CN114438265A

CN114438265A - Nucleic acid composition, kit and detection method for simultaneously detecting porcine delta coronavirus, reovirus and porcine kobuvirus

Info

Publication number: CN114438265A
Application number: CN202210244238.3A
Authority: CN
Inventors: 王利丽; 李秀丽; 江珊; 鄢明华; 路超; 张莉; 任卫科; 董志民; 田向学; 朱琪; 李富强
Original assignee: Tianjin Academy of Agricultural Sciences
Current assignee: Tianjin Academy of Agricultural Sciences
Priority date: 2022-03-14
Filing date: 2022-03-14
Publication date: 2022-05-06
Anticipated expiration: 2042-03-14
Also published as: CN114438265B

Abstract

Nucleic acid composition, kit and detection method for simultaneously detecting porcine delta coronavirus, reovirus and porcine kobuvirus. The invention belongs to the technical field of porcine pathogen detection, and particularly relates to a nucleic acid composition, a kit and a detection method for simultaneously detecting porcine delta coronavirus, reovirus and porcine kobuvirus. The nucleic acid composition provided by the invention can specifically identify the porcine delta coronavirus, the reovirus and the porcine kobuvirus, and the triple fluorescence quantitative PCR detection method established by utilizing the nucleic acid composition has the characteristics of rapidness, convenience, high efficiency and high sensitivity, can be widely applied to epidemiological investigation, laboratory identification and detection and related scientific research of the porcine diarrhea disease, and has a high practical application value.

Description

Nucleic acid composition, kit and detection method for simultaneously detecting porcine delta coronavirus, reovirus and porcine kobuvirus

Technical Field

The invention belongs to the technical field of porcine pathogen detection, and particularly relates to a nucleic acid composition, a kit and a detection method for simultaneously detecting porcine delta coronavirus, reovirus and porcine kobuvirus.

Background

In recent years, the prevalence of pig diarrhea is more and more extensive, and the complexity of the disease is increasingly increased, which causes serious economic loss to the animal husbandry in China. Porcine Epidemic Diarrheal Virus (PEDV), Porcine transmissible gastroenteritis virus (TGEV), and Porcine rotavirus (Porcine rotavirus, PoRV) are three important viral pathogens that cause Porcine diarrhea. In recent years, new diarrhea pathogens are continuously reported to appear, so that the porcine diarrhea pathogens are complicated, and the identification and the detection are difficult.

Porcine delta coronaviruses (PDCoV), also known as Porcine delta coronaviruses, belong to the genus Coder coronavirus, which is a single-stranded plus-strand RNA virus with an envelope. The virus can infect pigs of different ages of days, the infection rate is about 20-30%, and the virus mainly causes clinical symptoms such as vomit, diarrhea, dehydration and the like. The suckling piglet has stronger susceptibility to the piglet, more serious clinical symptoms and the fatality rate after infection is as high as 50 to 100 percent.

Mammalian Reoviruses (MRV) are widely spread and infect almost all mammals and are generally of low pathogenicity. Including human, mink, pig, cow, bat, dog, etc., and has zoonosis. The porcine reovirus is detected from a suspected diarrhea-in-winter piglet belly stool sample in 2005 (Zeng Zhi Yong Wan zhu, Xushiwen, Song Zhenghui, Yinhua Ping, Wanxin, Wan Xiaoyu, separation and identification of the porcine reovirus in a piglet diarrhea stool sample, and veterinary science of animal husbandry 2007,38(6): 574-580), which indicates that the porcine reovirus is possibly related to the disease and has certain pathogenicity to the pig. Therefore, the rapid detection of the pathogen can provide scientific basis for the early prevention and control of the disease.

Porcine Kobuvirus (PKV) is a member of the genus chikungunya of the picornaviridae family, and this virus mainly damages piglets up to 3 weeks of age and can cause gastroenteritis, severe diarrhea, and even death in piglets. The positive detection rate of PKV in China is gradually increased year by year from 2009. The phenomenon of mixed infection of PKV with other viruses is also common and aggravates the disease, and the infection is not limited to the intestinal tract, resulting in so-called "poor vaccine use".

The current methods for detecting the pathogeny of the pig diarrhea disease comprise virus separation and identification, serological methods, molecular biological methods and the like, symptoms caused by the pig diarrhea disease are all characterized by diarrhea, and differential diagnosis is difficult only according to clinical symptoms and epidemiology, so that laboratory detection technology is required. The common diagnostic techniques such as virus separation and serology have the problems of complex operation, high difficulty and the like, and a diagnosis conclusion is difficult to obtain at the early stage of the disease; the serology method has lower accuracy and the single PCR method has low detection efficiency. Therefore, establishing a sensitive, rapid and efficient method for simultaneously detecting three pathogens of the porcine delta coronavirus, the mammalian reovirus and the porcine kobuvirus aiming at the porcine diarrhea pathogen is the key for rapidly identifying and early preventing and controlling the porcine diarrhea pathogen.

