CN110964848A - RAA amplification primer and probe for rapidly detecting carp herpesvirus II, detection kit and use method - Google Patents
RAA amplification primer and probe for rapidly detecting carp herpesvirus II, detection kit and use method Download PDFInfo
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Abstract
The invention discloses an RAA amplification primer and a probe for rapidly detecting a carp herpesvirus II type, a detection kit and a using method. The RAA isothermal amplification system has the advantages of rapid reaction and wide temperature range, can realize effective amplification of target genes under the condition of 34-40 ℃, and has the lowest detection limit of 8.14 multiplied by 100Copy/mu L, the sensitivity is higher than that of the common PCR of CyHV-2, and the cross reaction with other aquatic animal epidemic diseases does not occur. The kit can quickly, efficiently and sensitively detect the carp herpesvirus II, has the characteristics of simple operation, high specificity, easy observation of reaction result, safety, no pollution and the like, not only provides an effective technical means for the on-site quick detection and screening of the carp herpesvirus II infected nucleic acid, but also controls the virus in crucian, goldfish and the likeThe infection transmission of the aquarium fishes and the inspection and quarantine of the aquarium fishes in epidemic areas and entry and exit ports are also of great significance.
Description
Technical Field
The invention relates to the field of aquatic animal epidemic diseases in aquaculture industry, in particular to an RAA amplification primer and a probe for rapidly detecting carp herpesvirus II, a detection kit and a use method.
Background
The carp herpes virus type II (Cyprinus carpioides 2, CyHV-2) is a highly pathogenic pathogen for causing hematopoietic necrosis caused by infecting goldfish and crucian (Carassius auratus), and seriously harms the fish breeding industries of goldfish, crucian and the like. The virus infects gill, kidney and spleen tissues of fish, and the infected fish has slow action, enlarged abdomen and bleeding on body surface and fin base. In 1995, the virus was first isolated from goldfish cultivated in japan, and then rapidly spread to countries such as the united states, australia, uk, and taiwan of china. After the virus is introduced into China, the virus is erupted and epidemic in the main culture area of crucian in China on a large scale, so that the cultured goldfishes and crucian are killed in a large scale, and huge economic loss is generated.
An accurate and rapid on-site detection method is established, which is the premise of effectively preventing and controlling the disease. Currently, the most widely used methods for CyHV-2 detection are PCR and fluorescence PCR, and in addition, virus isolation methods, immunohistochemistry methods and blood smears. However, these methods all have certain limitations: cell lines capable of separating viruses need to be improved, and an immunohistochemical method established on the basis of monoclonal antibodies needs to be verified. In order to better detect and discover potential virus infection as early as possible so as to take active prevention and control measures to control disease spread, a new method which is rapid, simple and convenient and suitable for detecting CyHV-2 on site is established as a powerful supplement to the existing method.
Disclosure of Invention
The invention aims to solve the technical problem of providing an RAA amplification primer and a probe for rapidly detecting the carp herpesvirus II, a detection kit and a using method, and has the advantages of high sensitivity, strong specificity, visualization, simple operation method, no need of expensive equipment and the like.
In order to solve the technical problems, the invention adopts the technical scheme that: a RAA amplification primer and probe for rapidly detecting carp herpesvirus II type comprises a pair of primers and probes of the following nucleotide sequences:
an upstream primer:
RAA-F：5’-ATCTTCAAGCCCAAGATCAATCACAATAAC-3’ SEQ ID NO:1
a downstream primer:
RAA-R：5’-AAACACTTGAGTTTGATATTTTAGTACGC-3’ SEQ ID NO:2
and (3) probe:
Probe：5’-ACGAGCGTAGAAAGTTTGCAGCCGTGGTCGTCAACGGTTCAAGCA-3’ SEQ ID NO:3。
the downstream primer modifies Biotin at the 5' end.
The 5 'end of the probe is marked with FAM, the middle position adopts tetrahydrofuran THF to replace one base, and the 3' end is processed by C3 spacer.
The kit for rapidly detecting the carp herpesvirus II comprises the RAA amplification primer and the probe for rapidly detecting the carp herpesvirus II.
