CN114058738A - Fluorescence quantitative PCR detection kit for detecting eriocheir sinensis reovirus - Google Patents
Fluorescence quantitative PCR detection kit for detecting eriocheir sinensis reovirus Download PDFInfo
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Abstract
The invention discloses a fluorescence quantitative PCR detection kit for detecting eriocheir sinensis reovirus by a probe method. The kit comprises a group of primer groups for detecting the eriocheir sinensis reovirus and a group of primer groups for detecting the eriocheir sinensis internal reference gene, wherein each of the two primer groups comprises a pair of specific primers and corresponding TaqMan probes, and the kit further comprises DNA polymerase, reverse transcriptase, UNG enzyme, 2 multiplied by reaction buffer solution, sealing liquid, a positive control reference substance and a negative control reference substance. The kit provided by the invention can be used for directly detecting the Eriocheir sinensis reovirus from a complex sample, has the advantages of strong specificity, high sensitivity, good experimental repeatability and convenience and rapidness in operation, can complete detection without expensive instruments, and can be used for rapidly and accurately detecting the Eriocheir sinensis reovirus on site.
Description
Technical Field
The invention relates to the technical field of microbial detection, in particular to a fluorescence quantitative PCR detection kit for detecting eriocheir sinensis reovirus.
Background
Reovirus is a double-stranded RNA virus with 9 to 12 linear genome segments that has been found in many hosts, including vertebrates, invertebrates and plants. In recent years, the breeding yield of the freshwater eriocheir sinensis in China is increased, and the eriocheir sinensis Tremor Disease (TD) caused by reovirus is one of the most serious diseases of the eriocheir sinensis. The disease first appeared in Jiangsu province in China in 1994. Crabs with TD exhibit tremor or bending of their legs, bradykinesia and loss of appetite, and reovirus has been isolated from diseased crabs by researchers.
At present, a Polymerase Chain Reaction (PCR) is more frequently applied in molecular biology detection, and a PCR instrument is also matched with an electrophoresis instrument and a gel imager for use in the detection process, so that the operation steps are more, the time consumption is long, and aerosol pollution is easily caused in the process. The Real-time fluorescent Quantitative PCR (Quantitative Real-time PCR) is a method which adds fluorescent dye or fluorescent probe on the basis of the conventional PCR technology, monitors the whole PCR process in Real time by utilizing fluorescent signal accumulation and carries out Quantitative analysis on a template with unknown concentration by a standard curve. Compared with the common PCR technology, the TaqMan probe of the real-time fluorescent quantitative PCR technology has strong specificity, can distinguish single nucleotide, and has simpler and more convenient operation and shorter detection time.
The prior art discloses a Eriocheir sinensis reovirus RT-LAMP detection kit and a detection method thereof, but the kit is low in sensitivity and complex in operation, and cannot meet the requirements of technical personnel.
Disclosure of Invention
The invention provides a probe method fluorescence quantitative PCR detection kit for detecting Eriocheir sinensis reovirus, which is an improvement measure on the basis of the prior art. The kit comprises a group of primer groups for detecting the eriocheir sinensis reovirus and a group of primer groups for detecting the eriocheir sinensis internal reference gene, wherein the two groups of primer groups respectively comprise a pair of specific primers and corresponding TaqMan probes, and the kit further comprises DNA polymerase, reverse transcriptase, UNG enzyme, 2 multiplied by reaction buffer solution, sealing liquid, a positive control reference substance and a negative control reference substance. The kit provided by the invention can be used for directly detecting the Eriocheir sinensis reovirus from a complex sample, has the advantages of strong specificity, high sensitivity, good experimental repeatability and convenience and rapidness in operation, and meanwhile, an internal reference gene primer is designed to be used as an internal reference, so that abnormal results caused by sample nucleic acid problems or reagent failure are eliminated, and the kit is suitable for rapidly and accurately detecting the Eriocheir sinensis reovirus on site.
The first purpose of the invention is to provide a primer group for detecting the eriocheir sinensis reovirus
The second purpose of the invention is to provide a primer group for detecting the internal reference gene of the eriocheir sinensis.
The third purpose of the invention is to provide a detection kit.