Disclosure of Invention

The invention aims to provide a nucleic acid composition, a kit and a detection method for simultaneously detecting porcine delta coronavirus, reovirus and porcine kobuvirus.

The invention provides a nucleic acid composition for simultaneously detecting porcine delta coronavirus, reovirus and porcine kobuvirus, which comprises a forward primer PDCoV-F, a reverse primer PDCoV-R and a probe PDCoV-P aiming at the porcine delta coronavirus, a forward primer PKV-F, a reverse primer PKV-R and a probe PKV-P aiming at the reovirus, and a forward primer MRV-R, a reverse primer MRV-R and a probe MRV-P aiming at the porcine kobuvirus;

the sequence of the forward primer aiming at the porcine delta coronavirus is shown as SEQ ID NO. 1;

the reverse primer sequence aiming at the porcine delta coronavirus is shown as SEQ ID NO. 2;

the probe sequence aiming at the porcine delta coronavirus is shown as SEQ ID NO. 3;

the forward primer sequence aiming at the reovirus is shown as SEQ ID NO. 4;

the reverse primer sequence aiming at the reovirus is shown as SEQ ID NO. 5;

the probe sequence aiming at the reovirus is shown as SEQ ID NO. 6;

the sequence of the forward primer aiming at the porcine kobuvirus is shown as SEQ ID NO. 7;

the reverse primer sequence aiming at the porcine kobuvirus is shown as SEQ ID NO. 8;

the probe sequence for the porcine kobuvirus is shown as SEQ ID NO. 9.

Preferably, the 5 'end of the probe sequence for the porcine delta coronavirus, the 5' end of the probe sequence for the reovirus and the 3 'end of the probe sequence for the porcine kobuvirus are modified by a fluorescent reporter group, and the 3' ends of the probe sequences are modified by a fluorescent quencher group.

Preferably, the 5 ' end of the probe sequence for porcine delta coronavirus, the 5 ' end of the probe sequence for reovirus and the 5 ' end of the probe sequence for porcine kobuvirus are respectively modified by a reporter group with different fluorescence wavelengths.

The invention also provides a kit for simultaneously detecting the porcine delta coronavirus, the reovirus and the porcine kobuvirus, which comprises the nucleic acid composition in the technical scheme.

The invention also provides application of the nucleic acid composition or the kit in the technical scheme in preparation of products for simultaneously detecting the porcine delta coronavirus, the reovirus and the porcine kobuvirus.

The invention also provides a method for simultaneously detecting the porcine delta coronavirus, the reovirus and the porcine kobuvirus, which comprises the following steps of:

extracting total RNA of a detection sample, and performing reverse transcription to obtain cDNA;

and (3) carrying out fluorescence PCR reaction by using the cDNA template and the nucleic acid composition according to the technical scheme, collecting a fluorescence signal and a Ct value, and judging the product to be negative when the Ct value is greater than 36.

Preferably, the reaction system of the fluorescent PCR reaction comprises, in 25 μ L: 12.5. mu.L of 2 XPromix Ex Taq, 0.5. mu.L to 1.4. mu.L of 10. mu. mol/L of PDCoV-F, PDCoV-R, PKV-F, PKV-R, MRV-F and MRV-R, 0.3. mu.L to 0.8. mu.L of 10. mu. mol/L of LPDCoV-P, PKV-P and MRV-PcDNA, 4. mu.L of cDNA and the balance of water.

Preferably, the procedure of the fluorescent PCR reaction is: first stage pre-denaturation at 95 ℃ for 3 min; the second stage is denaturation at 94 ℃ for 10s and extension at 55.8 ℃ for 30s for 40 cycles, and the fluorescence signal is collected during the extension stage.

The invention provides a nucleic acid composition for simultaneously detecting porcine delta coronavirus, reovirus and porcine kobuvirus, which comprises a forward primer PDCoV-F, a reverse primer PDCoV-R and a probe PDCoV-P aiming at the porcine delta coronavirus, a forward primer PKV-F, a reverse primer PKV-R and a probe PKV-P aiming at the reovirus, and a forward primer MRV-R, a reverse primer MRV-R and a probe MRV-P aiming at the porcine kobuvirus. According to the conserved gene sequences of the porcine delta coronavirus, the reovirus and the porcine kobuvirus registered in GenBank and the difference among different strains, the conserved nucleotide sequence is selected as an amplification region, and three pairs of specific primers and probes are designed. The three pairs of specific primers and probes can simultaneously, quickly, conveniently and efficiently specifically identify and detect the porcine delta coronavirus, the reovirus and the porcine kobuvirus.

