CN109234458B - Complete set of reagents for detecting infectious pancreatic necrosis virus - Google Patents
Complete set of reagents for detecting infectious pancreatic necrosis virus Download PDFInfo
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Abstract
The invention discloses a reagent set for detecting infectious pancreatic necrosis virus. The invention discloses a reagent set for detecting infectious pancreatic necrosis virus, which consists of six single-stranded DNAs with the names of B2-F3, B2-B3, B2-FIP, B2-BIP, B2-LooF and B2-loB respectively; B2-F3, B2-B3, B2-FIP, B2-BIP, B2-LooF and B2-loB are single-stranded DNA shown in SEQ ID No.1-6 in the sequence table respectively. Experiments prove that the kit for detecting the infectious pancreatic necrosis virus has good specificity, and does not have cross reaction with Infectious Hematopoietic Necrosis Virus (IHNV) and Viral Hemorrhagic Septicemia Virus (VHSV); the sensitivity is high and is 0.0008fg/25 muL reaction system; the detection is simple and convenient and saves time.
Description
Technical Field
The invention relates to a reagent set for detecting infectious pancreatic necrosis virus in the field of biotechnology.
Background
Infectious Pancreatic Necrosis Virus (IPNV) is a pathogen causing infectious pancreatic necrosis disease (IPN) of salmon and trout, belongs to Birnaviridae (Birnaviridae), aquatic Birnaviridae (Aquaninavirus), and has a mortality rate of up to 90% for susceptible salmon and trout, survivors are potential infection sources and carry the virus for life, the disease is wide in susceptibility range, high in lethality rate and strong in infectivity, and the fish farming industry all over the world is seriously damaged. The virus has spread to most rainbow trout farms in China from the last 80 th century to the present, causing serious economic loss.
The detection means aiming at IPNV mainly comprises enzyme-linked immunosorbent assay (ELISA), RT-PCR, fluorescent quantitative PCR and probe fluorescent quantitative RT-PCR. The methods can be completed only by using professional equipment in a laboratory, and the requirements on test conditions are high.
Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid isothermal amplification technology developed by Notomi and the like, and the reaction can be completed within 1 hour under the isothermal condition. The product obtained by LAMP amplification can be detected by agarose gel electrophoresis, and can be stained by a fluorescent stain SYBR Green I, and then the result of the sample is judged according to the color change of the product by visual observation.
Disclosure of Invention
The invention aims to solve the technical problem of how to detect the infectious pancreatic necrosis virus.
In order to solve the technical problems, the invention firstly provides a reagent set which consists of six single-stranded DNAs with the names of B2-F3, B2-B3, B2-FIP, B2-BIP, B2-LooF and B2-looB;
the B2-F3 is any one of the following single-stranded DNA from a1) to a 4):
a1) a single-stranded DNA represented by SEQ ID No.1 in the sequence Listing;
a2) single-stranded DNA obtained by adding one or several nucleotides to the 5 'end and/or 3' end of a 1);
a3) a single-stranded DNA having 85% or more identity to the single-stranded DNA defined in a1) or a 2);
a4) single-stranded DNA which hybridizes with the single-stranded DNA defined in a1) or a2) under stringent conditions;
the B2-B3 is any one of the following single-stranded DNA from B1) to B4):
b1) a single-stranded DNA represented by SEQ ID No.2 in the sequence Listing;
b2) single-stranded DNA obtained by adding one or several nucleotides to the 5 'end and/or 3' end of b 1);
b3) a single-stranded DNA having 85% or more identity to the single-stranded DNA defined in b1) or b 2);
b4) single-stranded DNA which hybridizes with the single-stranded DNA defined in b1) or b2) under stringent conditions;
the B2-FIP is any one single-stranded DNA of c1) to c4) as follows:
c1) a single-stranded DNA represented by SEQ ID No.3 of the sequence Listing;
c2) single-stranded DNA obtained by adding one or several nucleotides to the 5 'end and/or 3' end of c 1);
c3) a single-stranded DNA having 85% or more identity to the single-stranded DNA defined in c1) or c 2);
c4) single-stranded DNA which hybridizes with the single-stranded DNA defined in c1) or c2) under stringent conditions;
the B2-BIP is any one of the following single-stranded DNA from d1) to d 4):
d1) a single-stranded DNA represented by SEQ ID No.4 of the sequence Listing;
d2) single-stranded DNA obtained by adding one or several nucleotides to the 5 '-end and/or 3' -end of d 1);
d3) a single-stranded DNA having 85% or more identity to the single-stranded DNA defined by d1) or d 2);
d4) single-stranded DNA which hybridizes with the single-stranded DNA defined by d1) or d2) under stringent conditions;
the B2-LooF is any one of the following single-stranded DNA from e1) to e 4):
e1) a single-stranded DNA represented by SEQ ID No.5 of the sequence Listing;
e2) single-stranded DNA obtained by adding one or several nucleotides to the 5 '-end and/or 3' -end of e 1);
e3) a single-stranded DNA having 85% or more identity to the single-stranded DNA defined by e1) or e 2);
e4) single-stranded DNA which hybridizes under stringent conditions with the single-stranded DNA defined in e1) or e 2);
the B2-looB is any one of the following single-stranded DNA from f1) to f 4):
f1) a single-stranded DNA represented by SEQ ID No.6 of the sequence Listing;
f2) single-stranded DNA obtained by adding one or several nucleotides to the 5 '-end and/or 3' -end of f 1);
f3) a single-stranded DNA having 85% or more identity to the single-stranded DNA defined by f1) or f 2);
f4) a single-stranded DNA which hybridizes with the single-stranded DNA defined in f1) or f2) under stringent conditions.