In order to achieve the purpose, the invention is realized by the following scheme:
the primer group comprises a forward primer F, a reverse primer R and a fluorescent probe Q, wherein the nucleotide sequence of the forward primer F is shown as SEQ ID NO. 3, the nucleotide sequence of the reverse primer R is shown as SEQ ID NO. 4, and the nucleotide sequence of the fluorescent probe Q is shown as SEQ ID NO. 5.
A primer group for detecting internal reference genes of eriocheir sinensis is characterized by consisting of a forward primer F, a reverse primer R and a fluorescent probe Q, wherein the nucleotide sequence of the forward primer F is shown as SEQ ID NO. 9, the nucleotide sequence of the reverse primer R is shown as SEQ ID NO. 10, and the nucleotide sequence of the fluorescent probe Q is shown as SEQ ID NO. 11.
Preferably, the 5 'end of the fluorescent probe Q is labeled with a fluorescent group, and the 3' end of the fluorescent probe Q is labeled with a quenching group.
More preferably, the 5 'end of the fluorescent probe Q is labeled with a 6-FAM fluorescent group, and the 3' end is labeled with an MGB quenching group.
A detection kit comprises a group of primer groups for detecting the eriocheir sinensis reovirus.
Preferably, the kit also comprises a primer group for detecting the eriocheir sinensis internal reference gene.
Preferably, the kit further comprises DNA polymerase, reverse transcriptase, UNG enzyme, 2 × reaction buffer, sealing liquid, positive control reference substance and/or negative control reference substance.
Preferably, the DNA polymerase is a mixture of Tth DNA polymerase and a hot start antibody, the reverse transcriptase is M-MLV reverse transcriptase, the UNG enzyme is uracil-N-glycosylase, the 2 × reaction buffer is a 2 × concentration reaction solution containing dATP, dCTP, dGTP and dUTP, and the sealing liquid is mineral oil.
Preferably, the positive control reference substance is plasmid DNA containing a Eriocheir sinensis reovirus detection target gene, and the negative control reference substance is deionized water without RNase.
Preferably, the reaction system of the kit is: in the primer group for detecting the eriocheir sinensis reovirus, the final concentrations of a forward primer F, a reverse primer R and a fluorescent probe Q primer in a reaction system are respectively 0.4-0.6 mu M, 0.4-0.6 mu M and 0.2-0.3 mu M; in the primer group for detecting the internal reference gene of the eriocheir sinensis, the final concentrations of a forward primer F, a reverse primer R and a fluorescent probe Q primer in a reaction system are respectively 0.2-0.4 mu M, 0.2-0.4 mu M and 0.1-0.2 mu M; 12.4-12.6 mu L of 2 multiplied reaction buffer solution, 2-4U of DNA polymerase, 0.3-0.5U of reverse transcriptase 2-4U, UNG enzyme, 2-5 mu L of sample to be detected, and 25 mu L of deionized water without RNA enzyme.
More preferably, the reaction system of the kit is: the final concentrations of a forward primer F, a reverse primer R and a fluorescent probe Q primer of the primer group of the Eriocheir sinensis reovirus in a reaction system are respectively 0.4 mu M, 0.4 mu M and 0.2 mu M; the final concentrations of the primer group forward primer F, the reverse primer R and the fluorescent probe Q primer of the internal reference gene of the eriocheir sinensis in the reaction system are respectively 0.24 mu M, 0.24 mu M and 0.16 mu M; 2 Xreaction buffer 12.5U L, DNA polymerase 3U, reverse transcriptase 3U, UNG enzyme 0.4U, test sample 5U L, adding no RNase deionized water to 25U L.
Preferably, the reaction condition of the reaction system is 50 ℃ for 5 min; 5min at 95 ℃; 15s at 95 ℃, 30s at 60 ℃ and 45 cycles.
The kit is applied to the detection of the Eriocheir sinensis reovirus.
The present invention judges the detection result based on the amplification curve. The amplification curve is S-shaped, the detection result is positive, namely the detected sample contains the Eriocheir sinensis reovirus; no S-shaped amplification curve appears, the detection result is negative, namely the detected sample does not contain the Eriocheir sinensis reovirus.
