CN114634996B - Primer probe combination and kit for detecting bovine respiratory disease and application of primer probe combination and kit - Google Patents
Primer probe combination and kit for detecting bovine respiratory disease and application of primer probe combination and kit Download PDFInfo
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Abstract
The invention belongs to the technical field of bovine respiratory tract pathogen detection, and discloses a primer probe combination, a kit and application thereof for detecting bovine respiratory tract diseases, in particular a reagent comprising a primer group and/or a probe group. The reagent provided by the invention can be used for simultaneously detecting infectious bovine rhinotracheitis virus, bovine viral diarrhea virus and mycoplasma bovis, and can detect the pathogens of 3 bovine respiratory diseases by performing PCR amplification for 1 time in one tube reaction.
Description
Technical Field
The invention belongs to the technical field of bovine respiratory virus detection, and particularly relates to a primer probe combination for detecting bovine respiratory diseases, a kit and application thereof.
Background
Bovine infectious rhinotracheitis virus (Infectious bovine rhinotracheitis virus, IBRV), also known as bovine herpes virus type I (BHV-1), is a DNA virus. IBRV-infected cattle often exhibit respiratory symptoms and are associated with clinical symptoms such as cow abortion, milk production decline, metritis, infectious vulvitis, and enteritis. Bovine Viral Diarrhea Virus (BVDV) is a single positive stranded, enveloped RNA virus that can present in infected cattle with acute and persistent infections and mucosal diseases such as pneumonia, diarrhea, abortion, and the like. Mycoplasma bovis (M.bovis) belongs to the Mycoplasma family, mycoplasma genus, and has no cell wall, double-stranded DNA and ribosomes present in the cytoplasm. Mycoplasma bovis can cause pneumonia, mastitis, keratitis, and arthritis in cattle, most frequently causing respiratory diseases. Infectious bovine rhinotracheitis virus, bovine viral diarrhea virus and mycoplasma bovis are the common 3 main pathogens causing bovine respiratory disease syndrome, and infectious diseases caused by the viruses seriously endanger the cattle industry, thus restricting the development of the cattle industry. Thus, establishing an effective and rapid detection method is critical for effective control and eradication of disease.
The current detection methods for infectious bovine rhinotracheitis virus, bovine viral diarrhea virus and mycoplasma bovis mainly comprise pathogen separation, ELISA, serum neutralization test, PCR and the like. Since the time required for the body to generate antibodies after the animals are infected with the viruses is usually ten days, the detection of the antibodies is unfavorable for the diagnosis of acute infection and early infection of bovine infectious rhinotracheitis virus, bovine viral diarrhea virus and mycoplasma bovis; the serum neutralization test and virus separation and identification have the problems of long time consumption and low sensitivity; the judgment of the common PCR detection result requires gel electrophoresis analysis of the product, and the operation is complicated. In addition, these methods are currently mainly used for detection of a single pathogen, and the sensitivity, specificity, simplicity and the like of these methods are still to be further improved. Real-time fluorescence PCR is a rapid, accurate and high-flux virus detection technology, and is widely applied to detection of various pathogens in recent years.
Disclosure of Invention
The object of the first aspect of the present invention is to provide a reagent.
The object of the second aspect of the present invention is to provide a kit.
The object of a third aspect of the present invention is to provide the use of a reagent according to the first aspect of the present invention or a kit according to the second aspect of the present invention.
