CN106987657A - For differentiating that the primer of bovine viral diarrhea virus and bovine rota is combined and its applied - Google Patents

Abstract

The invention discloses a combination of primers for distinguishing bovine viral diarrhea virus and bovine rotavirus and its application. The primer combination provided by the present invention is composed of a single-stranded DNA molecule shown in sequence 1-8 of the sequence table, a fluorescent group A is connected to the 5' end of the single-stranded DNA molecule shown in sequence 3, and a fluorescent group A is connected to the 5' end of the single-stranded DNA molecule shown in sequence 7. A fluorescent group B is attached to the 5' end of the single-stranded DNA molecule. The present invention introduces a fluorescent group into the LAMP method for the first time, and establishes a dual fluorescent RT-LAMP method for identifying BVDV and BRV. The two viruses can be differentially diagnosed simultaneously by observing the color of the amplified product. The dual RT-LAMP method established by the present invention has good specificity, can efficiently amplify the target gene, has no amplification for other pathogenic nucleic acids, has good sensitivity, and can detect at least 100 mixed template copies/reaction, which is a simple and convenient method. , a rapid and low-cost diagnostic method suitable for large-scale epidemiological investigations.

Description

Primer combination and application for differentiating bovine viral diarrhea virus and bovine rotavirus

technical field

The invention relates to a combination of primers for distinguishing bovine viral diarrhea virus and bovine rotavirus and its application.

Background technique

Bovine viral diarrhea virus (BVDV) and bovine rotavirus (Bovine Rotavirus, BRV) are two common bovine diarrhea viruses with similar symptoms and difficult to distinguish. Clinically, BVDV often presents with fever, diarrhea, mucosal injury, and toe injury, but most BVDV-infected cattle do not show clinical symptoms due to persistent infection. disease with a 100% mortality rate. Persistently infected cattle can transmit the virus horizontally through blood, excrement and other channels, and can also transmit the virus vertically through the reproductive tract. They carry the virus for life and continue to shed the virus, becoming an important source of infection and a potential hazard to the cattle industry. Bovine rotavirus infection of calves aged 1-7 days can cause digestive tract dysfunction in calves, which is characterized clinically by vomiting, diarrhea, dehydration and acid-base balance. Both viruses will cause serious economic losses after infection. Therefore, it is urgent to establish a rapid detection technology for bovine viral diarrhea and bovine rotavirus to provide technical support for the prevention and control of BVDV and BRV in my country.

Currently commonly used diagnostic methods for BVDV and BRV mainly include: pathogen isolation, serological methods and molecular biological methods (RT-PCR and fluorescent RT-PCR). Loop-mediated in vitro isothermal amplification detection technology (Loop-mediated isothermal amplification, LAMP) is a new nucleic acid detection technology developed on the basis of PCR method, which breaks through the technical difficulties of constant temperature amplification. It has good specificity and has been applied in the detection of various diseases. Multiple LAMP is a highly efficient form of LAMP. One LAMP reaction can detect and identify multiple pathogens at the same time. However, the current multiple LAMP methods in China have certain limitations, and it is impossible to determine which pathogen caused the positive result, and cannot achieve a true differential diagnosis.

Contents of the invention

The purpose of the present invention is to provide a combination of primers for distinguishing bovine viral diarrhea virus and bovine rotavirus and its application.

The present invention first protects the primer combination, which consists of primer group I and primer group II;

The primer set I is composed of primer BVDV-F3, primer BVDV-B3, primer BVDV-FIP and primer BVDV-BIP;

The primer BVDV-F3 is as follows (a1) or (a2):

(a1) a single-stranded DNA molecule shown in Sequence 1 of the sequence listing;

(a2) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to Sequence 1 and has the same function as Sequence 1;

The primer BVDV-B3 is as follows (a3) or (a4):

(a3) a single-stranded DNA molecule shown in sequence 2 of the sequence listing;

(a4) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to Sequence 2 and has the same function as Sequence 2;

The primer BVDV-FIP is as follows (a5) or (a6):

(a5) a single-stranded DNA molecule shown in sequence 3 of the sequence listing;

(a6) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to Sequence 3 and has the same function as Sequence 3;

The primer BVDV-BIP is as follows (a7) or (a8):

(a7) a single-stranded DNA molecule shown in sequence 4 of the sequence listing;

(a8) a DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 4 and has the same function as sequence 4;

