1. A reverse transcription multiple nested PCR detection primer for identifying the genotype of a prawn yellow head virus is characterized by comprising two outer primers 736F and 736R and three inner primers 307F, 194F and 307/194R, wherein the outer primers can amplify YHV-1 and YHV-8 and generate 763bp target bands; three inner primers are matched to respectively amplify YHV-1 and YHV-8 specifically to generate target bands of 307bp and 194bp respectively;

the sequence of the outer primer 736F is: SEQ ID NO: 1; the sequence of the outer primer 736R is: SEQ ID NO: 2;

the sequence of the inner primer 307F is: SEQ ID NO: 3; the sequence of the inner primer 194F is: SEQ ID NO: 4; the sequence of the inner primer 307/194R is: SEQ ID NO: 5.

2. the reverse transcription multiple nested PCR detection primer for identifying the genotype of the prawn yellow head virus of claim 1 is characterized by being capable of detecting the pathogenic genotypes YHV-1 and YHV-8 of the yellow head virus with high sensitivity.

3. The reverse transcription nested PCR detection primer for identifying the genotype of the prawn yellow head virus of claim 2, wherein YHV-8 comprises the virus of which the new species name is Okavirus 1 and the abbreviation is OKV 1.

4. The reverse transcription multiple nested PCR detection primer for identifying the genotype of the prawn yellow head virus according to claim 1, which is characterized in that the primer comprises 1 or more primers of the primer set and sequences covered by the primers, and is a reverse transcription multiple nested PCR reagent and a kit for identifying, detecting and monitoring YHV-1 and YHV-8.

Background

The prawn is a high-quality aquatic product recognized in the world, creates considerable economic benefit for China, and is a high-quality food protein source for the nation at the same time. The Yellow head disease of the prawns is an infectious disease caused by Yellow Head Virus (YHV), generally affects the prawns cultured for 50-70 days, the morbidity reaches 100% after 3-5 days of infection, and the mortality reaches 80-90%. Infected sick shrimps have a process of eating a large amount of food and then stopping suddenly, and generally have the appearance of food deprivation, yellowing of head and chest and whitening of the whole body within 2 to 4 days, and the hepatopancreas turns soft and turns from brown to yellow. Many of the endangered shrimps gather on the water surface in the corners of the pond and die in a large number in a short period of time.

The yellowhead virus belongs to the order of nested viruses, the family of baculovirus and the genus of cephalo-alpha virus, 10 genotypes are known at present, wherein YHV-1 and YHV-8 are pathogenic genotypes which are proved to cause the disease of prawns, and in addition, 8 genotypes (2 type to 7 type, 9 type and 10 type) rarely or never cause the disease, and the popular report in the culture of prawns in China is not available. Therefore, the method for sensitively detecting and identifying the pathogenic genotypes YHV-1 and YHV-8 of the yellow head virus has important significance for epidemic disease prevention and control and etiology research in actual epidemic disease monitoring and epidemiological investigation of the prawn aquaculture industry.

Currently, PCR detection methods for YHV have been reported: the method comprises a reverse transcription PCR method (Wong teerarapaya et al 1997) aiming at YHV-1, a reverse transcription multiplex nested PCR method (Cowley et al 2004) aiming at YHV-1 and YHV-2, a reverse transcription multiplex PCR method (Wuriqin et al 2011) aiming at IHHNV, WSSV, YHV and TSV, and a multiplex PCR detection primer and a kit (CN105567875A) for simultaneously detecting IMNV, YHV and TSV of prawns. The 4 methods mainly aim at the pathogenic genotype YHV-1 of the yellow head virus, and no target sequence which is completely combined with the 4 PCR primers exists on the YHV-8 genome, so that the detection sensitivity of the method on the YHV-8 genotype is not enough, and the detection rate is low. Meanwhile, the above methods cannot realize the identification of YHV-1 and YHV-8 because of the limitation of primer design. Similarly, the conventional YHV detection method based on isothermal amplification and gene chip technology has the above problems that the detection rate of YHV-8 is low and two genotypes cannot be identified.

Through the above analysis, the problems and defects of the prior art are as follows:

(1) the existing detection method is established in an early time, and a primer aims at the early discovered pathogenic genotype YHV-1 of the yellow head virus, so that the high-sensitivity detection of a new pathogenic genotype YHV-8 cannot be met.

