CN114574633A - RHDV2 fluorescent quantitative RT-PCR detection method - Google Patents
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Abstract
The invention provides a primer probe set, a kit and a detection method for detecting RHDV2 by fluorescent quantitative RT-PCR, belonging to the technical field of molecular biology. The primer probe group of the invention consists of a primer F, a primer R and a probe T: the sequence of the primer F is as follows: 5 '-GAGTGTTGATGGRTACTTC-3', wherein the sequence of the primer R is as follows: 5'-CCCAAGTTGTACACAAGC-3', the sequence of the probe T is: 5 '-AGCCACCCTCATYGACCTGT-3', the 5 'end of the probe T is marked with a fluorescence reporter group FAM, and the 3' end is marked with a fluorescence quenching group BHQ 1. The primer probe set and the detection method thereof provided by the invention can be used for rapidly detecting the rabbit viral hemorrhagic disease type 2, and have the advantages of accurate result, good specificity and high sensitivity.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a primer, a kit and a detection method for detecting rabbit viral hemorrhagic disease type 2 (RHDV2) by using fluorescent quantitative RT-PCR.
Background
The rabbit breeding has a long history in China, and rabbit meat is always popular with the masses because of good taste and high nutritive value. Since the 90 s of the 20 th century, the rabbit meat yield in China has been increased at a rate of about 8.5% every year, and in 2016, the quantity of rabbits in China is about 5.62 hundred million, which accounts for 55.4% of the world and is a major rabbit breeding country in the world. The Sichuan rabbit industry is in the front of the country, nearly 3 hundred million rabbits are sold in Sichuan in 2016, which accounts for nearly 7 percent of the national market, according to the statistics of the rabbit-industry division of the Chinese animal husbandry Association, and the rabbit industry becomes an important way for Sichuan farmers to become rich.
The rabbit hemorrhagic disease type 2, also called rabbit plague type 2, is an acute, highly contact and highly pathogenic rabbit infectious disease caused by the virus of rabbit hemorrhagic disease type 2 (RHDV2), the death rate is up to 90 percent, especially the death rate of young rabbits is up to 100 percent. Since 2010, the disease is firstly appeared in France and gradually spread to Italy, Spain, portugal and other countries, which has a very important influence on the rabbit industry. In 4 months of 2020, a certain rabbit farm in Sichuan has a rabbit plague type 2 epidemic for the first time, and although a series of strict treatment measures do not cause more economic loss, at present, the rabbit plague vaccine in China can only prevent classical rabbit plague, lacks targeted protective power for the rabbit plague type 2, and does not have a commercially available rabbit plague type 2 vaccine, so the rabbit plague type 2 still has a great threat to the rabbit farming industry in China.
At present, the detection method for the disease still has no unified national standard, local standard and industrial standard, and only has a group standard issued by the Chinese animal husbandry and animal husbandry association. However, although many diagnostic techniques are specified by the standard, the operation is complicated, and the clinical requirement for rapid diagnosis of the disease cannot be met.
Therefore, a method for rapidly detecting rabbit viral hemorrhagic disease type 2 is urgently needed to be developed so as to solve the problem that the clinical judgment of classical rabbit viral hemorrhagic disease and rabbit viral hemorrhagic disease type 2 is difficult by naked eyes.
Disclosure of Invention
The invention aims to solve the technical problems, and provides a primer, a kit and a detection method for detecting rabbit viral hemorrhagic disease type 2 (RHDV2) by using fluorescent quantitative RT-PCR. The detection method provided by the invention can be used for rapidly detecting the rabbit viral hemorrhagic disease type 2, has accurate result and high sensitivity, and can be used for clinically and rapidly judging the classical rabbit viral hemorrhagic disease and the rabbit viral hemorrhagic disease type 2.
One of the purposes of the invention is to provide a primer probe set for detecting rabbit viral hemorrhagic disease type 2 by fluorescent quantitative RT-PCR, wherein the primer probe set consists of a primer F, a primer R and a probe T:
the sequence of the primer F is as follows: 5 '-GAGTGTTGATGGRTACTTC-3',
the sequence of the primer R is as follows: 5'-CCCAAGTTGTACACAAGC-3' the flow of the air in the air conditioner,
the sequence of the probe T is as follows: 5 '-AGCCACCCTCATYGACCTGT-3', the 5 'end of the probe T is marked with a fluorescence reporter group FAM, and the 3' end is marked with a fluorescence quenching group BHQ 1.
The invention also aims to provide a time-fluorescence quantitative RT-PCR detection kit for rabbit viral hemorrhagic disease type 2, which comprises the primer probe set.