The primer group II is composed of primer BRV-F3, primer BRV-B3, primer BRV-FIP and primer BRV-BIP;

The primer BRV-F3 is as follows (b1) or (b2):

(b1) a single-stranded DNA molecule shown in sequence 5 of the sequence listing;

(b2) a DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 5 and has the same function as sequence 5;

The primer BRV-B3 is as follows (b3) or (b4):

(b3) a single-stranded DNA molecule shown in sequence 6 of the sequence listing;

(b4) a DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 6 and has the same function as sequence 6;

The primer BRV-FIP is as follows (b5) or (b6):

(b5) a single-stranded DNA molecule shown in sequence 7 of the sequence listing;

(b6) a DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 7 and has the same function as sequence 7;

The primer BRV-BIP is as follows (b7) or (b8):

(b7) a single-stranded DNA molecule shown in sequence 8 of the sequence listing;

(b8) A DNA molecule having the same function as that of Sequence 8 by substituting and/or deleting and/or adding one or several nucleotides to Sequence 8.

The 5' end of the primer BVDV-FIP is connected with a fluorescent group A.

The 5' end of the primer BRV-FIP is connected with a fluorescent group B.

The fluorescent group A can be FITC.

The fluorescent group B can be CY5.5.

The use of the primer combination is any one of the following (c1) to (c6):

(c1) distinguishing between bovine viral diarrhea virus and bovine rotavirus;

(c2) preparing a kit for distinguishing bovine viral diarrhea virus and bovine rotavirus;

(b3) Detect whether the pathogenic microorganism to be tested is bovine virus diarrhea virus or bovine rotavirus;

(c4) preparing a test kit for detecting whether the pathogenic microorganism to be tested is bovine virus diarrhea virus or bovine rotavirus;

(c5) Detect whether the sample to be tested contains bovine virus diarrhea virus and/or bovine rotavirus;

(c6) preparing a kit for detecting whether the sample to be tested contains bovine viral diarrhea virus and/or bovine rotavirus.

The present invention also protects the application of the primer combination, which is any one of the following (c1) to (c6):

(c1) distinguishing between bovine viral diarrhea virus and bovine rotavirus;

(c2) preparing a kit for distinguishing bovine viral diarrhea virus and bovine rotavirus;

(b3) Detect whether the pathogenic microorganism to be tested is bovine virus diarrhea virus or bovine rotavirus;

(c4) preparing a test kit for detecting whether the pathogenic microorganism to be tested is bovine virus diarrhea virus or bovine rotavirus;

(c5) Detect whether the sample to be tested contains bovine virus diarrhea virus and/or bovine rotavirus;

(c6) preparing a kit for detecting whether the sample to be tested contains bovine viral diarrhea virus and/or bovine rotavirus.

The present invention also protects the kit containing the primer combination; the use of the kit is at least one of the following (d1)-(d3):

(d1) distinguish between bovine viral diarrhea virus and bovine rotavirus;

(d2) Detect whether the pathogenic microorganism to be tested is bovine virus diarrhea virus or bovine rotavirus;

(d3) Detecting whether the sample to be tested contains bovine viral diarrhea virus and/or bovine rotavirus.

The invention also protects the preparation method of the kit, including the step of packaging each primer separately.

The invention also protects a method for distinguishing bovine virus diarrhea virus and bovine rotavirus, comprising the following steps: extracting the nucleic acid of the virus to be tested; using the nucleic acid as a template, using the primer combination to perform double fluorescent RT-LAMP, and then Carry out following judgment: if can detect the amplification product that has the fluorescence corresponding to fluorescent group A, the virus to be tested is bovine virus diarrhea virus, if can detect the amplification product that has the fluorescence corresponding to fluorescent group B, the virus to be tested for bovine rotavirus.

In the method, when the fluorophore A is FITC and the fluorophore B is CY5.5, if the amplification product can observe a green DNA fragment under 520nm ultraviolet light, the The virus is bovine virus diarrhea virus, and if the amplified product can observe a red DNA fragment under 670nm ultraviolet light, the virus to be tested is bovine rotavirus.

In the method, the virus to be tested is bovine viral diarrhea virus or bovine rotavirus.

