CN103451157B - Method for separating new type duck reovirus from hybrid virus sample - Google Patents
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Abstract
The invention relates to a method for separating new type duck reovirus from hybrid virus sample. The method comprises the following steps of: preparing sucrose solutions with continuous gradient, preparing hybrid virus solution, preparing virus suspension, and performing density gradient centrifugation. The separation method provided by the invention can effectively obtain the single purified virus, and provides an achievable separation method for separation and purification of a clinical sample subjected to mixed infection of various viruses, particularly, in case of failure in separating an emerging strain or a variant by a conventional method, virus separation can be finished accurately and quickly by means of sucrose density gradient centrifugation, thus, the foundation of research and prevention and control on epidemic disease is laid.
Description
Technical field
The present invention relates to the separation method of virus, particularly relate to a kind of Novel duck reovirus and exhale with kind duck the method being separated Novel duck reovirus in the hybrid virus sample of the lonely vaccine strain of intestines.
Background technology
Kind duck exhales the lonely vaccine of intestines to be one of attenuated vaccine be most widely used in current kind of duck group, and the situation of this virus with poison in kind duck body is very general.Muscovy duck reovirus as RNA viruses variation quickly, is difficult to prevention and control.Therefore, the Novel duck reovirus in separation and purification biased sample exhales the lonely sick monitoring of intestines most important to kind duck.
The method of conventional separation and purification virus has serum Neutralizing test and different hosts cell proliferation experiment at present, but these two kinds of experimental programs all can not be separated the high virus strain of homology.But the homology of exhaling the antigenic determinant of the lonely vaccine strain of intestines due to Novel duck reovirus and kind duck is about 60%, and existing serum Neutralizing test and different hosts cell proliferation experiment all can not isolate separately the Novel duck reovirus in biased sample.
Serum Neutralizing test is this kind of antigen in utilizing the high antibody of specific antigen in serum and in biased sample, thus reaches the object isolating other antigens.The method be applicable to not between synantigen without the situation of antibody cross reaction, because Novel duck reovirus and kind duck exhale the antigenic determinant of the lonely vaccine strain of intestines to have the homology of about 60%, cause the probability of antibody cross reaction larger, therefore be difficult to find suitable positive serum extent of dilution can neutralize separately a kind duck and exhale the lonely vaccine strain of intestines, and retain Novel duck reovirus.
Different hosts cell proliferation experiment utilizes different virus inconsistent and wherein a kind of virus can be bred smoothly to different host's preferendums, and other virus can not be bred, through several take turns go down to posterity after, other miscellaneous diseases poison can be filtered out, obtains the single virus of purifying.Novel duck reovirus and kind duck exhale the lonely vaccine strain of intestines can breed on the chicken embryo of specific pathogen free bacterium, common duck embryo, DEF, culture condition and growth characteristics no significant difference, therefore cannot obtain the Novel duck reovirus of purifying with this separation method.
Summary of the invention
In view of this, be necessary to exhale the lonely vaccine strain of intestines to be difficult to the problem be separated for Novel duck reovirus and kind duck, a kind of method being separated Novel duck reovirus from hybrid virus sample is provided.
From hybrid virus sample, be separated a method for Novel duck reovirus, comprise the following steps:
1) prepare the sucrose solution of continuous density gradient: adopt successively from the top down concentration be 20%, 30%, 40% and 50% sucrose solution, 4 DEG C are spent the night and obtain the sucrose solution of continuous density gradient;
2) hybrid virus liquid is prepared: to the hybrid virus being separated the Novel duck reovirus that obtains and kind duck in pathological material of disease and exhaling the lonely vaccine strain of intestines, carry out enlarged culturing, obtain hybrid virus liquid;
3) prepare viral suspension: in evaporating pipe, add hybrid virus liquid, then at the bottom of pipe, add lower concentration sucrose solution, frozen centrifugation, after concentrated, precipitation is dissolved in a small amount of PBS and makes viral suspension;
4) density gradient centrifugation: viral suspension step 3) obtained adds in the sucrose solution of the continuous density gradient in step 1), after frozen centrifugation, is divided into some parts by centrifugate, obtains the sample containing single Novel duck reovirus.
