CN112921118A - Accurate prevention and control method for infectious spleen and kidney necrosis virus disease of micropterus salmoides - Google Patents
Accurate prevention and control method for infectious spleen and kidney necrosis virus disease of micropterus salmoides Download PDFInfo
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Abstract
The invention discloses an accurate prevention and control method for infectious spleen and kidney necrosis virus diseases of micropterus salmoides. The scheme is that a magnetic bead method virus DNA/RNA nucleic acid extraction kit (LBT-VDEK) is adopted, a 16-hole fluorescence quantitative PCR instrument (LBT-16QPCR) and matched equipment thereof are utilized, a PCR-fluorescent probe technology is adopted, quantitative detection is carried out on a specific DNA nucleic acid fragment of the Micropterus salmoides infectious spleen and kidney necrosis virus, and the method is simple, convenient and rapid, good in specificity and high in sensitivity; the modern traditional Chinese medicine technology and the microecological technology are adopted to prevent and treat the micropterus salmoides carrying the infectious spleen and kidney necrosis virus, so that the death rate can be effectively reduced, the accurate prevention and control can be achieved, the infectious spleen and kidney necrosis virus disease of the micropterus salmoides can be comprehensively, systematically and integrally solved, the application prospect is very wide, and the practical value is very high.
Description
Technical Field
The invention relates to a aquatic animal disease prevention and control technology, and mainly relates to an accurate prevention and control method for infectious spleen and kidney necrosis virus diseases of micropterus salmoides.
Background
The Micropterus salmoides is a famous and high-quality fresh water culture variety in China. With the success of artificial propagation and seedling raising technology of micropterus salmoides, the micropterus salmoides breeding industry is developed rapidly, and meanwhile, the disease problem is increasingly serious. The micropterus salmoides diseases mainly comprise viral diseases, bacterial diseases and parasitic diseases. Wherein, the virus diseases have fast prevalence and high incidence, and no effective control measure exists at present, thus seriously influencing the development of the micropterus salmoides breeding industry. In recent years, large-area fulminant epidemic disease occurs in the cultured micropterus salmoides, which leads to massive death of micropterus salmoides and causes serious loss. At present, the breeding yield of the micropterus salmoides is large, the disease occurs in main breeding areas, the disease occurs in summer, the water temperature is 25-30 ℃ during the disease, the adult fish is damaged, the lethality rate is high, great economic loss is caused to the micropterus salmoides breeding industry, and the healthy development of the industry is seriously threatened. Among them, infectious spleen and kidney necrosis virus (infectious spleens and kidney necrosis virus) has a serious influence on the culture of Micropterus salmoides. After the micropterus salmoides are infected with the virus, symptoms are usually not obvious, or bleeding, exophthalmos or obvious blood coagulation on the liver exist on the body surface. How to prevent and treat infectious spleen and kidney necrosis virus diseases of micropterus salmoides becomes an important problem which is urgently needed to be solved by the micropterus salmoides breeding industry at present.
At present, no specific treatment method for the virus disease of the micropterus salmoides exists, and the promotion of healthy culture of the micropterus salmoides is the most effective prevention measure, and the method mainly depends on early rapid detection. Symptoms of different diseases of aquatic animals can be similar, symptoms are difficult to observe in the early stage of the disease occurrence, and the disease occurrence is difficult to accurately predict mostly by experience or pathogen detection. The traditional aquatic disease detection method has the defects of long detection time, few detection indexes, untimely information transmission and the like, the detection level and means are different, the detection method and the result are unstable, the rapid and accurate detection of the aquatic epidemic disease is limited, the disease treatment is delayed, and the great economic loss is caused. At present, the detection method for the micropterus salmoides virus mainly comprises the traditional histology method, the electron microscopy technology, the biological indication method, the biochemical detection method, the immunological method (mainly comprising the fluorescent antibody technology, the enzyme-linked immunosorbent assay (ELISA), the immunoelectron microscopy technology, the cell culture method, the molecular biology method (mainly comprising the molecular hybridization method and the Polymerase Chain Reaction (PCR)), and the like.