The invention also aims to provide application of the primer probe set in preparation of a kit for detecting rabbit viral hemorrhagic disease type 2 by fluorescent quantitative RT-PCR.
The fourth purpose of the invention is to provide a fluorescent quantitative RT-PCR detection method for rabbit viral hemorrhagic disease type 2, which comprises the following steps:
(1) extracting RHDV2 virus genome RNA according to a virus nucleic acid extraction kit and a reverse transcriptase instruction, performing reverse transcription by taking the extracted genome RNA as a template to synthesize cDNA, performing PCR amplification on a VP60 gene by using a primer F and a primer R, cloning to a T vector, constructing a positive recombinant T vector, and performing sequencing identification. The method comprises the following specific steps: performing gel purification and recovery on the PCR amplification product according to a kit, preparing a reaction system by taking 4 mu L of gel recovery product according to the instruction of a T carrier connection kit, and connecting overnight at 16 ℃; taking 100 mu L of DH5a competent cells melted on ice, adding the ligation product, gently mixing, and standing on ice for 25 min; carrying out water bath heat shock at 42 ℃ for 45-60 s, quickly transferring to an ice bath, and standing for 2 min; adding 700 mu L of LB culture solution into a centrifuge tube, mixing uniformly, and performing shake culture at 37 ℃ and 200rpm for 1 h; centrifuging at 3000rpm for 5min, discarding supernatant 700 μ L, mixing the rest bacteria solution gently, sucking out, spreading on LB agar plate (50 μ g/ml Amp +), and culturing at 37 deg.C overnight in incubator; the white single colonies were picked up and cultured overnight in a 37 ℃ incubator in 5mL of LB liquid medium (50. mu.g/mL Amp +). Taking 2 mu L of culture as a template, and carrying out PCR identification; and (4) sending the colony culture with positive PCR identification to a biotechnology sequencing company for sequencing identification.
(2) Diluting the positive recombinant vector constructed in the step (1) by 10 times of gradient, performing real-time fluorescent quantitative PCR amplification by using a primer F and a primer R, and making a standard curve by using a Log value of template concentration as a vertical coordinate and a Ct value obtained by amplification as a horizontal coordinate;
(3) and (3) extracting genome RNA of various samples, carrying out reverse transcription to synthesize cDNA, carrying out real-time fluorescent quantitative PCR amplification by using a primer F, a primer R and a probe T to obtain a Ct value, and then calculating the copy number of the target fragment according to the standard curve obtained in the step (2).
Specifically, the reverse transcription step for synthesizing cDNA comprises the following steps:
the reaction system is 10 μ L, and the following reactants are sequentially added into a PCR reaction tube: 2 μ L of 5 × AMV reaction buffer; reverse transcription primer 50. mu. mol/L Oligo dT 0.5. mu.L; AMV reverse transcriptase 0.5 μ L; random 6 nucleotide primer 100. mu. mol/L0.5. mu.L; 4.0 mu L of nuclease-free water; 2.5 mu L of RNA template; after mixing, the mixture was centrifuged instantaneously and then subjected to reverse transcription at 37 ℃ for 15min and 65 ℃ for 5 s.
Specifically, the fluorescence RT-PCR reaction system adopted by the PCR amplification is as follows:
18 μ L of fluorescent RT-PCR reaction mix consisting of:
specifically, the PCR amplification step is as follows: adding 18 mu L of fluorescent RT-PCR reaction mixed solution into a PCR amplification tube, adding 2 mu L of cDNA into the PCR amplification tube, adding a template, sealing the PCR tube, performing instant centrifugation, and placing the PCR amplification tube in a PCR instrument, wherein the amplification procedure is as follows:
pre-denaturation at 95 ℃ for 3 min;
denaturation at 95 ℃ for 10 s;
annealing at the temperature of 59 ℃ for 10s,
extension at 72 ℃ for 25s, 45 cycles.
When the rabbit hemorrhagic disease virus type 2 is identified by adopting the detection method, the method comprises the following steps: the Ct value of the detected sample is less than or equal to 36, and a specific amplification curve appears, and then the rabbit hemorrhagic disease virus type 2 nucleic acid is judged to be positive; when the Ct value is not present or is more than or equal to 40, the rabbit hemorrhagic disease virus type 2 nucleic acid is judged to be negative; when the Ct value is more than 36 and less than 40 and a specific amplification curve appears, determining that the sample is suspected; and (4) repeatedly detecting the suspected sample, and judging that the rabbit hemorrhagic disease virus type 2 nucleic acid is positive if the result is still suspected.