The kit can be used for detecting the infectious pancreatic necrosis virus or preparing a kit for detecting the infectious pancreatic necrosis virus.
Each single-stranded DNA in the kit can be independently packaged. The molar ratio of the B2-F3, the B2-B3, the B2-FIP, the B2-BIP, the B2-LooF and the B2-looB in the kit can be 1:1:4:4:2: 2.
In the kit, the addition of one or several nucleotides may be one to ten nucleotides.
The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences having 85% or more, or 90% or more, or 95% or more identity to the nucleotide sequence shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5 or SEQ ID No.6 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.
In the above kit, the stringent conditions are hybridization and membrane washing at 68 ℃ for 2 times and 5min each in a solution of 2 XSSC, 0.1% SDS, and hybridization and membrane washing at 68 ℃ for 2 times and 15min each in a solution of 0.5 XSSC, 0.1% SDS; alternatively, hybridization was carried out at 65 ℃ in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS, and the membrane was washed.
The above-mentioned identity of 85% or more may be 85%, 90% or 95% or more.
The invention also provides the application of the kit in the preparation of the kit; the use of the kit is g1) or g2) as follows:
g1) detecting or detecting the infectious pancreatic necrosis virus in an auxiliary way;
g2) is used for preparing a kit for detecting or assisting in detecting the infectious pancreatic necrosis virus.
The invention also provides a kit containing the kit, and the use of the kit is g1) or g2) as follows:
g1) detecting or detecting the infectious pancreatic necrosis virus in an auxiliary way;
g2) is used for preparing a kit for detecting or assisting in detecting the infectious pancreatic necrosis virus.
The kit may further contain a strand displacement-type DNA polymerase. The strand displacement type DNA polymerase may be Bst DNA polymerase.
The kit may also contain a reverse transcriptase. The reverse transcriptase may be AMV reverse transcriptase.
The kit may further comprise betaine, dNTPs and/or Mg2+。
The kit can be prepared from the kit and the strand displacement type DNA polymerase, the reverse transcriptase, betaine, dNTPs and/or Mg2+And (4) forming.
The kit can be a loop-mediated isothermal amplification reagent or a kit.
The invention also provides application of the kit in detection or auxiliary detection of the infectious pancreatic necrosis virus.
The invention also provides a method for detecting or assisting in detecting infectious pancreatic necrosis virus, the method comprising: and (3) performing LAMP by using the complete set of reagents by using the genome RNA of a sample to be detected as a template, and determining whether the sample to be detected contains the infectious pancreatic necrosis virus or is the infectious pancreatic necrosis virus according to a reaction product in a reaction system.
The concentrations of the B2-F3 and the B2-B3 in the reaction system are both 0.4 mu M, the concentrations of the B2-FIP and the B2-BIP are both 1.6 mu M, and the concentrations of the B2-LooF and the B2-looB are both 0.8 mu M.
Concentration of the strand displacement type DNA polymerase in the reaction systemThe degree was 8U/25. mu.L. The concentration of the reverse transcriptase in the reaction system may be 10U/25. mu.L. The concentration of betaine in the reaction system is 0-1.2M, such as 0.4M. The concentration of dNTPs in the reaction system is 0.5-1.5 mM, such as 1mM, i.e., the concentration of dATP, dTTP, dCTP and dGTP in the reaction system is 0.5-1.5 mM (such as 1 mM). Mg in the reaction system2+May be 6 mM.