The fourth aspect of the present invention is to provide a multiplex fluorescence PCR detection method for simultaneously detecting infectious bovine rhinotracheitis virus, bovine viral diarrhea virus and mycoplasma bovis.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
in a first aspect of the present invention, there is provided a reagent comprising a primer set and/or a probe set, the nucleotide sequences of the primer set and probe set being as follows:
The primer group is used for detecting infectious bovine rhinotracheitis virus, bovine viral diarrhea virus and/or mycoplasma bovis;
wherein, the primer for detecting the infectious bovine rhinotracheitis virus is as follows:
IBRV-F1:5'-ACGACGGACGATGTGTAC-3' (SEQ ID NO. 1), or a complement of the sequence;
IBRV-R1:5'-GAACGAGTCGTACGGGTA-3' (SEQ ID NO. 2), or a complement of the sequence;
The primers for detecting bovine viral diarrhea virus are as follows:
BVDV-F1:5'-GGCCCACTGTATTGCTACT-3' (SEQ ID NO. 8), or a complement of the sequence;
BVDV-R1:5'-CTGGTCGTAAACAGGTTCC-3' (SEQ ID NO. 9), or a complement of the sequence;
the primer for detecting mycoplasma bovis is as follows:
Mb-F2:5'-AAAGAACACCCAGAAGCTAAGA-3' (SEQ ID NO. 17), or a complement of the sequence;
Mb-R2:5'-AACTGGATATGAAGCTGTGGC-3' (SEQ ID NO. 18), or a complement of the sequence;
the probe set is used for detecting infectious bovine rhinotracheitis virus, bovine viral diarrhea virus and/or mycoplasma bovis;
wherein, the probe for detecting infectious bovine rhinotracheitis virus is as follows:
IBRV-P:5'-CGCCTCCACTTCTTCCACGATGC-3' (SEQ ID NO. 7), or a complement of the sequence;
The probe for detecting bovine viral diarrhea virus comprises the following components:
BVDV-P:5'-CTGTACATGGCACATGGAGTTGA-3' (SEQ ID NO. 14), or a complement of the sequence;
The probe for detecting mycoplasma bovis comprises:
Mb-P:5'-ACTTCTGGTGAACCCGTTGCAGTTGCTGC-3' (SEQ ID NO. 21), or a complement of the sequence.
Preferably, the two ends of the sequence of the probe for detecting the infectious bovine rhinotracheitis virus, the probe for detecting the bovine viral diarrhea virus and the probe for detecting mycoplasma bovis are respectively marked with a fluorescent group and a quenching group, and the marked fluorescent groups are different among the probe sequences.
Preferably, the fluorophore is at least one of FAM, VIC, TAMRA, ROX and CY 5.
Preferably, the quenching group is at least one of BHQ1, BHQ2 and BHQ 3.
Further preferably, the fluorescent group is attached to the 5' end of the primer.
Further preferably, the quenching group is attached to the 3' end of the primer.
Further preferably, the 5 'end of the IBRV-P is connected with a fluorescent group FAM and the 3' end is connected with a quenching group BHQ1.
Further preferably, the 5 'end of BVDV-P is connected with a fluorescent group ROX and the 3' end is connected with a quenching group BHQ2.
Further preferably, the Mb-P is linked at its 5 'end to a fluorophore VIC and at its 3' end to a quencher BHQ2.
In a second aspect, the invention provides a kit comprising a reagent according to the first aspect of the invention.
Preferably, the kit further comprises a PCR reaction solution, an enzyme, a negative reference and a positive reference.
Preferably, the enzyme comprises TaqDNA polymerase and reverse transcriptase.
In a third aspect of the present invention, there is provided the use of the primer set and the probe set of the first aspect of the present invention or the kit of the second aspect of the present invention in any one of (1) to (6);
(1) Identifying infectious bovine rhinotracheitis virus, bovine viral diarrhea virus and/or mycoplasma bovis;
(2) Preparing a product for detecting infectious bovine rhinotracheitis virus, bovine viral diarrhea virus and/or mycoplasma bovis;
(3) Detecting whether a sample to be detected is infectious bovine rhinotracheitis virus, bovine viral diarrhea virus or mycoplasma bovis;
(4) Preparing a sample to be tested whether the sample is a product of infectious bovine rhinotracheitis virus, bovine viral diarrhea virus or mycoplasma bovis;
(5) Detecting whether a sample to be detected is infected with infectious bovine rhinotracheitis virus, bovine viral diarrhea virus and/or mycoplasma bovis;
(6) Preparing a product for detecting whether a sample to be detected is infected with infectious bovine rhinotracheitis virus, bovine viral diarrhea virus and/or mycoplasma bovis;
The above-mentioned applications are for diagnosis and treatment of non-diseases.
In a fourth aspect, the present invention provides a multiplex fluorescence PCR detection method for simultaneously detecting infectious bovine rhinotracheitis virus, bovine viral diarrhea virus and mycoplasma bovis, comprising the steps of:
1) Extracting nucleic acid from a sample to be tested;
2) Using the nucleic acid of step 1) as a template, performing multiplex fluorescence PCR amplification reaction with the reagent of the first aspect of the invention or the kit of the second aspect of the invention, and collecting fluorescence signals;
3) Judging whether the sample to be tested contains infectious bovine rhinotracheitis virus, bovine viral diarrhea virus and mycoplasma bovis according to the fluorescent signal;
The above methods are useful for diagnosis and treatment of non-diseases.
Preferably, the multiplex fluorescence PCR amplification reaction system in the step 2) is as follows:
preferably, the multiplex fluorescence PCR amplification reaction procedure in step 2) is: 40-45 ℃ for 4-6 min; 94-96 deg.c for 25-35 s; 4-6 s at 90-95 deg.c, 30s at 52-62 deg.c and 35-40 cycles.