The invention has the following beneficial effects:
the invention provides a universal fluorescent quantitative RT-PCR detection method capable of quickly identifying RHDV and RHDV2, which has the characteristics of strong primer specificity, high sensitivity, simple and convenient operation, good repeatability, easy judgment and the like, can be used for quickly carrying out clinical diagnosis on rabbit viral hemorrhagic disease, and provides effective technical support for epidemiological investigation and prevention and control of the disease.
Drawings
FIG. 1 is a diagram of fluorescent quantitative RT-PCR primer validation;
FIG. 2 is a diagram showing the results of the reaction temperature optimization, where the annealing gradients are set at 60 deg.C, 59.6 deg.C, 58.8 deg.C, 57.5 deg.C, 55.8 deg.C, 54.4 deg.C, 53.5 deg.C, and 53 deg.C;
FIG. 3 is a graph showing the results of optimization of primer concentrations, with primer amounts of 0.25, 0.5, 0.75 and 1.0. mu.L, respectively;
FIG. 4 is a graph showing the results of optimization of probe concentrations, with probe amounts of 0.25, 0.5, 0.75 and 1.0. mu.L, respectively;
FIG. 5 is a standard graph;
FIG. 6 is a graph showing the results of a sensitivity test;
FIG. 7 is a graph showing the results of the specificity test.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more clearly understood, the present invention is described in detail below with reference to the following embodiments, and it should be noted that the following embodiments are only for explaining and illustrating the present invention and are not intended to limit the present invention. The invention is not limited to the above embodiments, but may be modified within the scope of the invention.
Example 1
1 reagent
RNA extraction reagent, nuclease-free water (see preparation method A.1) and fluorescent PCR premix (2X).
2 instrumentation
A fluorescent PCR amplification instrument, a desk type low-temperature high-speed centrifuge (the maximum centrifugal force is over 12000 g), a micro adjustable pipette (different specifications such as 2.5-1000 mu L), a non-nuclease centrifuge tube and a suction head, a PCR amplification tube and an automatic nucleic acid extraction instrument.
3 primers and probes
3.1 primer and Probe design
Designing primer and probe sequences aiming at the conserved sequence of the RHDV2 VP60 gene:
an upstream primer F: 5 '-GAGTGTTGATGGRTACTTC-3',
a downstream primer R: 5'-CCCAAGTTGTACACAAGC-3', respectively;
TaqMan probe: FAM-5 '-AGCCACCCTCATYGACCTGT-3' BHQ 1.
The designed primer was verified and shown in FIG. 1, and it can be seen that the primer is specific to RHDV 2.
3.2 reaction temperature optimization
The annealing temperature is set from high to low as: the optimization results of the annealing reactions at 60 ℃, 59.6 ℃, 58.8 ℃, 57.5 ℃, 55.8 ℃, 54.4 ℃, 53.5 ℃ and 53 ℃ are shown in FIG. 2, and finally 59 ℃ is determined as the optimal annealing temperature.
3.3 primer concentration optimization
According to the concentration of the upstream primer F and the downstream primer R which are both 5 mu mol/L, respectively adding 0.25, 0.5, 0.75 and 1.0 mu L, the result is shown in figure 3, and the primer amount in the system is determined as that the upstream primer F and the downstream primer R are respectively added with 1.0 mu L because the difference of the Cq value of the final result is not large.
3.4 Probe concentration optimization
According to the probe concentration of 5 mu mol/L, 0.5, 1.0, 1.5 and 2 mu L are respectively added, the result is shown in figure 4, and the addition amount of the probe in the reaction system is finally determined to be 1 mu L because the Cq values of the result are not obviously different.
3.5 drawing of Standard Curve
Diluting the positive recombinant T vector constructed according to the step (1) by 10 times, constructing a standard curve according to the established reaction temperature and reaction system, and selecting 59 ℃ when determining the annealing temperature for increasing the specificity of the reaction because the nucleic acid homology of several viruses of RCV is relatively high as shown in FIG. 5.
3.6 sensitivity test
Constructing positive recombinant T vector, diluting by 10 ploidy degree, performing test detection according to the established reaction temperature and reaction system, and determining that the lowest detectable template copy number is 43copies as shown in FIG. 6.
3.7 specificity assay
After the experimental conditions are optimized, different samples are detected, and the steps are as follows:
3.7.1RNA extraction
Extracting virus RNA in various samples by adopting a nucleic acid extraction reagent or an automatic nucleic acid extractor. The extracted RNA is immediately subjected to subsequent experiments or stored below-60 ℃.
