CN110616281A - Method for quantitatively detecting Luo lake virus by using real-time fluorescent RT-PCR - Google Patents
Method for quantitatively detecting Luo lake virus by using real-time fluorescent RT-PCR Download PDFInfo
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
Abstract
The invention discloses a method for quantitatively detecting lake ro virus by using real-time fluorescence RT-PCR, and particularly relates to the technical field of lake ro virus detection, and the method comprises the following specific steps: s1, separating the Luo lake virus; s2, extracting virus RNA in the sample; s3, establishing a RT-PCR detection method; s4, establishing a real-time fluorescent quantitative PCR detection method; s5, establishing morphological and anatomical characteristics to identify the infected individual. The virus molecular biology inspection and identification method can provide the virus detection service and issue an inspection report for aquaculture processing and trade enterprises, can provide technical service for government supervision departments to carry out epidemic situation monitoring and prevention, participate in epidemic prevention and control work of official and aquatic enterprises and provide technical support and epidemic situation confirmation, and can be used for cooperating with the aquaculture enterprises and prevention and control agent production enterprises to develop the Rou lake virus prevention and treatment agent after the inspection and identification method is established, so that the method has wide development prospect.
Description
Technical Field
The invention relates to the technical field of lake Luo virus detection, in particular to a method for quantitatively detecting lake Luo virus by using real-time fluorescence RT-PCR.
Background
Tilapia mossambica is a very important edible fish, and the second largest aquaculture species in the world. The worldwide tilapia breeding and catching yield reaches 640 million tons in 2015, the value is about 98 hundred million dollars, the global trade amount reaches 18 hundred million dollars, the yield is continuously increased in 2016 and 2017, as an important animal protein source, the tilapia has the advantages of wide distribution range, strong adaptability, short growth period, wide eating quality, strong disease resistance and the like, is considered as one of the 'pillars' for global food safety and nutrition, and provides food, employment, domestic and export income for millions of people including many young farmers.
Recently, the food and agriculture organization of the united nations issues a special alarm of tilapia lake viruses (TiLV), lake viruses are epidemic in six countries around the world, in the areas of Galega, south America, Asia and middle east, at present, whether the lake viruses can be transmitted by freezing tilapia products is uncertain, but the food and agriculture organization indicates that the lake viruses are possibly more widely distributed than the known lake viruses at present and pose serious threat to the global tilapia culture, the lake viruses can grow at 24-33 ℃ and have the optimal temperature of 25 ℃, so the attack can be caused in 5-10 months every year, the Food and Agriculture Organization (FAO) indicates that tilapia producing countries need to keep alert, the world animal health organization aquatic animal health code is followed when tilapia trade is carried out, the countries should implement an active monitoring plan to determine whether the lake viruses exist or not and determine the geographical range of infection, and to determine risk factors that help contain transmission, CraigWatson, university of florida, university of farming, indicates that there is a high probability that the world animal health Organization (OIE) will list the lake luo virus as a "reporter virus". Once implemented, the importer of aquatic products will carry out import inspection on the susceptible species, such as live fish. According to the feedback of a plurality of tilapia exports in Yangjiang local city, the tilapia in countries such as African CoteDewa which is exported at present requires the release of Chinese official quarantine certification.
In view of great economic significance of tilapia in China and the world and serious harmfulness of Luo lake viruses, the tilapia has been listed as a quarantine item for product entry in multiple countries in the world, but no Luo lake virus quarantine identification method and no effective treatment method exist in published documents at present, according to the current research situation, systematic research on Luo lake viruses is not deep enough, a relevant immunology and molecular biology detection method is not formally established, and in view of the great transmissibility and harmfulness of the viruses, investigation and research on epidemic propagation conditions of the viruses are necessary to be carried out, and technical support of rapid screening and accurate identification methods is needed, so a method for quantitatively detecting the Luo lake viruses by using real-time fluorescence RT-PCR is needed.