The present invention also protects a method for detecting whether the pathogenic microorganism to be tested is bovine virus diarrhea virus or bovine rotavirus, comprising the following steps: extracting the nucleic acid of the pathogenic microorganism to be tested; using the nucleic acid as a template, using the primer combination to carry out Double fluorescent RT-LAMP, and then make the following judgments: if it is possible to detect the amplification product corresponding to the fluorescence of the fluorescent group A, the pathogenic microorganism to be tested is bovine virus diarrhea virus, and if it can detect the amplification product corresponding to the fluorescent group B Fluorescence amplification products, pathogenic microorganisms to be detected are bovine rotavirus, if the amplification products with fluorescence corresponding to fluorophore A cannot be detected and the amplification products with fluorescence corresponding to fluorophore B cannot be detected, wait for The pathogenic microorganisms tested were non-bovine viral diarrhea virus and non-bovine rotavirus.

In the method, when the fluorophore A is FITC and the fluorophore B is CY5.5, if the amplification product can observe a green DNA fragment under 520nm ultraviolet light, the The pathogenic microorganism is bovine virus diarrhea virus, if the amplified product can observe red DNA fragments under the ultraviolet light of 670nm, and the pathogenic microorganism to be tested is bovine rotavirus, if the amplified product cannot be detected under the ultraviolet light of 520nm Green DNA fragments were observed under 670nm ultraviolet light and red DNA fragments could not be observed under 670nm ultraviolet light. The pathogenic microorganisms to be tested were non-bovine virus diarrhea virus and non-bovine rotavirus.

In the method, the pathogenic microorganism to be detected is Mycoplasma bovis, bovine viral diarrhea virus, bovine infectious rhinotracheitis virus, foot-and-mouth disease virus, vesicular stomatitis virus, bluetongue virus, bovine rotavirus or small ruminant virus Epizootic virus.

The Mycoplasma bovis can specifically be the Mycoplasma bovis GL-1 strain.

The bovine viral diarrhea virus can specifically be bovine viral diarrhea virus Oregon strain or bovine viral diarrhea virus GX-041 strain (BVDV-2 type).

The foot-and-mouth disease virus can specifically be type A of foot-and-mouth disease virus.

The vesicular stomatitis virus can specifically be vesicular stomatitis virus NJ type or vesicular stomatitis virus IND type.

The bluetongue virus may specifically be bluetongue virus serotype 4.

Specifically, the bovine rotavirus can be NCDV strain of bovine rotavirus.

Specifically, the Peste des petits ruminants virus can be Nigeria75/1 strain of Peste des pets ruminants virus.

The present invention also protects a method for detecting whether bovine virus diarrhea virus and/or bovine rotavirus is contained in the sample to be tested, comprising the following steps: extracting the nucleic acid of the sample to be tested; using the nucleic acid as a template, using the primer combination Perform double fluorescent RT-LAMP, and then make the following judgments: if the amplification product with fluorescence corresponding to fluorophore A can be detected, the sample to be tested contains bovine virus diarrhea virus, and if the amplification product with fluorophore B can be detected If the fluorescent amplification product of the fluorescent group to be detected contains bovine rotavirus in the sample to be tested, if the fluorescent amplification product corresponding to the fluorescent group A can be detected and if the fluorescent amplification product corresponding to the fluorescent group B can be detected , the sample to be tested contains bovine virus diarrhea virus and contains bovine rotavirus, if the amplification product with the fluorescence corresponding to the fluorescent group A cannot be detected and the amplification product with the fluorescence corresponding to the fluorescent group B cannot be detected, The sample to be tested does not contain bovine viral diarrhea virus and does not contain bovine rotavirus.

In the method, when the fluorophore A is FITC and the fluorophore B is CY5.5, if the amplification product can observe a green DNA fragment under 520nm ultraviolet light, the The sample contains bovine virus diarrhea virus, if the amplified product can observe red DNA fragments under the ultraviolet light of 670nm, and the sample to be tested contains bovine rotavirus, if the amplified product can be observed under the ultraviolet light of 520nm Green DNA fragments can be observed under 670nm ultraviolet light and red DNA fragments can be observed under 670nm ultraviolet light. The sample to be tested contains bovine virus diarrhea virus and contains bovine rotavirus. Green DNA fragments can be observed under 670nm ultraviolet light, and red DNA fragments cannot be observed under 670nm ultraviolet light. The sample to be tested does not contain bovine virus diarrhea virus and does not contain bovine rotavirus.

In the method, the sample to be tested is an isolated animal tissue, such as beef, food processed from beef, bovine viscera, food processed from bovine viscera, and the like.

