CN110894550A - RAA constant temperature fluorescence detection method and reagent for eel Herpes Virus (HVA) - Google Patents

Abstract

The invention discloses a constant-temperature fluorescence detection method and a detection kit for herpes anguilla virus (HVA) RAA. The detection kit comprises a forward primer SEQ ID NO.1, a reverse primer SEQ ID NO.2, a specific fluorescent probe SEQ ID NO.3, reaction liquid, recombinant polymerase and a reference substance. The kit has strong specificity; the detection sensitivity is high and can reach 0.4 fg/mu L; the accuracy is high and reliable; the method is simple, convenient and quick to operate, is suitable for field detection, and has wide application scenes.

Description

RAA constant temperature fluorescence detection method and reagent for eel Herpes Virus (HVA)

Technical Field

The invention belongs to the technical field of molecular biology, relates to a detection method of marine aquaculture industry, and particularly relates to an RAA constant-temperature fluorescence detection method and a kit for herpes viruses of eel.

Background

Since the introduction of eel culture technology in China at the end of the 70 s in the 20 th century, a mature industrial chain integrating culture, processing, export and feed production has been formed, and the output value of the industrial chain plays an important role in the trade of aquatic products in China. Due to large-scale intensive culture, serious diseases of the eels occur more and more frequently, and the healthy development of the industry is seriously hindered. Eel Herpes Virus (HVA) is a viral pathogen that is ubiquitous and causes great economic loss to cultured freshwater eels. Herpes viruses were first isolated from Japanese eels in Sano et al 1990; in 1993, Ueno et al isolated HVA from Japanese eel bred in Taiwan Lugang.

The clinical symptoms of eel in outbreak of HVA are very different, such as increase of fish mucus secretion, eye protrusion and abdominal distension of sick fish, liver fading, kidney enlargement, gall bladder enlargement, enteritis, skin ulceration, perforation and viscera exposure, and increase of death rate. Latent infection is also an important characteristic of HVA, viruses can be detected in wild eels without disease symptoms in Europe, and the positive detection rate reaches 48 percent. As few researches on HVA are carried out in China, so that once a disease outbreak occurs, few countermeasures are taken to cause huge economic loss, and the prevention of the HVA is important.

The method mainly comprises microscopic observation, molecular detection and immunological detection. The PCR molecular detection method is one of the most commonly used detection methods, and is sensitive, accurate, rapid and widely applied, but is not suitable for field detection and popularization due to the requirement of expensive instruments and equipment, higher detection cost and higher technical requirements for detection personnel. The invention establishes a method for detecting eel herpes virus by RAA constant temperature fluorescence, is quick, convenient, accurate and reliable, is suitable for the times of rapid port detection and big customs, and has important effect on promoting the Chinese aquaculture and the product trade thereof.

The Recombinase-aid Amplification (RAA) technique is also a method by which nucleic acids can be rapidly amplified at a constant temperature. Unlike RPA, RAA amplification uses a recombinase obtained from bacteria or fungi, which binds tightly to the primer DNA at a constant temperature of 37 ℃ to form an aggregate of the enzyme and the primer, when the primer searches for a sequence on the template DNA that is completely complementary to the primer, the template DNA is melted with the help of single-strand DNA binding protein (SSB), and a new complementary strand of DNA is formed under the action of DNA polymerase, and the reaction product is exponentially increased, and usually an amplified fragment that can be detected by agarose gel electrophoresis can be obtained within 1 hour. The fluorescent group is added into the RAA reaction system, the whole RAA amplification process is monitored in real time by utilizing the accumulation of fluorescent signals, and the quantitative and qualitative analysis of the initial template can be realized within 20 minutes. The whole reaction is simple and quick, and high-temperature circulation is not needed, so the method is particularly suitable for being used in non-laboratory detection places with a large number of samples, and is suitable for the field of quick detection of foods.

Disclosure of Invention

In view of the above, the present invention aims to provide a RAA constant temperature fluorescent nucleic acid detection kit and a detection method for herpes viruses of eel.