The temperature of the LAMP reaction may be 63 ℃ to 67 ℃.
Experiments prove that the kit for detecting the infectious pancreatic necrosis virus has good specificity, and does not have cross reaction with Infectious Hematopoietic Necrosis Virus (IHNV) and Viral Hemorrhagic Septicemia Virus (VHSV); the sensitivity is high and is 0.0008fg/25 muL reaction system. The detection is only carried out in a conventional water bath kettle, the reaction can be completed within 60min, the detection result can be judged by naked eyes according to the color change of the product by adding the fluorescent dye after the reaction is finished, and the detection is simple, convenient and time-saving.
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FIG. 1 is Mg2+Influence of concentration on amplification effect of RT-LAMP. Wherein A is an electrophoresis detection result, and B is a color change of the reaction system. M is DL2000DNA molecular mass standard; 1: negative control; 2:2 mM; 3: 4 mM; 4: 6 mM; 5: 8 mM.
FIG. 2 shows the effect of betaine concentration on the amplification effect of RT-LAMP. Wherein A is an electrophoresis detection result, and B is a color change of the reaction system. M is DL2000DNA molecular mass standard; 1: negative control; 2: 0.0M; 3: 0.4M; 4: 0.8M; 5: 1.2M.
FIG. 3 shows the effect of dNTPs concentration on RT-LAMP amplification. Wherein A is an electrophoresis detection result, and B is a color change of the reaction system. M is DL2000DNA molecular mass standard; 1: negative control; 2: 0.5 mM; 3: 1.0 mM; 4: 1.5 mM.
FIG. 4 shows the effect of temperature on the amplification effect of RT-LAMP. Wherein A is an electrophoresis detection result, and B is a color change of the reaction system. M is DL2000DNA molecular mass standard; 1: negative control; 2: 63 ℃; 3: 65 ℃; 4: 67 ℃.
FIG. 5 shows the results of specific detection of the kit of example 1. Wherein A is an electrophoresis detection result, and B is a color change of the reaction system. M is DL2000DNA molecular mass standard; 1: negative control; 2: IHNV; 3: VHSV; 4: IPNV.
FIG. 6 shows the results of the sensitive detection of the reagent set of example 1. Wherein, M is DL2000DNA molecular mass standard; 1: negative control; 2: the content of IPNV RNA in the reaction system is 8X 10-1ng; 3: the content of IPNV RNA in the reaction system is 8X 10-2ng; 4: the content of IPNV RNA in the reaction system is 8X 10-3ng; 5: the content of IPNV RNA in the reaction system is 8X 10-4ng; 6: the content of IPNV RNA in the reaction system is 8X 10-5ng; 7: the content of IPNV RNA in the reaction system is 8X 10-6ng; 8: the content of IPNV RNA in the reaction system is 8X 10-7ng; 9: the content of IPNV RNA in the reaction system is 8X 10- 8ng; 10: the content of IPNV RNA in the reaction system is 8X 10-9ng; 11: the content of IPNV RNA in the reaction system is 8X 10-10ng; 12: the content of IPNV RNA in the reaction system is 8X 10-11ng。
FIG. 7 shows the results of the sensitivity test of the reagent set of comparative example 1. Wherein, M is DL2000DNA molecular mass standard; 1: negative control; 2: the content of IPNV RNA in the reaction system is 8X 10-1ng; 3: the content of IPNV RNA in the reaction system is 8X 10-2ng; 4: the content of IPNV RNA in the reaction system is 8X 10-3ng; 5: the content of IPNV RNA in the reaction system is 8X 10-4ng; 6: the content of IPNV RNA in the reaction system is 8X 10-5ng; 7: the content of IPNV RNA in the reaction system is 8X 10-6ng; 8: the content of IPNV RNA in the reaction system is 8X 10-7ng; 9: the content of IPNV RNA in the reaction system is 8X 10- 8ng; 10: the content of IPNV RNA in the reaction system is 8X 10-9ng; 11: the content of IPNV RNA in the reaction system is 8X 10-10ng; 12: the content of IPNV RNA in the reaction system is 8X 10-11ng。
Fig. 8 is the result of the test of comparative example 2. Wherein A is an electrophoresis detection result, and B is a color change of the reaction system. M is DL2000DNA molecular mass standard; 1: negative control; 2: kit 3; 3: kit 1; 4: reagent set 2; 5: the kit of example 1.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA, and the last position is the 3' terminal nucleotide of the corresponding DNA.
