CN115725782A

CN115725782A - Eel herpesvirus RPA primer and detection kit

Info

Publication number: CN115725782A
Application number: CN202210885686.1A
Authority: CN
Inventors: 林而舒; 卓玉琛; 陈斌; 林煜; 钟全福; 樊海平; 曾占壮
Original assignee: FRESHWATER FISHERIES RESEARCH INSTITUTE OF FUJIAN PROVINCE
Current assignee: FRESHWATER FISHERIES RESEARCH INSTITUTE OF FUJIAN PROVINCE
Priority date: 2022-07-26
Filing date: 2022-07-26
Publication date: 2023-03-03

Abstract

The invention relates to eel herpesvirus RPA primers and a detection kit, and belongs to the technical field of biological detection. The eel herpes virus RPA primer comprises an upstream primer and a downstream primer; the upstream primer is any one of primers shown by SEQ ID NO. 1-SEQ ID NO. 8; the downstream primer is any one of primers shown by SEQ ID NO. 9-SEQ ID NO. 11. The detection kit obtained by the design of the primer of the eel herpes virus RPA has higher detection sensitivity and greatly shortens the detection time.

Description

Eel herpesvirus RPA primer and detection kit

Technical Field

The invention belongs to the technical field of biological detection, and particularly relates to eel herpesvirus RPA primers and a detection kit.

Background

Eel herpesvirus (Anguillar herpesvirus, angHV) is a linear double stranded DNA virus with a genome length of 248kb containing 136 Open Reading Frames (ORFs) and belongs to the Herpesviridae family of Herpesviridae (Herpesviridae) and the genus Cyprinivirus (Cyprinivirus). The eel herpes virus is one of common diseases in the process of eel culture, the diagnosis and control difficulty of the virus is higher than that of diseases caused by pathogens such as bacteria, parasites and the like, and huge economic loss is caused to the eel industry.

At present, several methods for detecting eel herpes virus are established, including immunoperoxidase monolayer assay (IPMA), in situ hybridization, PCR, and indirect immunofluorescence antibody assay (IFAT), but these methods all have the disadvantages of complex operation and time consumption, and ordinary PCR usually takes 3 hours. The Chinese invention patent with application publication number CN110894550A discloses an RAA constant temperature fluorescence detection method and detection kit for eel herpes virus, which comprises a specific fluorescence probe, but the method is expensive, needs a precise and expensive special instrument, and is not suitable for field detection and basic research units.

The Recombinase Polymerase Amplification (RPA) technology is an isothermal nucleic acid amplification technology, and has been currently applied to rapid diagnosis of medical pathogens, detection of transgenic crops, and detection of plant diseases. RPA was successfully developed in 2006 by twist dx Inc. Although the RPA isothermal amplification method is established later, the development speed is faster. The major advantages of RPA over other isothermal amplification techniques are as follows: (1) The amplification reaction can be carried out at the temperature of 23-45 ℃, the optimal temperature is 37-42 ℃, the amplification process can be completed at normal temperature, and thermal denaturation is not needed; (2) The target amplification product can be obtained within 20-40 min, and the nucleic acid amplification speed is high; (3) Does not need very precise and expensive temperature control equipment, and is more suitable for basic scientific research and field detection; (4) RPA has higher sensitivity and specificity compared with other isothermal amplification methods because of its relative simplicity of use.

Therefore, the research and development of the RPA primer and the detection kit applied to the eel herpesvirus can improve the detection confirmation rate of the eel herpesvirus, can popularize the detection capability of basic research units, and has important significance for early warning the spread of the eel herpesvirus.

Disclosure of Invention

In order to overcome the defects of the prior art, the technical problems to be solved by the invention are as follows: provides eel herpes virus RPA primers and a detection kit which have high detection accuracy and can be popularized in basic scientific research units.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows: an eel herpes virus RPA primer comprises an upstream primer and a downstream primer;

the upstream primer is any one of primers shown by SEQ ID NO. 1-SEQ ID NO. 8;

the downstream primer is any one of primers shown by SEQ ID NO. 9-SEQ ID NO. 11 (see Table 1).