Preferably, the result determination method of the detection method is as follows: on the basis of the establishment of the experiment, if the detection result of the sample to be detected is less than or equal to 35 in the Ct value of the FAM channel and has an amplification curve, judging that the nucleic acid of the infectious bovine rhinotracheitis virus is positive; if the Ct value of the ROX channel is less than or equal to 34 and an amplification curve exists, judging that the bovine viral diarrhea virus nucleic acid is positive; if the Ct value of the VIX channel is less than or equal to 35 and an amplification curve exists, judging that the mycoplasma bovis nucleic acid is positive; if the detection of the three channels has no Ct value or the Ct value is more than 38, the detection is that the infectious bovine rhinotracheitis virus, the bovine viral diarrhea virus and the bovine mycoplasma nucleic acid are all negative; if the Ct value of the sample to be detected is less than or equal to 38 in any channel 35, the sample is detected again after extracting the nucleic acid again, and if the repeated detection result has no Ct value, the sample is negative, otherwise, the sample is positive.
The beneficial effects of the invention are as follows:
The reagent provided by the invention can be used for simultaneously detecting infectious bovine rhinotracheitis virus, bovine viral diarrhea virus and mycoplasma bovis, and can detect the pathogens of 3 bovine respiratory diseases by performing PCR amplification for 1 time in one tube reaction.
The detection kit provided by the invention breaks through the limitation of single fluorescence PCR detection, can simultaneously detect the contents of infectious bovine rhinotracheitis virus, bovine viral diarrhea virus and mycoplasma bovis in a sample in real time, has high sensitivity, strong specificity and good repeatability, is low in cost, can simultaneously detect a large number of samples, has no cross reaction with other bovine pathogens, is very suitable for detection and epidemic situation monitoring of a large number of clinical samples, and provides a scientific and reliable method for identifying pathogenic microorganisms of bovine respiratory diseases and reducing economic loss of cultivation.
The detection kit provided by the invention can be used for rapidly detecting the infectious bovine rhinotracheitis virus, the bovine viral diarrhea virus and the mycoplasma bovis of various samples such as serum, secretion, tissue culture, nasal swab, fecal swab and the like, has a wide sample application range, is low in reagent infectivity, has no biological potential safety hazard component and is high in use safety.
Drawings
FIG. 1 is a graph of amplification curves for a triple fluorescence PCR detection kit for amplifying a positive reference and a negative reference.
FIG. 2 is a graph showing the sensitivity amplification of the triple fluorescence PCR detection kit for amplifying infectious bovine rhinotracheitis virus.
FIG. 3 is a graph showing the sensitivity amplification of bovine viral diarrhea virus by triple fluorescence PCR detection kit.
FIG. 4 is a plot of the sensitivity amplification of Mycoplasma bovis by triple fluorescence PCR detection kit.
Detailed Description
The invention will now be described in detail with reference to specific examples, without limiting the scope of the invention.
The materials, reagents and the like used in this example are commercially available materials and reagents unless otherwise specified.
Example 1 design and screening of primer and Probe set
The performance of the primer determines the quality of the detection effect of the kit. In the embodiment, a plurality of groups of primer/probe combinations are screened aiming at detection indexes of infectious bovine rhinotracheitis virus, bovine viral diarrhea virus and mycoplasma bovis, and finally, a group of optimal primer/probe combinations is screened. This example is intended to illustrate the design of primer/probe combinations and the screening process.
Primer and probe design: the gB gene (SEQ ID NO: MF 287966.1) of bovine infectious rhinotracheitis virus, the 5' -UTR gene (SEQ ID NO: AF 091605.1) of bovine viral diarrhea virus and the P48 gene (SEQ ID NO: KX 772800.1) of Mycoplasma bovis registered on GenBank are downloaded, specific primers and fluorescent probes for amplifying the above gene fragments are designed by using Primer expression 5.0 and oligo6.0 design software, and BLAST verification is performed on the primers and probes designed to ensure high specificity of the primers, and 9 sets of primers and 3 sets of probes shown in Table 1 are designed through verification.