IPNV ChRtm213 strain (serotype Jasper, titer about 1X 10) in the examples below5TCID50/ml)(Ji F,Zhao J Z,Liu M,et al.Complete genomic sequence of an infectious pancreatic necrosis virus isolated from rainbow trout(Oncorhynchus mykiss)in China[J]The Virus Genes,2017,53(2):1-11.) the public has available from the applicant this biomaterial only for use in repeating the experiments associated with the present invention, and not for other uses.
The biological material was available to the public from the applicant for the isolation and identification of the oncorhynchus mykiss virus (CHSE-214) (liu\2815638, zurich, zhao jing, et al. rainbow trout infectious pancreatic necrosis virus [ J ] university of gangrene, 2017,32(1):56-61.) in the following examples, which was used only for the repetition of the experiments related to the present invention and was not used for other purposes.
Betaine is a product of Sigma company; bst 2.0DNA polymerase is a product of NEB company; AMV reverse transcriptase is a product of Promega corporation; SYBR Green I is a product of Invitrogen corporation; DL2000Marker is a product of Takara corporation; the gel imaging system Universal Hood II is a BIO-RAD product; the DKZW-D-2 electric heating constant temperature water bath is a product of Yongguanming medical instruments Limited company in Beijing.
Example 1 preparation of kit for detecting infectious pancreatic necrosis Virus
The embodiment provides a reagent set for detecting infectious pancreatic necrosis virus by using a loop-mediated isothermal amplification method, which consists of six single-stranded DNAs (deoxyribonucleic acids) named as B2-F3, B2-B3, B2-FIP, B2-BIP, B2-LooF and B2-looB; B2-F3 is single-stranded DNA shown in SEQ ID No.1 in the sequence table; B2-B3 is single-stranded DNA shown in SEQ ID No.2 in the sequence table; B2-FIP is single-stranded DNA shown in SEQ ID No.3 in the sequence table; B2-BIP is single-stranded DNA shown in SEQ ID No.4 in the sequence table; B2-LooF is a single-stranded DNA shown by SEQ ID No.5 in the sequence table; B2-looB is a single-stranded DNA shown by SEQ ID No.6 in the sequence table.
In the reagent set, each single-stranded DNA is independently packaged, wherein the molar ratio of B2-F3, B2-B3, B2-FIP, B2-BIP, B2-LooF and B2-looB is 1:1:4:4:2: 2.
Example 2 establishment of a method for detecting infectious pancreatic necrosis Virus
The total RNA of IPNV ChRtm213 strain and Cannabis sativa Ha embryo cell (CHSE-214) is respectively extracted, and the method for detecting the infectious pancreatic necrosis virus by using the reagent set of the embodiment 1 is optimized.
1. Mg in the reaction system2+Optimization of content
Preparing a 25 mu L RT-LAMP reaction system, wherein the reaction system comprises the following substances in percentage by weight: both B2-F3 and B2-B3 were 0.4. mu.M in concentration, both B2-FIP and B2-BIP were 1.6. mu.M in concentration, both B2-LooF and B2-loB were 0.8. mu.M in concentration, dNTPs were 1.0mM in concentration (dNTPs contained four dNTPs each at 1.0mM in the reaction system), and 2.5. mu.L of 10 × Isotermal Amplification Buffer [ containing 2mM MgSO 2 in final concentration4](NEB, cat # B0537S), Betaine (Betaine) concentration of 0.4mM, Bst 2.0DNA polymerase concentration of 8U/25. mu.L, AMV reverse transcriptase concentration of 10U/25. mu.L, 2. mu.L IPNV ChRtm213 total RNA, no RNase water to 25. mu.L, and mixing. Total RNA from Cannabis har embryonic cells (CHSE-214) was used as a negative control.
Using 100mM MgSO 2 according to the above reaction system4MgSO in the reaction system4Are adjusted to final concentrations of 2mM, 4mM, 6mM and 8m, respectivelyM, obtaining different Mg2+Concentration of the reaction system.
The Bst DNA polymerase is inactivated by placing the reaction systems in a water bath kettle with a constant temperature of 63 ℃ for reaction for 60min and immediately placing the reaction systems in a water bath with a temperature of 80 ℃ for 2 min. After the reaction is finished, adding 5 mu L of SYBR Green I diluted by 100 times into the amplification product, lightly shaking, and directly observing the color change judgment result of the reaction solution. Then, 5. mu.L of the product was subjected to 2% agarose gel electrophoresis, and the result was observed.