Disclosure of Invention
In order to overcome the defects of the prior art, the embodiment of the invention provides a method for quantitatively detecting the Luo lake virus by using real-time fluorescence RT-PCR, and the method can provide detection service for detecting the virus and issuing an inspection report for aquaculture processing and trade enterprises, can provide technical service for carrying out epidemic situation monitoring and prevention and control for government supervision departments, participates in epidemic prevention and control work of official and aquatic enterprises and provides technical support and epidemic situation confirmation, and can be used for cooperating with the aquaculture enterprises and preventive and therapeutic agent production enterprises to develop a preventive and therapeutic agent for the Luo lake virus after the method to be detected is established, so that the method has a wide development prospect.
In order to achieve the purpose, the invention provides the following technical scheme: a method for quantitatively detecting Luo lake virus by using real-time fluorescence RT-PCR comprises the following specific steps:
s1, separating the Luo lake virus:
s1.1, a treatment mode of suspicious case samples: the collected suspected sample needs to be transported at low temperature in an insulation box with more than half of the volume of ice bags and good insulation effect, and the temperature is kept at 0-4 ℃;
s1.2, a virus separation method: liver, kidney, spleen, intestine, gill and brain tissue of suspected individuals were obtained under laboratory sterile conditions, and the samples were homogenized with a tissue grinder into a paste according to a 1: suspending the dilution of 10 in appropriate cell culture solution, incubating at 30-37 deg.C for 10-15min, centrifuging in a centrifuge, and collecting the supernatant containing cells and placing in an EP tube;
s2, extracting virus RNA in the sample: viral RNA was extracted using the CTAB method or commercial kit;
s3, establishment of RT-PCR detection method:
s3.1, designing upstream and downstream primers according to the Luo lake virus registered in Genbank;
s3.2, carrying out reverse transcription on the extracted RNA, and carrying out nucleic acid amplification and identification after synthesizing DNA;
s4, establishment of a real-time fluorescent quantitative PCR detection method:
s4.1, designing a primer and a probe according to a specific sequence of the Rou lake virus gene NC-029930 registered in Genbank;
s4.2, synthesizing single-chain small fragment DNA according to the DNA sequence, splicing the single-chain small fragment DNA into a complete double-chain DNA fragment by a PCR method, synthesizing an NC-029930 (Tipapia) gene (465bp), connecting the synthesized gene fragment to a pMD19-T vector, and constructing a positive plasmid; taking the In-Fusion product, converting the In-Fusion product into E.coli CompetentCellJM109, and storing for later use;
carrying out PCR on E.coli CompetentCellJM109 colonies by using M13-47/RV-M primers, and detecting the length of an insert fragment in a contained plasmid;
coli compentcelljmm 109 colonies were subjected to plasmid DNA extraction and sequencing to verify positive plasmid sequences;
s4.3, under the condition that a positive sample of the Luo lake virus cannot be obtained, the positive plasmid can be used as a positive control of a detection experiment;
s4.4, amplifying by using the positive plasmid as a template and the designed primer and probe, and verifying the correctness of the primer, the probe and the template;
s4.5, carrying out gene sequencing on the amplified product, inputting the amplified product into a Genbank for comparison, and determining the amplified product as a specific sequence of the Luo lake virus;
s4.6, confirming through experiments, designing primers and probes; positive controls were constructed and all were effective. The detection method can be used for carrying out molecular biological identification on the Luo lake virus;
s5, establishing a morphological anatomical feature to identify the infected individual: finally establishing the quarantine identification method of the lake Luo virus by combining the morphological anatomy and molecular biology methods through the method.
In a preferred embodiment, whether antibiotics are required to be added to prevent contamination or not can be considered according to the cell culture result in step S1.2, so as to improve the cell survival rate.
In a preferred embodiment, the centrifugation temperature in step S1.2 is 0-4 ℃, the centrifuge rotation speed is 8000-.
In a preferred embodiment, in step S3, the double-labeled fluorescent probe method is used, and the primers designed in step S3.1 are: an upstream primer: TiLV-PFSeq: 5'TCACCCCACTCTATATTATC3', downstream primer: TiLV-PRSeq: 5'AGTCGGAAGATCAAGAAG3', primers, probes designed in step S4.1: an upstream primer: TiLV-PFSeq: 5'TCACCCCACTCTATATTATC3', downstream primer: TiLV-PRSeq: 5'AGTCGGAAGATCAAGAAG3', probe: (FAM) CCGCTCTCGTCAGCACCATA(TAMRA).