In order to achieve the purpose, the invention adopts the following technical scheme:

a detection kit for eel herpes virus nucleic acid comprises: the specific fluorescent probe comprises an eel herpes virus forward primer, a reverse primer and a specific fluorescent probe, wherein the nucleotide sequence of the eel herpes virus forward primer is shown as SEQ ID No.1, the nucleotide sequence of the eel herpes virus reverse primer is shown as SEQ ID No.2, the nucleotide sequence of the specific fluorescent probe is shown as SEQ ID No.3, a fluorescent reporter group is marked at the 5 'end of the specific fluorescent probe, and a fluorescent quenching group is marked at the 3' end of the specific fluorescent probe.

In some embodiments, the fluorescent reporter group of the specific fluorescent probe is selected from one of FAM, VIC, JOE, TET, CY3, CY5, ROX, Texas Red, or LC Red460, and the fluorescence quenching gene is selected from one of BHQ1, BHQ2, BHQ3, Dabcy1, or Tamra.

In some embodiments, the nucleic acid detection kit further comprises a primer mixture, a specific fluorescent probe, an A Buffer, a B Buffer, a RAA dry powder reagent, an eel herpesvirus standard and ddH₂At least one of O.

In some embodiments, the kit of (a), wherein the a Buffer is 20% PEG; b Buffer is 280mM MgAc.

In some embodiments, the kit, wherein the composition of the RAA dry powder reagent is as follows: 1mmol/L dNTP, 90ng/μ L SSB protein, 120ng/μ L recA recombinase protein (SC-recA/BS-recA) or 30ng/μ LRad51, 30ng/μ L Bsu DNA polymerase, 100mmol/LTricine, 20% PEG, 5mmol/L dithiothreitol, 100ng/μ L creatine kinase, Exo exonuclease.

In some embodiments, the nucleic acid detection kit and the eel herpesvirus standard are positive plasmids containing a partial sequence of eel herpesvirus DNA polymerase gene.

In some embodiments, the kit, the positive plasmid containing the partial sequence of eel herpes virus DNA polymerase gene is shown as SEQ ID No. 4.

The invention also provides an RAA constant-temperature fluorescence detection method of eel herpes virus, which comprises the steps of extracting DNA of a sample to be detected, taking the DNA of the sample to be detected as a template, and performing fluorescence detection on a forward primer, a reverse primer, a specific fluorescent probe, an RAA dry powder reagent, an A Buffer, a B Buffer and a ddH of the eel herpes virus₂Carrying out real-time fluorescence RAA reaction in the presence of O, and analyzing a sample to be detected according to a real-time fluorescence RAA amplification curve; the nucleotide sequence of the eel herpes virus forward primer is shown as SEQ ID NO.1, the nucleotide sequence of the eel herpes virus reverse primer is shown as SEQ ID NO.2, the nucleotide sequence of the specific fluorescent probe is shown as SEQ ID NO.3, the 5 'end of the specific fluorescent probe is marked with a fluorescent reporter group, and the 3' end of the specific fluorescent probe is marked with a fluorescent quenching group.

In some embodiments, the eel herpesvirus DNA extraction is performed using a marine animal tissue DNA extraction kit or using an equivalent DNA extraction kit.

In some embodiments, the performing a fluorescent RAA reaction procedure is: at 37 ℃ for 40 s; the temperature of the mixture is controlled at 37 ℃ for 20min,

a total of 40 cycles;

according to the detection method, after the real-time fluorescence RAA reaction is required to be finished, the to-be-detected sample is analyzed according to the amplification curve of the real-time fluorescence RAA by using the analysis software of the real-time fluorescence RAA instrument. Preferably, the FAM channel fluorescence curve of the sample to be tested is S-shaped, the CT value is less than or equal to 35, and the sample to be tested is judged to be the positive result of the eel herpesvirus; and when the curve of the sample to be detected is not S-shaped or the CT value is more than 35, judging the curve as the eel herpes virus negative result.