Compared with the prior art, the invention has the following beneficial effects:
(1) the specificity is strong: the Eriocheir sinensis reovirus VP9 gene sequence used for primer design is a section of specific segment, the nucleotide sequence of the specific segment is shown as SEQ ID NO. 1, a pair of specific primers and a fluorescent probe are designed for the target gene segment for amplification, and the specificity is strong.
(2) The sensitivity is high: for positive plasmids containing the eriocheir sinensis reovirus detection target gene, the lowest detection limit can reach 101copies/μL；
(3) The experimental repeatability is high: for positive control reference (plasmid concentration 10)6copies/. mu.L and 102copies/mu L) are respectively carried out for 20 times of repeated experiments, and the variation coefficient of the Ct value is less than or equal to 5 percent; the amplification curves all present typical "S" type curves.
(4) The operation is simple, and the result is direct and objective: the method has simple operation steps, can directly detect the Eriocheir sinensis reovirus from a complex sample, directly judges whether the Eriocheir sinensis reovirus is negative or positive by observing an amplification curve, has direct and objective results, does not need any other analysis steps such as fussy electrophoresis and the like, has low experience requirement on operators, and is suitable for rapid and accurate field detection.
(5) Setting an internal reference primer: abnormal results caused by sample nucleic acid problems or reagent failures can be eliminated according to whether the amplification result of the internal reference primer is normal or not.
Drawings
Fig. 1 is a schematic diagram of results of screening a eriocheir sinensis reovirus primer set by a fluorescence quantitative PCR method in example 1, wherein 1 is a eriocheir sinensis reovirus primer set 1 positive control, 2 is a eriocheir sinensis reovirus primer set 2 positive control, 3 is a eriocheir sinensis reovirus primer set 1 negative control, and 4 is a eriocheir sinensis reovirus primer set 2 negative control.
Fig. 2 is a schematic diagram of the results of screening the primer set of the internal reference gene of the eriocheir sinensis by the fluorescent quantitative PCR method in example 1, wherein 1 is a positive control of the primer set 1 of the internal reference gene of the eriocheir sinensis, 2 is a positive control of the primer set 2 of the internal reference gene of the eriocheir sinensis, 3 is a negative control of the primer set 1 of the internal reference gene of the eriocheir sinensis, and 4 is a negative control of the primer set 2 of the internal reference gene of the eriocheir sinensis.
FIG. 3 is a diagram showing the results of the fluorescence quantitative PCR method for detecting the Eriocheir sinensis reovirus in example 2, in which 1 is a positive control, 2 is a sample 2, 3 is a sample 1, and 4 is a negative control.
FIG. 4 is a graph showing the results of sensitivity detection of plasmid DNA of Eriocheir sinensis reovirus by the fluorescent quantitative PCR method in example 3, wherein 1 is 107copies/. mu.L, 2 is 106copies/. mu.L, 3 is 105copies/. mu.L, 4 is 104copies/. mu.L, 5 is 103copies/. mu.L, 6 is 102copies/. mu.L, 7 is 101copies/. mu.L, 8 is 100 copies/. mu.L, and 9 is a negative control.
Fig. 5 is a schematic diagram of the results of detecting reference genes in an actual sample by the fluorescent quantitative PCR method in example 4, in which 1 is a eriocheir sinensis reovirus sample, 2 is a grass carp reovirus sample, 3 is a positive control sample, 4 is a blue crab cistronic virus sample, 5 is a type i herpesvirus sample, and 6 is a negative control.
Fig. 6 is a schematic diagram of the result of detecting the specificity of the actual sample of the eriocheir sinensis reovirus by the fluorescent quantitative PCR method in example 4, wherein 1 is a positive control sample, 2 is the eriocheir sinensis reovirus sample, 3 is the grass carp reovirus sample, 4 is the blue crab cistron virus sample, 5 is the herpes virus type i sample, and 6 is a negative control.
FIG. 7 is a graph showing the results of the reproducibility of the plasmid DNA for detecting Eriocheir sinensis reovirus by the fluorescent quantitative PCR method in example 5, in which 1 is 106copies/. mu.L, 2 is 102copies/. mu.L, 3 is a negative control.