The invention also establishes a triple fluorescence quantitative PCR detection method of the delta coronavirus, the porcine kobuvirus and the mammalian reovirus, has the characteristics of rapidness, convenience, high efficiency and high sensitivity, can be widely applied to epidemiological investigation, laboratory identification and detection and related scientific research of the porcine diarrhea disease, and has high practical application value.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments will be briefly described below.

FIG. 1 shows the amplification result of detecting porcine delta coronavirus standard by the triple fluorescence quantitative PCR detection method of the present invention;

FIG. 2 is the amplification result of the porcine kobuvirus standard detected by the triple fluorescence quantitative PCR detection method of the present invention;

FIG. 3 is the amplification result of a mammalian reovirus standard substance detected by the triple fluorescence quantitative PCR detection method of the present invention;

FIG. 4 is a standard curve for detecting porcine delta coronavirus using the triple fluorescent quantitative PCR detection method of the present invention;

FIG. 5 is a standard curve for detecting porcine kobuvirus using the triple fluorescent quantitative PCR detection method of the present invention;

FIG. 6 is a standard curve for detecting mammalian reovirus using the triple fluorescent quantitative PCR detection method of the present invention;

FIG. 7 shows the results of specific tests of the triple fluorescent quantitative PCR detection method for porcine delta coronavirus, porcine kobuvirus and mammalian reovirus;

FIG. 8 shows the results of the sensitivity test for detecting the porcine delta coronavirus standard by the triple fluorescence quantitative PCR detection method of the present invention;

FIG. 9 shows the results of the sensitivity test for detecting porcine kobuvirus standard using the triple fluorescent quantitative PCR detection method of the present invention;

FIG. 10 shows the results of a sensitivity test using the triple fluorescent quantitative PCR detection method of the present invention for mammalian reovirus standards;

FIG. 11 shows the result of the repetitive tests for detecting the porcine delta coronavirus standard by the triple fluorescence quantitative PCR detection method of the present invention;

FIG. 12 is a result of a repetitive test for detecting porcine kobuvirus standards using the triple fluorescence quantitative PCR detection method of the present invention;

FIG. 13 is a result of a reproducibility test of a mammalian reovirus standard using the triple fluorescent quantitative PCR detection method of the present invention.

Detailed Description

the forward primer sequence for the porcine delta coronavirus is shown as SEQ ID NO.1, and specifically comprises the following steps: 5'-GTTCTGATATACCAGGGTG-3', respectively;

the reverse primer sequence aiming at the porcine delta coronavirus is shown as SEQ ID NO.2, and specifically comprises the following steps: 5'-ATATACTTATACAGGCGAGC-3', respectively;

the probe sequence for the porcine delta coronavirus is shown as SEQ ID NO.3, and specifically comprises the following steps: 5'-GCTTCAAACGCTGGACTTCACACCATAACC-3', respectively;

the forward primer sequence for reovirus is shown as SEQ ID NO.4, and specifically comprises the following steps: 5'-CCAAACTCCTACCCGACA-3', respectively;

the reverse primer sequence for reovirus is shown as SEQ ID NO.5, and specifically comprises the following steps: 5'-TCTACTCATGGGCGAGCC-3';

the probe sequence for reovirus is shown as SEQ ID NO.6, and specifically comprises the following steps: 5'-CGTGTCCGCGTGCTGAGTAATGGGAT-3', respectively;

the forward primer sequence for the porcine kobuvirus is shown as SEQ ID NO.7, and specifically comprises the following steps: 5'-TGCAGAATCCGGAGTTAAAGT-3', respectively;

the reverse primer sequence aiming at the porcine kobuvirus is shown as SEQ ID NO.8, and specifically comprises the following steps: 5'-TCGAATAATGACGGAGGGTT-3', respectively;

the probe sequence for the porcine kobuvirus is shown as SEQ ID NO.9, and specifically comprises the following steps: 5 '-GTAGTGAGTGGGAGAARTATGGAGCGGGDA-3'. D in the sequence shown in SEQ ID NO.9 is degenerate basic group, and can be G or A or T.