Advantageous effects

1. Fast and efficient: the whole amplification can be completed within 20-30min, and the amplification yield can reach 10⁹-10¹⁰A copy;

2. the operation is simple: no special reagent is needed, complicated steps such as deformation of double-stranded DNA and the like are not needed in advance, only a constant-temperature fluorometer is needed, and the conditions are mild;

3. high specificity: the invention does not amplify other fish diseases IPNV, ISAV, SAV, VNNV, FBS and CEV plasmids.

4. High sensitivity: the detection limit of the invention can reach 0.4 fg/reaction

5. The identification is simple: and the amplification result is directly judged according to the real-time fluorescence data, electrophoresis detection is not needed, and the method is suitable for field detection.

Drawings

FIG. 1 is a graph of 4 pairs of primer HVA amplification involved in the present invention.

FIG. 2 is a sensitivity test chart of the RAA detection method to HVA, and the amplification results of the positive standard samples are 4pg/μ L, 400fg/μ L, 40fg/μ L, 4fg/μ L and 0.4fg/μ L from left to right in sequence.

FIG. 3 is a diagram showing the specificity of the RAA detection method for HVA.

Detailed description of the invention

The present invention is further illustrated by the following specific examples, but is not limited thereto.

Example 1:

the invention searches eel herpes virus DNA polymerase gene sequences in a Genebank database, and compares the multiple sequences by using DNMAN 6.0 software to find out conserved segments. 4 sets of primers and probes were designed in the conserved regions and BLAST alignments were performed in the NCBI database, with the sequences of the primers and probes as shown in Table 1. The positive sample amplification curve is shown in FIG. 1.

TABLE 1 primer and Probe sequences

As can be seen from the results in FIG. 1, the amplification curves for the fourth set of primers and probes are most typical, with distinct exponential and plateau phases, with higher fluorescence intensity (ordinate values), and smaller CT values (abscissa corresponding to the intersection of the curve with the threshold line) and the results are analyzed in Table 2. The rise height of other primer probe curves is lower, the CT value is larger, and the plateau period is not obvious; or no amplification occurs and missed detection occurs. The fourth group of primers and the target products of the probes have higher replication speed, more quantity and higher amplification reaction efficiency.

TABLE 2 analysis of primer Probe screening results

Group \ result	CT value	Intensity of fluorescence
			First group	4.49	412,500
Second group	5.82	492,500
			Third group	5.13	460,000
Fourth group	4.36	515,000

Real-time example 2: the kit eel herpes virus

The nucleic acid detection kit also comprises primer mixed liquor, a specific fluorescent probe, an A Buffer, a BBbuffer, an RAA dry powder reagent, an eel herpesvirus standard and ddH₂O。

The kit of the invention, wherein the ABuffer is 20% PEG; b Buffer is 280mM MgAc.

The kit of the invention, wherein the RAA dry powder reagent comprises the following components: 1mmol/L dNTP, 90ng/μ L SSB protein, 120ng/μ L recA recombinase protein (SC-recA/BS-recA) or 30ng/μ L Rad51, 30ng/μ L Bsu DNA polymerase, 100mmol/LTricine, 20% PEG, 5mmol/L dithiothreitol, 100ng/μ L creatine kinase, Exo exonuclease.

In the primer mixture, the base sequence of the forward primer is shown as SEQ ID NO.1, the base sequence of the reverse primer is shown as SEQ ID NO.2, and the molar ratio of the forward primer to the reverse primer is SEQ ID NO. 1: SEQ ID NO.2 is 1: 1.

The base sequence of the specific probe for the eel herpesvirus is shown in SEQ ID NO.3, the 5 'end of the probe is marked with FAM fluorescent reporter group, and the 3' end of the probe is marked with BHQ1 fluorescent quenching group.

The eel herpesvirus standard provided by the invention comprises a positive plasmid of a gene sequence of a conserved region of eel herpesvirus, and the base sequence of the plasmid is shown as SEQ ID NO. 4.