The kit is a recombinase mediated amplification kit and contains the RAA amplification primer and the probe, a lateral flow test strip for rapid nucleic acid detection, a positive control sample and a negative control sample of a carp herpesvirus II helicase gene, an RAA dry powder reagent, a hydrolysis Buffer solution A Buffer and a magnesium acetate solution B Buffer.
The use method of the kit for rapidly detecting the carp herpesvirus II comprises the following steps:
(1) adding 40.9 mu L of hydrolysis Buffer solution A Buffer, 10 mu M and 2 mu L of carp herpes virus II type upstream primer SEQ ID NO:1, 10 mu M and 2 mu L of carp herpes virus II type downstream primer SEQ ID NO:2, 10 mu M and 0.6 mu L of carp herpes virus II type probe SEQ ID NO:3 and 2 mu L of template into RAA freeze-dried enzyme powder, adding 280mM and 2.5 mu L of magnesium acetate B Buffer on a reaction tube cover, and turning the reaction tube upside down to fully mix the mixture and then instantly separate the mixture; reacting for 10min at constant temperature of 37 ℃;
(2) reaction: detecting the product obtained after the constant-temperature reaction in the step (1) by using a nucleic acid rapid detection test strip, and observing the result for 3-5 min;
(3) and (4) interpretation of results: direct visual interpretation
① is negative, a band appears only on the quality control line, and no band appears on the detection line, which indicates that the detected sample has no carp herpesvirus II infection;
②, when the test line and the quality control line are positive, both have strips, the test sample is proved to be the carp herpesvirus type II infection;
③ is invalid, the quality control line and the detection line have no strip, which indicates that the test is unsuccessful and the test paper for rapid detection of nucleic acid is invalid.
The invention has the beneficial effects that: the RAA isothermal amplification system disclosed by the invention is rapid in reaction and wide in temperature range, and can realize effective amplification of a target gene under the condition of 34-40 ℃, the kit disclosed by the invention can be used for rapidly, efficiently and sensitively detecting CyHV-2, has the characteristics of simplicity in operation, high specificity, easiness in observing a reaction result, safety, no pollution and the like, not only provides an effective technical means for on-site rapid detection and screening of CyHV-2 infected nucleic acid, but also has very important significance for controlling the detection and quarantine of CyHV-2 in goldfish and crucian infection transmission and in epidemic areas and entry and exit ports.
Drawings
FIG. 1 is a gel result of the RAA detection primer screening of CyHV-2 (1: first set of primers; 2: second set of primers; 3: third set of primers; 4: fourth set of primers);
FIG. 2 is a graph showing the sensitivity of the RAA-LFD method to CyHV-2 (1. negative control; 2-10 is 8.14X 10)0-8.14×108Amplification result of the positive standard of (1);
FIG. 3 shows a primer specificity test chart of the present invention (1: negative control; 2: helicase gene positive plasmid control of CyHV-2; 3: Infectious Hematopoietic Necrosis Virus (IHNV) nucleic acid; 4: cyprinivirus blood disease virus (SVC) nucleic acid; 5: grass carp hemorrhagic disease virus (GV-9014) nucleic acid; 6: Cyprinivirus Edema Virus (CEV) nucleic acid; 7: cyprinid herpesvirus disease (KHV) nucleic acid; 8: Epidemic Hematopoietic Necrosis Virus (EHNV) nucleic acid; 9: Channel Catfish Virus (CCV) nucleic acid).
Detailed Description
The invention is described in further detail below with reference to the following figures and detailed description:
materials, reagents and the like used in the following examples are conventional ones unless otherwise specified.
The experimental procedures used in the following examples are conventional unless otherwise specified.
The RAA amplification kit in the following examples was purchased from Hangzhou Mass Biotechnology Ltd.
The portable lateral flow test strip (LFD) for rapid nucleic acid detection in the following examples is a product of Yosida Biotechnology, Hangzhou.
The viral genomic DNA extraction kit in the following examples was purchased from QIAGEN.
The primers and probes in the following examples were synthesized by Shanghai Bioengineering services, Inc.
In the following examples, helicase gene-positive plasmids of CyHV-2 were synthesized based on GenBank published sequences (GenBank accession Nos.: MK260012.1 and KU199244.1) and cloned in pEASY-T1 vector as a standard of the kit of the present invention.