TABLE 1

The invention adopts another technical scheme that: a detection kit for eel herpes virus comprises eel herpes virus RPA primers and a positive standard substance.

The invention has the beneficial effects that: the Eel herpes virus RPA primer has high sensitivity and stable detection effect, does not have cross reaction with Eel circovirus (Eel circovirus), eel double RNA virus (Eel virus Europe) and Tilapia lake virus (Tilapia lake virus), and shows good specificity. The detection kit designed based on the eel herpesvirus RPA has high detection sensitivity and can detect the herpes virus RPA to 10 at least ^-3 Mu g/mL, which is 10 times higher than PCR detection sensitivity of herpes virus of eel under the same condition, the detection time only needs 20-40 min, greatly shortens the detection time, and can be used for virus detection of grass-roots scientific research units and disease sites.

Drawings

FIG. 1 is a gel electrophoresis chart of a part of primer combinations of eel herpesvirus RPA in the third embodiment of the present invention;

FIG. 2 is a gel electrophoresis chart of a part of primer combinations of eel herpesvirus RPA in the third embodiment of the present invention;

FIG. 3 is a gel electrophoresis diagram of specific detection of eel herpesvirus RPA primers in an embodiment of the present invention;

FIG. 4 is a gel electrophoresis diagram of the repetitive detection of the RPA primer of eel herpesvirus in the embodiment of the present invention;

FIG. 5 is a gel electrophoresis chart of different reaction times of the primer RPA of herpes anguilla virus according to the embodiment of the present invention;

FIG. 6 is a gel electrophoresis diagram of the sensitivity detection of the primer RPA of herpes anguilla virus according to the embodiment of the present invention;

FIG. 7 is a diagram of a sensitive detection gel electrophoresis of a general PCR reaction system in accordance with an embodiment of the present invention.

Detailed Description

In order to explain technical contents, achieved objects, and effects of the present invention in detail, the following description is made with reference to the accompanying drawings in combination with the embodiments.

The most key concept of the invention is as follows: by designing eel herpesvirus RPA primers and a detection kit, the detection sensitivity is improved, and the detection time is shortened.

Referring to fig. 1 to fig. 3, the eel herpesvirus RPA primer of the present invention includes an upstream primer and a downstream primer;

the upstream primer is any one of primers shown by SEQ ID NO. 1-SEQ ID NO. 8;

the downstream primer is any one of primers shown by SEQ ID NO. 9-SEQ ID NO. 11.

As can be seen from the above description, the beneficial effects of the present invention are: the eel herpesvirus RPA primer is designed based on a conserved sequence, the size of an amplified fragment is about 200-400 bp, and the design needs to meet the following requirements: (1) Selecting 30-36 bases from the virus conserved sequence single strand as an upstream or downstream primer; (2) the amplified product fragment does not exceed 500bp generally; (3) controlling the GC content to be between 20 and 70 percent; (4) the maximum allowable length of the single nucleotide repeat is 5. The design can avoid mismatching, ensure effective and rapid amplification and ensure stable amplification.

The Eel herpes virus RPA primer has high sensitivity and stable detection effect, does not have cross reaction with Eel circovirus (Eel circovirus), eel double RNA virus (Eel virus Europe) and Tilapia lake virus (Tilapia lake virus), and shows good specificity.

Further, the upstream primer is shown as SEQ ID NO. 7, and the downstream primer is shown as SEQ ID NO. 10;

or the upstream primer is shown as SEQ ID NO. 1, and the downstream primer is shown as SEQ ID NO. 9;

or the upstream primer is shown as SEQ ID NO. 2, and the downstream primer is shown as SEQ ID NO. 9;

or the upstream primer is shown as SEQ ID NO. 5, and the downstream primer is shown as SEQ ID NO. 9;

or the upstream primer is shown as SEQ ID NO. 6, and the downstream primer is shown as SEQ ID NO. 9.