Any of the above nucleic acids is DNA, RNA, or a mixture of DNA and RNA.

The nucleic acid extracted from the sample to be tested or the nucleic acid of the virus to be tested described above is the nucleic acid extracted by using the RNA/DNA co-extraction kit.

The initial reaction system of any of the above dual fluorescent RT-LAMP is (25 μL): template 1 μL, 10× buffer 2.5 μL, Bst DNA polymerase 15U, AMV reverse transcriptase 20U, primer BVDV-FIP-2 40pmol, primer BVDV-BIP40pmol, primer BRV-FIP-2 40pmol, primer BRV-BIP 40pmol, primer BVDV-F3 5pmol, primer BVDV-B35pmol, primer BRV-F3 5pmol, primer BRV-B3 5pmol, and the balance is water.

The composition of the 10×buffer is: Tris-HCl (pH 8.8) 200mM, KCl 100mM, MgSO ₄ 80mM, (NH ₄ ) ₂ SO ₄ 100mM, 1% (volume percentage) Tween 20, betaine 8M, dNTPs 14M.

The reaction procedure of any one of the above dual fluorescent RT-LAMPs is: 42°C for 20 minutes, 62°C for 90 minutes, and 80°C for 5 minutes.

The present invention introduces a fluorescent group into the LAMP method for the first time, and establishes a dual fluorescence RT-LAMP method for identifying BVDV and BRV, and can simultaneously differentiate and diagnose the two viruses by observing the color of the amplification product. The dual RT-LAMP method established by the present invention has good specificity, can efficiently amplify the target gene, but has no amplification for other pathogenic nucleic acids, has good sensitivity, and can detect at least 100 mixed template copies/reaction, which is a simple and convenient method. , a rapid and low-cost diagnostic method suitable for large-scale epidemiological investigations.

Description of drawings

Figure 1 is a schematic diagram of primer set I and primer set II.

Fig. 2 is the result of embodiment 3.

Fig. 3 is the electrophoresis result of embodiment 5.

Fig. 4 is the turbidity detection result of embodiment 5.

Fig. 5 is the result of embodiment 6.

detailed description

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. Quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged.

The strains used in the examples are shown in Table 1.

Table 1

10×buffer: SIGMA company. The ingredients are: Tris-HCl (pH8.8) 200mM, KCl 100mM, MgS0 ₄ 80mM, (NH ₄ ) ₂ SO ₄ 100mM, 1% (volume percentage) Tween 20, betaine 8M, dNTPs 14M.

Bst DNA polymerase: new England.

AMV reverse transcriptase: Takara.

pEASY-T1 vector: Quanshijin Biochemical Technology (Beijing) Co., Ltd.

Bovine kidney cells (MDBK): China Veterinary Drug Control Institute.

Embodiment 1, design and preparation of primer combinations

A large number of sequence analyzes and comparisons were carried out to obtain several primers for the identification of bovine viral diarrhea virus (BVDV) and bovine rotavirus (BRV). Preliminary experiments were carried out on each primer to compare performances such as sensitivity and specificity, and finally two sets of primers for identifying BVDV and BRV were obtained. Each set of primers consists of outer primer F3, outer primer B3, inner primer FIP (Flc+F2) and inner primer BIP (Blc+B2).

The primer set used to identify BVDV consists of the following four primers (5'→3'):

BVDV-F3 (SEQ ID NO: 1 of the SEQUENCE LISTING): TGCCCTTAGTAGGACTAGCA;

BVDV-B3 (SEQ ID NO: 2 of the SEQUENCE LISTING): AGCACCCTATCAGGCTGTA;

BVDV-FIP (SEQ ID NO: 3 of the SEQUENCE LISTING): CGAACCACTGACGACTACCCTGGGTAGCAACAGTGGTGAGTT;

BVDV-BIP (SEQ ID NO: 4 of the SEQUENCE LISTING): CAAGCCTCGAGATGCCACGTCCGTTTTCACCTGAACGACC;

The fluorophore FITC was attached to the 5' end of the primer BVDV-FIP.