Example 1 establishment of RAA-LFD Rapid detection kit for CyHV-2
Design and Synthesis of RAA primer and Probe for CyHV-2
Taking the helicase gene conserved region of CyHV-2 in NCBI database as a target site, adopting DNAman 6.0 software to perform sequence comparison analysis according to the RAA primer design principle, selecting a fragment with high homology, and designing 4 pairs of primers (shown in Table 1) by using primer primer 5.0.
TABLE 1 primers used for RAA
Screening of CyHV-2RAA detection primers
With CyHV-2 positive plasmid (8.14X 10)6copies/. mu.L) as template, and after the reaction is finished, identifying by 2% agarose electrophoresis (see figure 1), screening the designed primers, and screening the optimal primer pair3 sets of primers, wherein the optimal primer pair is F and R:
an upstream primer:
RAA-GFHNV-3-F：5’-ATCTTCAAGCCCAAGATCAATCACAATAAC-3’ SEQ ID NO:1
a downstream primer:
RAA-GFHNV-3-R：5’-AAACACTTGAGTTTGATATTTTAGTACGC-3’ SEQ ID NO:2
establishment of CyHV-2RAA-LFD kit
The CyHV-2RAA-LFD kit comprises a nucleic acid detection kit and a lateral flow test strip for rapidly detecting nucleic acid.
The nucleic acid detection kit comprises a primer mixed solution, a specific probe, an A Buffer, a BBuffer, an RAA dry powder reagent, a CyHV-2 standard substance and a negative sample.
Wherein, the A Buffer is 20 percent of PEG; b Buffer is 280mM MgAc; the components of the RAA dry powder reagent were as follows: 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/L Tricine, 20% PEG, 5mmol/L dithiothreitol, 100ng/μ L creatine kinase, nfo exonuclease, 30ng/μ L RTE reverse transcriptase.
In the primer mixture, the base sequence of the upstream primer is shown as SEQ ID NO. 1, the base sequence of the downstream primer is shown as SEQ ID NO. 2, and the molar ratio of the upstream primer to the downstream primer is 1: 1. Wherein the downstream primer modifies Biotin at the 5' end.
RAA-R：5’-Biotin-AAACACTTGAGTTTGATATTTTAGTACGC-3’
The base sequence of the specific probe of CyHV-2 provided by the invention is shown in SEQ ID NO. 3, FAM is marked at the 5 'end of the probe, THF (tetrahydrofuran) is adopted to replace one base at the middle position, and C3spacer treatment is carried out at the 3' end.
And (3) Probe: 5' -FAM-ACGAGCGTAGAAAGTTTGCAGCCGTGGTC [ THF ] GTCAACGGTTCAAGCA-C3spacer the CyHV-2 standard substance provided by the invention is a positive plasmid of the sequence of the helicase gene conserved region of CyHV-2.
Example 2 method for detection Using RAA-LFD Rapid detection kit of CyHV-2 described in example 1
1. Tissue pretreatment and DNA extraction: 30-80mg of gill and kidney tissues of goldfish or crucian are taken, after low-temperature homogenization, PBS buffer solution or M199 cell culture solution is added according to the proportion of 1:10, freeze thawing is carried out for 1 time, after centrifugation is carried out for 10min at 1000r/min, 200 mu L of supernatant is taken, and DNA extraction is carried out by adopting a traditional phenol-chloroform reagent or an equivalent DNA extraction kit.
RAA reaction system configuration of CyHV-2: 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 2.
Table 2: formula table of RAA reaction system
A Buffer is 20% PEG; b Buffer is 280mM MgAc
RAA reaction conditions: placing the RAA reaction tube with the prepared reaction system in a water bath kettle at 37 ℃ for 10 min;
4. and (3) test strip detection: and detecting the amplification product by a lateral flow test strip device for rapid nucleic acid detection. In order to prevent aerosol generated by reaction products from influencing detection results, 50 mu L of RAA reaction products are directly placed in a device, are fully mixed with vacuole buffer liquid of the device, are erected on a horizontal operation platform, and are observed and photographed after 3-5min to record results.