Aquatic diseases have the characteristics of easy infection, high epidemic speed and the like, and pathogenic microorganisms of the aquatic diseases are various and are updated and mutated quickly. The traditional detection methods such as microscopic examination, plate coating and the like cannot carry out rapid diagnosis and quantitative detection on pathogens due to technical limitations, and cannot meet the detection requirements of aquatic pathogens at the present stage. The LAMP method which is a rapid detection technology and appears in the market at present cannot accurately and comprehensively detect the existing diseases due to the problems of high false positive rate, difficult primer design and the like. By referring to a large number of relevant documents, patents, standards and the like related to molecular detection, the principle, reaction time, detection mode, operability and the like of the detection methods such as common PCR, RAA, LAMP, qPCR and the like are compared, analyzed and summarized, and the qPCR is determined to be the detection method which is most suitable for rapid and accurate diagnosis of the important epidemic diseases of the aquatic products in the basic-level farms. The fluorescent quantitative PCR detector has the advantages of simple operation, quick detection, touch screen operation of the detector, friendly human-computer interface and capability of simultaneously completing detection of various pathogens. The detection instrument is movable and portable, is operated in the field, is convenient to transport by a matched box body, and does not need to be corrected before the detection of the whole machine. The detection reagent is developed and customized for service, the cost of subsequent consumables is low, and the consumables can be independently developed according to the requirements of users.
The existing aquatic animal disease prevention and control technologies including micropterus salmoides mostly only meet the requirements of detecting and detecting related pathogens, a series of comprehensive technologies of corresponding prevention and treatment measures are seriously lacked, and an accurate disease prevention and control method of quantitative rapid detection and traditional Chinese medicine technical intervention (or microecological intervention) is urgently needed to be established.
Disclosure of Invention
The invention aims to provide an accurate prevention and control method for infectious spleen and kidney necrosis virus diseases of micropterus salmoides aiming at the defects in the prior art.
The invention realizes the aim through the following technical scheme:
the method for accurately preventing and controlling the infectious spleen and kidney necrosis virus disease of the micropterus salmoides comprises the following steps:
1. extracting virus DNA/RNA;
2. fluorescent quantitative rapid detection;
3. the modern Chinese medicine technology and the micro-ecological technology intervene.
Wherein, the following operation method is preferably selected for the extraction of the virus DNA/RNA in the step 1: :
(1) transfer 1mL of lysis solution to a centrifuge tube containing 100. mu.L or 100mg of sample. Vortex well to dissolve the mixed sample.
(2) Centrifuging at 10,000rpm for 3 minutes, transferring 200 mu L of supernatant into a new centrifuge tube, adding 15 mu L of magnetic bead mixed solution and 400 mu L of lysis solution, vortexing for 30 seconds, mixing uniformly, standing for 3 minutes, putting on a magnetic rack after mixing uniformly, standing for aggregating magnetic beads, and discarding the supernatant.
(3) The centrifuge tube was removed from the magnetic stand, 500. mu.L of Wash 1 was added, and vortexed for 10 seconds.
(4) The centrifuge tube was placed on a magnetic stand and left to stand for 30 seconds, and after the magnetic beads were completely adsorbed, the liquid was carefully aspirated away.
(5) The centrifuge tube was removed from the magnetic stand, 500. mu.L of Wash 2 was added, and vortexed for 10 seconds.
(6) The centrifuge tube was placed on a magnetic stand and left to stand for 30 seconds, and after the magnetic beads were completely adsorbed, the liquid was carefully aspirated away.
(7) And (4) centrifuging for a short time, placing the centrifugal tube on a magnetic frame, standing for 10 seconds, and completely sucking residual liquid.
(8) The centrifuge tubes were placed on a magnetic stand, the lid was opened, and the tubes were air-dried at room temperature for 5 minutes.
(9) The tube was removed, 100. mu.L of eluent was added, and vortexed for 10 seconds.
(10) Placing the centrifuge tube on a magnetic frame, standing, absorbing magnetism for 2 min, transferring the eluent to a new centrifuge tube, and storing at-20 deg.C.
(1) after the detection kit was taken out and sufficiently thawed, 23. mu.L of each PCR reaction solution was added to each PCR reaction tube.
(2) Then respectively adding 2 mul of DNA/RNA extracting solution of the sample to be detected, respectively adding 2 mul of negative control and positive control into the remaining two PCR reaction tubes,
(3) finally, 20. mu.L of mineral oil was added to each reaction tube.
