CN111793723A

CN111793723A - Duplex RT-PCR detection primer for duck hepatitis C virus and novel duck picornavirus

Info

Publication number: CN111793723A
Application number: CN202010882032.4A
Authority: CN
Inventors: 陈翠腾; 刘斌琼; 陈珍; 朱春华; 蔡国漳
Original assignee: Institute of Animal Husbandry and Veterinary of Fujian Academy of Agricultural Sciences
Current assignee: Institute of Animal Husbandry and Veterinary of Fujian Academy of Agricultural Sciences
Priority date: 2020-08-28
Filing date: 2020-08-28
Publication date: 2020-10-20
Anticipated expiration: 2040-08-28
Also published as: CN111793723B

Abstract

The invention relates to a dual RT-PCR detection primer for duck hepatitis C virus and novel duck picornavirus, which comprises the following steps: specific primer sequences aiming at the duck hepatitis C virus are shown as SEQ.ID.No.1 and SEQ.ID.No.2, and the size of a target fragment is 502 bp; specific primers for the novel duck picornavirus are shown as SEQ.ID.No.3 and SEQ.ID.No.4, and the size of a target fragment is 255 bp. The primer can be used for establishing a dual RT-PCR detection method for differential diagnosis of DuHCV and DuMV, the method can be used for simultaneously detecting DuHCV and DuMV infection in duck groups, the operation procedure is simplified, the repeated detection of conventional PCR is avoided, and the method has the advantages of low cost, high efficiency and the like.

Description

Duplex RT-PCR detection primer for duck hepatitis C virus and novel duck picornavirus

Technical Field

The invention belongs to the field of animal virology, and particularly relates to a dual RT-PCR (reverse transcription-polymerase chain reaction) detection primer for duck hepatitis C virus and novel duck picornavirus.

Background

The aquatic bird breeding amount of China is the first in the world and is an important component of poultry industry, however, in recent years, various new infectious diseases appear in the aquatic birds of China in succession without breaking off, and the occurrence of the new infectious diseases causes huge economic loss to the aquatic bird industry of China and becomes a key factor which seriously restricts the healthy and stable development of the aquatic bird industry of China.

According to the latest classification of the International Committee for viral classifications (International Committee on Taxonomy of viruses), 4 virus genera were set under the Flaviviridae Family (Flaviviridae Family): flaviviruses (Flavivirus), 53 virus species, some of which are zoonotic pathogens such as epidemic encephalitis b virus, dengue virus; hepacivirus type c (hepacivirus), there are 14 virus species; pegivirus genus, 11 species; pestiviruses (pestiviruses) are 11 species, and are commonly exemplified by classical swine fever virus and bovine viral diarrhea virus. Hepatitis C Virus (HCV) is a single-stranded positive-stranded RNA virus belonging to the genus hepatitis C virus of the family flaviviridae, the virion of which is spherical and has a diameter of about 40-60nm, and the nucleocapsid is icosahedral in symmetry and surrounds a lipid-containing envelope with spikes. Duck hepatitis c virus (DuHCV) is a newly discovered hepatitis c virus from a duck group suffering from egg laying abnormality, the genome of the duck hepatitis c virus has the characteristics of general hepatitis c, is single-stranded positive-strand RNA, has the length of 11422bp, encodes an Open Reading Frame (ORF) with the length of 10824bp, and encodes a polyprotein with the length of 3607 amino acids.

Viruses of the Picornaviridae Family (Picornaviridae Family) are important zoonotic pathogens that cause subclinical infections or severe illnesses ranging from mild fever to the heart, liver and central nervous system in humans and animals. Picornaviridae viruses are a class of icosahedral, symmetrical, spherical, single-stranded, positive-stranded RNA viruses of about 20-30nm diameter that are membrane-free. The viruses of the family picornaviridae include 63 virus genera, and commonly include the genus foot-and-mouth disease virus (Aphthovirus), the genus Enterovirus (Enterovirus), the genus Cardiovirus (Cardiovirus), the genus hepacivirus (Hepatovirus), and the like. The length of the gene of the virus in the family of picornaceae is generally 6.6kb-9.8kb, the genome generally only contains one large Open Reading Frame (ORF), the 3 'end is a Poly A tail, the 5' end is covalently bound with VPg protein, and the genome RNA is infectious. In recent years, with the progress of viral metagenomics, pathogens belonging to the genus Megrivirus of the family picornaceae have been discovered in the past from turkeys, chickens, pigeons, and ducks. Genomic analysis shows that the genome structural characteristics of the duck novel picornavirus (DuMV) are similar to those of turkey-derived megrivus and chicken-derived megrivus, and the polyprotein of the duck novel picornavirus contains characteristic motifs of typical picornaviridae viruses. Genetic evolutionary analysis shows that the novel duck picornavirus (DuMV) and the novel Megrivirus (Megrivirus) turkey, chicken and pigeon source picornaviruses belong to the same genetic evolutionary branch.