The primer set used to identify BRV consists of the following four primers (5'→3'):

BRV-F3 (Sequence 5 of the Sequence Listing): ACTCATGTGCAATAAACGC;

BRV-B3 (Sequence 6 of the Sequence Listing): GTTTAGTAGAAACTCTATTTCAACG;

BRV-FIP (SEQ ID NO: 7 of the Sequence Listing): TCTTTCTGCATCTGGTAAAAGAGTTTAATACGCAACAATTTGAGCA;

BRV-BIP (SEQ ID NO: 8 of the SEQUENCE LISTING): CCAAGAGTGATTAATTCAGCTGACGGGTCTAAGAATCACTGGATTG;

The fluorophore CY5.5 was attached to the 5' end of the primer BRV-FIP.

The primer set used to identify BVDV was named primer set I, and the schematic diagram is shown in Figure 1A.

The primer set used to identify BVDV was named primer set II, and the schematic diagram is shown in Figure 1B.

Each of the above primers constitutes a primer combination.

Embodiment 2, establishment of detection method

1. Use the RNA/DNA co-extraction kit to extract the nucleic acid of the sample to be tested.

2. Take the nucleic acid obtained in step 1 as a template, and use the primer combination prepared in Example 1 to perform dual fluorescent RT-LAMP.

The initial reaction system is (25 μL): template 1 μL, 10×buffer 2.5 μL, Bst DNA polymerase 15U, AMV reverse transcriptase 20U, primer BVDV-FIP 40pmol, primer BVDV-BIP 40pmol, primer BRV-FIP 40pmol, primer BRV- BIP 40pmol, primer BVDV-F3 5pmol, primer BVDV-B3 5pmol, primer BRV-F3 5pmol, primer BRV-B35pmol, the balance is water.

The reaction program is: 42°C for 20min, 62°C for 90min, 80°C for 5min.

3. Take the product of step 2, perform 1% agarose gel electrophoresis, and observe under ultraviolet lamps with wavelengths of 520nm and 670nm respectively.

Embodiment 3, specificity experiment

The samples to be tested are: bovine viral diarrhea virus Oregon strain in Table 1, bovine viral diarrhea virus GX-041 strain (BVDV-2 type), bovine rotavirus NCDV strain, bovine viral diarrhea virus Oregon strain and bovine rotavirus Mixture of viruses NCDV strains (mixture I), foot-and-mouth disease virus type A, vesicular stomatitis virus type NJ, vesicular stomatitis virus type IND, bluetongue virus serotype 4, Peste des petits ruminants virus Nigeria75/1 strain, bovine infection Rhinotracheitis virus and Mycoplasma bovis GL-1 strain.

Detection was carried out according to the method established in Example 2.

Set ddH ₂ 0 as a template as a blank control for the sample to be tested.

The nucleic acid of bovine kidney cells (MDBK) was set as the negative control of the sample to be tested.

The result is shown in Figure 2. Figure 2A is the electropherogram of the 520nm channel, the bands are yellow-green. Figure 2B is the electrophoresis result of the 670nm channel, and the band is bright red. Figure 2C is the electrophoresis result of dual channels of 520nm and 670nm, and the bands are mixed colors of red and green. Lane 1 corresponds to bovine viral diarrhea virus Oregon strain, lane 2 corresponds to bovine viral diarrhea virus GX-041 strain (BVDV-2 type), lane 3 corresponds to bovine rotavirus NCDV strain, and lane 4 corresponds to bovine viral diarrhea virus Oregon strain Mixture of Wagyu rotavirus NCDV strains (mixture I), lane 5 corresponds to foot-and-mouth disease virus type A, lane 6 corresponds to vesicular stomatitis virus type NJ, lane 7 corresponds to vesicular stomatitis virus type IND, and lane 8 corresponds to bluetongue virus Serum type 4, lane 9 corresponds to Peste des petits ruminants virus Nigeria75/1 strain, lane 10 corresponds to bovine infectious rhinotracheitis virus, lane 11 corresponds to Mycoplasma bovis GL-1 strain, lane 12 corresponds to the blank control, and lane 13 corresponds to the negative control.

The results show that the method established in Example 2 can only amplify BVDV and BRV nucleic acids, as well as BVDV and BRV mixed templates. BVDV positive is yellow-green and can only be seen under channel 520; BRV positive is red and can only be seen under channel 670; mixed templates of BVDV and BRV are red and green, and can be observed under both 520 and 670 channels arrive. However, there is no amplification for other bovine viruses and negative controls, and the specificity is good.

