CN113234860A - Dual detection primer and probe combination for bovine viral diarrhea virus and bovine parainfluenza virus type 3 - Google Patents
Dual detection primer and probe combination for bovine viral diarrhea virus and bovine parainfluenza virus type 3 Download PDFInfo
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Abstract
The invention relates to the technical field of biology, in particular to a dual detection primer and probe combination for bovine viral diarrhea virus and bovine parainfluenza virus type 3. The invention combines recombinase amplification technology and lateral flow chromatography technology, and the designed primer and probe composition can realize quick, sensitive and accurate detection of BVDV and BPIV 3. Experiments show that the primer probe provided by the invention can detect 36copies of nucleic acid with the lowest BVDV, can detect 34copies of nucleic acid with BPIV3, and can obtain a detection result at the earliest 10min after the reaction starts.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a dual detection primer and probe combination for bovine viral diarrhea virus and bovine parainfluenza virus type 3.
Background
Bovine viral diarrhea, also known as bovine viral diarrhea-mucosal disease (BVD-MD), is a strong infectious disease caused by Bovine Viral Diarrhea Virus (BVDV). The virus can infect pigs, deer, sheep, camels, rabbits and other wild ruminants besides cattle, more than 40 animals are reported to be infected at present, and hosts are quite wide. Bovine parainfluenza, also known as "shipping fever", is an acute respiratory disease caused by Bovine parainfluenza virus type 3 (BPIV 3). Infected animals are characterized by high fever, dyspnea, and cough, which can severely lead to cattle death. BPIV3 is frequently co-infected with BVDV, and together constitutes the Bovine respiratory disease syndrome (BRDC).
BRDC is the main cause of morbidity and mortality of barn feeding cattle in the century range, and economic loss brought to cattle herds is huge. Infection with BVDV and BPIV3 can cause a variety of clinical conditions and also creates conditions for infection of animals with other pathogens to cause secondary infections due to immunosuppression caused by viral infection, and thus the risk to the herd of cattle is not negligible.
With the development of national economy, the demand structure of domestic consumers for animal food such as meat and the like is changed, the domestic beef productivity is insufficient, and in order to meet the consumption requirements of domestic consumers for animal food such as beef, milk and the like, in recent years, the number of cattle, dairy cattle and beef products imported from Australia and New Zealand in China is increased sharply, and the possibility that BVDV and BPIV3 are introduced into China is increased along with the frequent import trade of cattle byproducts. Therefore, the quarantine and the prevention and the treatment of epidemic diseases of the large-scale cattle farm in China are strengthened. Therefore, the early detection of the pathogen is of great significance for reducing and treating the disease.
However, the two pathogens are usually detected separately at present, and a complicated experimental instrument such as a PCR instrument is needed, and the detection result is often not visualized and needs a professional instrument for interpretation. Therefore, the existing detection technology has complex required conditions and high requirements on the professional level of technicians, needs long time and is difficult to meet the detection requirements under the condition of a farm.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a primer and a probe combination for dual detection of bovine viral diarrhea virus and bovine parainfluenza virus type 3.
The invention provides an RPA detection primer and a probe combination of bovine viral diarrhea virus, which comprises the following components:
1, a forward primer of a nucleic acid sequence shown in SEQ ID NO;
a reverse primer of the nucleic acid sequence shown in SEQ ID NO. 2;
3, and a probe of a nucleic acid sequence shown in SEQ ID NO.
In the RPA detection primer and probe combination of the bovine viral diarrhea virus, the 5' end of the reverse primer is modified with Bio; 6-FAM is modified at the 5 'end of the probe, dSpacer is modified at the middle part of the probe, and C3Spacer is modified at the 3' end of the probe. The dSpacer modified in the middle refers to the dSpacer modified between the 32 th nucleic acid and the 33 th nucleic acid of the probe of the nucleic acid sequence shown in SEQ ID NO. 3.
The invention provides a primer and probe combination for detecting bovine parainfluenza virus type 3 RPA, which comprises the following components:
a forward primer of the nucleic acid sequence shown as SEQ ID NO. 4;
a reverse primer of the nucleic acid sequence shown in SEQ ID NO. 5;
a probe of a nucleic acid sequence shown as SEQ ID NO. 6.
In the RPA detection primer and probe combination of the bovine parainfluenza virus type 3, the 5' end of the reverse primer is modified with Dig; 6-FAM is modified at the 5 'end of the probe, dSpacer is modified at the middle part of the probe, and C3Spacer is modified at the 3' end of the probe. The dSpacer modified in the middle refers to the dSpacer modified between the 33 th nucleic acid and the 34 th nucleic acid of the probe of the nucleic acid sequence shown in SEQ ID NO. 6.