Primer screening: the 3 groups of primers of the infectious bovine rhinotracheitis virus, the bovine viral diarrhea virus and the mycoplasma bovis are respectively subjected to single fluorescence PCR reaction by taking a dilution of 10-time gradient dilution of a mixture of nucleic acid extracted from pathogens such as the infectious bovine rhinotracheitis virus, the bovine viral diarrhea virus and the mycoplasma bovis as a template, and the optimal primer probe combination is screened. The initial reaction system of the single fluorescence PCR is as follows: taqDNA polymerase (5U/. Mu.L) 0.4. Mu.L, reverse transcriptase (5U/. Mu.L) 0.4. Mu.L, 2 XPCR Buffer 10. Mu.L, upstream and downstream primers (10. Mu.M) 0.4. Mu.L, probe (10. Mu.M) 0.2. Mu.L, no ribozyme water 6.2. Mu.L, template 2. Mu.L. The reaction procedure: reverse transcription at 42℃for 5min; pre-denaturation at 94℃for 30s; denaturation at 94℃for 5s, annealing at 60℃for 30s,40 cycles. As can be seen from Table 2, the optimal primer sets for finally screening the infectious bovine rhinotracheitis virus, the bovine viral diarrhea virus and the mycoplasma bovis according to the sensitivity and the amplification efficiency are ：IBRV-F1(SEQ ID NO.1)、IBRV-R1(SEQ ID NO.2),BVDV-F1(SEQ ID NO.8)、BVDV-R1(SEQ ID NO.9),Mb-F2(SEQ ID NO.17)、Mb-R2(SEQ ID NO.18), primer sets for the three primer sets as the primer sets for the subsequent multiplex PCR reaction of the present invention.
TABLE 1 primer and probe sequences
TABLE 2 primer screening results
Note that: 0 represents no Ct value.
EXAMPLE 2 preparation of Positive reference
The cDNA of infectious bovine rhinotracheitis virus, the DNA of bovine viral diarrhea virus and the DNA of bovine mycoplasma are used as templates respectively, the optimal primer group screened in the example 1 is used for common PCR amplification, and target fragments of 117bp, 105bp and 132bp are obtained respectively after electrophoresis. The PCR product was recovered and purified using an agarose gel DNA recovery kit (Tiangen Biotechnology Co., ltd.), the purified DNA fragment was cloned into pMD 19-T Vector (Takara), then transformed into DH 5. Alpha. (Shanghai Weidi Biotechnology Co., ltd.), and positive clones were selected for sequencing verification, and further amplified and cultured after passing the verification. The positive bacterial solutions were each subjected to plasmid miniprep kit (Tiangen Biotechnology Co., ltd.) to extract recombinant plasmids. The protein concentration was measured using a nucleic acid protein meter, and the copy number was calculated according to the formula [ 6.02X10 23 (copies/moL) ]× [ concentration (g/μl) ]/[ MW (g/moL) ]=copy number (copies/μl), the concentrations of the three recombinant plasmids were adjusted to 1× 6 copies/μl, and then the three recombinant plasmids were mixed at 1:1:1 to obtain a positive reference.
Example 3 triple fluorescence PCR detection method was established and optimized
Taking a positive reference (prepared in example 2) diluted by 3 times as a template, respectively carrying out a single fluorescent PCR primer (the concentration of an upper primer and a lower primer is 1:1) and a probe concentration gradient test on the infectious bovine rhinotracheitis virus, the bovine viral diarrhea virus and the mycoplasma bovis respectively, and respectively determining the concentration ranges of the infectious bovine rhinotracheitis virus, the bovine viral diarrhea virus and the mycoplasma bovis primer/probe according to the amplification efficiency and the sensitivity (the final concentration range of the primer is 0.2-0.6 mu M and the final concentration range of the probe is 0.1-0.3 mu M); and then selecting three concentrations of the infectious bovine rhinotracheitis virus, the bovine viral diarrhea virus and the bovine mycoplasma primer according to the determined primer concentration range, sequentially designing 8 groups of primer concentration ratio combinations (table 3), and screening out the optimal ratio combination of the primer concentration after considering the reagent cost on the basis of ensuring the amplification efficiency. The same method is used to screen out the optimal ratio combination of probe concentration. After the optimal ratio combination of primer and probe concentrations was determined, the annealing temperature (52 to 62 ℃) was further optimized (Table 5), and finally the reaction system and reaction procedure of the triple fluorescence PCR were determined. As can be seen from tables 3 to 5, the optimal primer combinations for bovine infectious rhinotracheitis virus, bovine viral diarrhea virus and mycoplasma bovis are ibrv+bvdv+mb=0.3 μΜ+0.4 μΜ+0.3 μΜ, the optimal probe combinations are ibrv+bvdv+mb=0.15 μΜ+0.2 μΜ+0.15 μΜ, and the optimal annealing temperature is 52 ℃, therefore, the final reaction system of the triple fluorescence PCR is shown in table 6, and the reaction procedure is specifically: reverse transcription is carried out at 42 ℃ for 5min; pre-denaturation at 94℃for 30s; denaturation at 94℃for 5s, annealing at 55℃for 30s,40 cycles. The optimal reaction system and the optimal reaction program are used for amplifying a positive reference and a negative reference (no ribozyme water), the amplification curve of the positive reference is shown in figure 1, the positive reference has an amplification curve, and the negative reference has no amplification curve, so that the reaction system and the reaction program have good amplification effects on mixed samples of bovine infectious rhinotracheitis virus, bovine viral diarrhea virus and mycoplasma bovis.