In the invention, the 5 'ends of the probe sequence aiming at the porcine delta coronavirus, the probe sequence aiming at the reovirus and the probe sequence aiming at the porcine kobuvirus are preferably modified by a fluorescence reporter group, and the 3' ends are preferably modified by a fluorescence quenching group; the 5 ' end of the probe sequence for the porcine delta coronavirus, the 5 ' end of the probe sequence for the reovirus and the 5 ' end of the probe sequence for the porcine kobuvirus are further preferably modified by reporter groups with different fluorescence wavelengths respectively. The 5 ' end of the probe sequence aiming at the porcine delta coronavirus is preferably modified by a red fluorophore, the 5 ' end of the probe sequence aiming at the reovirus is preferably modified by a green fluorophore, and the 5 ' end of the probe sequence aiming at the porcine kobuvirus is preferably modified by a purple fluorophore.

The reovirus of the present invention is preferably a mammalian reovirus.

According to the conserved gene sequences (porcine delta coronavirus accession number KY065120.1, reovirus accession number KC462151.1 and porcine kobuvirus accession number MT125685) of the porcine delta coronavirus, the reovirus and the porcine kobuvirus which are logged in GenBank, the difference among different strains is considered, the conserved nucleotide sequence is selected as an amplification region, and three pairs of specific primers and probes are designed. The three pairs of specific primers and probes can specifically identify the porcine delta coronavirus, the reovirus and the porcine kobuvirus, and can simultaneously, rapidly, conveniently and efficiently detect the three pathogens. Sensitivity test results of triple fluorescence quantitative PCR detection method of porcine delta coronavirus, porcine kobuvirus and mammal reovirus show that the primer is applied to the delta coronavirus and the porcine kobuvirusThe PCR detection of virus and mammal reovirus pathogen has the lowest detection limit of 2.7X 10¹copies·μL^-1、1.22×10¹copies·μL^-1And 1.26X 10²copies·μL^-1And has high sensitivity.

The invention does not strictly require the other components contained in the kit, and the reagents for detecting the porcine delta coronavirus, the reovirus and the porcine kobuvirus belong to the protection scope of the invention, such as enzyme mixture, PCR reaction reagent and the like. The enzyme mixture of the invention preferably comprises a DNA polymerase and a reverse transcriptase; the PCR reaction reagent comprises PCR buffer solution, dNTPs and cations.

The total RNA is preferably extracted from feces and/or intestinal tracts of a subject to be detected, and the method for extracting the total RNA is not particularly limited, and the total RNA is preferably extracted by using a kit. The specific source of the nucleic acid extraction kit is not strictly required, and the nucleic acid extraction kit can be purchased conventionally, such as a DNA/RNA extraction kit produced by Rayji ocean Biotechnology GmbH.

Before reverse transcription of the extracted total RNA, the method preferably further comprises purification, and more preferably comprises removing DNA in the total RNA; the removal ofThe reaction system of DNA in total RNA is preferably 10. mu.L, and includes: 5 XgDNA Eraser Buffer 2.0. mu.L, gDNA Eraser 1.0. mu.L, total RNA 5.0. mu.L, RNase Free dH₂O is complemented to 10 mu L; the reaction process is as follows: enzymolysis at 42 deg.C for 2 min. The invention does not strictly require the source of the reagent used in the reaction system, and the reagent can be purchased conventionally. In the specific implementation process of the invention, the used reagent is purchased from precious bioengineering (Dalian) Co.

The present invention carries out reverse transcription on purified total RNA to obtain cDNA, wherein the reaction system used for the reverse transcription is 20 μ L, and preferably comprises:

buffer 4.0. mu.L, PrimeScriptRT Enzyme Mix 1.0. mu.L, RT PrimeMix Total RNA 1.0. mu.L, Total RNA depleted of DNA 10.0. mu.L, RNase Free ddH₂O is complemented to 20.0 mu L; the reaction process preferably comprises: extension at 37 ℃ for 15min, reverse transcription at 85 ℃ for 5sec, and storage at 4 ℃. The invention does not strictly require the source of the reagent used in the reaction system, and the reagent can be purchased conventionally. In the specific implementation process of the invention, the used reagent is purchased from precious bioengineering (Dalian) Co.