From the above description, the eel herpesvirus RPA primer combinations above performed well specifically.

Further, the upstream primer is shown as SEQ ID NO. 4, and the downstream primer is shown as SEQ ID NO. 9.

As can be seen from the above description, the specificity of the eel herpesvirus RPA primers combined as SEQ ID NO. 4 and SEQ ID NO. 9 performed best.

As can be seen from the above description, the eel herpesvirus detection kit,has high detection sensitivity, and can detect at least 10 ^-3 Mu g/mL, which is 10 times higher than PCR detection sensitivity of herpes anguilla viruses under the same condition, the detection time is only 20-40 min, the detection time is greatly shortened, and the method can be used for virus detection of grass-roots scientific research units and disease sites.

Furthermore, the nucleotide sequence of the positive standard is shown as SEQ ID NO. 12.

Further, the enzyme dry powder mixture, the rehydration buffer solution and the magnesium acetate solution are also included.

As can be seen from the above description, the RPA reaction is performed by means of the enzyme dry powder mixture, and the rehydration buffer provides a favorable reaction environment for the amplification reaction of the magnesium acetate solution, and also plays a role in starting the RPA amplification reaction. The addition of the enzyme dry powder mixture, the rehydration buffer solution and the magnesium acetate solution mainly affects the speed of the amplification reaction, the reaction is inhibited when the addition is too large, and the reaction cannot be started when the addition is too small.

Further, the enzyme dry powder mixture comprises a recombinase, a single-strand binding protein, and a strand-displacing DNA polymerase.

From the above description, the recombinase and the primer form a protein/single-stranded nucleotide complex, rec/ssDNA, that invades the double-stranded DNA template with the aid of a strand-displacing DNA polymerase and a single-stranded binding protein; forming a D-loop region at the invasion site, and starting to scan the DNA double strand; after the region of interest complementary to the primer is found, the polymerase binds to the 3' end of the primer to initiate strand extension while the complex Rec/ssDNA is cleaved.

Further, the magnesium acetate concentration was 280mM.

As can be seen from the above description, the magnesium acetate solution mainly affects the speed of the amplification reaction. The proper amount of magnesium acetate solution not only can ensure the rapid starting of the RPA reaction, but also can avoid the situation that the reaction is inhibited or can not be started due to too high or too low content.

The invention adopts another technical scheme that: a use method of a herpes anguilla virus detection kit comprises the following steps:

s1: extracting DNA of a sample to be detected;

s2: carrying out RPA amplification by using eel herpesvirus RPA primers to obtain an RPA amplification product;

s3: purifying RPA amplification products, and detecting and verifying herpes zoster virus in a sample to be detected through agarose gel electrophoresis.

Further, S2 specifically is: firstly, preparing an RPA rehydration solution by using eel herpes virus RPA primers, a rehydration buffer solution, DNA of a sample to be detected and water; oscillating and centrifuging the RPA rehydration solution, transferring the solution into an enzyme dry powder mixture, and uniformly blowing and mixing the solution until the whole microspheres are resuspended; adding magnesium acetate, mixing uniformly, oscillating, centrifuging again, and incubating at constant temperature of 23-45 ℃ for 20-40 min to obtain an RPA amplification product.

Preferably, the constant temperature is 37 ℃ to 42 ℃.

As can be seen from the above description, the detection method can complete the amplification process at normal temperature without thermal denaturation; the target amplification product can be obtained within 20-40 min, and the nucleic acid amplification speed is high; does not need very precise and expensive temperature control equipment, and is more suitable for basic scientific research and field detection.

Further, the DNA of the sample to be tested is extracted from the mixed tissues of liver, kidney, spleen and gill suspected of being the disease of the viral disease of eel.