Base sequence of plasmid (SEQ ID NO.4):

GTGTCGGGCCTTTGTGGTGAACAAGGTGGTGTTGGATTGGAAAAGAATGCCGGCCATCTCCGGCCTCATAGAGAATAGGGAGTACGGGGAGGCCGAGAACCTCTACACCATCCTCCTACACAAGAGTGTGGAGACCGGGTGGACCAGGTTTACGACGTACACGTCTTCCAGCCTCCGCCACTACCTGAGCATGCGAAACAAATACAAGGGCGACATGAAGACGGCCAAGAGCCCCGGACTGAAAAAATACTTCAACCAGCTGCAGAACGAGATGAAGATCTGCGCCAACTCGCACTACGGCGTATCGGATCGCATCTGTCAGATGTTGACCACGCTCTCGGGGCGACAAAAGATCCTTATAGTGGAGGACGTGATCAAAAAGACTCCCGGCATGACTGTGATTTACGGGGACACGGACTCCATTATGTTCCAGGTGCCACCCTCCGACAGAGAGGTGATGAACACGAACACTTTCAAACCGATCTCCGT

example 3: the kit of the invention relates to eel herpes virus

1. Extraction of nucleic acids from Positive samples

Cutting a small amount of tissue, extracting nucleic acid DNA with marine animal tissue kit, and standing at-20 deg.C

2. Configuration of RAA reaction system: one RAA reaction dry powder tube was used for each test sample, and the reaction components and the added volume in each RAA reaction dry powder tube are shown in Table 3.

Table 3:

a Buffer is 20% PEG; b Buffer is 280mM MgAc

TABLE 3RAA reaction Dry powder tube reaction Components and addition volumes

3. Placing the RAA reaction tube with the prepared reaction system in an ABI7500 amplification instrument, and carrying out RAA amplification according to the following procedures: at 39 ℃ for 40 s; at 39 ℃ for 20min, for a total of 40 cycles. Fluorescence of FAM channels was collected for each cycle.

4. And after the amplification is finished, judging the positive or negative result of the eel herpes virus according to the fluorescence curve judgment and the CT value. And (4) judging the result: the fluorescence curve of the FAM channel is S-shaped, the CT value is less than or equal to 35, and the curve is judged as the eel herpes virus positive result; and when the curve of the sample to be detected is not S-shaped or the CT value is more than 35, judging the curve as the eel herpes virus negative result.

Example 4: evaluation of RAA detection kit of the invention in clinical practical application

The kit is adopted to carry out clinical blind sample experiments, and 25 parts of eel are detected; of 25, 19 were positive and 6 were negative as measured by the RAA method, and the detection rate was 76%.

Test example 5: sensitivity test of the kit of the invention

The eel herpesvirus standard plasmid provided by the kit in theembodiment 2 of the invention is extracted to obtain a positive plasmid, the concentration of the positive plasmid is measured by using NanoDrop, and the positive plasmid is respectively diluted to 5 concentration gradients of 4 pg/muL, 400 fg/muL, 40 fg/muL, 4 fg/muL and 0.4 fg/muL to carry out sensitivity test.

The detection results are shown in figure 2, and are amplification results of positive standard substances of 4 pg/muL, 400 fg/muL, 40 fg/muL, 4 fg/muL and 0.4 fg/muL from left to right in sequence, so that the RAA fluorescence amplification reagent and the detection sensitivity can reach 0.4 fg/muL, the accuracy is superior to that of the common PCR detection method, and the RAA constant temperature fluorescence detection kit and the detection method have high sensitivity on HVA diagnosis.

Test example 6: specificity test of the kit of the present invention

In order to detect the specificity of the kit, the detection methods in example 3 are adopted to detect the IPNV, ISAV, SAV, VNNV, FBS and CEV samples respectively, and the detection conditions of the kit on other common viruses of fish are analyzed.

The detection result shows that: normal amplification occurred only for HVA samples, negative control (ddH)₂O) and IPNV, ISAV, SAV, VNNV, FBS and CEV samples were not amplified (as shown in figure 3). The results show that the RAA constant temperature fluorescence detection kit of the invention can specifically amplify the target sequence in HVA without cross reaction with other virus nucleic acid. The method and the kit have good specificity and do not generate false negative.