Detailed Description
The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
Example 1 influence of primer set on fluorescent quantitative PCR for detecting Eriocheir sinensis reovirus
First, design primer
The Eriocheir sinensis reovirus VP9 gene is used as a specific target gene, and the nucleotide sequence is shown as SEQ ID NO. 1; the Eriocheir sinensis 18S rRNA partial gene sequence is taken as an internal reference gene, and the nucleotide sequence is shown as SEQ ID NO. 2. Designed by PrimeExpress3.0.1 using primer design software, and synthesized by Biotechnology engineering (Shanghai) Ltd.
1. Primer group of Eriocheir sinensis reovirus
In the primer group 1 of the Eriocheir sinensis reovirus:
forward primer F (SEQ ID NO: 3): 5'-AGTTTGTTTGCCCCGGAATAC-3', respectively;
reverse primer R (SEQ ID NO: 4): 5'-AAGGTGAGATCTTTGGCTAATTCTG-3', respectively;
fluorescent probe Q (SEQ ID NO: 5): 5'-ATGGTCTAGTGCCACATC-3', respectively;
the 5 'end of the fluorescent probe Q is marked with a 6-FAM fluorescent group, and the 3' end is marked with a quenching group MGB capable of quenching a fluorescent signal emitted by the fluorescent group.
In the primer group 2 of the Eriocheir sinensis reovirus:
forward primer F (SEQ ID NO: 6): 5'-TCCACTGAGTCATACTTTTGGTTACC-3', respectively;
reverse primer R (SEQ ID NO: 7): 5'-CACAGTGGCGTCATAATCGTAATC-3', respectively;
fluorescent probe Q (SEQ ID NO: 8): 5'-ACTTCTCGCACAAAAG-3', respectively;
the 5 'end of the fluorescent probe Q is marked with a 6-FAM fluorescent group, and the 3' end is marked with a quenching group MGB capable of quenching a fluorescent signal emitted by the fluorescent group.
2. Primer group of internal reference gene of eriocheir sinensis
In the primer group 1 of the internal reference gene of the eriocheir sinensis:
forward primer F (SEQ ID NO: 9): 5'-ACGTCCCTGCCCTTTGTACAC-3', respectively;
reverse primer R (SEQ ID NO: 10): 5'-GGCCTCACTAAATCATTCAATCG-3', respectively;
fluorescent probe Q (SEQ ID NO: 11): 5'-CGCCCGTCGCTAC-3', respectively;
the 5 'end of the fluorescent probe Q is marked with a 6-FAM fluorescent group, and the 3' end is marked with a quenching group MGB capable of quenching a fluorescent signal emitted by the fluorescent group.
In the primer group 2 of the internal reference gene of the eriocheir sinensis:
forward primer F (SEQ ID NO: 12): 5'-CCCAGTAAGCGCAAGTCATGA-3', respectively;
reverse primer R (SEQ ID NO: 13): 5'-GACGGGCGGTGTGTACAAAG-3', respectively;
fluorescent probe Q (SEQ ID NO: 14): 5'-TGCGTTGATTACGTCCCT-3', respectively;
the 5 'end of the fluorescent probe Q is marked with a 6-FAM fluorescent group, and the 3' end is marked with a quenching group MGB capable of quenching a fluorescent signal emitted by the fluorescent group.
Secondly, carrying out PCR reaction
The reaction system is as follows: the final concentrations of the primer group forward primer F, the reverse primer R and the fluorescent probe Q primer of the Eriocheir sinensis reovirus in the reaction system are 0.4 mu M, 0.4 mu M and 0.2 mu M respectively. The final concentrations of the primer group forward primer F, the reverse primer R and the fluorescent probe Q primer of the eriocheir sinensis internal reference gene in the reaction system are respectively 0.24 mu M, 0.24 mu M and 0.16 mu M. 2 Xreaction buffer 12.5U L, DNA polymerase 3U, reverse transcriptase 3U, UNG enzyme 0.4U, test sample 5U L, adding no RNase deionized water to 25U L.
The reaction procedure is as follows: 5min at 50 ℃; 1min at 95 ℃; 95 ℃ for 15s, 60 ℃ for 45s, 45 cycles.