(2) The existing detection method can not realize the identification of pathogenic genotypes YHV-1 and YHV-8 of the yellow head virus.

The difficulty in solving the above problems and defects is: according to the gene sequences of the existing yellow head virus genotypes YHV-1 and YHV-8, a special primer combination is redesigned, a reaction system and a reaction program are optimized, one-step reverse transcription multiple nested PCR detection with high sensitivity on two pathogenic genotypes is met, and the two genotypes are rapidly identified by the molecular weight of a product generated by amplification.

The significance of solving the problems and the defects is as follows: a reverse transcription multiple-nested PCR primer is used for simultaneously realizing high-sensitivity detection on pathogenic genotypes YHV-1 and YHV-8 of the known yellow head virus, and the genotype of sample infection is quickly identified through the molecular weight of a product of the reaction, thereby providing technical support for prevention and control of epidemic diseases and etiology research in the breeding industry. The identification method is not only accurate, but also reduces the reaction cost and reaction time.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a reverse transcription multiple nested PCR detection primer. The primer can sensitively detect pathogenic genotypes YHV-1 and YHV-8 of the yellow head virus, and can rapidly identify the two genotypes according to the size of the product, thereby providing simple, convenient and reliable technical support for clinical detection and epidemiological research of the virus. The method not only has accurate result, but also saves time and reaction cost.

In order to solve the technical problem, the invention discloses a reverse transcription multiple nested PCR detection primer for identifying the genotype of a prawn yellow head virus, which comprises two outer primers (736F and 736R) and three inner primers (307F, 194F and 307/194R), wherein the outer primers can amplify YHV-1 and YHV-8 to generate 763bp target bands; the three inner primers are matched with each other to specifically amplify YHV-1 and YHV-8, so as to generate 307bp and 194bp target bands respectively, and the target sequences of YHV-1 and YHV-8 are shown in FIG. 2.

The sequence of the outer primer 736F is: SEQ ID NO: 1; the sequence of the outer primer 736R is: SEQ ID NO: 2;

the sequence of the inner primer 307F is: SEQ ID NO: 3; the sequence of the inner primer 194F is: SEQ ID NO: 4; the sequence of the inner primer 307/194R is: SEQ ID NO: 5.

furthermore, the pathogenic genotypes YHV-1 and YHV-8 of the yellow head virus can be detected with high sensitivity.

Further, YHV-8 includes viruses designated by their new species name Okavirus 1 and their abbreviation OKV 1.

Furthermore, the primer comprises 1 or more primers of the primer set and sequences covered by the primers, and is used for reverse transcription multiple-set PCR reagent and kit for identification, detection and monitoring of YHV-1 and YHV-8.

By combining all the technical schemes, the invention has the advantages and positive effects that: YHV has been reported to have 10 genotypes, only two genotypes of YHV-1 and YHV-8 may cause prawn morbidity, and the remaining 8 genotypes are not reported to be prevalent in our country. In order to simultaneously detect YHV-1 and YHV-8 two pathotype yellow head viruses and simultaneously quickly identify the genotype of the infected virus in a sample according to the size of a reaction product, the invention establishes a reverse transcription multiple-nested PCR detection primer. Two outer primers and three inner primers were designed by comparing genomic sequences of YHV-1 and YHV-8. Wherein, the external primer can amplify target genes of YHV-1 and YHV-8, and both generate 763bp target bands. Three inner primers are matched to amplify YHV-1 and YHV-8 specifically respectively to generate target bands of 307bp and 194 bp. Tong (Chinese character of 'tong')Through optimization of the reaction program, the optimal annealing temperature of the two-step PCR reaction is determined. Specificity tests show that the method can accurately detect the RNAs of YHV-1 and YHV-8 to generate target bands which are not similar to White Spot Syndrome Virus (WSSV), Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) and vibrio (V) causing acute hepatopancreatic necrosis_AHPND) And RNA templates of Taura Syndrome Virus (TSV), Macrobrachium rosenbergii nodavirus (MrNV), infectious myonecrosis virus (IMNV) and healthy prawns undergo non-specific reaction. The sensitivity test result shows that the lower limit of the detection of YHV-1 and YHV-8RNA by the method reaches 1.2 multiplied by 10¹And (6) copying. The results show that the multiple nested PCR detection primers established by the invention can detect pathogenic genotypes YHV-1 and YHV-8 of the yellow head virus, can rapidly identify the two genotypes according to the size of the product, and provide simple, convenient and reliable technical support for clinical detection and epidemiological research of the virus.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiments of the present application will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present application, and it is obvious for those skilled in the art that other drawings can be obtained from the drawings without creative efforts.