Embodiment 4, standard product preparation

BVDV standard product: connect the double-stranded DNA molecule shown in Sequence 9 of the sequence table with the pEASY-T1 vector to obtain a recombinant plasmid, linearize the recombinant plasmid by referring to the instructions of the T7 in vitro transcription kit (Fermentas), and eliminate it with DNase The DNA contamination was transcribed into RNA in vitro, the RNA concentration was measured, and the concentration was converted into copy number according to the Avogadro constant to obtain the BVDV standard.

BRV standard product: Ligate the double-stranded DNA molecule shown in sequence 10 of the sequence table with the pEASY-T1 vector to obtain a recombinant plasmid, linearize the recombinant plasmid by referring to the instructions of the T7 in vitro transcription kit (Fermentas), and eliminate it with DNase The DNA contamination was transcribed into RNA in vitro, the RNA concentration was measured, and the concentration was converted into copy number according to the Avogadro constant to obtain the BRV standard.

Embodiment 5, sensitivity test

1. Mix the BVDV standard prepared in implementation 4 and the BRV standard with equal copy numbers to obtain a mixed solution.

2. Dilute the mixture obtained in step 2 with ddH ₂ O in a 10-fold gradient to obtain each dilution.

3. Using the dilution obtained in step 2 as a template, the method established in Example 2 is used for detection.

Due to the different dilutions of the diluents used, the following different reaction systems are formed:

In reaction system 1, the initial concentrations of BVDV standard and BRV standard were both 10 ⁶ copies/μL;

In reaction system 2, the initial concentrations of BVDV standard and BRV standard were both 10 ⁵ copies/μL;

In reaction system 3, the initial concentrations of BVDV standard and BRV standard were both 10 ⁴ copies/μL;

In reaction system 4, the initial concentrations of BVDV standard and BRV standard were both 10 ³ copies/μL;

In reaction system 5, the initial concentrations of BVDV standard and BRV standard were both 10 ² copies/μL;

In reaction system 6, the initial concentrations of the BVDV standard and BRV standard were both 10 copies/μL.

In reaction system 7, the initial concentrations of BVDV standard and BRV standard were both 1 copy/μL.

Set up a blank control using ddH ₂ O as a template.

The result is shown in Figure 3. Figure 3A is the electropherogram of the 520nm channel, the bands are yellow-green. Figure 3B is the electrophoresis result of the 670nm channel, and the band is bright red. Figure 3C is the electrophoresis result of dual channels of 520nm and 670nm, and the bands are mixed colors of red and green. Swimming lanes 1-7 correspond to reaction systems 1-7 in turn, and swimming lane 8 corresponds to the blank control.

The real-time turbidity diagram at 650 nm was generated by using the real-time turbidimeter loopam LA-320C of the reaction product, as shown in FIG. 4 . Curves 1-7 correspond to reaction systems 1-7 in turn, and curve 8 corresponds to the blank control.

The above results show that when the method established in Example 2 is used to detect the mixed template standard of BVDV and BRV, when the template concentration in the detection system is as low as 100 copies/μL, BVDV and BRV can also be detected, and the method has high sensitivity.

Embodiment 6, interfering experiment

1. Mix the BVDV standard prepared in implementation 4 and the BRV standard with equal copy numbers to obtain a mixed solution.

2. Dilute the mixture obtained in step 2 with ddH ₂ O in a 10-fold gradient to obtain each dilution.

3. Using the dilution obtained in step 2 as a template, the method established in Example 2 is used for detection.

Due to the different dilutions of the diluents used, the following different reaction systems are formed:

In reaction system 1, the initial concentration of BVDV standard was 10 ⁵ copies/μL, and the initial concentration of BRV standard was 10 ² copies/μL;

In reaction system 2, the initial concentration of the BVDV standard was 10 ⁷ copies/μL, and the initial concentration of the BRV standard was 10 ³ copies/μL;

In reaction system 3, the initial concentration of the BVDV standard was 10 ² copies/μL, and the initial concentration of the BRV standard was 10 ⁶ copies/μL;

In reaction system 4, the initial concentration of the BVDV standard was 10 ³ copies/μL, and the initial concentration of the BRV standard was 10 ⁶ copies/μL.

The result is shown in Figure 5. Figure 5A is the electropherogram of the 520nm channel, the bands are yellow-green. Figure 5B is the electrophoresis result of the 670nm channel, and the band is bright red. Figure 5C is the electrophoresis result of dual channels of 520nm and 670nm, and the bands are mixed colors of red and green. Lanes 1-4 correspond to reaction systems 1-4 in turn.