In a preferred embodiment, in step S3.2, reverse transcription and amplification can be performed in a single reaction system by using a one-step method, and reaction conditions are designed and optimized according to the characteristics of equipment and reverse transcriptase.
In a preferred embodiment, the reverse transcription step in step 3.2 is:
a. adding DEPC water, primers and extracted viral RNA to an EP tube;
b. centrifuging in a centrifuge, and heating in 100 deg.C water bath for 1 min;
c. then adding 0.5ul dNTP, 2ul 5 Xbuffer and 1ul AMV reverse transcriptase, then placing the mixture into a centrifuge for slight centrifugation, sealing the opening of the EP pipe by using a sealing film, then carrying out water bath at 42 ℃, and then carrying out RT at 90 ℃;
d. inactivating AMV in 100 deg.C boiling water bath for 3 min.
In a preferred embodiment, nucleic acid amplification is performed in step S3.2 using a nucleic acid amplification apparatus.
The invention has the technical effects and advantages that:
the invention establishes a sampling method, a nucleic acid extraction method and a molecular biology detection series method of the suspected sample of the Luo lake virus, combines a molecular biology detection confirmation result, establishes an apparent and anatomical feature identification method of the virus, provides a virus detection service and issues an inspection report for aquaculture processing and trade enterprises, can provide a technical service for the government supervision department to carry out epidemic situation monitoring and prevention, and participates in epidemic prevention and control work of official and aquatic enterprises and provides technical support and epidemic situation confirmation.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1:
the invention provides a method for quantitatively detecting lake Luo virus by using real-time fluorescence RT-PCR, which comprises the following steps:
s1, separating the Luo lake virus:
s1.1, a treatment mode of suspicious case samples: more than half of the volume of the collected suspected sample needs to be provided with an ice bag, the heat preservation with good heat preservation effect can consider whether antibiotics need to be added according to the cell culture result to prevent and control pollution, the cell survival rate is improved, the suspected sample is transported in a box at low temperature, and the temperature is kept at 0 ℃;
s1.2, a virus separation method: liver, kidney, spleen, intestine, gill and brain tissue of suspected individuals were obtained under laboratory sterile conditions, and the samples were homogenized with a tissue grinder into a paste according to a 1: suspending the dilution of 10 in a proper cell culture solution, wherein the centrifugation temperature is 0 ℃, the rotation speed of a centrifuge is 8000r/min, the centrifugation time is 10min, incubating at 30 ℃ for 10min, placing in the centrifuge for centrifugation, and sucking the supernatant containing cells and placing in an EP tube after centrifugation;
s2, extracting virus RNA in the sample: extracting virus RNA by using a CTAB method;
s3, establishment of RT-PCR detection method:
s3.1, designing an upstream primer and a downstream primer according to the Rou lake virus registered in Genbank by using a double-label fluorescent probe method: an upstream primer: TiLV-PFSeq: 5'TCACCCCACTCTATATTATC3', downstream primer: TiLV-PRSeq: 5'AGTCGGAAGATCAAGAAG 3';
s3.2, carrying out reverse transcription on the extracted RNA, synthesizing DNA, and then carrying out nucleic acid amplification and identification by using a nucleic acid amplification instrument, or completing the reverse transcription and amplification in a reaction system by adopting a one-step method, and designing and optimizing reaction conditions according to the characteristics of equipment and reverse transcriptase, wherein the reverse transcription step is as follows:
a. adding DEPC water, primers and extracted viral RNA to an EP tube;
b. centrifuging in a centrifuge, and heating in 100 deg.C water bath for 1 min;
c. then adding 0.5ul dNTP, 2ul 5 Xbuffer and 1ul AMV reverse transcriptase, then placing the mixture into a centrifuge for slight centrifugation, sealing the opening of the EP pipe by using a sealing film, then carrying out water bath at 42 ℃, and then carrying out RT at 90 ℃;