Preferably, in step 1), concentration is the volume ratio of the sucrose solution of 20%, 30%, 40% and 50% is 1:1:1:1.
Preferably, in step 3), lower concentration sucrose solution is the sucrose solution of 15-25% concentration.
Preferably, the frozen centrifugation in step 3) is 40,000rpm, 4 DEG C of centrifugal 60min.
Preferably, the frozen centrifugation in step 4) is 26,500rpm, 4 DEG C of centrifugal 120min.
Preferably, the volume adding viral suspension in step 4) is the 10%-15% of the sucrose solution volume adding continuous density gradient.
Density gradient centrifugation, utilize certain medium (as cesium chloride, sucrose and saccharosan) in centrifuge tube, form continuous or discrete density gradient, virus, albumen, DNA suspension or other homogenate are placed in the top of medium, make the material aggregation of same buoyant density at same layer by the effect of gravity or centrifuge field, thus obtain certain single composition.Density gradient centrifugation can be divided into again discontinuous gradient density and continuous gradient density 2 kinds.The sucrose of different concns is normally slowly filled in centrifuge tube by discontinuous gradient density successively, forms obvious interface between each concentration, has the abrupt slope of density gradient; Continuous density gradient is that the discontinuous density gradient sucrose configured is placed on 4 DEG C of refrigerator overnight, relies on the effect of molecular diffusion, forms the density gradient of steady and continuous, and between each concentration, interface disappears, and medium density gradient is mild.
Density gradient centrifugation can be used for virus, DNA and protein purification, but is all a kind of composition and other component separating are opened.When density gradient centrifugation is used for viral purification, virus can only be separated with other non-viral impurity, can not by different virus strain high for homology separately.
Different virus varies in size due to nucleic acid substances and protein, can cause between buoyant density variant, and in separation method of the present invention, they can be dispersed in different density gradients, can obtain single virus by the sample after Fractional Collections ultracentrifugation.
The method that the present invention is separated Novel duck reovirus from hybrid virus sample effectively can obtain the virus of single purifying, for the separation and purification of the clinical sample of the multiple virus of polyinfection clinically provides practicable separation method, particularly for emerging strain or variant when ordinary method cannot be separated, the centrifugal work that can complete virus purification accurately and rapidly of sucrose continuous density gradient, for the research of epidemic disease and prevention and control lay the first stone.
Accompanying drawing explanation
Fig. 1 is that sucrose density gradient centrifugation sample RT-PCR identifies electrophorogram.
Figure 1A is that a kind duck exhales intestines lonely vaccine strain Auele Specific Primer RT-PCR to identify electrophorogram, and M:DL2000DNA Marker, Y are that a kind duck exhales the lonely vaccine strain positive control of intestines, and N1 is that a kind duck exhales the lonely vaccine strain negative control of intestines, and No. 1-28 is sample.
Figure 1B is that Novel duck reovirus Auele Specific Primer RT-PCR identifies electrophorogram, and M:DL2000DNA Marker, P are Novel duck reovirus positive control, and N2 is Novel duck reovirus negative control, and No. 1-28 is sample.
Fig. 2 is that the Novel duck reovirus sample RT-PCR of purifying identifies electrophorogram, M:DL2000DNAMarker, P is Novel duck reovirus positive control, N1 is Novel duck reovirus negative control, Y is that a kind duck exhales the lonely vaccine strain positive control of intestines, N2 is that a kind duck exhales the lonely vaccine strain negative control of intestines, and X1, Z1 are that purification of samples Novel duck reovirus Auele Specific Primer detects, and X2, Z2 are that purification of samples muscovy duck reovirus vaccine strain Auele Specific Primer detects.
Embodiment
For a better understanding of the present invention, be described further below in conjunction with embodiment and accompanying drawing.It should be noted that, in order to make the present invention more succinct, the experimental technique known by those of ordinary skill in the art, step and condition etc. can operate routinely, do not record in detail in the present invention.
The main agents used in the present invention and instrument have: sucrose, Thermo PCR instrument, AxyPrep body fluid viral DNA/RNA small volume of reagent box (Cat No.:AP-MN-BF-VNA-250), TaKaRa PrimeScript One Step RT-PCR Kit Ver.2 (TaKaRa Code:DRR055A).