TABLE 3 primer concentration combination optimization results
TABLE 4 Probe concentration combination optimization results
TABLE 5 annealing temperature optimization results
Table 6 triple fluorescence PCR detection reaction system
Example 4 triple fluorescence PCR detection kit
A triple fluorescence PCR detection kit comprises the following components: the PCR reaction premix comprises a2 XPCR Buffer, non-ribozyme water, IBRV-F1, IBRV-R1, IBRV-P, BVDV-F1, BVDV-R1, BVDV-P, mb-F2, mb-R2 and Mb-P. The reaction system, reaction procedure and assembly conditions of the detection kit are shown in Table 7.
Table 7 triple fluorescence PCR detection kit reaction system and reaction program
Example 5 specificity test of triple fluorescence PCR detection kit
Extracting nucleic acid of swine fever, bovine epidemic fever, bovine parainfluenza virus type 3, bovine respiratory syncytial virus, non-lactating mycoplasma, bovine rhinomycoplasmas, ovine mycoplasma pneumoniae, mycoplasma hyopneumoniae, bovine infectious rhinotracheitis virus, bovine viral diarrhea virus, bovine mycoplasma and other pathogens (all the pathogens are isolated, identified and stored by the institute of animal medicine of the sea institute), and performing triple fluorescence PCR amplification by using the nucleic acid as a template and adopting the detection kit of the embodiment 4, evaluating the specificity of the detection kit, wherein the specific reaction system is shown in table 6, and the reaction procedure is as follows: reverse transcription at 42℃for 5min; pre-denaturation at 94℃for 30s; denaturation at 94℃for 5s, annealing at 55℃for 30s,40 cycles.
The result shows that the positive reference products all have Ct values, corresponding amplification curves appear, and the negative reference products all have no amplification curve, thus indicating that the experiment is successful; in addition to the negative reference and the positive reference, the detection kit has an amplification curve and Ct value of 17.355 only for bovine infectious rhinotracheitis virus samples in FAM channel, has an amplification curve and Ct value of 15.448 only for bovine viral diarrhea virus samples in ROX channel, has an amplification curve and Ct value of 21.336 only for bovine mycoplasma samples in VIC channel, and has good specificity in swine fever, bovine epidemic fever, bovine parainfluenza virus type 3, bovine respiratory syncytial virus, mycoplasma bovis, mycoplasma rhino, mycoplasma ovinii, mycoplasma hyopneumoniae and other samples in FAM, ROX, VIC channels.
Example 6 sensitive detection of triple fluorescence PCR detection kit
The positive plasmids of bovine infectious rhinotracheitis virus, bovine viral diarrhea virus and mycoplasma bovis are respectively diluted in a 10-fold gradient, mixed in equal proportion, and subjected to triple fluorescence PCR amplification by taking a diluted mixed sample of 1X 10 5～100 copies/. Mu.L as a template, wherein the sensitivity of the detection kit of the example 4 is evaluated, the specific reaction system is shown in Table 6, and the reaction procedure is as follows: reverse transcription at 42℃for 5min; pre-denaturation at 94℃for 30s; denaturation at 94℃for 5s, annealing at 55℃for 30s,40 cycles. The amplification results are shown in FIGS. 2, 3 and 4.
The detection results are shown in Table 8, and on the basis that the negative reference and the positive reference are established, the detection kit can detect the infectious bovine rhinotracheitis virus by 10 copies/mu L, the infectious bovine diarrhea virus by 10 copies/mu L and the mycoplasma bovis by 10 copies/mu L, so that the detection kit has higher sensitivity for detecting the infectious bovine rhinotracheitis virus, the infectious bovine diarrhea virus and the mycoplasma bovis in the sample.
TABLE 8 sensitivity results of triple fluorescence PCR detection kit
Note that: 0 represents no Ct value.