The results show that when the concentration of one template is high and the other template is low, the dual fluorescent RT-LAMP can still detect the two templates at the same time, without affecting the amplification efficiency of each other. small.

Embodiment 7, clinical sample detection

The samples to be tested are: 144 clinical samples (fecal cotton swabs from cows with diarrhea).

The nucleic acid of the sample to be tested is extracted and detected according to the method in Example 2. At the same time, the positive amplification products were sequenced to verify the correctness of the results.

The method in Example 2 was used for detection, and a total of 14 BVDV positive samples and 9 BRV positive samples were detected. The positive samples were sequenced, and all positive results were true positives, and there were no false positives of non-specific amplification.

<110> Veterinary Research Institute of Guangxi Zhuang Autonomous Region

<120> Primer combination and application for differentiating bovine viral diarrhea virus and bovine rotavirus

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<211> 20

<212>DNA

<213> Artificial sequence

<220>

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tgcccttagt aggactagca 20

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<213> Artificial sequence

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agcaccctat caggctgta 19

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<212>DNA

<213> Artificial sequence

<220>

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cgaaccactg acgactaccc tgggtagcaa cagtggtgag tt 42

<210> 4

<211> 40

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<213> Artificial sequence

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caagcctcga gatgccacgt ccgttttcac ctgaacgacc 40

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<213> Artificial sequence

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actcatgtgc aataaacgc 19

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gtttagtaga aactctattt caacg 25

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<213> Artificial sequence

<220>

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tctttctgca tctggtaaaa ggtttaata cgcaacaatt tgagca 46

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<211> 46

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ccaagagtga ttaattcagc tgacgggtct aagaatcact ggattg 46

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<213> Artificial sequence

<220>

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ctgaagccct gagtacaggg tagtcgtcag tggttcgacg ctttgtgcga caagcctcga 120

gatgccacgt ggacgagggc atgcccacag cacatcttaa cctgagcggg ggtcgttcag 180

gtgaaaacgg tttaaccaac cgctacgaat acagcctgat agggtgct 228

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actcatgtgc aataaacgcg ccagctaata cgcaacaatt tgagcatatt gtacagcttc 60

gaggggtgtt gactacagct acaataactc ttttaccaga tgcagaaaga tttagttttc 120

caagagtgat taattcagct gacggagcga ctacatggta cttcaatcca gtgattctta 180

gaccaaataa cgttgaaata gagtttctac taaac 215

Claims (9)

1. primer is combined, it is made up of primer sets I and primer sets II；

The primer sets I is made up of primer BVDV-F3, primer BVDV-B3, primer BVDV-FIP and primer BVDV-BIP；

The primer BVDV-F3 is following (a1) or (a2)：

(a1) single strand dna shown in the sequence 1 of sequence table；

(a2) have by the substitution of one or several nucleotides of the process of sequence 1 and/or missing and/or addition and with sequence 1 identical The DNA molecular of function；

The primer BVDV-B3 is following (a3) or (a4)：

(a3) single strand dna shown in the sequence 2 of sequence table；

(a4) have by the substitution of one or several nucleotides of the process of sequence 2 and/or missing and/or addition and with sequence 2 identical The DNA molecular of function；

The primer BVDV-FIP is following (a5) or (a6)：

(a5) single strand dna shown in the sequence 3 of sequence table；

(a6) have by the substitution of one or several nucleotides of the process of sequence 3 and/or missing and/or addition and with sequence 3 identical The DNA molecular of function；

The primer BVDV-BIP is following (a7) or (a8)：

(a7) single strand dna shown in the sequence 4 of sequence table；

(a8) have by the substitution of one or several nucleotides of the process of sequence 4 and/or missing and/or addition and with sequence 4 identical The DNA molecular of function；

The primer sets II is made up of primer BRV-F3, primer BRV-B3, primer BRV-FIP and primer BRV-BIP；

The primer BRV-F3 is following (b1) or (b2)：

(b1) single strand dna shown in the sequence 5 of sequence table；

(b2) have by the substitution of one or several nucleotides of the process of sequence 5 and/or missing and/or addition and with sequence 5 identical The DNA molecular of function；

The primer BRV-B3 is following (b3) or (b4)：

(b3) single strand dna shown in the sequence 6 of sequence table；

(b4) have by the substitution of one or several nucleotides of the process of sequence 6 and/or missing and/or addition and with sequence 6 identical The DNA molecular of function；

The primer BRV-FIP is following (b5) or (b6)：

(b5) single strand dna shown in the sequence 7 of sequence table；

(b6) have by the substitution of one or several nucleotides of the process of sequence 7 and/or missing and/or addition and with sequence 7 identical The DNA molecular of function；

The primer BRV-BIP is following (b7) or (b8)：

(b7) single strand dna shown in the sequence 8 of sequence table；

(b8) have by the substitution of one or several nucleotides of the process of sequence 8 and/or missing and/or addition and with sequence 8 identical The DNA molecular of function.

