines of hog cholera, BVDV pollution condition happens occasionally, and brings very large threat to vaccine quality.Therefore, set up special, responsive BVDV detection kit, the BVDV that not only can carry out the susceptible animal such as pig, ox detects, and can also be applied to production of vaccine, for China's animal vaccine quality provides sound assurance as a kind of exogenous virus detection method.RT-PCR, as a kind of conventional nucleic acid detection method, has that specificity is good, susceptibility advantages of higher, is applicable to the detection of BVDV.
Summary of the invention
The object of the invention is BVDV and RT in reference GenBank, devising a pair can the primer of specific detection BVDV institute toxic strain, the object fragment length of amplification is 470bp, RT-PCR reaction solution and reaction conditions etc. are optimized, develop bovine viral diarrhea virus RT-PCR detection kit.
Technical program of the present invention lies in:
1. this test kit contains: positive control, negative control, RT-PCR reaction solution, RT-PCR reaction enzymes, primer mixed solution, without enzyme water, wherein:
Positive control: be BVDV Oregon strain MDBK passage product, virus titer is 10
5.5tCID
50/ 0.1ml, packing after formaldehyde 37 DEG C of deactivations;
Negative control: be Pestivirus suis Lapinized strain bull testis cell cultures product, through formaldehyde 37 DEG C of deactivations;
RT-PCR reaction solution and RT-PCR reaction enzymes are the precious biotech firm in Dalian product;
The preparation of primer mixed solution: be diluted to 20 μMs respectively with without enzyme water after primer synthesis, then press 1:1 mixing, the sequence of primer is:
Sequence 1, BVU:5 '-GGGGGGTAGC AACAGTGGTG AGTTCGT-3 ',
Sequence 2, BVL:5 '-TTACCCGACC TACAGTCACC TCTTTTTG-3 ';
Without enzyme water: after ultrapure water autoclaving, add the diethylpyrocarbonate (DEPC) that final concentration is 0.1%, process is spent the night, and next day, autoclaving was to remove DEPC.
2. the application of bovine viral diarrhea virus RT-PCR detection kit, is mainly used in the detection of bovine viral diarrhea virus in the detection of BVDV in suspected infection pathological material of disease and veterinary biologics ox source raw material and finished product.
Detailed description of the present invention
1. reference substance
Positive control: (virus titer is 10 for BVDV Oregon strain MDBK passage product
5.5tCID
50/ 0.1ml), after formaldehyde 37 DEG C of deactivations, be packed as 300 μ l/ manage ,-20 DEG C are frozen for subsequent use.
Negative control: be Pestivirus suis Lapinized strain (HCLV) bull testis cell culture, be packed as 300 μ l/ and manage after formaldehyde 37 DEG C of deactivations ,-20 DEG C frozen for subsequent use.
2. reagent
RT-PCR reaction solution (PrimeScript One Step RT-PCRKit) is the precious biotech firm in Dalian product; RT-PCR reaction enzymes is the precious biotech firm in Dalian product.
3. the design of primer and screening
Carry out global alignment according to 29 that GenBank downloads the CSFV full length sequences recorded both at home and abroad, 18 bovine viral diarrhea virus-I (BVDV-I) genom sequences, compare, avoid and CSFV analogous parts, find out BVDV conserved regions, the primer for conserved regions design, respectively the RNA of BVDV and CSFV is increased, finishing screen selects a pair specific amplification BVDV RNA, and with the primer pair of CSFV RNA no cross reaction.Primer sequence is as follows:
BVU:5 '-GGGGGGTAGC AACAGTGGTG AGTTCGT-3 ' (sequence 1),
BVL:5 '-TTACCCGACC TACAGTCACC TCTTTTTG-3 ' (sequence 2).
4. the determination of reaction system and condition
Get positive reference substance 200 μ l ordinary method or commercial kit extraction RNA.Carry out system preparation according to table 1, by RT-PCR product 1% agarose gel electrophoresis after reaction terminates, deposition condition is 100v30min.