After obtaining cDNA, the invention preferably uses the cDNA as a template, utilizes the nucleic acid composition of the technical scheme to prepare a reaction system for carrying out fluorescence PCR reaction, collects a fluorescence signal and a Ct value, and judges as negative when the Ct value is more than 36. The reaction system of the present invention preferably comprises, in 25. mu.L: 12.5. mu.L of 2 XPremix Ex Taq, 0.5. mu.L-1.4. mu.L of 10. mu.mol/LPDCoV-F, PDCoV-R, PKV-F, PKV-R, MRV-F and MRV-R respectively, 0.3. mu.L-0.8. mu.L of 10. mu.mol/L of PDCoV-P, PKV-P and MRV-P respectively, 4. mu.L of cDNA and the balance of water. The volumes of the PDCoV-F, PDCoV-R, PKV-F, PKV-R, MRV-F and the MRV-R are preferably 0.8-1.2 mu L respectively, and more preferably 1.2 mu L respectively; the volumes of the PDCoV-P, PKV-P and the MRV-P are preferably 0.4 to 0.6. mu.L, and more preferably 0.45. mu.L, respectively. In the process of preparing the reaction system, 2 XPremix Ex Taq, PDCoV-F, PDCoV-R, PKV-F, PKV-R, MRV-F, MRV-R, PDCoV-P, PKV-P, MRV-P and water are preferably mixed to obtain a premixed solution, and then the premixed solution is mixed with cDNA to obtain the PCR reaction system. The invention does not strictly require the source of the reagent used in the reaction system, and the reagent can be purchased conventionally. In the specific implementation process of the invention, the used reagent is purchased from the company Daojie bioengineering (Dalian) Limited for production. In the specific implementation process, the volume of the reaction system is adjusted according to the needs, and the volume ratio of the premixed solution to the cDNA is only required to be ensured to be 21: 4, the product is obtained.

The invention has no strict requirement on the concentration of the cDNA and ensures the OD of the cDNA₂₆₀/OD₂₈₀The ratio is 1.8-2.0. In the specific implementation process of the invention, 2 XPremix Ex Taq, PDCoV-F, PDCoV-R, PKV-F, PKV-R, MRV-F, MRV-R, PDCoV-P, PKV-P and MRV-P are mixed according to the concentration and the volume, then are mixed with cDNA, are centrifuged at 500r/min to 1000r/min for 30s, and then are subjected to fluorescence PCR reaction. The fluorescence PCR reaction of the invention is preferably carried out in a Gentier 96E full-automatic medical PCR instrument (Saan Tianlong science and technology Co., Ltd.).

The procedure of the fluorescent PCR reaction of the present invention is preferably:

the first stage is as follows: pre-denaturation at 95 ℃ for 3 min;

and a second stage: denaturation at 94 ℃ for 10s and annealing at 55.8 ℃ for 30s for 40 cycles.

After the fluorescent PCR reaction is finished, the invention collects the extension stage fluorescent signal and Ct value, and judges whether the PCR reaction contains PDCoV, MRV and PKV according to the Ct value. And when the Ct value is more than 36, the test sample is judged to be negative, namely the test sample does not contain PDCoV, MRV and PKV.

As long as any one of the 3 viruses including the porcine delta coronavirus, the reovirus and the porcine kobuvirus is contained in the sample to be detected, the method for simultaneously detecting the porcine delta coronavirus, the reovirus and the porcine kobuvirus provided by the invention can be used for detecting, and the sample which only contains the 3 viruses cannot be detected. The method of the invention improves the sensitivity of detection and simultaneously reduces the workload of conventional PCR, thereby improving the working efficiency. Provides an effective technical means for scientific research, laboratory detection and clinical prevention and control of the porcine diarrhea.

In order to further illustrate the present invention, the following detailed description of the technical solutions provided by the present invention is made with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.

Example 1

1. Reaction system and reaction program of triple fluorescence quantitative PCR detection method

(1) Primer and probe design

Genomic sequences of PDCoV, PKV and MRV were downloaded from NCBI, respectively, and after sequence analysis by MEGA7(version7.0.21), primers and probes were designed in conserved regions using primer set (version 7.1.0(44)) of primer design software NDASTAR laser, and synthesized by sumau, inc.

TABLE 1 nucleotide sequences of primers and probes

(2) Preparation of plasmid Standard

Based on the information that genomic sequences of PDCoV, PKV and MRV have been analyzed and the design positions of primers and probes in Table 1, the respective plasmids pPDCoV-M, pPKV, and pMRV, were synthesized by CinzoZhi Biotech, Suzhou. Calculating the copy number of the plasmid, and using ddH to obtain three plasmid standards₂The gradient dilution of O was carried out to obtain seven concentrations (2.7X 10 each)⁷copies·μL^-1To 2.7X 10¹copies·μL^-1、1.22×10⁷copies·μL^-1To 1.22X 10¹copies·μL^-1And 1.26X 10⁷copies·μL^-1To 1.26X 10¹copies·μL^-1) The recombinant plasmid standard of (4) is stored at-20 ℃ for later use.

