CN110904272A - Primer probe combination, kit and method for simultaneously detecting multiple pathogenic microorganisms - Google Patents
Primer probe combination, kit and method for simultaneously detecting multiple pathogenic microorganisms Download PDFInfo
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Abstract
The invention provides a primer probe combination, a kit and a method for simultaneously detecting a plurality of pathogenic microorganisms, which comprises the following steps: the primer pair and the probe sequence for detecting the reovirus are shown as SEQ ID NO: 1-3; the primer pair and the probe sequence for detecting the bovine parvovirus are shown as SEQ ID NO: 4-6; the primer pair and the probe sequence for detecting the bluetongue virus are shown as SEQ ID NO: 7-9; the primer pair and the probe sequence for detecting the bovine diarrhea virus are shown as SEQ ID NO: 10-12; the primer pair and the probe sequence for detecting the bovine polyoma virus are shown as SEQ ID NO: 13-15; and a primer pair for detecting the rabies virus, and a probe sequence of the primer pair is shown as SEQ ID NO: 16-18. According to the invention, a primer probe combination, a kit and a method for detecting multiple pathogenic microorganisms simultaneously, which are simple, rapid and high in sensitivity, are provided.
Description
Technical Field
The invention relates to the technical field of microorganism detection, in particular to a primer probe combination, a kit and a method for simultaneously detecting multiple pathogenic microorganisms.
Background
The emergence of biologics is of particular interest for the improvement of human health and quality of life. With the development of biological medicine in China, the demand for serum is increased at a rate of 25% per year, and the external dependence is higher. Bovine serum is used in a large amount in vaccine production, the quality of the bovine serum directly affects the quality safety of biological products, and particularly, the quality safety of the products can be seriously affected by the pollution of virus bioactive substances. Harasawa et al, Tokyo university, Japan reported that bovine diarrhea virus (BVDV) RNA was detected in 5 batches of human leprosy and rubella vaccine preparations. Wessman et al reported that contamination of various cell lines, such as fetal bovine kidney cells, bovine turbinate cells, porcine kidney cells, feline kidney nucleus Vero cells, has resulted from the use of fetal bovine serum contaminated with BVDV.
Bovine diarrhea virus is one of common pollutants and serious exogenous pathogens in vaccine production, so that the detection of BVDV in bovine serum is particularly important. In addition, Reovirus (REOV), Bovine Parvovirus (BPV), bluetongue virus (BTV), bovine polyoma virus (BPyV), and Rabies virus (Rabies virus, RABV) are also important pathogenic exogenous factors in bovine serum, and the establishment of a rapid detection method for exogenous viruses is helpful for controlling the bovine serum quality and preventing the pollution of exogenous viruses because the quality of bovine serum is important in biological product quality.
However, traditional detection methods can only detect a single pathogen at a time, and therefore establishing a rapid, high-throughput detection method is crucial to the safety of biological products. The trend of research at present is further turning to the detection of multiple pathogenic microorganisms or the detection of the same pathogenic microorganism of multiple animals on one detection platform, so as to shorten the detection period and reduce the detection cost. Therefore, a high-throughput detection technology capable of simultaneously, rapidly and effectively detecting various pathogenic microorganisms has become a hotspot of research in the fields of biological product pathogen detection, public safety and the like.
Disclosure of Invention
The invention aims to provide a primer probe combination, a reagent kit and a method for simultaneously detecting multiple pathogenic microorganisms, so as to solve the problem that the prior art lacks a method for quickly and effectively simultaneously detecting reovirus, bovine parvovirus, bluetongue virus, bovine diarrhea virus, bovine polyoma virus and rabies virus.
In order to solve the technical problems, the invention adopts the following technical scheme:
according to a first aspect of the present invention, there is provided a primer probe combination for simultaneously detecting a plurality of pathogenic microorganisms, the primer probe combination comprising: for detecting reovirus as set forth in SEQ ID NO: 1-2, as shown in SEQ ID NO: 3, a specific probe; as shown in SEQ ID NO: 4-5, as shown in SEQ ID NO: 6, a specific probe; the nucleotide sequence shown as SEQ ID NO: 7-8, as shown in SEQ ID NO: 9, a specific probe; the nucleotide sequence shown as SEQ ID NO: 10-11, as shown in SEQ ID NO: 12, or a specific probe; for detecting bovine polyoma virus as shown in SEQ ID NO: 13-14, as shown in SEQ ID NO: 15, a specific probe; and a nucleic acid sequence as shown in SEQ ID NO: 16-17, as shown in SEQ ID NO: 18, or a specific probe as shown in figure 18.