As is clear from the above description, when eel suffers from eel herpes virus disease, the main clinical features are "de-viscous sepsis", "gill rot", "red head", "liver blood loss", because the virus is easily parasitic in organs such as liver, kidney, spleen and gill, and thus DNA of the sample to be tested is extracted from the liver, kidney, spleen and gill, and a large amount of virus DNA can be easily obtained.

Referring to fig. 1 to fig. 3, a first embodiment of the present invention is:

eel herpes virus RPA primers comprise samples 1-8 consisting of different combinations of upstream primers and downstream primers, and the combinations are shown in Table 2.

TABLE 2

Sample 1

Sample 2

Sample 3

Sample No. 4

Sample No. 5

Sample No. 6

Sample 7

Sample 8

Combination of

F1/R1

F2/R1

F3/R1

F4/R1

F5/R1

F6/R1

F7/R7

F8/R8

The second embodiment of the invention is as follows: a kit for detecting eel herpesvirus comprises a positive standard substance, an enzyme dry powder mixture, a rehydration buffer solution, a magnesium acetate solution and a sample 1.

The enzyme dry powder mixture contains recombinase, single-strand binding protein and strand-displacing DNA polymerase, and the concentration of magnesium acetate is 280mM.

The third embodiment of the invention is as follows:

a use method of a herpes anguilla virus detection kit comprises the following steps:

s1: 20mg of a mixed tissue sample of liver, kidney, spleen and gill suspected of suffering from eel viral diseases is taken, and DNA of a sample to be detected is extracted according to the instruction of a marine animal genome DNA extraction kit. The genome was stored at-20 ℃ until use.

S2: 47.5 μ L of RPA rehydration solution was prepared separately using RPA primer samples 1-8 according to the instructions of the twist Amp Basic RPA kit: includes 2.4. Mu.L of the forward primer, 2.4. Mu.L of the reverse primer, 29.5. Mu.L of the primer-free rehydration buffer, 2. Mu.L of the DNA of the sample to be tested and 11.2. Mu.L of water.

S2: shaking and centrifuging 47.5 μ L of the rehydrated solution, transferring to enzyme dry powder mixture, and blowing and mixing until the whole microsphere is resuspended; adding 2.5 mu L of 280mM magnesium acetate, and uniformly mixing to activate the reaction; oscillating again and centrifuging, and then placing the test tube at 39 ℃ for incubation for 30min at constant temperature to obtain RPA amplification products 1-8 respectively;

s3: and respectively purifying the RPA amplification products 1-8 by using a PCR product purification kit, and detecting and verifying the herpes zoster viruses in the RPA amplification products 1-8 by using 1% agarose gel electrophoresis.

Primer-free rehydration buffer, enzyme dry powder mixture, magnesium acetate solution were purchased from TABASO3KIT KIT from TwistAmp; the twist Amp Basic RPA kit is purchased from twist Amp company, and the marine animal genome DNA extraction kit is purchased from Tiangen Biochemical technology (Beijing) limited company; the PCR product purification kit was purchased from Biotechnology engineering (Shanghai) Ltd.

The sizes of the RPA amplification products 1-8 are shown in Table 3.

TABLE 3

	Combination of	RPA amplification product size (bp)
			Sample 1	F1/R1	394
Sample 2	F2/R1	394
			Sample 3	F3/R1	394
Sample No. 4	F4/R1	394
			Sample No. 5	F5/R1	394
Sample No. 6	F6/R1	394
			Sample 7	F7/R7	317
Sample 8	F8/R8	251

The results of gel electrophoresis detection of the RPA amplification products 1-8 are shown in FIG. 1 (1 represents a primer combination F4/R1,2 represents a negative control, 3 represents a primer combination F7/R7,4 represents a primer combination F8/R8, and M represents a positive standard) and FIG. 2 (1 represents a primer combination F1/R1,2 represents a primer combination F2/R1,3 represents a primer combination F3/R1,4 represents a primer combination F4/R1,5 represents a primer combination F5/R1,6 represents a primer combination F6/R1, and M represents a positive standard).