Meanwhile, the same specificity experiment is carried out on the primers 1, 3 and 4 designed by the invention, and the primers can not distinguish different samples well in a specific way, so that the specificity is not good (the specific experimental data is little).

The invention shown and described herein may be practiced in the absence of any element or elements, limitation or limitations, which is specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, and it is recognized that various modifications are possible within the scope of the invention. It should therefore be understood that although the present invention has been specifically disclosed by various embodiments and optional features, modification and variation of the concepts herein described may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

The contents of the articles, patents, patent applications, and all other documents and electronically available information described or cited herein are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other documents.

Sequence listing

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Claims (9)

1. A detection kit for eel Herpes Virus (HVA) nucleic acid, comprising: the specific fluorescent probe comprises an eel herpes virus forward primer, a reverse primer and a specific fluorescent probe, wherein the nucleotide sequence of the eel herpes virus forward primer is shown as SEQ ID No.1, the nucleotide sequence of the eel herpes virus reverse primer is shown as SEQ ID No.2, the nucleotide sequence of the specific fluorescent probe is shown as SEQ ID No.3, a fluorescent reporter group is marked at the 5 'end of the specific fluorescent probe, and a fluorescent quenching group is marked at the 3' end of the specific fluorescent probe.

2. The nucleic acid detection kit according to claim 1, wherein the fluorescence reporter group of the specific fluorescent probe is selected from one of FAM, VIC, JOE, TET, CY3, CY5, ROX, Texas Red or LC RED460, and the fluorescence quenching gene is selected from one of BHQ1, BHQ2, BHQ3, Dabcy1 or Tamra.

3. The nucleic acid detection kit according to claims 1 and 2, further comprising a primer mixture, a specific fluorescent probe, ABuffer, B Buffer, RAA dry powder reagent,Eel herpesvirus standards and ddH₂At least one of O.

4. The kit according to claim 3, wherein the A Buffer is 20% PEG; b Buffer is 280mM MgAc.

5. The kit of claim 3, wherein the RAA dry powder reagent is comprised of: 1mmol/LdNTP, 90 ng/muL SSB protein, 120 ng/muL recA recombinase protein (SC-recA/BS-recA) or 30 ng/muL Rad51, 30 ng/muL Bsu DNA polymerase, 100mmol/L Tricine, 20% PEG, 5mmol/L dithiothreitol, 100 ng/muL creatine kinase, and Exo exonuclease.

6. The nucleic acid detection kit according to any one of claims 1 to 5, wherein the eel herpesvirus standard is a positive plasmid containing a partial sequence of eel herpesvirus DNA polymerase gene.

7. The kit according to claim 6, wherein the positive plasmid containing the partial sequence of herpes anguilla virus DNA polymerase gene is represented by SEQ ID No. 4.

8. RAA constant temperature fluorescence detection method for herpes anguilla virus comprises the steps of extracting DNA of a sample to be detected, taking the DNA of the sample to be detected as a template, and detecting the DNA in a forward primer, a reverse primer, a specific fluorescent probe, an RAA dry powder reagent, A Buffer, BBuffer and ddH of the herpes anguilla virus₂Carrying out real-time fluorescence RAA reaction in the presence of O, and analyzing a sample to be detected according to a real-time fluorescence RAA amplification curve; the nucleotide sequence of the eel herpes virus forward primer is shown as SEQ ID NO.1, the nucleotide sequence of the eel herpes virus reverse primer is shown as SEQ ID NO.2, the nucleotide sequence of the specific fluorescent probe is shown as SEQ ID NO.3, the 5 'end of the specific fluorescent probe is marked with a fluorescent reporter group, and the 3' end of the specific fluorescent probe is marked with a fluorescent quenching group.

9. According to claim 8, the eel herpesvirus DNA extraction is performed by using a marine animal tissue DNA extraction kit or an equivalent DNA extraction method.

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