d. inactivating AMV in 100 deg.C boiling water bath for 3 min;
s4, establishment of a real-time fluorescent quantitative PCR detection method:
s4.1, designing primers and probes according to specific sequences of NC-029930 gene of the Rou lake virus registered in Genbank: an upstream primer: TiLV-PFSeq: 5'TCACCCCACTCTATATTATC3', downstream primer: TiLV-PRSeq: 5'AGTCGGAAGATCAAGAAG3', probe: (FAM) CCGCTCTCGTCAGCACCATA(TAMRA);
s4.2, synthesizing single-chain small fragment DNA according to the DNA sequence, splicing the single-chain small fragment DNA into a complete double-chain DNA fragment by a PCR method, synthesizing an NC-029930 (Tipapia) gene (465bp), connecting the synthesized gene fragment to a pMD19-T vector, and constructing a positive plasmid; taking the In-Fusion product, converting the In-Fusion product into E.coli CompetentCellJM109, and storing for later use;
carrying out PCR on E.coli CompetentCellJM109 colonies by using M13-47/RV-M primers, and detecting the length of an insert fragment in a contained plasmid;
coli compentcelljmm 109 colonies were subjected to plasmid DNA extraction and sequencing to verify positive plasmid sequences;
s4.3, under the condition that a positive sample of the Luo lake virus cannot be obtained, the positive plasmid can be used as a positive control of a detection experiment;
s4.4, amplifying by using the positive plasmid as a template and the designed primer and probe, and verifying the correctness of the primer, the probe and the template;
s4.5, carrying out gene sequencing on the amplified product, inputting the amplified product into a Genbank for comparison, and determining the amplified product as a specific sequence of the Luo lake virus;
s4.6, confirming through experiments, designing primers and probes; positive controls were constructed and all were effective. The detection method can be used for carrying out molecular biological identification on the Luo lake virus;
s5, establishing a morphological anatomical feature to identify the infected individual: finally establishing the quarantine identification method of the lake Luo virus by combining the morphological anatomy and molecular biology methods through the method.
Example 2:
the invention provides a method for quantitatively detecting lake Luo virus by using real-time fluorescence RT-PCR, which comprises the following steps:
s1, separating the Luo lake virus:
s1.1, a treatment mode of suspicious case samples: more than half of the volume of the collected suspected sample needs to be provided with an ice bag, the heat preservation effect is good, whether antibiotics need to be added to prevent and control pollution or not can be considered according to the cell culture result, the cell survival rate is improved, the suspected sample is transported at low temperature in a box, and the temperature is kept at 2 ℃;
s1.2, a virus separation method: liver, kidney, spleen, intestine, gill and brain tissue of suspected individuals were obtained under laboratory sterile conditions, and the samples were homogenized with a tissue grinder into a paste according to a 1: suspending 10 dilution in appropriate cell culture solution, centrifuging at 2 deg.C at 10000r/min for 12.5min, incubating at 34 deg.C for 12.5min, centrifuging in a centrifuge, and collecting supernatant containing cells and placing in an EP tube;
s2, extracting virus RNA in the sample: extracting virus RNA by using a CTAB method;
s3, establishment of RT-PCR detection method:
s3.1, designing an upstream primer and a downstream primer according to the Rou lake virus registered in Genbank by using a double-label fluorescent probe method: an upstream primer: TiLV-PFSeq: 5'TCACCCCACTCTATATTATC3', downstream primer: TiLV-PRSeq: 5'AGTCGGAAGATCAAGAAG 3';
s3.2, carrying out reverse transcription on the extracted RNA, synthesizing DNA, and then carrying out nucleic acid amplification and identification by using a nucleic acid amplification instrument, or completing the reverse transcription and amplification in a reaction system by adopting a one-step method, and designing and optimizing reaction conditions according to the characteristics of equipment and reverse transcriptase, wherein the reverse transcription step is as follows:
a. adding DEPC water, primers and extracted viral RNA to an EP tube;