Preferably, the active organic group modified at the 5' end of the specific probe is an amino group.
Preferably, the sequence of SEQ ID NO: the 5' ends of 1, 5, 7, 10, 13 and 16 are modified with biotin labels.
According to a second aspect of the present invention, there is provided a kit for simultaneously detecting a plurality of pathogenic microorganisms, the kit comprising a primer probe combination as described above for simultaneously detecting a plurality of pathogenic microorganisms.
Most preferably, the molar concentration ratio of the 12 specific primers is: SEQ ID NO. 1: 2: 4: 5: 7: 8: 11: 13: 14: 16: 1.5: 2.0: 2.5: 2.0.
According to the primer probe combination and the reagent kit for simultaneously detecting multiple pathogenic microorganisms, the corresponding accession number of the reovirus to be targeted on Genbank is NC-015135.1, the accession number of bovine parvovirus is NC-001540.1, the accession number of bluetongue virus is MK348546.1, the accession number of bovine diarrhea virus is U18059.1, the accession number of bovine polyoma virus is NC-001442.1, and the accession number of rabies virus is NC-001542.1.
According to a third aspect of the present invention, there is provided a method for detecting microorganisms by liquid chip technology, comprising the steps of: s1, respectively designing and synthesizing corresponding reovirus probes with nucleotide sequences shown as SEQ ID NO.3, bovine parvovirus probes with nucleotide sequences shown as SEQ ID NO.6, bluetongue virus probes with nucleotide sequences shown as SEQ ID NO.9, bovine diarrhea virus probes with nucleotide sequences shown as SEQ ID NO.12, bovine polyoma virus probes with nucleotide sequences shown as SEQ ID NO.15 and rabies virus probes with nucleotide sequences shown as SEQ ID NO.18-19 according to the gene sequences of reovirus, bovine parvovirus, bluetongue virus, bovine enterovirus, bovine polyoma virus and rabies virus; s2, connecting a reovirus probe, a bovine parvovirus probe, a bluetongue virus probe, a bovine diarrhea virus probe, a bovine polyoma virus probe and a rabies virus probe with first, second, third, fourth, fifth and sixth fluorescent coding microspheres respectively to obtain probe solutions of first, second, third, fourth, fifth and sixth coupling microspheres respectively; s3, carrying out DNA extraction and PCR amplification on reovirus, bovine parvovirus, bluetongue virus, bovine diarrhea virus, bovine polyoma virus and rabies virus negative samples to obtain PCR products, mixing the probe solutions of the first, second, third, fourth, fifth and sixth coupling microspheres to form a probe solution of mixed coupling microspheres, adding the PCR products into the probe solution of the mixed coupling microspheres, reading signals in a Luminex instrument, and determining a threshold value according to the signals; s4, obtaining a PCR product to be detected by DNA extraction and PCR amplification of a sample to be detected, adding the PCR product to be detected into a probe solution of the hybrid coupling microsphere, reading a signal in a Luminex instrument, and comparing the signal with a threshold value to judge whether the sample to be detected is negative or positive.
Preferably, the Tm value of the reovirus probe, the bovine parvovirus probe, the bluetongue virus probe, the bovine diarrhea virus probe, the bovine polyoma virus probe and the rabies virus probe is kept between 56 ℃ and 58 ℃.
Preferably, the specific method for obtaining the PCR product to be detected comprises the following steps: providing SEQ ID NO: 1-2 for detecting bovine parvovirus SEQ ID NO: 4-5, and is used for detecting the nucleotide sequence shown in SEQ ID NO: 7-8 for detecting bovine diarrhea virus of SEQ ID NO: 10-11, for detecting the nucleotide sequence shown in SEQ ID NO: 13-14, and the nucleotide sequence shown in SEQ ID NO: 16-17, wherein the nucleotide sequence shown in SEQ ID NO: the 5' ends of 1, 5, 7, 10, 13 and 16 are modified with biotin labels; p2, amplifying by using the specific primer pair to respectively obtain a reovirus target gene, a bovine parvovirus target gene, a bluetongue virus target gene, a bovine diarrhea virus target gene, a bovine polyoma virus target gene, a rabies virus target gene and a multiple PCR product containing the reovirus target gene, the bovine parvovirus target gene, the bluetongue virus target gene, the bovine diarrhea virus target gene, the bovine polyoma virus target gene and the rabies virus target gene.