However, no research report of primers for carrying out a dual RT-PCR detection method on two new duck group viruses, namely duck hepatitis C virus (DuHCV) and duck novel picornavirus (DuMV), is available at present, and the establishment of the invention can fill the blank of related fields at home and abroad. The dual RT-PCR detection method established by the invention can be used for carrying out differential diagnosis on DuHCV and DuMV which are popular in duck groups, lays a foundation for developing new DuHCV and DuMV epidemiological investigation and subsequent scientific prevention and control of related diseases in duck groups, and has very important research significance.

Disclosure of Invention

The invention aims to provide a dual RT-PCR detection primer and a kit for duck hepatitis C virus and duck novel picornavirus, and establishes a dual RT-PCR detection method capable of carrying out differential diagnosis on DuHCV and DuMV prevalent in a duck group, thereby laying a foundation for developing new DuHCV and DuMV epidemiological investigation and subsequent scientific prevention and control related diseases in the duck group and having very important research significance.

The purpose of the invention is realized by the following technical scheme:

a dual RT-PCR detection primer for duck hepatitis C virus and novel duck picornavirus comprises the following primers:

specific primers for duck hepatitis C virus:

DuHCV-F：5’-GTTACTACCTATCTACGCAATT-3’；

DuHCV-R：5’-AGATGGCACTGGGATCGATGTA-3’；

the size of the target fragment is 502 bp;

specific primers aiming at the novel duck picornavirus:

DuMV-F：5’-TGTCAGCGCTATGATGGAGA-3’；

DuMV-R：5’-AATCCTTCCACCATCATCCAGT-3’；

the size of the target fragment was 255 bp.

The double RT-PCR detection method for detecting the duck hepatitis C virus and the novel duck picornavirus by using the primers has the PCR reaction system as follows: the following components were contained in a 50. mu.L system:

component name	Volume (μ L)
		2 × Multiple PCR Buffer PCR amplification reaction solution	25
DuHCV-F(10μmol/L)	0.4
		DuHCV-R(10μmol/L)	0.4
DuMV-F(10μmol/L)	0.6
		DuMV-R(10μmol/L)	0.6
PCR amplification enzyme	0.25
		cDNA template	1.0
Sterilizing deionized water	21.75

；

The PCR reaction conditions are as follows: carrying out pre-denaturation at 94 ℃ for 120s, then carrying out circulation, carrying out denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 45s, and extending at 72 ℃ for 45s, carrying out final extension at 72 ℃ for 10min after 35 cycles are finished, and carrying out identification according to conventional agarose gel electrophoresis after the reaction is finished.

If the detection sample only contains DuHCV, only one amplified target fragment is provided, and the size is 502 bp; if the detection sample only contains DuMV, only one amplified target fragment is provided, and the size of the amplified target fragment is 255 bp; if the detection sample contains DuHCV and DuMV, two amplified target fragments are provided, namely 502bp and 255bp respectively; if DuHCV and DuMV are not present in the test sample, no amplified band is observed.

The invention also provides application of the primer in preparation of a novel duck hepatitis C virus and duck picornavirus detection kit.

The invention also provides a kit for detecting duck hepatitis C virus and novel duck picornavirus, and the kit comprises the primer.

Compared with the prior art, the invention has the advantages that:

1. and (3) simultaneously detecting: the invention can establish a dual RT-PCR detection method for differential diagnosis of DuHCV and DuMV by using the primer, the method can simultaneously detect DuHCV and DuMV infection in duck groups, simplifies operation procedures, avoids repeated detection of conventional PCR, has the advantages of low cost, high efficiency and the like, and directly judges an amplification result by using different lengths of amplification fragments in the design of the primer, so that the method is simpler and more convenient in result judgment. If the detection sample only contains DuHCV, only one amplified target fragment is provided, and the size is 502 bp; if the detection sample only contains DuMV, only one amplified target fragment is provided, and the size of the amplified target fragment is 255 bp; if the detection sample contains DuHCV and DuMV, two amplified target fragments are provided, namely 502bp and 255bp respectively; if DuHCV and DuMV are not present in the test sample, no amplified band is observed.