Pathological material of disease in the present invention is the tissue such as liver, spleen picking up from Guangdong Province 5-10 age in days morbidity kind duck performance duck hemorrhagic necrotic hepatitis typical cytopathic in the first half of the year in 2012, and a kind duck exhales the lonely vaccine strain of intestines to be that DRV Attenuate vaccine is purchased from the smart biotechnology institute in Qingdao.
Embodiment 1
From hybrid virus sample, be separated a method for Novel duck reovirus, comprise the following steps:
1) prepare the sucrose solution of continuous density gradient: adopt successively from the top down isopyknic concentration be 20%, 30%, 40% and 50% sucrose solution, 4 DEG C are spent the night and obtain the sucrose solution of continuous density gradient;
2) prepare hybrid virus liquid: adopt ordinary method to be separated from pathological material of disease and obtain the hybrid virus that Novel duck reovirus and kind duck exhale the lonely vaccine strain of intestines, employing cell cultures mode carries out enlarged culturing to above-mentioned hybrid virus, obtains hybrid virus liquid;
3) viral suspension is prepared: in evaporating pipe, add the sucrose solution that then hybrid virus liquid add 20% concentration at the bottom of pipe be about 4-5mL, be advisable to cover at the bottom of pipe; At 40,000rpm, the centrifugal 60min of Superfreezing under the condition of 4 DEG C, is dissolved in precipitation in a small amount of PBS after concentrated and makes viral suspension; Sucrose solution wherein can play the effect of filtration to macromole impurity;
4) density gradient centrifugation: viral suspension step 3) obtained adds in the sucrose solution of the continuous density gradient in step 1), the volume adding viral suspension is the 10%-15% of the sucrose solution volume adding continuous density gradient; At 26,500rpm, under 4 DEG C of conditions, the centrifugal 120min of Superfreezing, is divided into some parts from top to bottom by centrifugate, and every part of 300 μ L, obtain the sample containing single Novel duck reovirus.
Embodiment 2RT-PCR identifies
By the sample obtained after embodiment 1 Midst density gradient centrifugation, number consecutively is No. 1-28 from top to bottom, carries out RT-PCR, and whether detect in sample is single Novel duck reovirus.
2.1RNA Template preparation
AxyPrep body fluid viral DNA/RNA small volume of reagent box (Cat No.:AP-MN-BF-VNA-250) is used to extract template ribonucleic acid ,-70 DEG C of preservations.
2.2PCR diagnostic primers
Novel duck reovirus Auele Specific Primer:
F1（SEQ?ID?No.1）：5'-ACCTCAGGATATCGCTGAAACT-3'
R1（SEQ?ID?No.2）：5'-CTCCATCCCTGCAGCACATGTAAAG-3'
Kind duck exhales the lonely vaccine strain Auele Specific Primer of intestines:
F2（SEQ?ID?No.3）：5'-TCGGACCTCGATGTGTGGTGGT-3'
R2（SEQ?ID?No.4）：5'-TTCGAGACCGACCGCACAGGTGAAT-3'
2.3PCR reaction
Reaction system: use TaKaRa PrimeScript One Step RT-PCR Kit Ver.2 (TaKaRa Code:DRR055A) PCR kit, reaction system is 25 μ L systems, specifically as shown in table 1.
Table 1PCR reaction system
Note: "---" represents nothing.
2.4PCR product is identified through 1% agarose gel electrophoresis
As shown in Figure 1, the size of target stripe is 586bp.Figure 1A is that a kind duck exhales intestines lonely vaccine strain Auele Specific Primer RT-PCR to identify electrophorogram, Y is that a kind duck exhales the lonely vaccine strain positive control of intestines, N1 is that a kind duck exhales the lonely vaccine strain negative control of intestines, yin and yang attribute contrast is all set up, in 28 samples, 1-10 sample fails to detect that a kind duck exhales the lonely vaccine strain of intestines, and 11-28 sample detection exhales the lonely vaccine strain of intestines to a kind duck.Figure 1B is that the novel lonely Auele Specific Primer RT-PCR of intestines that exhales of kind duck identifies electrophorogram, and P is Novel duck reovirus positive control, and N2 is Novel duck reovirus negative control, and yin and yang attribute contrast is all set up, and 1-28 sample all can detect Novel duck reovirus.