According to the primer probe combination and the kit for simultaneously detecting multiple pathogenic microorganisms provided by the invention, specific primers and specific probes for the six viruses are respectively designed aiming at the gene sequences of the selected reovirus, bovine parvovirus, bluetongue virus, bovine diarrhea virus, bovine polyoma virus and rabies virus, the multiple PCR technology is adopted, the six pathogens are quickly and effectively detected simultaneously, the sample demand is small, the method for detecting multiple pathogenic microorganisms is simple, quick, high in sensitivity and easy to popularize, the cost of the kit is low, the application range is wide, and the clinical diagnosis requirements of diseases and the pathogen detection requirements of the sample can be met simultaneously. The invention also establishes a liquid chip detection method capable of specifically detecting the REOV, BPV, BTV, BVDV, BPyV and RABV by using a liquid chip technology, and the detection method can be used for quickly detecting the target gene, the detection sensitivity of the detection method reaches 50 copies/reaction, and the specificity is good.
In a word, the invention provides a primer probe combination, a kit and a method for detecting various pathogenic microorganisms simultaneously, which are simple, rapid, high in sensitivity and easy to popularize.
Drawings
FIG. 1 shows the results of the detection of reovirus, bovine parvovirus, bluetongue virus, bovine diarrhea virus, bovine polyoma virus and rabies virus by single primer pair and multiple primer pair, wherein the following steps are performed from left to right: the kit comprises a standard, REOV, BPV, BTV, BVDV, BPyV and RABV bands amplified by a single pair of primers, a negative control, the standard, REOV, BPV, BTV, BVDV, BPyV and RABV bands amplified by a plurality of primers and the negative control.
Detailed Description
The present invention will be further described with reference to the following specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions. The reagents and starting materials used in the present invention are commercially available.
Streptomycin-avidin-phycoerythrin (SAPE) was purchased from Invitrogen, usa; surface carboxylated fluorescent-encoded microspheres were purchased from Bio-Rad, USA; the Luminex 200 liquid phase chip detector is a product of Luminex company of America; REOV, BPV, BTV, BVDV, BPyV and RABV plasmids are stored in Shanghai customs animal quarantine laboratories, all plasmids are cloned on a PUC57 vector, the prepared plasmids are prepared into 500 copies/mu L concentration by double-pure water and stored at-20 ℃ for later use.
Example 1: preparation of primers and probes
Sequence analysis, Primer and probe design are carried out by applying Primer 5.0 sequence analysis software according to the gene sequences of reovirus, bovine parvovirus, bluetongue virus, bovine diarrhea virus, bovine polyoma virus and rabies virus issued by GenBank, and the method comprises the following steps: SEQ ID NO: 1-18, wherein the primer SEQ ID NO: 1, 5, 7, 10, 13, 16, which are labeled with biotin at the 5 'end and poly-T long arms at the 5' end, and are simultaneously modified with amino groups, were synthesized by Shanghai Biotechnology (Shanghai) GmbH.
TABLE 1 primer and Probe sequences
Example 2: detection of amplification Performance of Single primer and multiplex primers
2.1 formulation of the Single PCR reaction solution was 10 × buffer (Mg)2+) 2.5. mu.L, 1.0. mu.L of dNTP (2.5 mol/L), 1.0. mu.L of primer, 2.0. mu.L of template, 0.5U of Hotstart Taq, ddH2O18.5. mu.L, 25. mu.L for the total PCR system.
2.2 establishment of multiplex PCR amplification System: the preparation scheme of the multiplex primer is that each primer is mixed according to the following optimized molar concentration ratio to prepare a primer stock solution, and the primer stock solution is stored at 100uM and 20 ℃ below zero.