FIG. 1 is a flow chart of the multiplex nested PCR detection method provided by the embodiment of the present invention.

FIG. 2 is a schematic diagram of YHV-1 and YHV-8 target sequence alignment and primer design provided in an embodiment of the invention.

FIG. 3 is a schematic diagram of annealing temperature optimization of multiplex nested PCR provided in the embodiment of the present invention; A. performing electrophoresis results on the first round PCR products; B. performing electrophoresis results on the second round PCR products; m: marker; FIG. A: lanes 1-8 are the reaction products at annealing temperatures of 49, 51, 53, 55, 57, 59, 61 and 63 ℃ respectively, and lane 9 is a blank control; and B: lanes 1-8 are the reaction products at annealing temperatures of 49, 51, 53, 55, 57, 59, 62, and 65 ℃ respectively, and lane 9 is a blank control.

FIG. 4 is a graph showing the sensitivity test results provided by the embodiment of the present inventionA drawing; A. performing electrophoresis results on the first round PCR products; B. performing electrophoresis results on the second round PCR products; m: marker; 1 to 8 lanes are 1.2X 10¹～1.2×10⁸Copies/. mu.L of the reaction products with YHV-1 and YHV-8RNA standards as templates; lane 9 is a blank control.

FIG. 5 is a schematic diagram of the results of a specificity test provided by an embodiment of the present invention; A. electrophoresis image of product using YHV-1 RNA standard as template; B. electrophoresis of products using YHV-8RNA standard as template; m: marker; lanes 1 to 10 are YHV-8, YHV-1, negative control, WSSV, IHHNV, V_AHPNDRNA of TSV, MrNV and IMNV is a reaction product of the template.

FIG. 6 is a schematic diagram of the results of a single-source detection of a diseased sample according to an embodiment of the present invention: this figure shows the results of the first and second rounds of PCR; m (first round): the 2000bp DNA Marker consists of DNA fragments of 2000bp, 1000bp, 750bp, 500bp, 250bp and 100 bp; m (second round): 50bp DNA Ladder, consisting of DNA fragments of 500bp, 400bp, 300bp, 250bp, 200bp, 150bp, 100bp and 50 bp; 1-30: a prawn sample; +: a positive control; -: and (5) negative control.

FIG. 7 is a diagram illustrating the results of genotyping a sample from a mixed source according to an embodiment of the present invention; the figure shows the electrophoresis results of the second round of PCR only; m: 50bp DNA Ladder, consisting of DNA fragments of 500bp, 400bp, 300bp, 250bp, 200bp, 150bp, 100bp and 50 bp; the reaction products of the samples of 8 lanes are respectively arranged at two sides of each Marker lane; the reaction product bands are indicated below by numerals 1 and 8 as positive for YHV-1 and YHV-8, respectively.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Aiming at the problems in the prior art, the invention provides a reverse transcription multiple-nested PCR detection primer, and the invention is described in detail with reference to the attached drawings.

As shown in fig. 1, the multiple nested PCR detection method provided by the present invention comprises the following steps:

s101: sample collection and RNA extraction, dissecting the sample in a laboratory, and storing in an ultra-low temperature refrigerator at-80 ℃; cutting about 30mg of gill silk tissue of each prawn, and extracting total RNA of a sample tissue by using an animal tissue/cell total RNA extraction kit;

s102: designing primers, namely two outer primers and three inner primers; wherein the external primer can simultaneously amplify target genes of YHV-1 and YHV-8 to generate 763bp target band. Three inner primers are matched to amplify YHV-1 and YHV-8 specifically respectively to generate target bands of 307bp and 194 bp;

s103: synthesizing two yellow head virus genotype target fragment RNA standard products;

s104: a25. mu.L round of reverse transcription PCR and a 25. mu.L round of PCR reaction were used.