2. primer combination as claimed in claim 1, it is characterised in that：5 ' the ends of the primer BVDV-FIP are connected with fluorescent base Group A；5 ' the ends of the primer BRV-FIP are connected with fluorophor B.

3. primer combination as claimed in claim 2, it is characterised in that：The fluorophor A is FITC, the fluorophor B For CY5.5.

4. the application of any described primer combinations of claim 1-3, is any of following (c1) to (c6)：

(c1) bovine viral diarrhea virus and bovine rota are differentiated；

(c2) kit for differentiating bovine viral diarrhea virus and bovine rota is prepared；

(b3) detect whether pathogenic microorganism to be measured is bovine viral diarrhea virus or bovine rota；

(c4) prepare for detect pathogenic microorganism to be measured whether be bovine viral diarrhea virus or bovine rota kit；

(c5) whether bovine viral diarrhea virus and/or bovine rota are contained in detection sample to be tested；

(c6) prepare for detect in sample to be tested whether the kit containing bovine viral diarrhea virus and/or bovine rota.

5. the kit containing any described primer combinations of claim 1-3；The purposes of the kit be following (d1)- At least one of (d3)：

(d1) bovine viral diarrhea virus and bovine rota are differentiated；

(d2) detect whether pathogenic microorganism to be measured is bovine viral diarrhea virus or bovine rota；

(d3) whether bovine viral diarrhea virus and/or bovine rota are contained in detection sample to be tested.

6. the preparation method of kit described in claim 5, including the step of each bar primer is individually packed.

7. a kind of method for differentiating bovine viral diarrhea virus and bovine rota, comprises the following steps：Extract viral core to be measured Acid；Using the nucleic acid as template, bifluorescence RT-LAMP is carried out using primer combination, then made the following judgment：If It is able to detect that the amplified production with the corresponding fluorescence of fluorophor A, virus to be measured are bovine viral diarrhea virus, if enough inspections It is bovine rota to measure the amplified production with the corresponding fluorescence of fluorophor B, virus to be measured.

8. it is a kind of detect virus whether be bovine viral diarrhea virus or bovine rota method, comprise the following steps：Extraction is treated Survey the nucleic acid of pathogenic microorganism；Using the nucleic acid as template, bifluorescence RT-LAMP is carried out using primer combination, then Make the following judgment：If being able to detect that the amplified production with the corresponding fluorescence of fluorophor A, pathogenic microorganism to be measured are Bovine viral diarrhea virus, be if enough detecting the amplified production with the corresponding fluorescence of fluorophor B, pathogenic microorganism to be measured Bovine rota, if the amplified production with the corresponding fluorescence of fluorophor A can not be detected and can not be detected with glimmering The amplified production of the corresponding fluorescence of light group B, pathogenic microorganism to be measured is non-bovine viral diarrhea virus and non-bovine rota.

9. in a kind of detection sample to be tested whether the method containing bovine viral diarrhea virus and/or bovine rota, it is including as follows Step：Extract the nucleic acid of sample to be tested；Using the nucleic acid as template, bifluorescence RT-LAMP is carried out using primer combination, Then make the following judgment：If being able to detect that the amplified production with the corresponding fluorescence of fluorophor A, containing in sample to be tested There is bovine viral diarrhea virus, if it is possible to detect the amplified production with the corresponding fluorescence of fluorophor B, contain in sample to be tested There is bovine rota, if it is possible to detect the amplified production with the corresponding fluorescence of fluorophor A and if be able to detect that It is containing bovine viral diarrhea virus and sick containing bull wheel shape in amplified production, sample to be tested with the corresponding fluorescence of fluorophor B Poison, if the amplified production with the corresponding fluorescence of fluorophor A can not be detected and can not be detected with B pairs of fluorophor Bovine viral diarrhea virus is not contained in the amplified production of the fluorescence answered, sample to be tested and does not contain bovine rota.