(4) Placing the mixture into a fluorescent quantitative PCR instrument after short-time centrifugation, and setting an operation reaction program according to the requirements of the detection kit: running for 5min at 55 ℃ for 1 cycle, running for 30s at 95 ℃ for 1 cycle, running for 40 cycles at 95 ℃ for 5s and running for 20s at 60 ℃, and finally collecting fluorescence.
(5) And after the operation reaction is finished, click data analysis is carried out, and whether the detected sample carries the disease gene and the carrying amount are judged according to the cycle value of the final amplification curve.
Step 3, the following operation method is preferably selected for the modern Chinese medicine technology and the micro-ecological technology intervention:
the infectious spleen and kidney necrosis virus treatment scheme of the Micropterus salmoides comprises the following steps:
in the culture process, if the cultured weever in the California is detected to carry infectious spleen and kidney necrosis virus and cause a large amount of death of the weever through fluorescent quantitative PCR detection, the treatment can be carried out in the following way.
The feed feeding is stopped first.
On days 1 and 2, pouring water for two days by 'Shanze Yihao 500 mL/mu Ten-materia medica Shenlu 1000 mL/mu'; (the process is mainly to improve the anti-stress capability and the immunity of the micropterus salmoides)
On days 3 and 4, pouring water for two days by 'Shang Du Qing No. 2 No. 40 g/mu'. (the process is mainly to specifically treat the Micropterus salmoides carrying infectious spleen and kidney necrosis virus to reduce the content of the Micropterus salmoides carrying virus.)
On days 5 and 6, povidone iodine solution (for aquatic products) is used for 330 mL/mu plus the herbal Shenlu for 1000 mL/mu, and splashing is continuously carried out for two days. (the process is mainly to further treat the infectious spleen and kidney necrosis virus of the micropterus salmoides to reduce the content of the infectious spleen and kidney necrosis virus and enhance the immunity of the micropterus salmoides, thereby increasing the antibody against the infectious spleen and kidney necrosis virus and reducing the death amount.)
After the fish with infectious spleen and kidney necrosis of the micropterus salmoides is treated in the above mode, the number of dead fish is greatly reduced, even reduced to zero death.
Step 4 is that the steps (1), (2) and (3) firstly provide a method for accurately preventing and controlling the infectious spleen and kidney necrosis virus diseases of the micropterus salmoides based on the quantitative rapid detection and the traditional Chinese medicine technical intervention (or microecological intervention).
Compared with the prior art, the invention has the following beneficial effects:
the method adopts a magnetic bead method virus DNA/RNA nucleic acid extraction kit (LBT-VDEK), utilizes a 16-hole fluorescence quantitative PCR instrument (LBT-16QPCR) and matched equipment thereof, adopts a PCR-fluorescent probe technology to carry out quantitative detection on the specific DNA nucleic acid fragment of the Micropterus salmoides infectious spleen and kidney necrosis virus, and has the advantages of simplicity, rapidness, good specificity and high sensitivity; the modern traditional Chinese medicine technology and the microecological technology are adopted to prevent and treat the micropterus salmoides carrying the infectious spleen and kidney necrosis virus, so that the death rate can be effectively reduced, the accurate prevention and control can be achieved, the infectious spleen and kidney necrosis virus disease of the micropterus salmoides can be comprehensively, systematically and integrally solved, the application prospect is very wide, and the practical value is very high. Description of the attached tables and drawings
FIG. 1 is a quantitative value table corresponding to the cycle peak value of the infectious spleen and kidney necrosis virus of Micropterus salmoides detected by fluorescent quantitative PCR;
FIG. 2 is a diagram of the peak cycle of infectious spleen and kidney necrosis virus of Micropterus salmoides detected by fluorescent quantitative PCR.
Detailed Description
Example 1
In 22 days 12 months in 2020, the death of the Micropterus salmoides cultured by a certain Foshan farmer for about 20 days is about 600 per day, the death caused by the infectious spleen and kidney necrosis virus is detected by fluorescent quantitative PCR, and the treatment is carried out according to the following treatment scheme:
the feed feeding is stopped first.
On days 1 and 2, pouring water for two days by 'Shanze Yihao 500 mL/mu Ten-materia medica Shenlu 1000 mL/mu';
on days 3 and 4, pouring water for two days by 'Shang Du Qing No. 2 No. 40 g/mu'.