2. The sensitivity is high: the duHCV minimum detection limit of the double RT-PCR detection method established by the invention is 3.79 multiplied by 10¹pg (i.e., 37.9pg), and the lowest detection limit of DuMV is 7.12X 10¹pg (i.e. 71.2 pg).

3. The specificity is strong: the dual RT-PCR detection method established by the primer has no reaction signals to common infectious diseases in duck groups such as DHAV-1, DHAV-3, AIV, APMV-1, MDRV and N-DRV, and only shows specific amplification bands to DuHCV and DuMV.

Drawings

FIG. 1 is an electrophoretogram showing the results of a specific test by the double RT-PCR method; wherein, M: DNA molecular weight DL 2000; 1: DuHCV; 2: DuMV; 3: DuHCV and DuMV co-infection; 4: DHAV-1; 5: DHAV-3; 6: an AIV; 7: APMV-1; 8: MDRV; 9: N-DRV.

FIG. 2 is an electrophoretogram of the results of the specificity test for DuHCV; wherein, M: DNA molecular weight DL 2000; 1: DuHCV; 2: DuMV; 3: DHAV-1; 4: DHAV-3; 5: an AIV; 6: APMV-1; 7: MDRV; 8: N-DRV.

FIG. 3 is an electrophoretogram of the results of the specificity test for DuMV; wherein, M: DNA molecular weight DL 2000; 1: DuMV; 2: DuHCV; 3: DHAV-1; 4: DHAV-3; 5: an AIV; 6: APMV-1; 7: MDRV; 8: N-DRV.

FIG. 4 is an electrophoretogram of the results of the double PCR duck hepatitis C virus susceptibility test; wherein, M: DNA molecular weight DL 2000; 1: 3.79X 10⁵pg；2：3.79×10⁴pg；3：3.79×10³pg；4：3.79×10²pg； 5：3.79×10¹pg；6：3.79×10⁰pg; 7: negative controls were tested.

FIG. 5Is an electrophoretogram of the double PCR duck novel picornavirus sensitivity test result; wherein, M: DNA molecular weight DL 2000; 1: 7.12X 10⁵pg；2：7.12×10⁴pg；3：7.12×10³pg；4：7.12×10²pg； 5：7.12×10¹pg；6：7.12×10⁰pg; 7: negative controls were tested.

Detailed Description

The invention is described in detail below with reference to the figures and examples of the specification. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1

1. Materials and methods

1.1 strains and strains

Pathogenic duck hepatitis C virus (DuHCV, strain H36a 8), novel duck picornavirus (DuMV, strain M19g 24), duck hepatitis 1 virus (DHAV-1), duck hepatitis 3 virus (DHAV-3), duck Avian Influenza Virus (AIV), duck paramyxovirus 1 (APMV-1), Muscovy Duck Reovirus (MDRV) and novel duck reovirus (N-DRV) for the test are identified and stored by animal husbandry and veterinary research institute of agricultural academy of sciences of Fujian province.

1.2 design of primers

According to the genomic characteristics encoded by DuHCV and DuMV in the National Center of Biotechnology information, NCBI database GenBank, primer design software Oligo (v7.0) was used to target specific primers for DuHCV and DuMV, respectively, which were synthesized by Biotechnology engineering (Shanghai) GmbH.

Specific primers for DuHCV:

DuHCV-F：5’-GTTACTACCTATCTACGCAATT-3’；

DuHCV-R：5’-AGATGGCACTGGGATCGATGTA-3’；

the size of the target fragment is 502 bp.

Specific primers for DuMV:

DuMV-F：5’-TGTCAGCGCTATGATGGAGA-3’；

DuMV-R：5’-AATCCTTCCACCATCATCCAGT-3’；

the size of the target fragment was 255 bp.

1.3 extraction of nucleic acids and preparation of cDNA

Nucleic acid RNAs of DuHCV, DuMV, DHAV-1, DHAV-3, AIV, APMV-1, MDRV and N-DRV were extracted according to the Viral nucleic acid extraction Kit (Easypure Viral DNA/RNA Kit). Using a reverse transcription kit (

One-Step gDNA Removal and cDNA Synthesis SuperMix) reverse transcribing the extracted RNA into cDNA for use.