Third, experimental results
As shown in FIG. 1, the results show that the time of appearance of the primer set 1 of the Eriocheir sinensis reovirus is earlier than that of the primer set 2, the fluorescence value of the primer set 1 is higher than that of the primer set 2, no non-specific amplification occurs in both primer sets, and the primer set 1 of the Eriocheir sinensis reovirus is selected as the primer set for detecting the Eriocheir sinensis reovirus.
As shown in figure 2, the results show that the internal reference gene primer group 1 of the eriocheir sinensis has a peak time earlier than that of the primer group 2, the repeatability of the primer group 1 is better than that of the primer group 2, and the two primer groups have no nonspecific amplification, so that the internal reference gene primer group 1 of the eriocheir sinensis is selected as a primer group for detecting the internal reference gene of the eriocheir sinensis.
Example 2 fluorescence quantitative PCR detection kit for detecting Eriocheir sinensis reovirus
The method comprises the following steps:
1. a group of fluorescence quantitative PCR primer groups (SEQ ID NO: 3-5) for detecting the Eriocheir sinensis reovirus in the embodiment 1;
forward primer F (SEQ ID NO: 3): 5'-AGTTTGTTTGCCCCGGAATAC-3', respectively;
reverse primer R (SEQ ID NO: 4): 5'-AAGGTGAGATCTTTGGCTAATTCTG-3', respectively;
fluorescent probe Q (SEQ ID NO: 5): 5'-ATGGTCTAGTGCCACATC-3', respectively;
the 5 'end of the fluorescent probe Q is marked with a 6-FAM fluorescent group, and the 3' end is marked with a quenching group MGB capable of quenching a fluorescent signal emitted by the fluorescent group.
2. A primer group (SEQ ID NO: 9-11) for detecting the internal reference gene of the Eriocheir sinensis in example 1;
forward primer F (SEQ ID NO: 9): 5'-ACGTCCCTGCCCTTTGTACAC-3', respectively;
reverse primer R (SEQ ID NO: 10): 5'-GGCCTCACTAAATCATTCAATCG-3', respectively;
fluorescent probe Q (SEQ ID NO: 11): 5'-CGCCCGTCGCTAC-3', respectively;
the 5 'end of the fluorescent probe Q is marked with a 6-FAM fluorescent group, and the 3' end is marked with a quenching group MGB capable of quenching a fluorescent signal emitted by the fluorescent group.
3. DNA polymerase, reverse transcriptase, UNG enzyme, 2 × reaction buffer solution, sealing solution, positive control reference substance and negative control reference substance.
The DNA polymerase is a mixed solution of Tth DNA polymerase and a hot start antibody, the reverse transcriptase is M-MLV reverse transcriptase, the UNG enzyme is uracil-N-glycosylase, the 2 × reaction buffer solution is a 2 × concentration reaction solution containing dATP, dCTP, dGTP and dUTP, and the sealing liquid is mineral oil.
The positive control reference substance is plasmid DNA containing a Eriocheir sinensis reovirus detection target gene SEQ ID NO. 1 and an internal reference gene sequence SEQ ID NO. 2, and the negative control reference substance is deionized water without RNase.
Second, use method
1. Grinding the sample to a uniform state, adding 400 mu L of lysis solution into a centrifuge tube, and uniformly mixing for 30s by oscillation. Standing at room temperature for 5-10 min, and centrifuging at 10000rpm for 3min to remove impurities.
2. The nucleic acid adsorption column sleeve in the kit is taken out, supernatant is transferred to the column as much as possible, and centrifugation is carried out at 10000rpm for 1 min. After the filtrate was discarded, the nucleic acid adsorbing column was put back into the collecting tube, 500. mu.L of the washing solution was added to the adsorbing column, and centrifugation was carried out at 10000rpm for 1 min. Then 400. mu.L of washing solution was added, centrifugation was carried out at 10000rpm for 1min, the filtrate was discarded, centrifugation was carried out at 10000rpm for 3min, the nucleic acid adsorption column was transferred to a new 1.5mL centrifuge tube, and 100. mu.L of eluent was added to the center of the membrane of the column. Standing at room temperature for 1min, and centrifuging at 10000rpm for 1 min.
3. Discarding the column, completing the extraction and storing at-20 deg.C for later use, and storing at-80 deg.C for a long time.