The invention verifies the software obtained by design, screens out several primers and probes with better effect, and then selects from the primers and probes, thereby obtaining the primer-probe combination with high sensitivity and strong specificity. The primers provided by the invention can realize accurate amplification of target fragments under a constant temperature condition, are not interfered with each other in the same system, and can be independently used or mixed in the same system.
The invention also provides a duplex detection kit for the bovine viral diarrhea virus and the bovine parainfluenza virus type 3, which comprises the primer and the probe combination.
In the kit of the present invention, the primer and the probe are present in the form of powder. Or in solution form, as the present invention is not limited in this respect. When it is present in the form of a solution, the concentration of each primer is 10. mu.M. The concentration of each probe was 10. mu.M.
The kit of the invention also comprises: RPA amplification reagent and lateral flow chromatography detection test paper;
the RPA amplification reagents comprise: rehydrationbuffer, ddH2O, magnesium acetate solution.
In some embodiments, the concentration of the magnesium acetate solution is 280 mM.
The lateral flow chromatography test strip is provided with a C line, a T1 test line and a T2 test line, wherein the C line is coated with anti-rabbit secondary antibody, the T1 test line is coated with anti-digoxin antibody, and the T2 test line is coated with avidin.
The kit also comprises an RNA extraction reagent and a reverse transcription reagent.
The invention also provides a non-diagnostic dual detection method of bovine viral diarrhea virus and bovine parainfluenza virus type 3, which is characterized by comprising the following steps:
extracting sample RNA and then carrying out reverse transcription to obtain cDNA;
mixing the cDNA with the primer and the probe combination, and carrying out amplification reaction to obtain an amplification product;
and detecting the amplification product by a lateral flow chromatography detection test strip, and judging whether the sample contains the virus or not according to the occurrence condition of the strip.
In the present invention, the sample is an environmental sample from a farm, and may be blood, tissue, secretion or excretion of a living animal.
The conditions of the amplification reaction include: reacting for 25min at 30-40 ℃;
the system of the amplification reaction comprises: 3. mu.L of cDNA, 2.1. mu.L of each of the upstream and downstream primers, and 0.6. mu. L, RehydrationBuffer 29.5.5. mu. L, ddH of each of the probes2O 5.4μL。
In the invention, the detection method also comprises the steps of detecting a positive control substance and a negative control substance;
the positive control substance is a plasmid containing a target fragment;
the negative control substance is water, PBS buffer solution or cell culture solution without virus;
the target fragment comprises a fragment obtained by amplifying the primer of the invention.
The detection method of the negative control substance or the positive control substance is the same as the detection method of the sample to be detected;
the judging whether the sample contains the virus according to the appearance condition of the strip comprises the following steps:
the lateral flow test strip for detecting the amplification product of the negative control sample only has a strip at the position of C, and the lateral flow test strip for detecting the amplification product of the positive control sample has strips at the positions of the C line, the T1 detection line and the T2 detection line, which indicates that the test is established, otherwise, the test fails;
two strips appear on the positions of a C line and a T1 detection line of a strip of a lateral flow test strip for detecting an amplification product of a sample to be detected, which indicates that the sample to be detected contains the bovine parainfluenza virus type 3;
two strips appear on the strip of the lateral flow test strip for detecting the amplification product of the sample to be detected at the positions of the C line and the T2 detection line, which indicates that the sample to be detected contains the bovine viral diarrhea virus;
strips appear on the positions of a C line, a T1 detection line and a T2 detection line of the lateral flow test strip for detecting the amplification product of the sample to be detected, which shows that the animal to be detected contains both bovine viral diarrhea virus and bovine parainfluenza virus type 3 virus;
a lateral flow test strip for detecting the amplification product of the sample to be detected only has a strip on the C line position, which shows that the sample to be detected is negative and does not contain bovine parainfluenza virus type 3 or bovine viral diarrhea virus.
The invention combines recombinase amplification technology and lateral flow chromatography technology, and the designed primer and probe composition can realize quick, sensitive and accurate detection of BVDV and BPIV 3. Experiments show that the primer probe provided by the invention can detect 36copies of nucleic acid with the lowest BVDV, can detect 34copies of nucleic acid with BPIV3, and can obtain a detection result at the earliest 10min after the reaction starts. The invention can be used for field detection and can take measures to treat clinical cases in the first time. The method is simple and convenient to operate, ordinary personnel can carry out detection, the technical threshold of detection is reduced, farmers and primary veterinarians outpatient doctors can carry out detection, and application and popularization are greatly facilitated. Has great clinical application value for the diagnosis, prevention and control of the bovine viral diarrhea virus and has wide market prospect.