(3) Triple fluorescent quantitative PCR detection method reaction annealing temperature test

The prepared plasmid is used as a template to carry out a fluorescent quantitative PCR test, and the reaction annealing temperature is searched, wherein the concentrations of the three plasmids are respectively 2.7 multiplied by 10⁵copies·μL^-1、1.22×10⁵copies·μL^-1And 1.26X 10⁵copies·μL^-1。

2 XPremix Ex Taq (Probe qPCR) used in this reaction system was produced by Takara Bio Inc.

The preparation of the reaction solution is carried out in a reaction mixture preparation area by applying a fluorescent PCR method.

The reaction system preparation is recommended to be carried out according to n +1 reactions. The sample detection reaction system is 25.0 mu L: 2 XPromix Ex Taq (Probe qPCR) 12.5. mu.L, PDCoV-F (10. mu. mol/L), PDCoV-R (10. mu. mol/L), PKV-F (10. mu. mol/L), PKV-R (10. mu. mol/L), MRV-F (10. mu. mol/L) and MRV-R (10. mu. mol/L) are respectively 1.2. mu.L, PDCoV-P (10. mu. mol/L), PKV-P (10. mu. mol/L) and MRV-P (10. mu. mol/L) are respectively 0.45. mu.L.

And (3) fully and uniformly mixing the prepared fluorescent PCR reaction solution, subpackaging the mixture into transparent fluorescent PCR tubes according to 21.0 mu L per tube, marking according to the sample adding sequence, and transferring the tubes to a nucleic acid extraction area for sample adding.

4.0 microliter of plasmid is added into each fluorescent PCR reaction tube, and the mixture is centrifuged for 30s at 500 r/min-1000 r/min. The sample was transferred to a detection zone, and annealing temperatures were set to 54.0 ℃, 55.8 ℃, 58.0 ℃, 60.5 ℃ and 62.0 ℃, respectively, and a specific reaction procedure was described below using 55.8 ℃ as an example.

Reaction procedure for sample detection: pre-denaturation at 95 ℃ for 3min, followed by a two-step procedure, denaturation at 94 ℃ for 10s, extension at 55.8 ℃ for 30s, 40 cycles, and fluorescence signal collection during the extension phase. The fluorescence channel of the PCR instrument is set as follows: channel 1: FAM, channel 2: CY5, channel 3: and ROX. The reaction was performed in a Gentier 96E full-automatic medical PCR apparatus (Saan Tianlong science and technology Co., Ltd.), and the detection results corresponding to different annealing temperatures are shown in Table 2 below.

TABLE 2 detection results corresponding to different annealing temperatures

As can be seen from Table 2, the Ct value of MRV changes significantly with the gradual increase of the annealing temperature, and the optimal annealing temperature of 55.8 ℃ is determined by considering the total detection effect of detecting PDCoV, PKV and MRV simultaneously.

(4) Test of reaction system of triple fluorescent quantitative PCR detection method

The prepared plasmid was used as a template to search for an optimal reaction system.

The reaction system preparation is recommended to be carried out according to n +1 reactions.

The sample detection reaction system is 25.0 mu L: 2 XPramix Ex Taq (Probe qPCR)12.5 muL, PDCoV-F (10 mumol/L), PDCoV-R (10 mumol/L), PKV-F (10 mumol/L), PKV-R (10 mumol/L), MRV-F (10 mumol/L) and MRV-R (10 mumol/L) are respectively 0.5 muL-1.4 muL in volume range, PDCoV-P (10 mumol/L), PKV-P (10 mumol/L) and MRV-P (10 mumol/L) are respectively 0.3 muL-0.8 muL in volume range. ddH₂O was made up to 21.0. mu.L (specific use cases of primers and probes are shown in Table 3 below).

4.0 mul of plasmid is added into each fluorescent PCR reaction tube, and the mixture is centrifuged for 30s at 500 r/min-1000 r/min. Transfer to a detection zone.

Reaction procedure: pre-denaturation at 95 ℃ for 3min, followed by a two-step procedure, denaturation at 94 ℃ for 10s, extension at 55.8 ℃ for 30s, 40 cycles, and fluorescence signal collection during the extension phase. The fluorescence channel of the PCR instrument is set as follows: channel 1: FAM, channel 2: CY5, channel 3: and ROX. The reaction was performed in a Gentier 96E fully automated medical PCR apparatus (Saan Tianlong science and technology Co., Ltd.), each set was designed in duplicate, and the average detection results corresponding to different reaction systems are shown in Table 3.