Example 7 repeated detection of triple fluorescence PCR detection kit
The positive reference in example 2 was 10-fold diluted by 3 gradients, 5 kits (markers: PN-1, PN-2, PN-3, PN-4, PN-5) in the same kit prepared in 2021 were randomly selected for triple fluorescence PCR amplification using the diluted positive reference as templates (markers: Y-1, Y-2, Y-3), each sample was repeatedly detected 3 times, and then a variation coefficient was calculated based on the detection result (Ct value), and the in-batch reproducibility of the kit was evaluated. In addition, any 1 kit (mark: PJ-1, PJ-2, PJ-3) in three kits prepared at different times in 2021 year is selected respectively, triple fluorescence PCR amplification is carried out by taking positive reference substances diluted by each gradient as templates, each sample is repeatedly detected for 3 times, then a variation coefficient is calculated according to a detection result (Ct value), and the batch-to-batch repeatability of the kit is evaluated.
As can be seen from tables 9 and 10, the triple fluorescence PCR detection kit provided in example 5 has a variation coefficient CV value in the batch of at most 3.77%, less than 5%, and a variation coefficient CV value in the batch of at most 4.09%, less than 5%, and thus has good reproducibility and high accuracy.
Table 9 results of in-batch reproducibility test of triple fluorescence PCR detection kit
Table 10 results of the batch-to-batch reproducibility test of triple fluorescence PCR detection kit
Example 8 interference assay of triple fluorescence PCR detection kit
1. Preparation of an interference test sample
Mixing 10 9 copies/. Mu.L of bovine infectious rhinotracheitis virus positive plasmid, 10 3 copies/. Mu.L of bovine viral diarrhea virus positive plasmid, and 10 3 copies/. Mu.L of mycoplasma bovis positive plasmid 1:1:1 to prepare an interference sample 1;
Mixing 10 3 copies/. Mu.L of bovine infectious rhinotracheitis virus positive plasmid, 10 9 copies/. Mu.L of bovine viral diarrhea virus positive plasmid, and 10 3 copies/. Mu.L of mycoplasma bovis positive plasmid 1:1:1 to prepare an interference sample 2;
Mixing 10 3 copies/. Mu.L of bovine infectious rhinotracheitis virus positive plasmid, 10 3 copies/. Mu.L of bovine viral diarrhea virus positive plasmid, and 10 9 copies/. Mu.L of mycoplasma bovis positive plasmid 1:1:1 to prepare an interference sample 3;
Mixing 10 3 copies/. Mu.L of infectious bovine rhinotracheitis virus positive plasmid and negative bovine nasal swab (infectious bovine rhinotracheitis virus, bovine viral diarrhea virus and mycoplasma bovis all negative) in a ratio of 1:1, and extracting nucleic acid to prepare an interference sample 4;
mixing 10 3 copies/. Mu.L of bovine viral diarrhea virus positive plasmid and negative bovine nasal swab 1:1, and extracting nucleic acid to prepare an interference sample 5;
Mixing 10 3 copies/. Mu.L of mycoplasma bovis positive plasmid and a negative bovine nasal swab 1:1, and extracting nucleic acid to prepare an interference sample 6;
Mixing 10 3 copies/mu L of bovine infectious rhinotracheitis virus positive plasmid and negative bovine serum (bovine infectious rhinotracheitis virus, bovine viral diarrhea virus and bovine mycoplasma are all negative) 1:1, and extracting nucleic acid to prepare an interference sample 7;
Mixing 10 3 copies/. Mu.L of bovine viral diarrhea virus positive plasmid and negative bovine serum 1:1, and extracting nucleic acid to prepare an interference sample 8;
The interfering sample 9 was prepared by mixing 10 3 copies/. Mu.L of Mycoplasma bovis positive plasmid with 1:1 of negative bovine serum and extracting nucleic acid.
2. Interference detection
Using 9 interfering samples prepared in step 1 as templates, performing triple fluorescence PCR amplification by using the detection kit of example 4, and performing an interfering test, wherein the specific reaction system is shown in table 6, and the reaction procedure is as follows: reverse transcription at 42℃for 5min; pre-denaturation at 94℃for 30s; denaturation at 94℃for 5s, annealing at 55℃for 30s,40 cycles.