On days 5 and 6, povidone iodine solution (for aquatic products) is used for 330 mL/mu plus the herbal Shenlu for 1000 mL/mu, and splashing is continuously carried out for two days.
Client feedback: the number of deaths within 6 days after administration varied significantly: the death rate is reduced from 600 death pieces per day at the beginning to several death pieces per day, and the death amount is finally reduced to zero.
Example 2
9 and 8 days in 2020, when some raiser in Henan breaks down and cultivates Lateolabrax japonicus for about 2 months, the death of the Lateolabrax japonicus occurs, the death number is about 800 per day, the death caused by the infectious spleen and kidney necrosis virus is detected by fluorescent quantitative PCR, and the treatment is carried out according to the following treatment scheme:
the feed feeding is stopped first.
On days 1 and 2, pouring water for two days by 'Shanze Yihao 500 mL/mu Ten-materia medica Shenlu 1000 mL/mu';
on days 3 and 4, pouring water for two days by 'Shang Du Qing No. 2 No. 40 g/mu'.
On days 5 and 6, povidone iodine solution (for aquatic products) is used for 330 mL/mu plus the herbal Shenlu for 1000 mL/mu, and splashing is continuously carried out for two days.
Client feedback: number of deaths within 6 days after dosing: 800 to end/400 to end/200 to end/100 to end/tens of ends/few ends, the death rate eventually falls to within a few ends.
Example 3
In 25 months 8 in 2020, the death of the Micropterus salmoides cultured by a certain farmer in Zhongshan in Guangdong for about 3 months occurs, the death number is about 400 per day, the death caused by the infectious spleen and kidney necrosis virus is detected by fluorescent quantitative PCR, and the treatment is carried out according to the following treatment scheme:
the feed feeding is stopped first.
On days 1 and 2, pouring water for two days by 'Shanze Yihao 500 mL/mu Ten-materia medica Shenlu 1000 mL/mu';
on days 3 and 4, pouring water for two days by 'Shang Du Qing No. 2 No. 40 g/mu'.
On days 5 and 6, povidone iodine solution (for aquatic products) is used for 330 mL/mu plus the herbal Shenlu for 1000 mL/mu, and splashing is continuously carried out for two days.
Client feedback: the number of deaths within 6 days after administration varied significantly: from 400 deaths per day at first to a final reduction in the amount of deaths to several days.
The above case analysis can yield: the quantitative rapid detection of the trocars on the infectious spleen and kidney necrosis viruses of the micropterus salmoides and the effective verification of the traditional Chinese medicine technical intervention treatment scheme in the actual culture process can be popularized and used in the aspects of prevention, judgment and treatment of the infectious spleen and kidney necrosis viruses of the micropterus salmoides, so that the loss of the infectious spleen and kidney necrosis viruses to the culture of the micropterus salmoides in the culture process can be greatly reduced.
Claims (5)
1. The precise prevention and control method for the infectious spleen and kidney necrosis virus disease of the micropterus salmoides is characterized by comprising the following steps:
(1) extracting virus DNA/RNA;
(2) fluorescent quantitative rapid detection;
(3) the modern Chinese medicine technology and the micro-ecological technology intervene.
2. The method for accurately preventing and controlling the infectious spleen and kidney necrosis virus disease of the micropterus salmoides according to claim 1, wherein the specific operation of the virus DNA/RNA extraction in the step (1) is as follows:
(1) transfer 1mL of lysis solution to a centrifuge tube containing 100. mu.L or 100mg of sample. Vortex well to dissolve the mixed sample.
(2) Centrifuging at 10,000rpm for 3 minutes, transferring 200 mu L of supernatant into a new centrifuge tube, adding 15 mu L of magnetic bead mixed solution and 400 mu L of lysis solution, vortexing for 30 seconds, mixing uniformly, standing for 3 minutes, putting on a magnetic rack after mixing uniformly, standing for aggregating magnetic beads, and discarding the supernatant.
(3) The centrifuge tube was removed from the magnetic stand, 500. mu.L of Wash 1 was added, and vortexed for 10 seconds.