TABLE 2 primer mixture formula
| Primer name | Serial number | Input amount |
| reoF | SEQ ID NO：1 | 1.5 |
| reoR | SEQ ID NO：2 | 1.5 |
| bpvF | SEQ ID NO：4 | 2.0 |
| bpvR | SEQ ID NO：5 | 2.0 |
| btvF | SEQ ID NO：7 | 3.0 |
| btvR | SEQ ID NO：8 | 3.0 |
| bvdvF | SEQ ID NO：10 | 2.0 |
| bvdvR | SEQ ID NO：11 | 2.0 |
| bpyvF | SEQ ID NO：13 | 1.5 |
| bpyvR | SEQ ID NO：14 | 1.5 |
| rabF | SEQ ID NO：16 | 2.0 |
| rabR | SEQ ID NO：17 | 2.0 |
TABLE 3 multiplex PCR reaction System
PCR amplification procedure: pre-denaturation at 95 ℃ for 10min, 30s at 95 ℃, 30s at 52 ℃, 20s at 72 ℃ for 35 cycles, and re-extension at 72 ℃ for 1 min. After the reaction, 5. mu.L of the LPCR product was electrophoresed on a 1.5% agarose gel in an upward direction.
As a result: the results of the detection of the reference products in the target region by single primer pair and multiple primer pair amplification electrophoresis are shown in fig. 1, and show that the multiple amplification system can effectively and specifically amplify the target gene, wherein the amplification products are 115bp, 79bp, 113bp, 143bp, 77bp and 117bp in sequence according to the sequence of reovirus, bovine parvovirus, bluetongue virus, bovine diarrhea virus, bovine polyoma virus and rabies virus.
Example 3: crosslinking of microsphere probes
Taking 100 mu L of corresponding fluorescent coding microspheres (table 2), centrifuging at 5000rpm for 10min, then discarding the supernatant, and washing with double distilled water for 2 times; resuspending with 100. mu.L of 0.1mol/L MES (pH6.0), adding 3. mu.L of 0.2 nmol/μ L probe, mixing, adding 15% EDC, and incubating at 37 deg.C in dark for 30 min; the supernatant was discarded by centrifugation, washed 2 times with 0.1% SDS, resuspended in 50. mu.L of TE buffer (pH8.0), and stored at 4 ℃ in the dark for further use.
TABLE 4 Probe names and corresponding encoded microspheres
| Name of probe | Encoded microspheres |
| REOVP | 21# |
| BPVP | 28# |
| BTVP | 32# |
| BVDVP | 37# |
| BPyVP | 38# |
| RABVP | 45# |
Example 4: performing hybridization capture detection, namely taking 5 mu L of PCR product, adding 25 mu L of microsphere solution (each microsphere is 2000/25 mu L), fully and uniformly mixing, then performing denaturation at 94 ℃ for 3min, and then performing incubation at 50 ℃ for 30 min; then 70. mu.L of SA-PE (concentration 0.2. mu.g/. mu.L) was added, incubation was continued for 20min, and the Luminex 200 hotplate was heated to 50 ℃ and adjusted to the degree state. After the incubation was complete, the signal was directly read by machine. According to the judgment standard recommended by Luminex company, when the number of each fluorescent coding microsphere is not less than 20 and the background blank fluorescence intensity is not higher than 300, the test is established, and the result judgment is carried out.
Example 5: detection of Probe specificity six types of microspheres were mixed for each 25. mu.L reaction system, and each pair of single primers was hybridized to detect the specificity of the probe.
TABLE 5 Probe specificity test results
| Reo | BPV | BTV | BVDV | BPyV | Rab | |
| REOV | 2537 | 13 | 22 | 19 | 33 | 35 |
| BPV | 23 | 1622 | 12 | 23 | 6 | 9 |
| BTV | 50 | 12 | 1591 | 25 | 6 | 14 |
| BVDV | 21 | 19 | 9 | 2718 | 21 | 12 |
| BPyV | 34 | 16 | 15 | 23 | 5601 | 16 |
| RABV | 35 | 24 | 10 | 24 | 6 | 1751 |
The data show that the mixed six probes can effectively perform identification capture on a single pair of primer amplification fragments, each probe has no obvious non-specific hybridization signal on the other two target products, and the average fluorescence intensity (MFI) of each probe is lower than 100. The designed probe sequence was shown to have better specificity for six amplification products.