As can be seen from table 11, on the basis that the negative reference and the positive reference are established, for the mixed plasmid standard solutions of bovine infectious rhinotracheitis virus, bovine viral diarrhea virus and mycoplasma bovis of different concentrations, when the concentration of part of templates is high and the concentration of part of templates is low, the detection kit of example 4 can still detect three templates simultaneously, without affecting the amplification efficiency of each other; when the low-concentration template sample contains the interfering substances such as bovine nasal swab, bovine serum and the like, the detection kit of the embodiment 4 can still detect normally, and the detection result is not influenced. Therefore, the detection kit of example 4 has good anti-interference performance, and is not easily interfered by other factors such as serum in practical detection.
Table 11 results of the interference test of the triple fluorescence PCR detection kit
Note that: 0 represents no Ct value.
Example 9 determination of the Yin-Yang Critical value of triple fluorescence PCR detection kit
The concentrations of positive plasmids of infectious bovine rhinotracheitis virus, bovine viral diarrhea virus and mycoplasma bovis are respectively regulated to 10 times of 5 multiplied by 10 2～100 copies/mu L by using different dilution methods, 10 parts of diluted samples of infectious bovine rhinotracheitis virus, bovine viral diarrhea virus and mycoplasma bovis are randomly selected as templates for triple fluorescence PCR amplification, each sample is repeated 5 times, and the yin-yang critical value of the triple fluorescence PCR detection kit is determined. The samples with Ct values and standard amplification curves were positive for 5 replicates, suspicious samples with no Ct values for one or more replicates, and negative samples with no Ct values for 5 replicates.
As can be seen from table 12, the detection kit detects bovine infectious rhinotracheitis virus with a yin-yang critical value of 35, bovine viral diarrhea virus with a yin-yang critical value of 34, and mycoplasma bovis with a yin-yang critical value of 35 on the basis that the negative reference and the positive reference are established. Therefore, the result judgment standard of the triple fluorescence PCR detection kit formulated according to the critical value of yin and yang is as follows: on the basis of the establishment of the experiment, if the detection result of the sample to be detected is less than or equal to 35 in the Ct value of the FAM channel and has an amplification curve, judging that the nucleic acid of the infectious bovine rhinotracheitis virus is positive; if the Ct value of the ROX channel is less than or equal to 34 and an amplification curve exists, judging that the bovine viral diarrhea virus nucleic acid is positive; if the Ct value of the VIX channel is less than or equal to 35 and an amplification curve exists, judging that the mycoplasma bovis nucleic acid is positive; if the detection of the three channels has no Ct value or the Ct value is more than 38, the detection is that the infectious bovine rhinotracheitis virus, the bovine viral diarrhea virus and the bovine mycoplasma nucleic acid are all negative; if the Ct value of the sample to be detected is less than 35 and less than or equal to 38 in any channel, the sample is detected again after extracting the nucleic acid again, and if the repeated detection result has no Ct value, the sample is negative, otherwise, the sample is positive.
Table 12 triple fluorescence PCR detection kit yin-yang critical value
Note that: 0 represents no Ct value.
Example 10 detection of clinical samples
20 Clinical samples (samples are collected in a certain cattle farm in Guangzhou of Guangdong) are detected by randomly taking one of a batch of detection kits prepared in 2021, and synchronously detecting by using the current standard (GB/T27981-2011 bovine infectious rhinotracheitis virus real-time fluorescence PCR detection method, GB/T18637-2018 bovine viral diarrhea/mucosal disease diagnosis technical specification and NY/T3234-2018 mycoplasma bovis PCR detection method). Sample types include bovine nasal swabs and serum, samples which are tested positive by the kit after the test is finished, and samples which do not accord with the current standard test results are subjected to gene sequencing.
The results show that the detection results of 1 part of bovine infectious rhinotracheitis virus positive samples, 3 parts of bovine viral diarrhea virus positive samples and 1 part of bovine mycoplasma positive samples (table 13) are mostly consistent with the detection results of the existing standards, and the sequencing results of the samples which are detected to be positive and the samples which are inconsistent with the two detection results are consistent with the detection results of the detection kit of the embodiment 4, so that the detection kit of the embodiment 4 can accurately and reliably detect the bovine infectious rhinotracheitis virus, the bovine viral diarrhea virus and the bovine mycoplasma in the clinical samples.
TABLE 13 clinical sample test results
Note that: 0 represents no Ct value.
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of one of ordinary skill in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.