1.4 Dual RT-PCR method establishment

Amplification was performed according to the 50. mu.L system recommended by the Multiplex PCR Kit (Multiplex PCR Assay Kit Ver.2), wherein 25. mu.L of 2 × Multiplex PCR Buffer PCR amplification reaction solution, 0.4. mu.L of each of primers for DuHCV (DuHCV-F and DuHCV-R) (both primer concentrations are 10. mu. mol/L), 0.6. mu. L, PCR of each of amplimers for DuMV (DuMV-F and DuMV-R) (both primer concentrations are 10. mu. mol/L), 1.0. mu.L of each of prepared cDNA templates, and sterile deionized water were supplemented to a final volume of 50. mu.L, followed by PCR amplification after mixing. The optimized amplification conditions were: carrying out pre-denaturation at 94 ℃ for 120s, then carrying out circulation, carrying out denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 45s, and extending at 72 ℃ for 45s, carrying out final extension at 72 ℃ for 10min after 35 cycles are finished, and carrying out identification according to conventional agarose gel electrophoresis after the reaction is finished.

The results are shown in FIG. 1: the size of the DuHCV target fragment is 502bp (lane 1); the size of the DuMV target fragment is 255bp (lane 2); the DuHCV and DuMV double positive amplified target fragments have two sizes, 502bp and 255bp respectively (lane 3).

1.5 specificity test

1.5.1 specificity test for Duck hepatitis C Virus

Utilizing specific primers (DuHCV-F and DuHCV-R) aiming at duck hepatitis C virus to perform PCR amplification on cDNA templates which are subjected to reverse transcription after RNA extraction of DuHCV, DuMV, DHAV-1, DHAV-3, AIV, APMV-1, MDRV and N-DRV; amplification was performed according to the 50. mu.L system recommended by the PCR kit (2 XEasyTaq PCR Supermix (+ dye)), wherein 25. mu.L of 2 XEasyTaq PCR Supermix Buffer PCR amplification reaction solution and 1.0. mu.L of 10. mu.M specific primers for duck hepatitis C virus (DuHCV-F and DuHCV-R) were added to 1. mu. L, cDNA template, respectively, and the final volume was 50. mu.L with sterile deionized water, and PCR amplification was performed after mixing. The amplification condition is pre-denaturation at 94 ℃ for 5min, then circulation is carried out, denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 30s and extension at 72 ℃ for 40s, after 35 cycles are finished, final extension at 72 ℃ for 10min is carried out, and after the reaction is finished, the agarose gel electrophoresis identification is carried out according to the prior method. The results are shown in FIG. 2: the size of the DuHCV target fragment is 502 bp; no specific amplification bands were observed for DuMV, DHAV-1, DHAV-3, AIV, APMV-1, MDRV and N-DRV.

1.5.2 specificity test for novel duck picornaviruses

Utilizing specific primers (DuMV-F and DuMV-R) aiming at the novel duck picornaviruses to perform PCR amplification on cDNA templates which are subjected to reverse transcription after RNA extraction of DuHCV, DuMV, DHAV-1, DHAV-3, AIV, APMV-1, MDRV and N-DRV; amplification was performed according to a 50. mu.L system recommended by a PCR kit (2 XEasyTaq PCR Supermix (+ dye)), wherein 25. mu.L of the 2 XEasyTaq PCR Supermix Buffer PCR amplification reaction solution and 1.0. mu.L of 10. mu.M primers specific to the novel duck picornavirus (DuMV-F and DuMV-R) were added to a final volume of 50. mu.L each of 1. mu. L, cDNA template, and PCR amplification was performed after mixing. The amplification condition is pre-denaturation at 94 ℃ for 5min, then circulation is carried out, denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 30s and extension at 72 ℃ for 40s, after 35 cycles are finished, final extension at 72 ℃ for 10min is carried out, and after the reaction is finished, the agarose gel electrophoresis identification is carried out according to the prior method. The results are shown in FIG. 3: the size of the target fragment of DuMV is 255 bp; no specific amplification bands were observed for DuHCV, DHAV-1, DHAV-3, AIV, APMV-1, MDRV and N-DRV.

1.5.3 specificity of the Dual RT-PCR method

According to the optimized multiplex PCR conditions of 1.4, the PCR amplification is carried out on the cDNA templates of DuHCV, DuMV, DHAV-1, DHAV-3, AIV, APMV-1, MDRV and N-DRV after RNA extraction and reverse transcription, the result is shown in figure 1, and the size of the DuHCV target fragment is 502bp (lane 1); the size of the DuMV target fragment is 255bp (lane 2); the DuHCV and DuMV double-positive amplified target fragments have two sizes, 502bp and 255bp respectively (lane 3), while DHAV-1 (lane 4), DHAV-3 (lane 5), AIV (lane 6), APMV-1 (lane 7), MDRV (lane 8) and N-DRV (lane 9) have no band amplification, which indicates that the method established by the invention has strong specificity.