Those skilled in the art can also use other steps to implement the reverse transcription multiplex PCR detection method provided by the present invention, and the multiplex PCR detection method provided by the present invention in FIG. 1 is only one specific example.

The technical solution of the present invention is further described with reference to the following specific examples.

Example 1 establishment of reverse transcription multiple nested PCR detection method for rapidly identifying pathogenic genotype of prawn yellow head virus:

1, materials and methods:

1.1 sample Collection and RNA extraction

The diseased Chinese prawn sample is collected from a prawn farm in Shandong province, and is stored in an ultra-low temperature refrigerator at-80 ℃ after being dissected in a laboratory. YHV-1 infected shrimp tissues were supplied by the Asia-Pacific Aquaculture center Net (NACA) and stored in ethanol.

About 30mg of gill silk tissue is cut from each Chinese prawn, total RNA of a sample tissue is extracted by using an animal tissue/cell total RNA extraction kit (Strongylocentrotus nudus, Beijing) and the operation is carried out according to the steps of a kit specification.

1.2 primer design

Genomic sequences of the Flaviviridae genotypes YHV-1 and YHV-8 (SEQ ID NO: FJ848673, KX947267) were downloaded from the National Center for Bioinformation (NCBI) database, aligned using the software Geneius Prime (version 2019.2.1), and primer design was performed using the software Oligo (version 7.60). The primer sequences, the target plant types and the sizes of the products are shown in Table 1. The primers were synthesized by Biotechnology engineering (Shanghai) Inc.

1.3 Synthesis of two Flaviviridae genotype target fragment RNA standards

To obtain RNA standards for YHV-1 and YHV-8 target fragments, reverse transcription and PCR amplification were performed using 763F and 763R as primers and YHV-1 and YHV-8RNA stored in the laboratory as templates, respectively, to obtain two 763 bp-sized amplification products. The two 763bp products were ligated to pGEM-T vector (Promega) containing the T7 promoter sequence and transformed into competent cells for culture, respectively. The purified plasmid was digested into a strand DNA with the restriction endonuclease Pst I (TaKaRa). Then in vitro transcription and DNA degradation are carried out by using a T7 transcription kit (all-gold), and purified YHV-1 and YHV-8 target RNA fragments are obtained after treatment by using an RNA purification kit (all-gold). The target RNA fragment was subjected to quality control and concentration determination using NanoDrop 2000(Thermo), and was diluted by 10-fold gradient to obtain 1.2X 10⁸～1.2×10¹Copies/. mu.LYHV-1 and YHV-8 of RNA standards.

1.4 optimization of reaction sequence of multiple nested PCR method

The first round of PCR was a 25 μ L reaction containing: 1 μ L of YHV-1 and YHV-8 mixed RNA template, 12.5 μ L of 2X 1Step Buffer, 400nM primers 763F and 763R, 1 μ L of PrimeScript 1Step Enzyme Mix (TaKaRa), RNase Free water was added to 25 μ L. The amplification program comprises reverse transcription at 50 ℃ for 30min, pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 30s, annealing temperature setting gradients of 49, 51, 53, 55, 57, 59, 61 and 63 ℃ for 30s respectively, extension at 72 ℃ for 50s, and 35 cycles; extension at 72 ℃ for 5 min. And (5) detecting the result of the amplification product by using 1% agarose gel electrophoresis, and selecting the optimal annealing temperature.

The second round of PCR was a 25 μ L reaction containing: mu.L of one round PCR reaction product template, 2.5. mu.L of 10 XEx Taq Buffer, 2. mu.L of dNTP (0.25mmol/L), 2. mu.L of Mg²⁺(2.5mmol/L), 400nM primer 307/194R, 200nM each of primer 307F and 194F, 0.625U of Ex Taq Enzyme (TaKaRa), PCR grade water was added to 25. mu.L. The amplification program is pre-denaturation at 95 ℃ for 2min, denaturation at 95 ℃ for 30s, annealing temperature setting gradients of 49, 51, 53, 55, 57, 59, 62 and 65 ℃ for 30s respectively, extension at 72 ℃ for 30s, and 35 cycles; extension at 72 ℃ for 2 min. And (5) detecting the result of the amplification product by using 1% agarose gel electrophoresis, and selecting the optimal annealing temperature.