b. centrifuging in a centrifuge, and heating in 100 deg.C water bath for 1 min;
c. then adding 0.5ul dNTP, 2ul 5 Xbuffer and 1ul AMV reverse transcriptase, then placing the mixture into a centrifuge for slight centrifugation, sealing the opening of the EP pipe by using a sealing film, then carrying out water bath at 42 ℃, and then carrying out RT at 90 ℃;
d. inactivating AMV in 100 deg.C boiling water bath for 3 min;
s4, establishment of a real-time fluorescent quantitative PCR detection method:
s4.1, designing primers and probes according to specific sequences of NC-029930 gene of the Rou lake virus registered in Genbank: an upstream primer: TiLV-PFSeq: 5'TCACCCCACTCTATATTATC3', downstream primer: TiLV-PRSeq: 5'AGTCGGAAGATCAAGAAG3', probe: (FAM) CCGCTCTCGTCAGCACCATA(TAMRA);
s4.2, synthesizing single-chain small fragment DNA according to the DNA sequence, splicing the single-chain small fragment DNA into a complete double-chain DNA fragment by a PCR method, synthesizing an NC-029930 (Tipapia) gene (465bp), connecting the synthesized gene fragment to a pMD19-T vector, and constructing a positive plasmid; taking the In-Fusion product, converting the In-Fusion product into E.coli CompetentCellJM109, and storing for later use;
carrying out PCR on E.coli CompetentCellJM109 colonies by using M13-47/RV-M primers, and detecting the length of an insert fragment in a contained plasmid;
coli compentcelljmm 109 colonies were subjected to plasmid DNA extraction and sequencing to verify positive plasmid sequences;
s4.3, under the condition that a positive sample of the Luo lake virus cannot be obtained, the positive plasmid can be used as a positive control of a detection experiment;
s4.4, amplifying by using the positive plasmid as a template and the designed primer and probe, and verifying the correctness of the primer, the probe and the template;
s4.5, carrying out gene sequencing on the amplified product, inputting the amplified product into a Genbank for comparison, and determining the amplified product as a specific sequence of the Luo lake virus;
s4.6, confirming through experiments, designing primers and probes; positive controls were constructed and all were effective. The detection method can be used for carrying out molecular biological identification on the Luo lake virus;
s5, establishing a morphological anatomical feature to identify the infected individual: finally establishing the quarantine identification method of the lake Luo virus by combining the morphological anatomy and molecular biology methods through the method.
Example 3:
the invention provides a method for quantitatively detecting lake Luo virus by using real-time fluorescence RT-PCR, which comprises the following steps:
s1, separating the Luo lake virus:
s1.1, a treatment mode of suspicious case samples: more than half of the volume of the collected suspected sample needs to be provided with an ice bag, the heat preservation effect is good, whether antibiotics need to be added to prevent and control pollution or not can be considered according to the cell culture result, the cell survival rate is improved, the suspected sample is transported at low temperature in a box, and the temperature is kept at 4 ℃;
s1.2, a virus separation method: liver, kidney, spleen, intestine, gill and brain tissue of suspected individuals were obtained under laboratory sterile conditions, and the samples were homogenized with a tissue grinder into a paste according to a 1: suspending the dilution of 10 in a proper cell culture solution, wherein the centrifugation temperature is 4 ℃, the rotation speed of a centrifuge is 12000r/min, the centrifugation time is 15min, incubating at 37 ℃ for 15min, placing the cell culture solution into the centrifuge for centrifugation, and sucking the supernatant containing the cells and placing the cell culture solution into an EP tube after centrifugation;
s2, extracting virus RNA in the sample: viral RNA was extracted using a commercial kit;
s3, establishment of RT-PCR detection method:
s3.1, designing an upstream primer and a downstream primer according to the Rou lake virus registered in Genbank by using a double-label fluorescent probe method: an upstream primer: TiLV-PFSeq: 5'TCACCCCACTCTATATTATC3', downstream primer: TiLV-PRSeq: 5'AGTCGGAAGATCAAGAAG 3';