3.7.2 reverse transcription reaction
The reaction system is 10 μ L, and the following reactants are sequentially added into a PCR reaction tube:
2 μ L of 5 × AMV reaction buffer; reverse transcription primer 50 mu mol/L Oligo dT 0.5 mu L; AMV reverse transcriptase 0.5 μ L; random 6 nucleotide primer 100. mu. mol/L0.5. mu.L; 4.0 mu L of nuclease-free water; 2.5 mu L of RNA template;
mixing, centrifuging instantaneously, reverse transcription circulating at 37 deg.c for 15min and 65 deg.c for 5s, and PCR amplifying the product or temporary storing at-20 deg.c for use.
3.7.3 amplification System (20. mu.L)
Each sample was prepared with 18. mu.L of fluorescent RT-PCR reaction mix consisting of:
adding 18 mu L of fluorescent PCR reaction mixed solution into a PCR amplification tube; mu.L of cDNA (reverse transcription product) was added to each PCR amplification tube. Positive control (positive recombinant plasmid is used as a template) and negative control (nuclease-free water is used as a template) are required to be set during each fluorescent RT-PCR amplification. After addition of the template, the PCR tube was sealed and centrifuged instantaneously. All PCR amplification tubes were placed in a PCR instrument and the amplification program was run under 7.4.3 conditions.
3.7.4 amplification conditions
Pre-denaturation at 95 ℃ for 3 min; the FAM fluorescence signal was collected at 95 ℃ denaturation for 10s, 59 ℃ annealing for 10s, and 72 ℃ extension for 25s, for 45 cycles, at 72 ℃ extension for each cycle.
Particularly, corresponding gene segments of European brown hare hemorrhagic syndrome (EBHSV) and RCV are artificially synthesized for specific detection.
3.7.5 determination of results
The Ct value of the positive control is less than or equal to 30, a specific amplification curve appears, the negative control has no Ct value or the Ct value of the negative control is more than or equal to 40, no specific amplification curve appears, and the test result is effective; otherwise, the test should be repeated. The Ct value of the detected sample is less than or equal to 36, and a specific amplification curve appears, and then the rabbit hemorrhagic disease virus type 2 nucleic acid is judged to be positive; when the Ct value is not present or is more than or equal to 40, the rabbit hemorrhagic disease virus type 2 nucleic acid is judged to be negative; when the Ct value is more than 36 and less than 40 and a specific amplification curve appears, the sample is judged to be suspected. And (4) repeatedly detecting the suspected sample, and determining that the rabbit hemorrhagic disease virus type 2 nucleic acid is positive if the result is still suspected.
Except for the positive control, the detection results of EBHSV, RHDV1, RCV, Escherichia coli, Pasteurella A, Pasteurella F and the like are negative, and the figure is 7.
Claims (8)
1. A primer probe group for detecting rabbit viral hemorrhagic disease type 2 by fluorescent quantitative RT-PCR is characterized in that the primer probe group consists of a primer F, a primer R and a probe T:
the sequence of the primer F is as follows: 5 '-GAGTGTTGATGGRTACTTC-3',
the sequence of the primer R is as follows: 5'-CCCAAGTTGTACACAAGC-3' the flow of the air in the air conditioner,
the sequence of the probe T is as follows: 5 '-AGCCACCCTCATYGACCTGT-3', the 5 'end of the probe T is marked with a fluorescence reporter group FAM, and the 3' end is marked with a fluorescence quenching group BHQ 1.
2. A kit for detecting rabbit viral hemorrhagic disease type 2 by time-fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction) comprising the primer probe set of claim 1.
3. The use of the primer probe set of claim 1 in the preparation of a kit for the fluorescent quantitative RT-PCR detection of rabbit viral hemorrhagic disease type 2.
4. A fluorescent quantitative RT-PCR detection method for rabbit viral hemorrhagic disease type 2 is characterized by comprising the following steps:
(1) RHDV2 virus genome RNA is extracted according to a virus nucleic acid extraction kit and a reverse transcriptase instruction, the extracted genome RNA is used as a template, reverse transcription is carried out to synthesize cDNA, a VP60 gene is amplified by PCR by using a primer F and a primer R, the gene is cloned to a T vector, a positive recombinant T vector is constructed, and sequencing identification is carried out. The method comprises the following specific steps: performing gel purification and recovery on the PCR amplification product according to a kit, preparing a reaction system by taking 4 mu L of gel recovery product according to the instruction of a T carrier connection kit, and connecting overnight at 16 ℃; taking 100 mu L of DH5a competent cells melted on ice, adding the ligation product, gently mixing uniformly, and standing on ice for 25 min; performing water bath heat shock at 42 ℃ for 45-60 s, quickly transferring the mixture into an ice bath, and standing for 2 min; adding 700 mu LLB culture solution into a centrifuge tube, uniformly mixing, and performing shake culture at 37 ℃ and 200rpm for 1 h; centrifuging at 3000rpm for 5min, discarding supernatant 700 μ L, mixing the rest bacteria liquid, sucking out, spreading on LB agar culture plate (50 μ g/ml Amp +), and culturing at 37 deg.C overnight in incubator; the white single colonies were picked up and cultured overnight in a 37 ℃ incubator in 5mL of LB liquid medium (50. mu.g/mL Amp +). Taking 2 mu L of culture as a template, and carrying out PCR identification; and (4) sending the colony culture with positive PCR identification to a biotechnology sequencing company for sequencing identification.