TABLE 3 detection results corresponding to different reaction systems

According to the results of comprehensive analysis of Ct values in Table 3, it was confirmed that the reaction results were optimal in the reaction systems in which PDCoV-F (10. mu. mol/L), PDCoV-R (10. mu. mol/L), PKV-F (10. mu. mol/L), PKV-R (10. mu. mol/L), MRV-F (10. mu. mol/L) and MRV-R (10. mu. mol/L) were 1.2. mu.L and PDCoV-P (10. mu. mol/L), PKV-P (10. mu. mol/L) and MRV-P (10. mu. mol/L) were 0.45. mu.L, respectively.

(5) Establishment of a Standard Curve

Six concentrations of standard plasmids (plasmid standards for PDCoV, PKV, and MRV, respectively, at 2.7X 10 concentrations) for each pathogen were determined using a triple fluorescent quantitative PCR reaction⁷copies·μL^-1To 2.7X 10²copies·μL^-1、1.22×10⁷copies·μL^-1To 1.22X 10¹copies·μL^-1And 1.26X 10⁷copies·μL^-1To 1.26X 10²copies·μL^-1) Amplification is performed).

2 XPremix Ex Taq (Probe qPCR) used in the reaction system is produced by Takara Bio-Rad, the preparation of reaction solution is carried out in a reaction mixture preparation area, the reaction system is the same as the step (4), wherein PDCoV-F (10 mu mol/L), PDCoV-R (10 mu mol/L), PKV-F (10 mu mol/L), PKV-R (10 mu mol/L), MRV-F (10 mu mol/L), MRV-R (10 mu mol/L) are respectively 1.2 mu L, PDCoV-P (10 mu mol/L), PKV-P (10 mu mol/L), MRV-F (10 mu mol/L) and MRV-P (10 mu mol/L) are respectively 0.45 mu L, the amplification is carried out according to the procedure of the step (4), the annealing temperature is 55.8 ℃, and the results are shown in figures 1-6 and Table 4.

TABLE 4 statistics of standard curve amplification efficiency results

As can be seen from FIGS. 1 to 6 and Table 4, the triple fluorescence PCR method has been successfully established.

(6) Specificity test

The triple fluorescence quantitative PCR method is applied to carry out specificity test, nucleic acids of positive samples such as PRRSV, CSFV, PCV2, PRV, PEDV, TGEV, RV, PDCoV, PKV, MRV and the like are taken as strains to carry out specificity detection, and the specifically used strains are shown in the following table 5.

TABLE 5 Strain species and sources for detection

Note: livestock epidemic prevention and control team laboratory address of animal husbandry veterinary research institute of Tianjin city agricultural science institute: livestock veterinary institute of academy of agricultural sciences at 17 km of Tianjin city Jingjing highway in the west Qing region of Tianjin city.

The fluorescent quantitative PCR detection was performed using the reaction system and reaction program of step (4), and the detection results are shown in FIG. 7 and Table 6.

TABLE 6 results of specificity test of triple fluorescent PCR

As can be seen from FIG. 7 and Table 6, the detection method of the present invention can be used to detect PRRSV, CSFV, PCV2, PRV, PEDV + TGEV + RV, PDCoV, PKV, MRV, and only PDCoV, PKV, and MRV, but also other pathogens, nucleic acid detection results are negative. The specificity of the triple fluorescence quantitative PCR method is good.

(7) Sensitivity test

Seven concentrations (2.7X 10) of three plasmid standards of PDCoV, PKV and MRV⁷copies·μL^-1To 2.7X 10¹copies·μL^-1、1.22×10⁷copies·μL^-1To 1.22X 10¹copies·μL^-1And 1.26X 10⁷copies·μL^-1To 1.26X 10¹copies·μL^-110-fold dilutions each), seven concentrations of standard plasmid per pathogen were amplified using a triple fluorescent quantitative PCR reaction. And (3) carrying out three repetitions at the same time, carrying out a sensitivity test by using the triple fluorescence quantitative PCR method, and detecting a reaction system and a procedure in the same step (4), wherein the test result is shown in FIGS. 11-13.

As can be seen from the descriptions in FIGS. 11 to 13, the minimum detection limits of PDCoV, PKV and MRV detection by the triple fluorescence quantitative PCR method of the present invention are 2.7X 10¹copies·μL^-1、1.22×10¹copies·μL^-1And 1.26X 10²copies·μL^-1Thus, the detection primers and the detection method of the present invention have high sensitivity.