(4) The centrifuge tube was placed on a magnetic stand and left to stand for 30 seconds, and after the magnetic beads were completely adsorbed, the liquid was carefully aspirated away.
(5) The centrifuge tube was removed from the magnetic stand, 500. mu.L of Wash 2 was added, and vortexed for 10 seconds.
(6) The centrifuge tube was placed on a magnetic stand and left to stand for 30 seconds, and after the magnetic beads were completely adsorbed, the liquid was carefully aspirated away.
(7) And (4) centrifuging for a short time, placing the centrifugal tube on a magnetic frame, standing for 10 seconds, and completely sucking residual liquid.
(8) The centrifuge tubes were placed on a magnetic stand, the lid was opened, and the tubes were air-dried at room temperature for 5 minutes.
(9) The tube was removed, 100. mu.L of eluent was added, and vortexed for 10 seconds.
(10) Placing the centrifuge tube on a magnetic frame, standing, absorbing magnetism for 2 min, transferring the eluent to a new centrifuge tube, and storing at-20 deg.C.
3. The method for accurately preventing and controlling the infectious spleen and kidney necrosis virus disease of the micropterus salmoides according to claim 1, wherein the specific operation of the fluorescence quantitative rapid detection in the step (2) is as follows:
(1) after the detection kit was taken out and sufficiently thawed, 23. mu.L of each PCR reaction solution was added to each PCR reaction tube.
(2) Then respectively adding 2 mu L of DNA/RNA extracting solution of the sample to be detected, and respectively adding 2 mu L of negative control and positive control into the rest two PCR reaction tubes.
(3) Finally, 20. mu.L of mineral oil was added to each reaction tube.
(4) Placing the mixture into a fluorescent quantitative PCR instrument after short-time centrifugation, and setting an operation reaction program according to the requirements of the detection kit:
running for 5min at 55 ℃ for 1 cycle, running for 30s at 95 ℃ for 1 cycle,
run for 40 cycles of 5s at 95 ℃ and 20s at 60 ℃ and finally fluorescence was collected.
(5) And after the operation reaction is finished, click data analysis is carried out, and whether the detected sample carries the disease gene and the carrying amount are judged according to the cycle value of the final amplification curve.
4. The method for accurately preventing and controlling the infectious spleen and kidney necrosis virus disease of the micropterus salmoides according to claim 1, wherein the step (3) of the intervention of modern Chinese medicine technology and microecology technology is as follows:
the infectious spleen and kidney necrosis virus treatment scheme of the Micropterus salmoides comprises the following steps:
in the culture process, if the cultured weever in the California is detected to carry infectious spleen and kidney necrosis virus and cause a large amount of death of the weever through fluorescent quantitative PCR detection, the treatment can be carried out in the following way.
The feed feeding is stopped first.
On days 1 and 2, pouring water for two days by 'Shanze Yihao 500 mL/mu Ten-materia medica Shenlu 1000 mL/mu'; (the process is mainly to improve the anti-stress capability and the immunity of the micropterus salmoides)
On days 3 and 4, pouring water for two days by 'Shang Du Qing No. 2 No. 40 g/mu'. (the process is mainly to specifically treat the Micropterus salmoides carrying infectious spleen and kidney necrosis virus to reduce the content of the Micropterus salmoides carrying virus.)
On days 5 and 6, povidone iodine solution (for aquatic products) is used for 330 mL/mu plus the herbal Shenlu for 1000 mL/mu, and splashing is continuously carried out for two days. (the process is mainly to further treat the infectious spleen and kidney necrosis virus of the micropterus salmoides to reduce the content of the infectious spleen and kidney necrosis virus and enhance the immunity of the micropterus salmoides, thereby increasing the antibody against the infectious spleen and kidney necrosis virus and reducing the death amount.)
After the fish with infectious spleen and kidney necrosis of the micropterus salmoides is treated in the above mode, the number of dead fish is greatly reduced, even reduced to zero death.
5. The method for accurately preventing and controlling the infectious spleen and kidney necrosis virus disease of the micropterus salmoides according to claim 1, wherein the steps (1), (2) and (3) firstly provide an overall solution based on the quantitative rapid detection and the technical intervention (or microecological intervention) of traditional Chinese medicine for accurately preventing and controlling the infectious spleen and kidney necrosis virus disease of the micropterus salmoides.
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