The purifying of embodiment 3 Novel duck reovirus
Get 2 single Novel duck reovirus samples, numbering X and Z respectively, inoculation kind DEF after filtering, in cultured continuously 3 generation, exhales the lonely vaccine strain Auele Specific Primer of intestines again to carry out RT-PCR qualification through Novel duck reovirus Auele Specific Primer and a kind duck.As shown in Figure 2, X and Z2 sample is single Novel duck reovirus sample, namely determines to be separated the virus obtaining purifying.
The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (5)
1. from hybrid virus sample, be separated a method for Novel duck reovirus, it is characterized in that, comprise the following steps:
1) prepare the sucrose solution of continuous density gradient: adopt successively from the top down concentration be 20%, 30%, 40% and 50% sucrose solution, 4 DEG C are spent the night and obtain the sucrose solution of continuous density gradient;
2) hybrid virus liquid is prepared: to the hybrid virus being separated the Novel duck reovirus that obtains and kind duck in pathological material of disease and exhaling the lonely vaccine strain of intestines, carry out enlarged culturing, obtain hybrid virus liquid;
3) prepare viral suspension: in evaporating pipe, add hybrid virus liquid, then at the bottom of pipe, add lower concentration sucrose solution, frozen centrifugation, after concentrated, precipitation is dissolved in a small amount of PBS and makes viral suspension; Described lower concentration sucrose solution is the sucrose solution of 15-25% concentration;
4) density gradient centrifugation: by step 3) obtained viral suspension adds step 1) in continuous density gradient sucrose solution in, after frozen centrifugation, centrifugate is divided into some parts, obtains the sample containing single Novel duck reovirus.
2. the method being separated Novel duck reovirus from hybrid virus sample according to claim 1, is characterized in that, step 1) in concentration be the volume ratio of the sucrose solution of 20%, 30%, 40% and 50% be 1:1:1:1.
3. the method being separated Novel duck reovirus from hybrid virus sample according to claim 1, is characterized in that, step 3) in frozen centrifugation be 40,000rpm, 4 DEG C of centrifugal 60min.
4. the method being separated Novel duck reovirus from hybrid virus sample according to claim 1, is characterized in that, step 4) in frozen centrifugation be 26,500rpm, 4 DEG C of centrifugal 120min.
5. the method being separated Novel duck reovirus from hybrid virus sample according to claim 1, is characterized in that, step 4) in the volume adding viral suspension be the 10%-15% of the sucrose solution volume adding continuous density gradient.
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| CN108977579A (en) * | 2018-08-10 | 2018-12-11 | 山东农业大学 | It is a kind of to detect the PCR kit for fluorescence quantitative for leading to the novel duck reovirus of duck spleen necrosis |
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| CN102068691A (en) * | 2010-12-24 | 2011-05-25 | 福尔生物制药股份有限公司 | Method for preparing high purity hand-foot-and-mouth disease vaccine with sucrose zonal ultracentrifugation |
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| CN102068691A (en) * | 2010-12-24 | 2011-05-25 | 福尔生物制药股份有限公司 | Method for preparing high purity hand-foot-and-mouth disease vaccine with sucrose zonal ultracentrifugation |
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| 新型鸭呼肠孤病毒RT-LAMP检测方法的建立;李兆龙 等;《第二届全国禽病分子生物技术青年学者论坛论文集》;20111101;388-394 * |
| 新型鸭呼肠孤病毒RT-PCR方法的建立与应用;王劭 等;《农业生物技术学报》;20111231;第19卷(第2期);388-392 * |
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Address after: 527439 Rugen Town, Xinxing County, Guangzhou, Guangdong Patentee after: Winson food group Limited by Share Ltd Address before: 527439 Rugen Town, Xinxing County, Guangzhou, Guangdong Patentee before: Guangdong Wens Foodstuff Group Co., Ltd. |
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