By observing the specificity and brightness of the electrophoretic bands obtained from the RPA primer samples 1-8 in fig. 1 and 2, it can be seen that the optimal primer combination is sample 4 (F4/R1), which has good specificity and the strongest band brightness.

The fourth embodiment of the invention is as follows:

the difference between the fourth embodiment and the third embodiment is that: the incubation time is 40min at 23 deg.C.

The fifth embodiment of the invention is as follows:

the difference between the fifth embodiment and the third embodiment is that: the incubation is carried out at a constant temperature of 45 ℃ for 20min.

The first comparative example of the present invention is:

the difference between the first comparative example and the third example is that: comparative example one method respectively adopts Eel circovirus (Eel circovirus), eel double RNA virus (Eel virus Europe) and Tilapia lake virus (Tilapia lake virus) as DNA of a sample to be detected.

And (3) specific detection:

ddH2O as a blank control, and the detection result of comparative example I is shown in figure 3 (1 represents a negative control, 2 represents eel circovirus, 3 represents eel double RNA virus, 4 represents eel herpes virus, 5 represents tilapia lake virus, and M represents a positive standard).

By observing figure 3, the eel herpes virus RPA primer and the detection kit have good specificity, the eel herpes virus has specific amplification and an electrophoresis band, and other pathogens do not have amplification bands.

And (3) repeatability detection:

repeatability tests were performed at three different time periods, using 10 ^-1 mu.g/mL of eel herpesvirus DNA was amplified using sample 4 (F4/R1) RPA primer as a template. The test results are shown in FIG. 4 (1 for the first replicate, 2 for the second replicate, 3 for the third replicate, and M for the positive standard).

As can be seen from FIG. 4, the three results are consistent, which shows that the eel herpes virus RPA primer and the detection kit have good repeatability and stability.

RPA reaction time optimization:

by 10 ^-1 mu.g/mL of eel herpesvirus DNA was amplified using sample 4 (F4/R1) RPA primer as a template. The incubation at constant temperature was carried out at 39 ℃ for 20, 40, 80 and 160 minutes, respectively, to compare the effects of different times on the amplification effect of RPA.

The results of the agarose gel electrophoresis are shown in FIG. 5 (1 represents 20 minutes, 2 represents 40 minutes, 3 represents 80 minutes, 4 represents 160 minutes, 5 represents a negative control, and M represents a positive standard).

As can be seen from the results in FIG. 5, the band is single when the reaction time is 20 to 40 minutes; when the reaction time is increased to 80-160 minutes, the appearance of the miscellaneous bands begins. This indicates that the detection effect is best when the reaction time is 20 to 40 minutes.

Sensitivity detection

Taking DNA templates of eel herpes virus positive samples for gradient dilution, wherein the dilution is respectively 10 ^-1 、10 ^-2 And 10 ^-3 Mu g/mL,3 dilution gradients, respectively carrying out RPA detection and common PCR detection, and analyzing and comparing the detection sensitivity of the two.

The RPA reaction was incubated at 39 ℃ for 40 minutes.

A common PCR reaction system (50. Mu.L) was as follows: 2 mu L of each upstream primer and downstream primer of 10 mu mol/L, and 25 mu L of rTaqTM Premix; 2 mu L of DNA template; 19 mu L of sterilized water; the reaction procedure was as follows: performing pre-denaturation at 94 deg.C for 5min; denaturation, at 94 ℃ for 30s; annealing at 54 ℃ for 30s; extension, 72 ℃,30s;30 cycles; extension, 72 ℃ for 10min.

Wherein, the primers used for the RPA detection and the common PCR detection are sample 4 (F4/R1).