4. Performing a PCR reaction
The reaction system is as follows: the final concentrations of the primer group forward primer F, the reverse primer R and the fluorescent probe Q primer of the Eriocheir sinensis reovirus in the reaction system are 0.4 mu M, 0.4 mu M and 0.2 mu M respectively. The final concentrations of the primer group forward primer F, the reverse primer R and the fluorescent probe Q primer of the eriocheir sinensis internal reference gene in the reaction system are respectively 0.24 mu M, 0.24 mu M and 0.16 mu M. 2 Xreaction buffer 12.5U L, DNA polymerase 3U, reverse transcriptase 3U, UNG enzyme 0.4U, test sample 5U L, adding no RNase deionized water to 25U L.
The reaction procedure is as follows: 5min at 50 ℃; 1min at 95 ℃; 95 ℃ for 15s, 60 ℃ for 45s, 45 cycles.
The extracted RNA is detected, and a negative control is set.
Thirdly, judging the result
The kit is used for detecting the actual sample of the Eriocheir sinensis reovirus, the sample presents typical S-shaped curve amplification (as shown in figure 3), and the result is a positive result, so that the Eriocheir sinensis reovirus is detected in the sample. Otherwise, the result is negative.
Example 3 sensitivity test
Plasmid DNA (positive control reference in the kit of example 2) was diluted 10-fold in gradient and each diluted 10-fold7copies/μL、106copies/μL、105copies/μL、104copies/μL、103copies/μL、102copies/μL、101copies/. mu.L and 100Eight gradient concentrations of copies/. mu.L DNA as template and negative control (RNase-free deionized water) were detected using the kit of example 2.
As shown in fig. 4, the results show that: after 10-fold gradient dilution of plasmid DNA, the kit of example 2 can detect 10 concentrations1Plasmid DNA at copies/. mu.L.
Example 4 specificity experiments
Samples of Eriocheir sinensis reovirus, grass carp reovirus, herpes virus type I and blue crab cistron virus were each detected using the kit of example 2.
As shown in FIG. 5, the results show that the reference genes in the test sample all show amplification curves, which indicate that the nucleic acids in the test sample are normal, and abnormal results caused by the nucleic acid problems or reagent failures in the test sample are eliminated.
As shown in FIG. 6, the results showed that only the sample for detecting the Eriocheir sinensis reovirus exhibited an amplification curve, and the remaining samples were not amplified, indicating that the detection specificity of the kit of example 2 was good.
EXAMPLE 5 repeatability experiments
At a concentration of 106copies/. mu.L and 102copies/. mu.L of Eriocheir sinensis reovirus plasmid DNA (positive control reference substance in the kit of example 2) as template DNA, each set 20 times in parallel, and set negative control (deionized water without RNase) for detection using the kit of example 2.
As shown in FIG. 7, the results show that the detection experiment was performed by repeating two concentrations of plasmid DNA 20 times, the reproducibility was good, and the Ct value variation coefficient was not more than 5%, demonstrating that the stability of the kit of example 2 was good.