The results are shown in FIG. 1 and show that when Mg is present2+No specific band at a concentration of 2mM, Mg2+The most clear and bright trapezoid band at the concentration of 6mM indicates Mg in the reaction system2+The optimal final concentration of (3) is 6 mM.
2. Optimization of Betaine (Betaine) content in reaction system
According to the reaction system of the step 1, MgSO in the reaction system4The concentration of (2) was set at 6mM, and the betaine concentration was adjusted to obtain reaction systems having betaine concentrations of 0.0M, 0.4M, 0.8M and 1.2M, respectively, while the remaining substances and concentrations were unchanged. And (3) putting each reaction system in a constant-temperature water bath kettle at 63 ℃ respectively for reacting for 60min, and immediately putting the reaction system in a water bath at 80 ℃ for 2 min. After the reaction is finished, adding 5 mu L of SYBR Green I diluted by 100 times into the amplification product, lightly shaking, and directly observing the color change judgment result of the reaction solution. Then, 5. mu.L of the product was subjected to 2% agarose gel electrophoresis, and the result was observed.
The results are shown in FIG. 2, and show that trapezoidal bands appear when the concentration of Betaine is changed from 0.0M to 1.2M, but the band is clearest when the concentration is 0.4M, and the amplification effect is the best, which indicates that the optimal concentration of Betaine in the reaction system is 0.4M.
3. Optimization of dNTPs concentration in reaction system
According to the reaction system of the step 1, MgSO in the reaction system4The concentration of dNTPs was adjusted to 6mM and the concentration of betaine was adjusted to 0.4M, so that reaction systems were obtained in which the concentration of each dNTP (i.e., dATP, dTTP, dCTP, or dGTP) in the dNTPs was 0.5mM, 1.0mM, and 1.5mM, respectively, and the remaining substances and concentrations were unchanged. Placing each reaction system in a water bath kettle with a constant temperature of 63 deg.C for reaction for 60min, and immediately placing untilWater bath at 80 deg.c for 2 min. After the reaction is finished, adding 5 mu L of SYBR Green I diluted by 100 times into the amplification product, lightly shaking, and directly observing the color change judgment result of the reaction solution. Then, 5. mu.L of the product was subjected to 2% agarose gel electrophoresis, and the result was observed.
As shown in FIG. 3, the results showed that trapezoidal bands appeared in the dNTPs at concentrations varying from 0.5 to 1.5mM, but the bands were most distinct at 1.0mM, and the amplification effect was the best. Indicating that the optimal concentration of dNTPs in the reaction system is 1.0 mM.
4. Optimization of reaction temperature
According to the reaction system of the step 1, MgSO in the reaction system4The concentration of (A) was set to 6mM, the concentration of betaine was set to 0.4M, dNTPs and the concentration was set to 1.0mM (i.e., the concentrations of dATP, dTTP, dCTP and dGTP in the reaction system were all 1.0mM), and the other substances and concentrations were not changed. The obtained reaction systems are respectively reacted in constant temperature water bath pots at 63 ℃, 65 ℃ and 67 ℃ for 60min, immediately placed in 80 ℃ water bath for 2min, and three repeated reaction systems are arranged at each reaction temperature. After the reaction is finished, adding 5 mu L of SYBR Green I diluted by 100 times into the amplification product, lightly shaking, and directly observing the color change judgment result of the reaction solution. Then, 5. mu.L of the product was subjected to 2% agarose gel electrophoresis, and the result was observed.
The results (FIG. 4) show that the amplification product shows fluorescent Green color after adding SYBR Green I when the reaction temperature is 63 ℃, 65 ℃ and 67 ℃, but the electrophoresis band of the amplification product is brightest when the reaction temperature is 63 ℃. Therefore, 63 ℃ is the optimum reaction temperature.
Example 3 specificity of kit for detecting infectious pancreatic necrosis Virus
The samples to be tested were: IPNV ChRtm213 strain, Infectious Hematopoietic Necrosis Virus (IHNV) Sn1203, and Viral Hemorrhagic Septicemia Virus (VHSV) RNA, wherein IHNV Sn1203(Xu L M, ZHao J Z, Liu M, et al. high throughput screening of Viral Infectious hematopoietic necrosis virus from Viral Infectious hematopoietic necrosis virus library [ J ]. Aquaculture,2016,460: 32-36.); VHSV is a product of China center for type culture Collection, the strain number is VR-1387, which is derived from China center for type culture Collection and is described in the establishment and application of RT-LAMP detection method for infectious hematopoietic necrosis virus of fish (Liu 28156, Xudaeng, Lutong rock, etc. [ J ] Chinese aquatic science, 2014,21(5):1065-1071 ]).