Example 6: the establishment of the liquid phase chip detection system uniformly mixes and hybridizes the probe with good specificity and the multiple PCR products, and an instrument is set to simultaneously detect 6 kinds of fluorescent coding microspheres. And (3) taking 20 negative samples, detecting by adopting an established liquid phase chip method, calculating the mean and standard deviation of the Mean Fluorescence Intensity (MFI) of each specific detection microsphere, taking the value obtained by the mean plus 3 times of the standard deviation as the cut-off value of the corresponding detection index, determining a threshold value, and determining the value as positive if the value is higher than the cut-off value.
As a result: 20 negative samples determined by other methods are subjected to DNA extraction and PCR amplification. The obtained PCR product was used for detection on a liquid phase chip, and the mean and standard deviation of MFI were measured by the liquid phase chip method, and the threshold was calculated to be 42.15+3 × 10.62 — 74.01, as shown in table 6 below.
TABLE 6 determination of threshold values for liquid-phase chip detection systems
Example 7: the liquid phase chip detection system specifically extracts positive data determined by other methods: DNA of bovine diarrhea virus, bovine parvovirus, bluetongue virus, reovirus, bovine polyoma virus, rabies virus, bovine parainfluenza virus type 3, infectious rhinotracheitis virus and akabane disease is used as a PCR amplification template, and hybridization detection is carried out by using an established liquid phase chip method.
As a result: as shown in the following table 7, the specific detection results show that the established liquid chip detection method has specific hybridization reactions on Reo, BPV, BTV, BVDV, BPyV and Rab, and is positive; and the method has no hybridization reaction with other important pathogenic virus plasmids of experimental animals, which shows that the method has good specificity.
TABLE 7 detection of specificity of liquid-phase chip detection system
| Target gene | Reo | BPV | BTV | BVDV | BPyV | Rab |
| Reo | 2342 | 14 | 7 | 51 | 6 | 32 |
| BPV | 27 | 1833 | 7 | 47 | 12 | 13 |
| BTV | 25 | 11 | 1292 | 43 | 7 | 19 |
| BVDV | 30 | 50 | 20 | 2111 | 10 | 29 |
| BPyV | 15 | 14 | 10 | 25 | 4784 | 12 |
| Rab | 34 | 41 | 18 | 34 | 9 | 1395 |
| BPI3 | 17 | 14 | 16 | 9 | 11 | 21 |
| AKAV | 29 | 33 | 15 | 25 | 17 | 35 |
| IBRV | 15 | 10 | 18 | 16 | 17 | 26 |
Example 8: the sensitivity of the liquid chip detection system is to dilute plasmids BVDV, BPV, BTV, REOV, BPyV and RABV by 5 times, 10 times and 50 times, respectively serve as PCR amplification templates, and the established liquid chip detection system is used for detection.
As a result: as shown in Table 8 below, the sensitivity of detection of the established liquid-phase chip detection system was approximately 50 copies/reaction.
TABLE 8 detection of sensitivity of liquid-phase chip detection system
| Sensitivity of the probe | REOV | BPV | BTV | BVDV | BPyV | RABV |
| 10 copies/reaction | 58 | 6 | 12 | 13 | 21 | 24 |
| 22 | 110 | 14 | 33 | 14 | 15 | |
| 12 | 9 | 89 | 18 | 20 | 29 | |
| 9 | 13 | 25 | 77 | 16 | 32 | |
| 21 | 13 | 17 | 16 | 211 | 15 | |
| 20 | 31 | 10 | 12 | 14 | 98 | |
| 50 copies/reaction | 125 | 13 | 31 | 35 | 28 | 16 |
| 17 | 279 | 25 | 13 | 11 | 19 | |
| 35 | 37 | 263 | 31 | 33 | 8 | |
| 32 | 17 | 36 | 303 | 31 | 25 | |
| 26 | 15 | 23 | 32 | 1036 | 18 | |
| 16 | 15 | 28 | 41 | 27 | 301 | |
| 100 copies/reaction | 1153 | 28 | 52 | 35 | 33 | 8 |
| 12 | 1360 | 25 | 22 | 28 | 18 | |
| 25 | 14 | 1423 | 25 | 26 | 22 | |
| 19 | 24 | 19 | 1989 | 31 | 38 | |
| 35 | 26 | 32 | 12 | 3306 | 25 | |
| 26 | 19 | 9 | 14 | 35 | 1075 | |
| 500 copies/reaction | 2078 | 22 | 23 | 51 | 45 | 27 |
| 35 | 1631 | 33 | 25 | 31 | 22 | |
| 44 | 15 | 1465 | 18 | 25 | 27 | |
| 19 | 18 | 25 | 2333 | 21 | 40 | |
| 31 | 33 | 21 | 25 | 4506 | 28 | |
| 33 | 25 | 26 | 15 | 35 | 1267 |
The above embodiments are merely preferred embodiments of the present invention, which are not intended to limit the scope of the present invention, and various changes may be made in the above embodiments of the present invention. All simple and equivalent changes and modifications made according to the claims and the content of the specification of the present application fall within the protection scope of the claims of the present patent application. The invention has not been described in detail in order to avoid obscuring the invention.