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FIG. 1 shows the results of different sample tests;
FIG. 2 shows the results of BVDV-RPA-LFD primer and probe screening;
FIG. 3 shows the results of the BPIV3-RPA-LFD primer and probe screening;
FIG. 4 shows the results of the BVDV-RPA-LFD sensitivity test;
FIG. 5 shows the results of the BPIV3-RPA-LFD sensitivity test;
FIG. 6 shows the result of BVDV-BPIV3-RPA-LFD specificity experiment.
Detailed Description
The invention provides a dual detection primer and probe combination of bovine viral diarrhea virus and bovine parainfluenza virus type 3, and a person skilled in the art can use the contents to refer to the contents and appropriately improve process parameters to realize the dual detection. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
Among them, Rehydrationbuffer was purchased from twist Amp NFO kit (twist DxTM, Cambridge, UK), and lateral flow chromatography test strip was purchased from hybrid 2T (Milenia Biotec GmbH, Germany).
The invention is further illustrated by the following examples:
example 1
1. Primer and probe design: downloading BVDV and BPIV3 virus gene sequences on GenBank, performing sequence alignment by molecular biology software (BVDV virus comprises 18 sequences of type 1, type 2 and type 3 genes, BPIV3 virus comprises 18 sequences of type A, type B and type C genes), and selecting conserved regions to design primers and probes aiming at BVDV and BPIV3 virus according to RPA primer and probe design principles, wherein the following table is as follows:
TABLE 1 BVDV primer Probe List
TABLE 2 BPIV3 primer Probe List
2. The RNA of BVDV and BPIV3 virus is extracted and is reversely transcribed into cDNA, and the conventional nucleic acid extraction and reverse transcription reagents are adopted.
RPA reaction system and conditions: 2.1. mu.L of each of the upstream and downstream primers (10. mu.M), 0.6. mu.L of each probe, 0.5. mu.L of each probe, and 0.4. mu.L of each probe, 2. O.4. mu.L of each probe, and 3. mu.L of each probe; and (3) uniformly mixing the reaction system, adding the mixture into an RPA enzyme reaction tube, adding 2.5 mu L of magnesium acetate, uniformly mixing, reacting at the constant temperature of 30-40 ℃ for 30min, detecting by using a flow measurement chromatography detection test strip, and judging the result.
And (3) judging standard:
if the lateral flow test strip in the negative control sample shows a strip only at the position of C, and the lateral flow test strip in the positive control sample shows a strip at the positions of C line, T1 detection line and T2 detection line, the test is established.
When two strips appear on the strip of the lateral flow test strip in the detection sample at the positions of the C line and the T1 detection line, the bovine parainfluenza virus type 3 is shown to be contained in the animal to be detected;
when two strips appear on the strip of the lateral flow test strip in the detection sample at the positions of the C line and the T2 detection line, the result shows that the animal to be detected contains the bovine viral diarrhea virus;
when the lateral flow test strip in the detection sample has strips at the positions of the C line, the T1 detection line and the T2 detection line, the result shows that the animal to be detected contains both bovine viral diarrhea virus and bovine parainfluenza virus type 3 virus;
when a strip of the lateral flow test strip in the detected sample only appears on the C line, the sample to be detected is negative and does not contain bovine parainfluenza virus type 3 or bovine viral diarrhea virus. For example, see FIG. 1 for BVDV-BPIV3-RPA-LFD test results.
3. Screening of primer probes
Pairwise matching and combining the designed primers and probes, carrying out tests according to the reaction system and conditions, and detecting and screening the optimal primer probe through a flow-measuring chromatography test strip:
the results of BVDV primer probe screening are shown in FIG. 2: the most clear band amplified by F3+ R3+ P2 is the best combination of primer and probe. The combination of F2+ R2+ P1 did not detect the amplification result, but F1+ R1+ P1 and F4+ R4+ P2 could amplify the result, but the band was not bright as F3+ R3+ P2, indicating that the amplification effect was not good. Therefore, the best primer and probe for detecting the BVDV is F3+ R3+ P2.