s3.2, carrying out reverse transcription on the extracted RNA, synthesizing DNA, and then carrying out nucleic acid amplification and identification by using a nucleic acid amplification instrument, or completing the reverse transcription and amplification in a reaction system by adopting a one-step method, and designing and optimizing reaction conditions according to the characteristics of equipment and reverse transcriptase, wherein the reverse transcription step is as follows:
a. adding DEPC water, primers and extracted viral RNA to an EP tube;
b. centrifuging in a centrifuge, and heating in 100 deg.C water bath for 1 min;
c. then adding 0.5ul dNTP, 2ul 5 Xbuffer and 1ul AMV reverse transcriptase, then placing the mixture into a centrifuge for slight centrifugation, sealing the opening of the EP pipe by using a sealing film, then carrying out water bath at 42 ℃, and then carrying out RT at 90 ℃;
d. inactivating AMV in 100 deg.C boiling water bath for 3 min;
s4, establishment of a real-time fluorescent quantitative PCR detection method:
s4.1, designing primers and probes according to specific sequences of NC-029930 gene of the Rou lake virus registered in Genbank: an upstream primer: TiLV-PFSeq: 5'TCACCCCACTCTATATTATC3', downstream primer: TiLV-PRSeq: 5'AGTCGGAAGATCAAGAAG3', probe: (FAM) CCGCTCTCGTCAGCACCATA(TAMRA);
s4.2, synthesizing single-chain small fragment DNA according to the DNA sequence, splicing the single-chain small fragment DNA into a complete double-chain DNA fragment by a PCR method, synthesizing an NC-029930 (Tipapia) gene (465bp), connecting the synthesized gene fragment to a pMD19-T vector, and constructing a positive plasmid; taking the In-Fusion product, converting the In-Fusion product into E.coli CompetentCellJM109, and storing for later use;
carrying out PCR on E.coli CompetentCellJM109 colonies by using M13-47/RV-M primers, and detecting the length of an insert fragment in a contained plasmid;
coli compentcelljmm 109 colonies were subjected to plasmid DNA extraction and sequencing to verify positive plasmid sequences;
s4.3, under the condition that a positive sample of the Luo lake virus cannot be obtained, the positive plasmid can be used as a positive control of a detection experiment;
s4.4, amplifying by using the positive plasmid as a template and the designed primer and probe, and verifying the correctness of the primer, the probe and the template;
s4.5, carrying out gene sequencing on the amplified product, inputting the amplified product into a Genbank for comparison, and determining the amplified product as a specific sequence of the Luo lake virus;
s4.6, confirming through experiments, designing primers and probes; positive controls were constructed and all were effective. The detection method can be used for carrying out molecular biological identification on the Luo lake virus;
s5, establishing a morphological anatomical feature to identify the infected individual: finally establishing the quarantine identification method of the lake Luo virus by combining the morphological anatomy and molecular biology methods through the method.
The invention aims to establish a sampling rule of suspected cases of the Luo lake virus, a sample pretreatment method, a virus separation and nucleic acid extraction technology, a molecular biology detection method of nucleic acid amplification and identification as early as possible, and establish an apparent disease sign identification method of the tilapia Luo lake virus by contrasting detection results, thereby providing detection service for aquatic enterprises and issuing a test report; providing technical service for government supervision departments to carry out epidemic monitoring and prevention and control; participating in epidemic prevention and control work of official and aquatic product enterprises and providing technical support and epidemic situation confirmation; after the method to be detected is established, the method is cooperated with aquaculture enterprises and chemical prevention and treatment production enterprises to develop the chemical for preventing and treating the Luo lake virus.
And finally: the above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that are within the spirit and principle of the present invention are intended to be included in the scope of the present invention.