Table 1 bovine viral diarrhea virus RT-PCR detection kit reaction system
Reaction conditions after optimizing is:
50 DEG C of 30min; 94 DEG C of 2min; 94 DEG C of 30sec, 55 DEG C of 30sec, 72 DEG C of 40sec, 35Cycles; 72 DEG C of 2min; 4 DEG C of preservations
Specificity and Study of Sensitivity
By bovine viral diarrhea virus RT-PCR detection kit, BVDVOregonC24V strain and NADL strain are increased, all amplify the band of 470bp size, conform to theoretical value; And HCLV strain and bull testis cell controls are without amplified band (Fig. 1).After PCR primer is checked order, compare with standard OregonC24V strain (AF091605) and NADL strain (M31182) sequence, prove that amplified fragments is BVDV distinguished sequence.
Positive reference substance MEM is carried out 10 times of gradient dilutions, each extent of dilution ordinary method extracting RNA, detects by bovine viral diarrhea virus RT-PCR detection kit.Result shows, the method can detect to 10-4 extent of dilution, then the limit of detection of this test kit is 10
1.5tCID
50/ 0.1ml.
Test kit preparations and applicatio
1. test kit preparation
1.1 reference substance
Positive control: (virus titer is 10 for BVDVOregon strain MDBK passage product
5.5tCID
50/ 0.1ml), after formaldehyde 37 DEG C of deactivations, be packed as 300 μ l/ manage ,-20 DEG C are frozen for subsequent use.
Negative control: be Pestivirus suis Lapinized strain (HCLV) bull testis cell culture, be packed as 300 μ l/ and manage after formaldehyde 37 DEG C of deactivations ,-20 DEG C frozen for subsequent use.
1.2 reagent
Without enzyme water: after ultrapure water autoclaving, add the diethylpyrocarbonate (DEPC) that final concentration is 0.1%, process is spent the night, and within second day, autoclaving is to remove DEPC, and be packed as 1mL/ pipe, 2 ~ 8 DEG C save backup.
RT-PCR reaction solution, be the precious biotech firm in Dalian product, aseptic subpackaged is that 800 μ l/ manage.-20 DEG C frozen for subsequent use.
RT-PCR reaction enzymes, be the precious biotech firm in Dalian product, aseptic subpackaged is that 50 μ l/ manage.-20 DEG C frozen for subsequent use.
The preparation of primer mixed solution: be diluted to 20 μMs respectively with without enzyme water after primer synthesis, then presses 1:1 mixing, is distributed into 50 μ l/ and manages.
2. test kit assembling (24 part/boxes)
Each reaction comprises RT-PCR reaction solution 25 μ l, and RT-PCR reaction enzymes 2 μ l, primer mixed solution 2 μ l, without enzyme water 11 μ l.Each test kit detects 24 reactions, and therefore each test kit comprises: positive control 2 is managed, and negative control 2 is managed, and RT-PCR reaction solution 1 is managed, and RT-PCR reaction enzymes 1 is managed, and primer mixed solution 1 is managed, and manages without enzyme water 1.
3. the application of test kit
The extracting of 3.1RNA and depositing
The extracting of RNA adopts commercial kit or ordinary method extracting RNA.The RNA extracted must increase in 2h; If need long-term preservation to place-70 DEG C of refrigerators.
3.2 reaction system preparations
Each component is taken out from test kit, after thaw at room temperature, the centrifugal 5s of 2000r/min.If have n sample to be checked, then required reaction sample number=n × each sample repeat number+1(positive control)+1(negative control), each test sample reaction system preparation is in table 1.
Table 1 bovine viral diarrhea virus RT--PCR detection kit reaction system
3.3 reaction conditions
50℃ 30min；94℃ 2min；
94℃ 30sec，55℃ 30sec，72℃ 40sec，35Cycles；
72 DEG C of 2min; 4 DEG C of preservations
3.4 electrophoresis
Get 10 μ l amplified production 1% sepharoses and carry out electrophoresis, deposition condition is 100v30min.