The results of the BPIV3 primer probe screening are shown in FIG. 3: the amplified band of F2+ R1+ P1 is clearest, and the composition is used as the optimal combination of the primer and the probe. F4+ R2+ P2 can detect amplification, but the result is slightly inferior to that of F2+ R1+ P1, while F1+ R1+ P1 and F3+ R2+ P2 can amplify the result, but the band is very weak, and the result is not easy to observe, so that the optimal primer and probe for detecting BPIV3 are F2+ R1+ P1.
4. Experiments were determined for different reaction temperatures of RPA-LFD: the reaction system was prepared as described above, and the reaction tube was placed under different temperature conditions (25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃) to determine the optimum reaction temperature. The results were obtained under the reaction conditions of 30 to 45 ℃, but the optimal temperature was determined to be 35 ℃ because the band could be detected more rapidly under the condition of 35 ℃.
5. Experiments were determined for different reaction times of RPA-LFD: the reaction system is prepared according to the method, the reaction is finished at different time after the preparation of the reaction system is finished, and the optimal reaction time is determined by applying the test result of the test paper strip. Results were obtained after 10min of reaction, but the optimal time was finally determined to be 25min in order to ensure the accuracy of the detection and avoid the occurrence of false negatives.
6. Sensitivity test of RPA-LFD:
preparing BVDV standard plasmid, obtaining target fragment of bovine viral diarrhea virus by primer BLT/BRT and RT-PCR method, wherein the sequence of the primer is
BLT: 5'CATGCCCATAGTAGGAC 3'; BRT: 5'CCATGTGCCATGTACAG3', the size of the amplified fragment is about 282bp, the target fragment is inserted into pMD18-T vector, standard plasmid is constructed and sequenced for verification, and the concentration is determined according to the formula (copy number copies/. mu.L ═ 6.02X 10)23X (concentration) ng/. mu.L.times.10-9DNA length × 660) determined standard plasmid copy number 3.6 × 107copies/. mu.L. The sensitivity of BVDV was determined by 10-fold dilution and RPA-LFD using this as template, and the result showed that the lowest detection rate was 36copy, as shown in FIG. 4.
BPIV3 standard plasmid was made by primer:
BPIV3-P-F:GGAAGAGAATCACATCCTGGAAC
BPIV3-P-F:CTTCTRTGTGATTCATCCAT，
the size of the amplified fragment is about 451bp, the target fragment is inserted into a pMD18-T vector, a standard plasmid is constructed and sequenced for verification, and the concentration is determined according to the formula (copy number copies/. mu.L ═ 6.02X 10)23X (concentration) ng/. mu.L.times.10-9DNA length × 660) determined standard plasmid copy number of 3.4 × 107copies/. mu.L. After 10-fold dilution, the sensitivity of BPIV3 was determined by using the template for RPA-LFD, and the result showed that the lowest detection rate was 34copy, which is shown in FIG. 5.
7. Specificity experiments for RPA-LFD: BVDV1 virus, BPIV3 virus, CSFV virus, IBRV virus, BRSV virus, BRV virus and BCOV virus are taken, the virus nucleic acid is extracted by the method, each reagent is added according to the requirement of a reaction system to carry out RPA-LFD experiment, and the specificity of the primer and the probe is determined. The result shows that only the BVDV1 and BPIV3 samples have bands, the result is positive, the rest samples are consistent with the negative samples, and no band is negative. The results are shown in FIG. 6, which shows that the primers and reagents of the present invention have good specificity.
6. And (3) detecting a clinical sample:
in order to examine the detection effect of the BVDV-BPIV3 dual RPA-LFP detection method established by the invention on actual samples, 50 clinical blood samples and nasal swabs are randomly collected from clinic, and BVDV and BPIV-3 detection is carried out by applying RT-PCR and the method established by the invention, and the detection result is shown in Table 3. The positive detection rate of the BVDV sample is higher than that of the traditional RT-PCR by applying the BVDV-BPIV3 dual RPA-LFP detection method, the positive detection rate of the BPIV3 is consistent with that of the traditional RT-PCR, but the method used in the research is simple and convenient, a PCR instrument and electrophoresis detection are not needed, the result judgment is visible by naked eyes, the whole detection time is obviously reduced compared with that of the traditional PCR, and the method is very suitable for detecting in the cattle field.
TABLE 3 test results of clinical samples
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.
Sequence listing
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Claims (10)
1. The RPA detection primer and probe combination of the bovine viral diarrhea virus comprises the following components:
1, a forward primer of a nucleic acid sequence shown in SEQ ID NO;
a reverse primer of the nucleic acid sequence shown in SEQ ID NO. 2;
3, and a probe of a nucleic acid sequence shown in SEQ ID NO.