The results of the RPA assay are shown in FIG. 6 (1 stands for 10) ^-1 μ g/mL,2 represents 10 ^-2 μ g/mL,3 for 10 ^-3 μ g/mL, M represents a positive standard), the detection result of the common PCR reaction system is shown in FIG. 7 (1 represents 10) ^-1 μ g/mL,2 represents 10 ^-2 μ g/mL, M for positive standard).

As is clear from FIGS. 6 and 7, the detection method of the herpes anguilla virus detection kit of the present invention has high detection sensitivity, and can detect at least 10 degrees of herpes anguilla virus ^-3 Mu g/mL, which is 10 times higher than the PCR detection sensitivity of eel herpes virus under the same condition.

In conclusion, the eel herpesvirus RPA primer provided by the invention has high sensitivity and stable detection effect, has no cross reaction with eel circovirus, eel double RNA virus and tilapia lake virus, and shows good specificity. The detection kit designed based on the eel herpesvirus RPA has high detection sensitivity and can detect the herpes virus RPA to 10 at least ^-3 Mu g/mL, which is 10 times higher than PCR detection sensitivity of herpes virus of eel under the same condition, the detection time only needs 20-40 min, greatly shortens the detection time, and can be used for virus detection of grass-roots scientific research units and disease sites.

The above description is only an embodiment of the present invention, and is not intended to limit the scope of the present invention, and all equivalent modifications made by the present invention and the contents of the accompanying drawings, which are directly or indirectly applied to the related technical fields, are included in the scope of the present invention.

Claims

1. An eel herpes virus RPA primer is characterized by comprising an upstream primer and a downstream primer;

the upstream primer is any one of primers shown by SEQ ID NO. 1-SEQ ID NO. 8;

2. The eel herpesvirus RPA primer according to claim 1, wherein the upstream primer is shown as SEQ ID NO. 7, and the downstream primer is shown as SEQ ID NO. 10;

3. The eel herpesvirus RPA primer according to claim 1, wherein the upstream primer is shown as SEQ ID NO. 4, and the downstream primer is shown as SEQ ID NO. 9.

4. A kit for detecting herpes anguilla virus, which is characterized by comprising the primer for herpes anguilla virus RPA according to any one of claims 1-3 and a positive standard substance.

5. The eel herpesvirus detection kit according to claim 4, wherein the nucleotide sequence of the positive standard is shown in SEQ ID NO. 12.

6. The eel herpesvirus detection kit according to claim 4, further comprising an enzyme dry powder mixture, a rehydration buffer and a magnesium acetate solution.

7. The eel herpesvirus detection kit according to claim 6, wherein the enzyme dry powder mixture comprises a recombinase, a single-strand binding protein and a strand displacement DNA polymerase.

8. The use method of the eel herpesvirus detection kit according to claim 4, characterized by comprising the following steps:

s1: extracting DNA of a sample to be detected;

s2: carrying out RPA amplification by using an eel herpesvirus RPA primer to obtain an RPA amplification product;

s3: purifying the RPA amplification product, and detecting and verifying the herpes simplex virus of the eel in the sample to be detected by agarose gel electrophoresis.

9. The use method of the eel herpesvirus detection kit according to claim 8, wherein S2 is specifically: firstly, preparing an eel herpes virus RPA primer, a rehydration buffer solution, DNA of a sample to be detected and water to obtain an RPA rehydration solution; oscillating and centrifuging the RPA rehydration solution, transferring the solution into an enzyme dry powder mixture, and uniformly blowing and mixing the solution until the whole microspheres are resuspended; adding magnesium acetate, mixing uniformly, oscillating, centrifuging again, and incubating at constant temperature of 23-45 ℃ for 20-40 min to obtain an RPA amplification product.

10. The use method of the eel herpesvirus detection kit according to claim 8, wherein the DNA of the sample to be tested is extracted from the mixed tissues of liver, kidney, spleen and gill which are suspected to be the disease of eel viral diseases.