Each viral RNA was detected according to the optimal reaction system and optimal reaction conditions obtained in example 2, using total RNA of Cannabis har embryo cells (CHSE-214) as a negative control. The optimum reaction system is as follows:
both B2-F3 and B2-B3 were 0.4. mu.M, both B2-FIP and B2-BIP were 1.6. mu.M, both B2-LooF and B2-LooB were 0.8. mu.M, and dNTPs were 1.0mM (dNTPs contained four dNTPs each at 1.0mM in the reaction system), 2.5. mu.L of 10 × Isotermal Amplification Buffer [ containing 2mM MgSO as the final concentration ]4]，1.0μL 100mM MgSO4Betaine (Betaine)0.4M, Bst 2.0DNA polymerase concentration of 8U/25. mu.L, AMV reverse transcriptase concentration of 10U/25. mu.L, 2. mu.L IPNV ChRtm213 total RNA, no RNase water to 25. mu.L, and mixing. Wherein MgSO4The concentration in the reaction system was 6 mM.
The obtained reaction system is put into a constant temperature water bath kettle at 63 ℃ for reaction for 60min, and immediately put into a water bath at 80 ℃ for 2 min. After the reaction is finished, adding 5 mu L of SYBR Green I diluted by 100 times into the amplification product, lightly shaking, and directly observing the color change judgment result of the reaction solution. Then, 5. mu.L of the product was subjected to 2% agarose gel electrophoresis, and the result was observed.
The results (FIG. 5) show that the RT-LAMP amplification product is fluorescent Green after adding SYBR Green I by using IPNV RNA as a template, and the gel result is a specific stepped strip; RT-LAMP detection products with IHNV and VHSV as templates are reddish brown, and the result is negative. The RT-LAMP detection method has good specificity and does not have cross reaction with IHNV and VHSV.
Example 4 sensitivity of kit for detecting infectious pancreatic necrosis Virus
The samples to be tested were: the total RNA of IPNV ChRtm213 strain is diluted by 10 times of gradient with the total RNA of 4 ng/. mu.L IPNV ChRtm213 strain as the initial concentration to obtain the concentration of 4 multiplied by 10-1ng/μL、4×10-2ng/μL、4×10-3ng/μL、4×10-4ng/μL、4×10-5ng/μL、4×10-6ng/μL、4×10-7ng/μL、4×10-8ng/μL、4×10-9ng/μL、4×10-10ng/μL、4×10-11ng/. mu.L of IPNV RNA solution.
Each viral RNA was detected according to the optimal reaction system and optimal reaction conditions obtained in example 2, using total RNA of Cannabis har embryo cells (CHSE-214) as a negative control. The optimum reaction system is the same as in example 3.
The obtained reaction system is put into a constant temperature water bath kettle at 63 ℃ for reaction for 60min, and immediately put into a water bath at 80 ℃ for 2 min. After the reaction is finished, adding 5 mu L of SYBR Green I diluted by 100 times into the amplification product, lightly shaking, and directly observing the color change judgment result of the reaction solution. Then, 5. mu.L of the product was subjected to 2% agarose gel electrophoresis, and the result was observed.
The result shows that when the content of RNA in the reaction system is more than or equal to 0.0008fg, the RT-LAMP product still becomes fluorescent Green after SYBR Green I is added, and a clear specific step-shaped band can be seen through agarose gel electrophoresis. The established RT-LAMP detection method is proved to have high sensitivity which can reach 0.0008fg/25 muL reaction system.
Comparative example 1 detection of IPNV ChRtm213 Strain with other primers
The primers used were as follows:
Sp-F3：5′-CCAATCTGCGGTGTAGACAT-3′
Sp-B3：5′-CATCAGCTCTCCCAGGTACT-3′
Sp-FIP：5′-CCTCCTCGTCCACTCCTGGT-TTTT-CCATCGCAGCCCATGAAC-3′
Sp-BIP：5′-TGCGAAACACATCCCTGGCC-TTTT-TCTTGTTGGAGCCCTTTGC-3′
Sp-LooF：5′-CGATGAGTGGCAGCCCTT-3′
Sp-LooB：5′-GATCCAGACCGGAACCCTG-3′
the samples to be tested were: 4X 10 of example 4-1ng/μL、4×10-2ng/μL、4×10-3ng/μL、4×10-4ng/μL、4×10-5ng/μL、4×10-6ng/μL、4×10-7ng/μL、4×10-8ng/μL、4×10-9ng/μL、4×10-10ng/μL、4×10-11ng/. mu.L of IPNV RNA solution.