2. The primer-probe combination according to claim 1,
the 5' end of the reverse primer is modified with Bio;
6-FAM is modified at the 5 'end of the probe, dSpacer is modified at the middle part of the probe, and C3Spacer is modified at the 3' end of the probe.
3. An RPA detection primer and probe combination of bovine parainfluenza virus type 3 comprises:
a forward primer of the nucleic acid sequence shown as SEQ ID NO. 4;
a reverse primer of the nucleic acid sequence shown in SEQ ID NO. 5;
a probe of a nucleic acid sequence shown as SEQ ID NO. 6.
4. The primer-probe combination according to claim 3,
the 5' end of the reverse primer is modified with Dig;
6-FAM is modified at the 5 'end of the probe, dSpacer is modified at the middle part of the probe, and C3Spacer is modified at the 3' end of the probe.
5. A dual detection kit for bovine viral diarrhea virus and bovine parainfluenza virus type 3, comprising the primer and probe combination of claims 1-4.
6. The duplex detection kit of claim 3, further comprising: RPA amplification reagent and lateral flow chromatography detection test paper;
the RPA amplification reagents comprise: rehydrationbuffer, ddH2O, magnesium acetate solution;
the lateral flow chromatography detection test strip is provided with a C line, a T1 detection line and a T2 detection line, wherein the C line is coated with anti-rabbit secondary antibody, the T1 detection line is coated with anti-Dig antibody, and the T2 detection line is coated with biotin ligand.
7. The dual assay kit of claim 3, further comprising an RNA extraction reagent and a reverse transcription reagent.
8. A dual detection method for non-diagnostic bovine viral diarrhea virus and bovine parainfluenza virus type 3, comprising:
extracting sample RNA and then carrying out reverse transcription to obtain cDNA;
mixing the cDNA with the primer and probe combination of claims 1-4, and performing an amplification reaction to obtain an amplification product;
and detecting the amplification product by a lateral flow chromatography detection test strip, and judging whether the sample contains the virus or not according to the occurrence condition of the strip.
9. The detection method according to claim 8,
the conditions of the amplification reaction include: reacting for 25min at 30-40 ℃;
the system of the amplification reaction comprises: 3 uL cDNA, 2.1 uL upstream and downstream primers, 0.6 u L, RehydrationBuffer 29.5.5 u L, ddH probe2O 5.4μL。
10. The assay of claim 8, further comprising detecting a positive control and a negative control;
the positive control substance is a plasmid containing a target fragment;
the negative control substance is water, PBS buffer solution or cell culture solution without virus;
the judging whether the sample contains the virus according to the appearance condition of the strip comprises the following steps:
the lateral flow test strip for detecting the amplification product of the negative control sample only has a strip at the position of C, and the lateral flow test strip for detecting the amplification product of the positive control sample has strips at the positions of the C line, the T1 detection line and the T2 detection line, which indicates that the test is established, otherwise, the test fails;
two strips appear on the positions of a C line and a T1 detection line of a strip of a lateral flow test strip for detecting an amplification product of a sample to be detected, which indicates that the sample to be detected contains the bovine parainfluenza virus type 3;
two strips appear on the strip of the lateral flow test strip for detecting the amplification product of the sample to be detected at the positions of the C line and the T2 detection line, which indicates that the sample to be detected contains the bovine viral diarrhea virus;
strips appear on the positions of a C line, a T1 detection line and a T2 detection line of the lateral flow test strip for detecting the amplification product of the sample to be detected, which shows that the animal to be detected contains both bovine viral diarrhea virus and bovine parainfluenza virus type 3 virus;
a lateral flow test strip for detecting the amplification product of the sample to be detected only has a strip on the C line position, which shows that the sample to be detected is negative and does not contain bovine parainfluenza virus type 3 or bovine viral diarrhea virus.
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| CN202110603732.XA Active CN113234860B (en) | 2021-05-31 | 2021-05-31 | Dual detection primer and probe combination for bovine viral diarrhea virus and bovine parainfluenza virus type 3 |
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| CN117106985A (en) * | 2023-10-17 | 2023-11-24 | 苏州依科赛生物科技股份有限公司 | BVDV rapid detection kit and detection method thereof |
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| CN117106985A (en) * | 2023-10-17 | 2023-11-24 | 苏州依科赛生物科技股份有限公司 | BVDV rapid detection kit and detection method thereof |
| CN117106985B (en) * | 2023-10-17 | 2024-01-26 | 苏州依科赛生物科技股份有限公司 | BVDV rapid detection kit and detection method thereof |
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