SEQUENCE LISTING
<110> Guangdong sea big animal husbandry veterinary research institute Co., ltd
<120> Primer probe combination for detecting bovine respiratory disease, kit and application thereof
<130>
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<170> PatentIn version 3.5
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Claims (10)
1. A reagent comprising a primer set and a probe set, the primer set and probe set having nucleotide sequences as follows:
the primer group is used for detecting infectious bovine rhinotracheitis virus, bovine viral diarrhea virus and mycoplasma bovis;
wherein, the primer for detecting the infectious bovine rhinotracheitis virus is as follows:
IBRV-F1:5'-ACGACGGACGATGTGTAC-3', or a complement of the sequence;
IBRV-R1:5'-GAACGAGTCGTACGGGTA-3', or a complement of the sequence;
The primers for detecting bovine viral diarrhea virus are as follows:
BVDV-F1:5'-GGCCCACTGTATTGCTACT-3', or a complement of the sequence;
BVDV-R1:5'-CTGGTCGTAAACAGGTTCC-3', or a complement of the sequence;
the primer for detecting mycoplasma bovis is as follows:
Mb-F2:5'-AAAGAACACCCAGAAGCTAAGA-3', or a complement of the sequence;
Mb-R2:5'-AACTGGATATGAAGCTGTGGC-3', or a complement of the sequence;
the probe set is used for detecting infectious bovine rhinotracheitis virus, bovine viral diarrhea virus and mycoplasma bovis;
wherein, the probe for detecting infectious bovine rhinotracheitis virus is as follows:
IBRV-P:5'-CGCCTCCACTTCTTCCACGATGC-3', or a complement of the sequence;
The probe for detecting bovine viral diarrhea virus comprises the following components:
BVDV-P:5'-CTGTACATGGCACATGGAGTTGA-3', or a complement of the sequence;
The probe for detecting mycoplasma bovis comprises:
Mb-P:5'-ACTTCTGGTGAACCCGTTGCAGTTGCTGC-3', or a complement of the sequence.
2. The reagent according to claim 1, wherein the probe for detecting infectious bovine rhinotracheitis virus, the probe for detecting bovine viral diarrhea virus and the probe for detecting mycoplasma bovis have a fluorescent group and a quenching group respectively labeled at both ends of the sequences of the probe, and the fluorescent groups labeled are different from each other.
3. The reagent of claim 2, wherein the fluorophore is at least one of FAM, VIC, TAMRA, ROX and CY 5; the quenching group is at least one of BHQ1, BHQ2 and BHQ 3.
4. A kit comprising the reagent of any one of claims 1 to 3.
5. The kit of claim 4, further comprising a PCR reaction solution, an enzyme, a negative reference, and a positive reference.
6. The kit of claim 5, wherein the enzymes comprise taq dna polymerase and reverse transcriptase.
7. Use of the reagent of any one of claims 1 to 3 or the kit of any one of claims 4 to 6 in any one of (1) to (5);
(1) Identifying infectious bovine rhinotracheitis virus, bovine viral diarrhea virus and mycoplasma bovis;
(2) Preparing a product for detecting infectious bovine rhinotracheitis virus, bovine viral diarrhea virus and mycoplasma bovis;
(3) Detecting whether a sample to be detected is infectious bovine rhinotracheitis virus, bovine viral diarrhea virus or mycoplasma bovis;
(4) Preparing a sample to be tested whether the sample is a product of infectious bovine rhinotracheitis virus, bovine viral diarrhea virus or mycoplasma bovis;
(5) Preparing a product for detecting whether a sample to be detected is infected with infectious bovine rhinotracheitis virus, bovine viral diarrhea virus and/or mycoplasma bovis;
The above-mentioned applications are for diagnosis and treatment of non-diseases.
8. A multiplex fluorescence PCR detection method for simultaneously detecting infectious bovine rhinotracheitis virus, bovine viral diarrhea virus and mycoplasma bovis comprises the following steps:
1) Extracting nucleic acid from a sample to be tested;
2) Performing multiplex fluorescence PCR amplification reaction with the nucleic acid of step 1) as a template and the reagent of any one of claims 1-3 or the kit of any one of claims 4-6, and collecting fluorescence signals;
3) Judging whether the sample contains infectious bovine rhinotracheitis virus, bovine viral diarrhea virus and mycoplasma bovis according to the fluorescent signal;
The above methods are useful for diagnosis and treatment of non-diseases.
9. The method according to claim 8, wherein the multiplex fluorescent PCR amplification reaction system in the step 2) is:
。
10. The method according to claim 9, wherein the multiplex fluorescent PCR amplification reaction procedure in step 2) is: 40-45 ℃ for 4-6 min; 94-96 ℃ for 25-35 s; 4-6 s at 90-95 ℃, 30s at 52-62 ℃ and 35-40 cycles.
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