The reaction system is as follows: Sp-F3 and Sp-B3 each had a final concentration of 0.4. mu.M, Sp-FIP and Sp-BIP each had a final concentration of 1.6. mu.M, Sp-LooF and Sp-LooB each had a final concentration of 0.8. mu.M, dNTPs each had a final concentration of 1.0mM (dNTPs contained four dNTPs each having a concentration of 1.0mM in the reaction system), and 2.5. mu.L of 10 × Isothermal Amplification Buffer [ containing a final concentration of 2mM MgSO 2mM4]，1.0μL 100mM MgSO4The final concentration of Betaine (Betaine) was 0.4M, the final concentration of Bst 2.0DNA polymerase was 8U/25. mu.L, the final concentration of AMV reverse transcriptase was 10U/25. mu.L, 2. mu.L of IPNV ChRtm213 RNA was supplemented to 25. mu.L with RNase-free water, and mixed well.
The reaction conditions are as follows: the obtained reaction system is put into a constant temperature water bath kettle at 63 ℃ for reaction for 60min, and immediately put into a water bath at 80 ℃ for 2 min. After the reaction is finished, adding 5 mu L of SYBR Green I diluted by 100 times into the amplification product, lightly shaking, and directly observing the color change judgment result of the reaction solution. Then, 5. mu.L of the product was subjected to 2% agarose gel electrophoresis, and the result was observed.
And after the reaction is finished, adding 5 mu L of SYBR Green I diluted by 100 times into the amplification product, lightly shaking, and directly observing the color change judgment result of the reaction solution. Then, 5. mu.L of the product was subjected to 2% agarose gel electrophoresis, and the result was observed.
The result (figure 7) shows that when the complete set of reagent is used for detecting the IPNV ChRtm213 strain, SYBR Green I is added into the product obtained when the RNA content of the IPNV ChRtm213 strain in the reaction system is more than or equal to 0.008fg, and the product is in fluorescent Green; the product obtained when the RNA content of the IPNV ChRtm213 strain in the reaction system is less than 0.008fg is orange-red after SYBR Green I is added, and no specific step-shaped band is seen in agarose gel electrophoresis. The result shows that the sensitivity of the complete set of reagents for detecting the IPNV ChRtm213 strain is 0.008fg/25 muL of reaction system.
Comparative example 2 detection of IPNV ChRtm213 Strain with other primers
The RNA of the IPNV ChRtm213 strain was detected using kit 1, kit 2, kit 3 and kit in example 1, respectively, according to the optimal reaction system and optimal reaction conditions obtained in example 2. Total RNA from Cannabis har embryonic cells (CHSE-214) was used as a negative control. The optimum reaction system is the same as in example 3.
The obtained reaction system is put into a constant temperature water bath kettle at 63 ℃ for reaction for 60min, and immediately put into a water bath at 80 ℃ for 2 min. After the reaction is finished, adding 5 mu L of SYBR Green I diluted by 100 times into the amplification product, lightly shaking, and directly observing the color change judgment result of the reaction solution. Then, 5. mu.L of the product was subjected to 2% agarose gel electrophoresis, and the result was observed. The sequences of the reagent sets are as follows:
as can be seen from FIG. 8, trapezoidal bands appear in the electrophoresis detection of the products of reagent set 1 and reagent set 2, the amplification efficiency of the reagent set in example 1 is better than that of reagent set 2, and neither reagent set 1 nor reagent set 3 can be used for detecting IPNV.