(2) Diluting the positive recombinant vector constructed in the step (1) by 10 times of gradient, performing real-time fluorescent quantitative PCR amplification by using a primer F and a primer R, and making a standard curve by using a Log value of template concentration as a vertical coordinate and a Ct value obtained by amplification as a horizontal coordinate;
(3) and (3) extracting genome RNA of various samples, carrying out reverse transcription to synthesize cDNA, carrying out real-time fluorescent quantitative PCR amplification by using a primer F, a primer R and a probe T to obtain a Ct value, and then calculating the copy number of the target fragment according to the standard curve obtained in the step (2).
5. The method of claim 4, wherein the reverse transcription to synthesize cDNA comprises:
the reaction system is 10 μ L, and the following reactants are sequentially added into a PCR reaction tube: 2 μ L of 5 × AMV reaction buffer; reverse transcription primer 50 mu mol/L Oligo dT 0.5 mu L; AMV reverse transcriptase 0.5 μ L; random 6 nucleotide primer 100. mu. mol/L0.5. mu.L; 4.0 mu L of nuclease-free water; 2.5 mu L of RNA template; after mixing, the mixture was centrifuged instantaneously and then subjected to reverse transcription at 37 ℃ for 15min and 65 ℃ for 5 s.
6. The detection method according to claim 4, wherein the fluorescence RT-PCR reaction system adopted by the PCR amplification is as follows:
18 μ L of fluorescent RT-PCR reaction mix consisting of:
7. the detection method according to claim 6, wherein the PCR amplification step comprises: adding 18 mu L of fluorescent RT-PCR reaction mixed solution into a PCR amplification tube, adding 2 mu L of cDNA into the PCR amplification tube, adding a template, sealing the PCR tube, performing instant centrifugation, and placing the PCR amplification tube in a PCR instrument, wherein the amplification procedure is as follows:
pre-denaturation at 95 ℃ for 3 min;
denaturation at 95 ℃ for 10 s;
annealing at the temperature of 59 ℃ for 10s,
extension at 72 ℃ for 25s, 45 cycles.
8. The method according to claim 6, wherein the rabbit hemorrhagic disease virus type 2 is identified by the following method: the Ct value of the detected sample is less than or equal to 36, and a specific amplification curve appears, and then the rabbit hemorrhagic disease virus type 2 nucleic acid is judged to be positive; when the Ct value is not present or is more than or equal to 40, the rabbit hemorrhagic disease virus type 2 nucleic acid is judged to be negative; when the Ct value is more than 36 and less than 40 and a specific amplification curve appears, determining that the sample is suspected; and (4) repeatedly detecting the suspected sample, and judging that the rabbit hemorrhagic disease virus type 2 nucleic acid is positive if the result is still suspected.
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| CN102154512A (en) * | 2011-03-15 | 2011-08-17 | 中国检验检疫科学研究院 | Fluorescence quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection method for rabbit hemorrhagic disease virus |
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| CN102154512A (en) * | 2011-03-15 | 2011-08-17 | 中国检验检疫科学研究院 | Fluorescence quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection method for rabbit hemorrhagic disease virus |
Non-Patent Citations (3)
| Title |
|---|
| AL-EBSHAHY,E.M.: "Rabbit hemorrhagic disease virus isolate RHDV/Egypt/Beh-9/2020 capsid protein (VP60) gene, partial cds，GenBank: MZ782084.1，539bp RNA linear", NCBI GENBANK, pages 1 * |
| SAIDI,A.: "Rabbit hemorrhagic disease virus isolate OTM.6 VP60 gene, partial cds，GenBank: MZ451406.1，710bp RNA linear", NCBI GENBANK, pages 1 * |
| 陈斌等: "四川兔病毒性出血症2型的分子生物学诊断", 《四川畜牧兽医》, no. 6, pages 30 - 32 * |
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