Claims (7)
1. A method for quantitatively detecting Luo lake virus by using real-time fluorescence RT-PCR is characterized by comprising the following steps: the method comprises the following specific steps:
s1, separating the Luo lake virus:
s1.1, a treatment mode of suspicious case samples: the collected suspected sample needs to be transported at low temperature in an insulation box with more than half of the volume of ice bags and good insulation effect, and the temperature is kept at 0-4 ℃;
s1.2, a virus separation method: liver, kidney, spleen, intestine, gill and brain tissue of suspected individuals were obtained under laboratory sterile conditions, and the samples were homogenized with a tissue grinder into a paste according to a 1: suspending the dilution of 10 in appropriate cell culture solution, incubating at 30-37 deg.C for 10-15min, centrifuging in a centrifuge, and collecting the supernatant containing cells and placing in an EP tube;
s2, extracting virus RNA in the sample: viral RNA was extracted using the CTAB method or commercial kit;
s3, establishment of RT-PCR detection method:
s3.1, designing upstream and downstream primers according to the Luo lake virus registered in Genbank;
s3.2, carrying out reverse transcription on the extracted RNA, and carrying out nucleic acid amplification and identification after synthesizing DNA;
s4, establishment of a real-time fluorescent quantitative PCR detection method:
s4.1, designing a primer and a probe according to a specific sequence of the Rou lake virus gene NC-029930 registered in Genbank;
s4.2, synthesizing single-chain small fragment DNA according to the DNA sequence, splicing the single-chain small fragment DNA into a complete double-chain DNA fragment by a PCR method, synthesizing an NC-029930 (Tipapia) gene (465bp), connecting the synthesized gene fragment to a pMD19-T vector, and constructing a positive plasmid; converting the In-Fusion product into E.coli component Cell JM109, and storing for later use;
coli component Cell JM109 was subjected to PCR using M13-47/RV-M primers, and the length of the insert in the contained plasmid was determined;
coli component Cell JM109 colonies were subjected to plasmid DNA extraction and sequencing to verify positive plasmid sequences;
s4.3, under the condition that a positive sample of the Luo lake virus cannot be obtained, the positive plasmid can be used as a positive control of a detection experiment;
s4.4, amplifying by using the positive plasmid as a template and the designed primer and probe, and verifying the correctness of the primer, the probe and the template;
s4.5, carrying out gene sequencing on the amplified product, inputting the amplified product into a Genbank for comparison, and determining the amplified product as a specific sequence of the Luo lake virus;
s4.6, confirming through experiments, designing primers and probes; positive controls were constructed and all were effective. The detection method can be used for carrying out molecular biological identification on the Luo lake virus;
s5, establishing a morphological anatomical feature to identify the infected individual: finally establishing the quarantine identification method of the lake Luo virus by combining the morphological anatomy and molecular biology methods through the method.
2. The method for quantitative detection of Luo lake virus by real-time fluorescence RT-PCR as claimed in claim 1, wherein: in step S1.2, whether antibiotics need to be added to prevent and treat pollution can be considered according to the cell culture result, and the cell survival rate is improved.
3. The method for quantitative detection of Luo lake virus by real-time fluorescence RT-PCR as claimed in claim 1, wherein: in step S1.2, the centrifugation temperature is 0-4 ℃, the rotation speed of the centrifuge is 8000-.
4. The method for quantitative detection of Luo lake virus by real-time fluorescence RT-PCR as claimed in claim 1, wherein: in step S3, the primers designed in step S3.1 were: an upstream primer: TiLV-PFSeq: 5'TCACCCCACTCTATATTATC3', downstream primer: TiLV-PRSeq: 5'AGTCGGAAGATCAAGAAG3', primers, probes designed in step S4.1: an upstream primer: TiLV-PFSeq: 5'TCACCCCACTCTATATTATC3', downstream primer: TiLV-PRSeq: 5'AGTCGGAAGATCAAGAAG3', probe: (FAM) CCGCTCTCGTCAGCACCATA(TAMRA).
5. The method for quantitative detection of Luo lake virus by real-time fluorescence RT-PCR as claimed in claim 1, wherein: in step S3.2, a one-step method may also be used to complete reverse transcription and amplification in one reaction system, and reaction conditions are designed and optimized according to the equipment and the characteristics of reverse transcriptase.
6. The method for quantitative detection of Luo lake virus by real-time fluorescence RT-PCR as claimed in claim 1, wherein: in step 3.2 the reverse transcription step is:
a. adding DEPC water, primers and extracted viral RNA to an EP tube;
b. centrifuging in a centrifuge, and heating in 100 deg.C water bath for 1 min;
c. then adding 0.5ul dNTP, 2ul 5 Xbuffer and 1ul AMV reverse transcriptase, then placing the mixture into a centrifuge for slight centrifugation, sealing the opening of the EP pipe by using a sealing film, then carrying out water bath at 42 ℃, and then carrying out RT at 90 ℃;
d. inactivating AMV in 100 deg.C boiling water bath for 3 min.
7. The method for quantitative detection of Luo lake virus by real-time fluorescence RT-PCR as claimed in claim 1, wherein: in step S3.2 nucleic acid amplification is performed using a nucleic acid amplifier.
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