5. The test paper is judged to be negative by direct visual results ①, namely, a strip appears only on a quality control line (C line), and no strip appears on a detection line (T line), which indicates that the detected sample is not infected by CyHV-2, ② positive results show that the detected sample is infected by CyHV-2 and the detected sample is proved to be infected by both the quality control line and the detection line (C line and T line), and ③ is invalid, namely, the test paper is proved to be unsuccessful and the lateral flow test paper strip for rapid nucleic acid detection fails.
Example 3 sensitivity test of the kit according to the invention
The concentration of the CyHV-2 positive plasmid standard substance provided by the kit of the embodiment 1 of the invention is determined by using NanoDrop, and the plasmid concentration is defined according to a formula (Degree (ng/. mu.L). times.10-9/[ 660X (plasmid Length + vector Length)]﹜×6.023×1023The plasmid copy number (copies/. mu.L) was converted to its copy number. The plasmids were each adjusted to 8.14X 108To 8.14X 100The copy/. mu.L 9 concentration gradient dilutions were subjected to sensitivity assay.
The results are shown in FIG. 2, except for the negative control, which is 8.14X 10 from left to right0～8.14 ×108As a result of amplification of the positive standard substance in copy/. mu.L, it can be seen that the RAA-LFD of the present invention has a lowest detection limit of 8.14X 10 in sensitivity0The copy/mu L and the sensitivity are superior to those of the common PCR detection method, which shows that the RAA-LFD rapid detection kit and the detection method have high sensitivity to the diagnosis of CyHV-2.
EXAMPLE 4 specificity test of the kit according to the invention
In order to detect the specificity of the kit, the detection method in example 2 is adopted to detect positive samples of viruses CyHV-2, IHNV, SVCV, GV, CEV, KHV, EHNV and CCV respectively, and the detection condition of the kit on common DNA viruses of CyHV-2 and other aquatic animals is analyzed.
The detection result shows that: normal amplification occurred only in the CEV samples, and no amplification occurred in the negative controls (DEPC-treated water) and in the KHV, GFHNV, EHNV, BIV, CCV, WSSV, EHP samples (as shown in fig. 3). The above results show that the RAA-LFD rapid detection kit of the invention can specifically amplify the target sequence in CEV without cross-reacting with other viral nucleic acids. The method and the kit have good specificity.
Example 5 evaluation of RAA-LFD Rapid test kit of the present invention in clinical practice
45 parts of suspected pathological material samples which are clinically detected are respectively detected by using the RAA-LFD method and the fluorescent quantitative PCR method.
The detection result shows that 22 positive samples and 23 negative samples are detected by RAA-LFD, the detection result is completely consistent with the common PCR result, and the positive coincidence rate of the two methods is 100%.
Compared with the common PCR, the method has simpler operation, shorter required time and lower technical requirements on operators; meanwhile, the detection conditions of the invention are more suitable for field and primary carp herpesvirus II screening, can be used for conveniently, quickly and accurately detecting and can be widely applied to instruments and equipment and areas with poor experimental conditions.
The kit is rapid and efficient, the whole amplification can be finished within 20-30min, and the amplification yield can reach 109More than one copy; the operation is simple, no special reagent is needed, complicated steps such as high-temperature denaturation of double-stranded DNA and the like are not needed in advance, only a constant-temperature water bath kettle or a metal bath or an incubator is needed, and the conditions are mild; the invention has high specificity, and the invention does not amplify nucleic acid of other aquatic animal viruses such as cyprinid herpesvirus III, cyprinid spring viremia virus and the like; high sensitivity, the detection limit of the invention can reach 8.14 multiplied by 100Copy/. mu.L; the identification is simple, the result is directly judged by naked eyes according to the test strip result, the detection such as electrophoresis and fluorescence quantification is not needed, and the method is suitable for field detection.
In conclusion, the RAA detection method established by the invention not only provides an effective technical means for the on-site rapid detection and screening of the herpes virus II infected with the carp, but also has very important significance for controlling the infection and spread of the herpes virus II of the carp in goldfish and crucian carp and the inspection and quarantine of the herpes virus II of the carp in an epidemic area and an entry and exit port.