(8) Repeatability test

Plasmids using PDCoV, PKV and MRV (concentrations of 2.7X 10, respectively)⁷copies·μL^-1To 2.7X 10²copies·μL^-1、1.22×10⁷copies·μL^-1To 1.22X 10²copies·μL^-1And 1.26X 10⁷copies·μL^-1To 1.26X 10²copies·μL^-1) As a template, the triple fluorescence quantitative PCR method of the invention is applied to carry out in-batch (three repetitions) and in-batch (three repetitions) tests respectively, the detection results are counted, the repeatability of the method is analyzed according to the coefficient of variation (CV%), the reaction system and procedure are detected in the same step (4), and the experimental results are shown in FIGS. 11-13 and tables 7-9.

TABLE 7 repeatability test results of PDCoV triple fluorescence PCR

TABLE 8 repeatability test results of PKV triple fluorescence PCR

TABLE 9 results of the reproducibility test of MRV triple fluorescence PCR

Note: CV% ((standard deviation SD/Mean) × 100%).

As can be seen from the contents recorded in FIGS. 11 to 13 and tables 7 to 9, the variation coefficient (CV%) of the in-batch and in-batch tests is between 0.004 and 0.02, and the triple fluorescence quantitative PCR method of the present invention has good stability.

The nucleic acid composition for simultaneously detecting the porcine delta coronavirus, the reovirus and the porcine kobuvirus, provided by the invention, can specifically identify the porcine delta coronavirus, the reovirus and the porcine kobuvirus, and can simultaneously, rapidly, conveniently and efficiently detect the three pathogens; the triple fluorescence quantitative PCR detection method established by using the nucleic acid composition has the characteristics of rapidness, convenience, high efficiency and high sensitivity, can be widely applied to epidemiological investigation, laboratory identification and detection and related scientific research of the porcine diarrhea disease, and has high practical application value.

Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.

Sequence listing

<110> Tianjin City academy of agricultural sciences

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Claims

1. A nucleic acid composition for simultaneously detecting porcine delta coronavirus, reovirus and porcine kobuvirus is characterized by comprising a forward primer PDCoV-F, a reverse primer PDCoV-R and a probe PDCoV-P aiming at the porcine delta coronavirus, a forward primer PKV-F, a reverse primer PKV-R and a probe PKV-P aiming at the reovirus, and a forward primer MRV-R, a reverse primer MRV-R and a probe MRV-P aiming at the porcine kobuvirus;

the forward primer sequence aiming at the reovirus is shown as SEQ ID NO. 4;

the reverse primer sequence aiming at the reovirus is shown as SEQ ID NO. 5;

the probe sequence aiming at the reovirus is shown as SEQ ID NO. 6;

the probe sequence for the porcine kobuvirus is shown as SEQ ID NO. 9.

2. The nucleic acid composition of claim 1, wherein the probe sequence for porcine delta coronavirus, the probe sequence for reovirus and the probe sequence for porcine kobuvirus are modified at the 5 'end with a fluorescent reporter group and at the 3' end with a fluorescent quencher group.

3. The nucleic acid composition of claim 2, wherein the 5 ' end of the probe sequence for porcine delta coronavirus, the 5 ' end of the probe sequence for reovirus, and the 5 ' end of the probe sequence for porcine kobuvirus are each modified with a reporter group of different fluorescent wavelengths.

4. A kit for simultaneously detecting porcine delta coronavirus, reovirus and porcine kobuvirus, comprising the nucleic acid composition of any one of claims 1-3.

5. Use of the nucleic acid composition of any one of claims 1 to 3 or the kit of claim 4 for the preparation of a product for simultaneous detection of porcine delta coronavirus, reovirus and porcine kobuvirus.

6. A method for simultaneously detecting porcine delta coronavirus, reovirus and porcine kobuvirus is characterized by comprising the following steps of:

performing a fluorescence PCR reaction using the nucleic acid composition according to any one of claims 1 to 3 using the cDNA template, and collecting a fluorescence signal and a Ct value, and determining negative when the Ct value is greater than 36.

7. The method of claim 6, wherein the reaction system of the fluorescent PCR reaction comprises, in 25 μ L: 12.5. mu.L of 2 XPimmix Ex Taq, 0.5. mu.L-1.4. mu.L of 10. mu.mol/L PDCoV-F, PDCoV-R, PKV-F, PKV-R, MRV-F and MRV-R respectively, 0.3. mu.L-0.8. mu.L of 10. mu.mol/L PDCoV-P, PKV-P and MRV-PcDNA respectively, 4. mu.L cDNA and the balance of water.

8. The method of claim 7, wherein the fluorescent PCR reaction is programmed by: first stage pre-denaturation at 95 ℃ for 3 min; the second stage is denaturation at 94 ℃ for 10s and extension at 55.8 ℃ for 30s for 40 cycles, and the fluorescence signal is collected during the extension stage.