3.5 results judge
3.5.1 establishment condition is tested
There is 470bp amplified band in positive control, and negative control is without amplified band.
3.5.2 result describes and judges
1) negative: without 470bp amplified band, represent in sample without BVDV.
2) positive: to occur 470bp amplified band, represent in sample to there is BVDV.
Accompanying drawing explanation
In the PCR primer electrophoresis result figure of the different sample of Fig. 1: M.DL2000DNA Marker; Lane1.Oregon C24V+HCLVMix; Lane2.HCLV; Lane3.OregonC24V strain; Lane4.NADL strain; Lane5. bull testis cell.
The microbial information that the present invention relates to
The microorganism that the present invention relates to has bovine viral diarrhea virus BVDV Oregon strain and the weak poison of Pestivirus suis rabbitization (HCLV) strain, identifying by there being China Veterinery Drug Inspection Office, preserving and distribution, write see China Veterinery Drug Inspection Office, Chinese veterinary microorganism culture presevation administrative center. Chinese animal doctor's bacterial classification catalogue second edition 2002, Scientia Agricultura Sinica technology press, 2002, p144-145.
Positive effect of the present invention
The present invention relates to bovine viral diarrhea virus RT-PCR detection kit.The present invention is with reference to BVDV and RT in GenBank, devising a pair can the primer of specific amplification BVDV, amplification object fragment length is 470bp, is optimized, has invented bovine viral diarrhea virus RT-PCR detection kit to RT-PCR reaction solution and reaction conditions.The present invention has the following advantages: 1) specificity is good: institute's toxic strain of specific amplification BVDV, with other cause of disease no cross reactions; 2) highly sensitive: limit of detection can reach 10
1.5tCID
50/ 0.1ml; 3) simple to operate: to adopt reaction premixed liquid, reduce operation steps, Reaction time shorten, decreasing pollution risk.The BVDV that this test kit can be used for pig and ox infects detection, also can be used for the BVDV pollution detection of bovine material in production of vaccine, for animal vaccine quality safety provides sound assurance.
Embodiment
The present embodiment is for better the present invention being described, is not construed as limiting the invention.
Embodiment 1
---clinical detection
Bovine viral diarrhea virus RT-PCR detection kit is adopted to produce 94 parts of swine Fever Vaccine finished products and the work in-process sample of live vaccines of hog cholera producer to 8, simultaneously using bovine viral diarrhea virus fluorescence quantitative RT-PCR detecting kit (BVDV-MGB-RT-PCRkit) (see note) as meeting, the clinical expansion feasibility of assessment the method.
The extracting of 1.RNA and depositing
The extracting of RNA adopts commercial kit or adopts conventional RNA method for extracting.
2. reaction system preparation
Each component is taken out from test kit, after thaw at room temperature, the centrifugal 5s of 2000r/min.Each test sample reaction system preparation sees the following form:
3. reaction conditions
50℃ 30min；94℃ 2min；
94℃ 30sec，55℃ 30sec，72℃ 40sec，35Cycles；
72 DEG C of 2min; 4 DEG C of preservations
4. electrophoresis
Get 10 μ l amplified production 1% agarose gel electrophoresis, deposition condition is 100v30min.
5. result judges
There is 470bp amplified band (shown in sequence 3 sequence) in positive control, and negative control is without amplified band.
To 94 parts of pattern detection, bovine viral diarrhea virus RT-PCR detection kit detects 18 parts of positives, and positive rate is 19.1%(18/94), wherein 8 parts is finished product vaccine, recall rate is 22.2%(8/36), 10 parts is work in-process, and recall rate is 17.2%(10/58); BVDV-MGB-RT-PCR kit detects 20 parts of BVDV positives altogether, and positive rate is 21.3%(20/94).The coincidence rate of the two is 97.9%, illustrates that this detection kit and BVDV-MGB-RT-PCR kit have higher coincidence rate.