In summary, the disclosure of the present invention is not limited to the above-mentioned embodiments, and persons skilled in the art can easily set forth other embodiments within the technical teaching of the present invention, but such embodiments are included in the scope of the present invention.
SEQUENCE LISTING
<110> popularization station for water production technology in Beijing
<120> RAA amplification primer and probe for rapidly detecting carp herpesvirus II, detection kit and use method
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<170>PatentIn version 3.5
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<213> Artificial Sequence (Artificial Sequence)
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aaacacttga gtttgatatt ttagtacgc 29
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<213> Artificial Sequence (Artificial Sequence)
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acgagcgtag aaagtttgca gccgtggtcg tcaacggttc aagca 45
Claims (6)
1. A RAA amplification primer and a probe for rapidly detecting the carp herpes virus type II are characterized by comprising a pair of primers and a probe with the following nucleotide sequences:
an upstream primer:
RAA-F: 5'-ATCTTCAAGCCCAAGATCAATCACAATAAC-3' SEQ ID NO:1 downstream primer:
RAA-R: 5'-AAACACTTGAGTTTGATATTTTAGTACGC-3' SEQ ID NO:2 Probe:
Probe：5’-ACGAGCGTAGAAAGTTTGCAGCCGTGGTCGTCAACGGTTCAAGCA-3’SEQ ID NO:3。
2. the RAA amplification primer and the probe for rapidly detecting the carp herpesvirus II according to claim 1, wherein the downstream primer is modified with Biotin at the 5' end.
3. The RAA amplification primer and probe for rapidly detecting the carp herpesvirus II type according to claim 1, wherein FAM is marked at the 5 'end of the probe, tetrahydrofuran THF is adopted to replace one base at the middle position, and C3spacer treatment is carried out at the 3' end.
4. A kit for rapidly detecting carp herpesvirus type II comprising the RAA amplification primer and probe for rapidly detecting carp herpesvirus type II according to any one of claims 1 to 3.
5. The kit for rapidly detecting the carp herpesvirus II according to claim 4, wherein the kit is a recombinase-mediated amplification kit, and comprises the RAA amplification primer and the probe according to any one of claims 1-3, a lateral flow test strip for rapid nucleic acid detection, a positive control sample and a negative control sample of the carp herpesvirus II helicase gene, a RAA dry powder reagent, a hydrolysis Buffer solution A Buffer and a magnesium acetate solution B Buffer.
6. The use method of the kit for rapidly detecting the carp herpesvirus type II according to claim 5, comprising the following steps:
(1) adding 40.9 mu L of hydrolysis Buffer solution A Buffer, 10 mu M and 2 mu L of carp herpes virus II type upstream primer SEQ ID NO:1, 10 mu M and 2 mu L of carp herpes virus II type downstream primer SEQ ID NO:2, 10 mu M and 0.6 mu L of carp herpes virus II type probe SEQ ID NO:3 and 2 mu L of template into RAA freeze-dried enzyme powder, adding 280mM and 2.5 mu L of magnesium acetate B Buffer on a reaction tube cover, and turning the reaction tube upside down to fully mix the mixture and then instantly separate the mixture; reacting for 10min at constant temperature of 37 ℃;
(2) reaction: detecting the product obtained after the constant-temperature reaction in the step (1) by using a nucleic acid rapid detection test strip, and observing the result for 3-5 min;
(3) and (4) interpretation of results: direct visual interpretation
① is negative, a band appears only on the quality control line, and no band appears on the detection line, which indicates that the detected sample has no carp herpesvirus II infection;
②, when the test line and the quality control line are positive, both have strips, the test sample is proved to be the carp herpesvirus type II infection;
③ is invalid, the quality control line and the detection line have no strip, which indicates that the test is unsuccessful and the test paper for rapid detection of nucleic acid is invalid.
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| CN111647690A (en) * | 2020-06-22 | 2020-09-11 | 华侨大学 | RT-RAA primer pair and diagnostic kit for detecting COVID-19 virus |
| CN113430302A (en) * | 2021-06-29 | 2021-09-24 | 宁波大学 | RT-RAA-LFS rapid visual detection primer, probe and kit for tomato mosaic virus and application |
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