1.6 sensitivity of the Dual RT-PCR method

DuHCV and DuMV extracted RNA were serially diluted 10-fold according to 1.4 optimized multiplex PCR conditions (DuHCV concentrations were 3.79X 10, respectively)⁵pg-3.79×10⁰The pg and DuMV concentrations are 7.12X 10⁵pg-7.12×10⁰pg) are subjected to reverse transcription and then amplification is performed.

The results are shown in FIGS. 4 and 5: the lowest detection limit of DuHCV is 3.79X 10¹pg (i.e., 37.9pg) (FIG. 4, lane 5); the lowest detection limit of DuMV is 7.12 multiplied by 10¹pg (i.e., 71.2pg) (FIG. 5, lane 5).

1.7 clinical applications

According to the method, after 145 parts of clinical inspection duck-origin disease material is treated according to a conventional method, corresponding nucleic acid RNA is extracted by using a virus nucleic acid extraction Kit EasyPure Viral DNA/RNA Kit and then is reversely transcribed into cDNA, and the DuHCV and DuMV infection is detected by using an established duplex RT-PCR method. As a result, 21 DuHCV infections are detected to be positive, and the positive rate is 14.45%; 9 DuMV infection positives are detected, and the positive rate is 6.21%; 2 DuHCV and DuMV co-infection positives were detected with a positive rate of 1.38%.

And (3) cutting and recovering the positive target fragments after the double RT-PCR reaction is finished by using an agarose gel recovery kit. Cloning the amplified gene fragment onto pEASY-T1 vector according to pEASY-T1 Simple Cloning Kit instruction, randomly picking 8 single colonies, culturing in LB liquid culture medium with ampicillin resistance (content 100 ug/mL) for 14h, and extracting corresponding plasmid by using rapid plasmid miniextraction Kit. The extracted plasmid is subjected to PCR identification by adopting primers and conditions during double RT-PCR amplification, and the screened positive recombinant plasmid is sent to the company of Biotechnology engineering (Shanghai) Ltd for sequencing. The sequencing results are verified by BLAST analysis on NCBI, and are corresponding DuHCV and DuMV gene fragments, and the nucleotide homology of related gene sequences is respectively over 98.3 percent (DuHCV) and 98.8 percent (DuMV), which shows that the established method accords with the experimental expectation.

Sequence listing

<110> animal husbandry and veterinary institute of agricultural academy of sciences of Fujian province

<120> duck hepatitis C virus and novel duck picornavirus dual RT-PCR detection primers

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<170>SIPOSequenceListing 1.0

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<213> Artificial sequence (Artificial sequence)

<400>1

gttactacct atctacgcaa tt 22

<210>2

<211>22

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>2

agatggcact gggatcgatg ta 22

<210>3

<211>20

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>3

tgtcagcgct atgatggaga 20

<210>4

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<213> Artificial sequence (Artificial sequence)

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aatccttcca ccatcatcca gt 22

Claims

1. A dual RT-PCR detection primer for duck hepatitis C virus and novel duck picornavirus is characterized in that: the primers are as follows:

specific primers for duck hepatitis C virus:

DuHCV-F：5’-GTTACTACCTATCTACGCAATT-3’；

DuHCV-R：5’-AGATGGCACTGGGATCGATGTA-3’；

the size of the target fragment is 502 bp;

specific primers aiming at the novel duck picornavirus:

DuMV-F：5’-TGTCAGCGCTATGATGGAGA-3’；

DuMV-R：5’-AATCCTTCCACCATCATCCAGT-3’；

the size of the target fragment was 255 bp.

2. The dual RT-PCR detection method for detecting duck hepatitis C virus and novel duck picornavirus by using the primer of claim 1 is characterized in that: the PCR reaction system is as follows: the following components were contained in a 50. mu.L system:

；

The PCR reaction conditions are as follows: carrying out pre-denaturation at 94 ℃ for 120s, then carrying out circulation, carrying out denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 45s, and extending at 72 ℃ for 45s, and carrying out final extension at 72 ℃ for 10min after 35 cycles are finished.

3. The use of the primer of claim 1 in the preparation of a novel kit for detecting duck hepatitis C virus and duck picornavirus.

4. A kit for detecting duck hepatitis C virus and novel duck picornavirus is characterized in that: the kit comprises the primer of claim 1.