Note:
Bovine viral diarrhea virus fluorescence quantitative RT-PCR detecting kit (BVDV-MGB-RT-PCRkit) operation instruction
(1) RNA extracting and deposit
The extracting of RNA adopts commercial kit or adopts conventional RNA method for extracting.The RNA extracted must increase in 2h; If need long-term preservation to place-70 DEG C of refrigerators.
(2) preparation of amplification system
Each component is taken out from test kit, after room temperature is melted, the centrifugal 5s of 2000r/min.If have n sample to be checked, then required reaction sample number=n × each sample repeat number+1(positive control)+1(negative control), each test sample reaction system preparation is in table 2.
Table 2BVDV-MGB-RT-PCRkit reaction system
(3) nucleic acid amplification and detection
Above-mentioned PCR pipe is put into fluorescent PCR detector, and order put by record sample.
Cycling condition is arranged:
First stage, reverse transcription 50 DEG C/30min;
Subordinate phase, denaturation 95 DEG C/3min;
Phase III, 88 DEG C/8s, 60 DEG C/35s, 40 circulations, 60 DEG C gather FAM fluorescent signal.
After testing inspection terminates, according to the Dynamic Fluorescence signal curve gathered and CT value synthetic determination result.
(4) result judges
1) setting of threshold value
Threshold setting principle adjusts according to noise of instrument situation, is as the criterion with the vertex of threshold line just above normal negative sample amplification curve.
2) establishment condition is tested
Negative control is without CT value and without amplification curve, and the CT value of positive control should occur typical amplification curve 15.0 ~ 25.0.Otherwise this time experiment is false.
3) result describes and judges
1. negative: without CT value and without typical amplification curve, represent in sample without Pestivirus suis.
2. positive: CT value≤35 and occur typical amplification curve, there is Pestivirus suis in expression sample.
3. suspicious: CT value >35 and occur that the sample of typical amplification curve should detect again.If CT value≤35 and typical amplification curve person are positive, otherwise be negative.
Claims (1)
1. a bovine viral diarrhea virus RT-PCR detection kit, is characterized in that this test kit contains: positive control, negative control, RT-PCR reaction solution, RT-PCR reaction enzymes, primer mixed solution, without enzyme water, wherein:
Positive control: be BVDV Oregon strain MDBK passage product, virus titer is 10
5.5tCID
50/ 0.1ml, packing after formaldehyde 37 DEG C of deactivations;
Negative control: be Pestivirus suis Lapinized strain bull testis cell cultures product, through formaldehyde 37 DEG C of deactivations;
RT-PCR reaction solution and RT-PCR reaction enzymes are the precious biotech firm in Dalian product;
The preparation of primer mixed solution: be diluted to 20 μMs respectively with without enzyme water after primer synthesis, then press 1:1 mixing, the sequence of primer is:
Sequence 1, BVU:5 '-GGGGGGTAGC AACAGTGGTG AGTTCGT-3 ',
Sequence 2, BVL:5 '-TTACCCGACC TACAGTCACC TCTTTTTG-3 ';
Without enzyme water: after ultrapure water autoclaving, add the diethylpyrocarbonate (DEPC) that final concentration is 0.1%, process is spent the night, and next day, autoclaving was to remove DEPC.
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| CN105695637A (en) * | 2016-04-19 | 2016-06-22 | 福清市默克兽医院 | RT-PCR-RFLP method for differentiating bovine viral diarrhea virus types 1 and 2 |
| CN107881260A (en) * | 2017-11-17 | 2018-04-06 | 扬州大学 | Bovine viral diarrhea virus carries out the method for separating identification in a kind of cow's serum |
| CN108251561A (en) * | 2018-04-04 | 2018-07-06 | 贾云云 | It is a kind of to be used for bovine viral diarrhea virus now separation strains and the primer sets and kit of vaccine strain antidiastole |
| CN113403256B (en) * | 2021-06-16 | 2023-04-18 | 中国农业科学院特产研究所 | Cell line capable of stably producing bovine viral diarrhea virus antigen and preparation method of antibody colloidal gold test strip |
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