<110> institute of aquatic products of Heilongjiang, China institute of aquatic science
<120> reagent set for detecting infectious pancreatic necrosis virus
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> DNA
<213> Artificial sequence
<400> 1
gagaccacaa acacccagg 19
<210> 2
<211> 16
<212> DNA
<213> Artificial sequence
<400> 2
gctgaggatg ctgccg 16
<210> 3
<211> 42
<212> DNA
<213> Artificial sequence
<400> 3
ttggggtcct tgtaggtccc cgtctccgaa gaaaaccctg ga 42
<210> 4
<211> 36
<212> DNA
<213> Artificial sequence
<400> 4
gagcgcagtg tggcgactgc ttcagcgccg aggctt 36
<210> 5
<211> 19
<212> DNA
<213> Artificial sequence
<400> 5
gatgttatgg cagctgtta 19
<210> 6
<211> 20
<212> DNA
<213> Artificial sequence
<400> 6
gaccaaagaa ccaaactccg 20
Claims (6)
1. The reagent set consists of six single-stranded DNAs with the names of B2-F3, B2-B3, B2-FIP, B2-BIP, B2-LooF and B2-looB;
B2-F3 is single-stranded DNA shown as SEQ ID No.1 in a sequence table;
the B2-B3 is single-stranded DNA shown as SEQ ID No.2 in a sequence table;
the B2-FIP is single-stranded DNA shown by SEQ ID No.3 in a sequence table;
the B2-BIP is single-stranded DNA shown by SEQ ID No.4 in a sequence table;
the B2-LooF is a single-stranded DNA shown by SEQ ID No.5 in the sequence table;
the B2-looB is a single-stranded DNA shown by SEQ ID No.6 in a sequence table;
the molar ratio of the B2-F3, the B2-B3, the B2-FIP, the B2-BIP, the B2-LooF and the B2-looB in the kit is 1:1:4:4:2: 2.
2. Use of a kit of parts according to claim 1 for the preparation of a kit; the use of the kit is g1) or g2) as follows:
g1) detecting or detecting the infectious pancreatic necrosis virus in an auxiliary way;
g2) is used for preparing a kit for detecting or assisting in detecting the infectious pancreatic necrosis virus.
3. A kit comprising the kit of parts according to claim 1 for the use of g1) or g2) as follows:
g1) detecting or detecting the infectious pancreatic necrosis virus in an auxiliary way;
g2) is used for preparing a kit for detecting or assisting in detecting the infectious pancreatic necrosis virus.
4. The kit of claim 3, wherein: the kit further contains a strand displacement-type DNA polymerase.
5. The kit of claim 4, wherein: the strand displacement type DNA polymerase is Bst DNA polymerase.
6. The kit according to any one of claims 3 to 5, wherein: the kit also contains reverse transcriptase.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103409563A (en) * | 2013-09-06 | 2013-11-27 | 青岛农业大学 | RT-LAMP (reverse transcription-loop-mediated isothermal amplification) detection kit for infectious pancreatic necrosis virus of rainbow trout |
| CN103937912A (en) * | 2014-05-13 | 2014-07-23 | 吴斌 | LAMP (Loop-Mediated Isothermal Amplification) primer composition for detecting infectious pancreas necrosis virus and application of LAMP primer composition |
| KR20170122933A (en) * | 2016-04-28 | 2017-11-07 | 한남대학교 산학협력단 | Primer set for detecting infectious pancreatic necrosis virus and uses thereof |
-
2018
- 2018-11-09 CN CN201811329310.2A patent/CN109234458B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103409563A (en) * | 2013-09-06 | 2013-11-27 | 青岛农业大学 | RT-LAMP (reverse transcription-loop-mediated isothermal amplification) detection kit for infectious pancreatic necrosis virus of rainbow trout |
| CN103937912A (en) * | 2014-05-13 | 2014-07-23 | 吴斌 | LAMP (Loop-Mediated Isothermal Amplification) primer composition for detecting infectious pancreas necrosis virus and application of LAMP primer composition |
| KR20170122933A (en) * | 2016-04-28 | 2017-11-07 | 한남대학교 산학협력단 | Primer set for detecting infectious pancreatic necrosis virus and uses thereof |
Non-Patent Citations (3)
| Title |
|---|
| Detection and phylogenetic analysis of infectious pancreatic necrosis virus in Chile;D Tapia等;《Dis Aquat Organ》;20151027;第116卷(第3期);第173-184页 * |
| Evaluation of rapid and sensitive reverse transcription loop-mediated isothermal amplification method for detecting infectious pancreatic necrosis virus in chum salmon (Oncorhynchus keta);Rungkarn Suebsing等;《J Vet Diagn Invest》;20110731;第23卷(第4期);第704-709页 * |
| Inter-laboratory ring trial to evaluate real-time reverse transcription polymerase chain reaction methods used for detection of infectious pancreatic necrosis virus in Chile;David Tapia等;《Electronic Journal of Biotechnology》;20170731;第28卷;第22页左栏第2、3段，表3 * |
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