It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Sequence listing
<110> Guangzhou double helix Gene technology, Inc
Quzhou city water production technology popularization center (Quzhou city fishery environment monitoring and epidemic disease prevention and control center)
<120> fluorescence quantitative PCR detection kit for detecting eriocheir sinensis reovirus
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atgtcagacc aagtcactaa tcgctcggga gctagtcttc cactgagtca tacttttggt 60
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acttggacac cattcgaatc taacgataag tatgggaatg cggatgttga tatatacttg 180
accttctatc agataggtgc taaagtattc ttcactgtca ctaaacaacc aacagaagat 240
gagcgtagtt tgccgttaag tgtcacctgg actagaaatg gaggtgttgg atttacagat 300
gacagaggtg cgattataaa ctggttgcag ctgatatcta aaactcaggc tacggttact 360
aaatctctaa ttttcctggt ttgcgataat ttcgatggtt tcttcgtcct cataccttgt 420
aatatgaatg atgctcatca gtgtggtagc tttgctgtca gaaagaaagt cgccaaaccg 480
ttagctgtac tcgctaggtt aatgaacata gtaccatcaa ctaaaggaga gagcaacctt 540
tacatacaaa atatgttaaa ggctctgcga ctcagaagtg ataataccac agagttgatc 600
ccatctgatg acggagctag agtcatggca ccatatctaa ctgatgagga tacagaatct 660
attctacgcg gtgcactttt aggactcgaa ccaggtaata aggttattcg ttggtgcaac 720
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atcgccagcg aaatgtggaa gacgcaaggt gtgcctatac ttactaacag tttgtttgcc 840
ccggaatacg atggtctagt gccacatctt ccagaattag ccaaagatct caccttatac 900
caagagtggg tgcacgaggc tatggagact gatcataatg gtgctttagt aagagctggt 960
aagtatggaa tcgaccaagg gttattcaca ctagacgatc ctttcatccc tgcatggttg 1020
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Claims (10)
1. A primer group for detecting the eriocheir sinensis reovirus is characterized by consisting of a forward primer F, a reverse primer R and a fluorescent probe Q, wherein the nucleotide sequence of the forward primer F is shown as SEQ ID NO. 3, the nucleotide sequence of the reverse primer R is shown as SEQ ID NO. 4, and the nucleotide sequence of the fluorescent probe Q is shown as SEQ ID NO. 5.
2. A primer group for detecting internal reference genes of eriocheir sinensis is characterized by consisting of a forward primer F, a reverse primer R and a fluorescent probe Q, wherein the nucleotide sequence of the forward primer F is shown as SEQ ID NO. 9, the nucleotide sequence of the reverse primer R is shown as SEQ ID NO. 10, and the nucleotide sequence of the fluorescent probe Q is shown as SEQ ID NO. 11.
3. The primer set according to claim 1 or 2, wherein the fluorescent probe Q is labeled with a fluorescent group at the 5 'end and a quenching group at the 3' end.
4. A detection kit comprising the primer set according to claim 1.
5. The detection kit according to claim 4, further comprising the primer set according to claim 2.
6. The kit of claim 4 or 5, further comprising a DNA polymerase, a reverse transcriptase, a UNG enzyme, a 2 Xreaction buffer, a sealing solution, a positive control reference and/or a negative control reference.
7. The kit according to claim 6, wherein the DNA polymerase is a mixture of Tth DNA polymerase and a hot start antibody, the reverse transcriptase is M-MLV reverse transcriptase, the UNG enzyme is uracil-N-glycosylase, the 2X reaction buffer is a 2X reaction solution containing dATP, dCTP, dGTP and dUTP, and the sealing liquid is mineral oil.
8. The kit of claim 6, wherein the positive control reference is plasmid DNA containing Eriocheir sinensis reovirus detection target gene and internal reference gene, and the negative control reference is deionized water without RNase.
9. The kit according to claim 6, wherein the reaction system of the kit is: the primer set according to claim 1, wherein the final concentrations of the forward primer F, the reverse primer R and the fluorescent probe Q primer in the reaction system are 0.4-0.6. mu.M, 0.4-0.6. mu.M and 0.2-0.3. mu.M, respectively; the primer set according to claim 2, wherein the final concentrations of the forward primer F, the reverse primer R and the fluorescent probe Q primer in the reaction system are 0.2-0.4. mu.M, 0.2-0.4. mu.M and 0.1-0.2. mu.M, respectively; 12.4-12.6 mu L of 2 multiplied reaction buffer solution, 2-4U of DNA polymerase, 0.3-0.5U of reverse transcriptase 2-4U, UNG enzyme, 2-5 mu L of sample to be detected, and 25 mu L of deionized water without RNA enzyme.
10. The kit according to claim 9, wherein the reaction conditions of the reaction system are 50 ℃ for 5 min; 5min at 95 ℃; 15s at 95 ℃, 30s at 60 ℃ and 45 cycles.
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| CN114438263A (en) * | 2022-02-22 | 2022-05-06 | 广州双螺旋基因技术有限公司 | Fluorescent quantitative PCR (polymerase chain reaction) primer group and kit for detecting European eel circovirus |
| CN114686601A (en) * | 2022-02-28 | 2022-07-01 | 天津师范大学 | Specific probe for detecting eriocheir sinensis hemocyanin gene expression and application |
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