1.5 sensitivity test

Using optimized reaction conditions, respectively at 1.2X 10¹～1.2×10⁸Copy/. mu.L of YHV-1 and YHV-8RNA standards as template, RNase-Free water as blank control, multiple nested PCR amplification was performed, and the sensitivity of the method was tested.

1.6 specificity test

YHV-8, YHV-1, White Spot Syndrome Virus (WSSV), Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV), Vibrio (V) causing acute hepatopancreatic necrosis_AHPND) RNA of Taura Syndrome Virus (TSV), Macrobrachium rosenbergii nodavirus (MrNV) and infectious myonecrosis virus (IMNV) is used as a template, and RNA of healthy prawns is used as negative control. And (3) performing specificity test by using an optimized multiplex PCR method.

2, results:

2.1 primer design

5 primers, including 2 outer primers 763F and 763R and 3 inner primers 307F, 194F and 307/194R (FIG. 2), were finally obtained by aligning YHV-1 and YHV-8 genomes and primer design. Wherein 763F, 763R and 307/194R primers can be matched with YHV-1 and YHV-8 by 100%, and 763bp of target band can be amplified. And the primer 307F and the primer 194F are specific inner primers of YHV-1 and YHV-8 respectively, target bands of 307bp and 194bp are generated respectively, and the type of the strain infected with YHV in the sample can be distinguished through the size of the product.

2.2 optimization of the reaction program

Through the optimization of the annealing temperature of the two PCR reactions, when the annealing temperature of the first PCR reaction is 51 ℃ (A in figure 3), the electrophoretic display product strip is clearest and bright, so 51 ℃ is the optimal annealing temperature; in the second PCR reaction, although the band was brightest when the annealing temperature was 57 ℃, 65 ℃ was selected as the annealing temperature in the second PCR reaction in order to ensure the specificity of the primer reaction, considering that the second PCR was multiplex PCR (FIG. 3B).

2.3 determination of sensitivity

In the first round of multiple nested PCR, the minimum concentration of the RNA standard in YHV-1 and YHV-8 was 1.2X 10⁴At copy/. mu.L, there was still a band visible to the naked eye (FIG. 4); in the second reaction, the minimum RNA standard concentration of YHV-1 and YHV-8 was 1.2X 10¹At copy/. mu.L, there was still a clear band (FIG. 4). Therefore, the detection sensitivity of the first round of PCR reaction of the method is 1.2X 10⁴The detection sensitivity of the copy and second round PCR reaction is 1.2X 10¹And (6) copying.

2.4 determination of specificity

Through specificity test, only YHV-1 and YHV-8RNA templates can amplify target bands in both PCR rounds, and the target bands cannot be amplified in the rest of RNA samples (FIG. 5). Therefore, the multiple PCR method can be proved to have high specificity.

Example 2 example of test results:

30 single-source diseased Chinese prawn sample tissues collected from Shandong sunshine one farm are detected by using the reverse transcription multiple nested PCR method developed by the invention. The detection result of the invention is shown in FIG. 6, and it can be seen from the figure that 24 samples generate a target band of more than 750bp after the first round of PCR amplification, 27 samples generate a target band of less than 200bp after the second round of PCR amplification, and 3 detection samples have no band after reverse transcription multiple nested PCR detection, so that 27 samples in the 30 samples of the batch are YHV-8 positive, and 3 samples are yellowhead virus pathogenic genotype negative.

Example 3 example of identification results:

96 parts of prawn sample tissues with unknown mixed sources and stored in a laboratory are detected by using the reverse transcription multiple nested PCR method developed by the invention. The identification result of the present invention is shown in FIG. 7, from which it can be obtained that 67 samples have no target bands after reverse transcription multiple nested PCR amplification and are negative to YHV-1 and YHV-8; after 16 samples are amplified, a target band larger than 300bp is generated and is positive in YHV-1; after amplification of 13 samples, a target band of less than 200bp was generated, which was positive for YHV-8. According to the size of the second reaction round strip, the pathogenic yellow head virus genotype infected by the prawn sample with unknown infection state is rapidly identified.

TABLE 1 multiple nested PCR primer information

The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.

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