SEQUENCE LISTING
<110> Shanghai customs animal and plant and food inspection and quarantine technology center
<120> primer probe combination, kit and method for simultaneously detecting multiple pathogenic microorganisms
<160>18
<170>PatentIn version 3.5
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Claims (8)
1. A primer probe combination for simultaneously detecting a plurality of pathogenic microorganisms is characterized by comprising:
for detecting reovirus as set forth in SEQ ID NO: 1-2, as shown in SEQ ID NO: 3, a specific probe;
as shown in SEQ ID NO: 4-5, as shown in SEQ ID NO: 6, a specific probe;
the nucleotide sequence shown as SEQ ID NO: 7-8, as shown in SEQ ID NO: 9, a specific probe;
the nucleotide sequence shown as SEQ ID NO: 10-11, as shown in SEQ ID NO: 12, or a specific probe;
as shown in SEQ ID NO: 13-14, as shown in SEQ ID NO: 15, a specific probe; and
as shown in SEQ ID NO: 16-17, as shown in SEQ ID NO: 18, or a specific probe as shown in figure 18.
2. The primer probe combination for simultaneously detecting multiple pathogenic microorganisms according to claim 1, wherein the active organic group modified at the 5' end of the specific probe is an amino group.
3. The primer probe combination for simultaneously detecting multiple pathogenic microorganisms according to claim 1, wherein the specific primers have the sequences shown in SEQ ID NO: the 5' ends of 1, 5, 7, 10, 13 and 16 are modified with biotin labels.
4. A kit for simultaneously detecting a plurality of pathogenic microorganisms, which comprises the primer probe combination for simultaneously detecting a plurality of pathogenic microorganisms according to any one of claims 1 to 3.
5. The kit according to claim 4, wherein the molar concentration ratio of the 12 specific primers is: SEQ ID NO. 1: 2: 4: 5: 7: 8: 11: 13: 14: 16: 1.5: 2.0: 2.5: 2.0.
6. A method for detecting microorganisms by a liquid-phase chip technology is characterized by comprising the following steps:
s1, respectively designing and synthesizing corresponding reovirus probes with the nucleotide sequence shown in SEQ ID NO.3, bovine parvovirus probes with the nucleotide sequence shown in SEQ ID NO.6, bluetongue virus probes with the nucleotide sequence shown in SEQ ID NO.9, bovine diarrhea virus probes with the nucleotide sequence shown in SEQ ID NO.12, bovine polyoma virus probes with the nucleotide sequence shown in SEQ ID NO.15 and rabies virus probes with the nucleotide sequence shown in SEQ ID NO.18 according to the gene sequences of reovirus, bovine parvovirus, bluetongue virus, bovine diarrhea virus, bovine polyoma virus and rabies virus;
s2, connecting a reovirus probe, a bovine parvovirus probe, a bluetongue virus probe, a bovine diarrhea virus probe, a bovine polyoma virus probe and a rabies virus probe with first, second, third, fourth, fifth and sixth fluorescent coding microspheres respectively to obtain probe solutions of first, second, third, fourth, fifth and sixth coupling microspheres respectively;
s3, carrying out DNA extraction and PCR amplification on reovirus, bovine parvovirus, bluetongue virus, bovine diarrhea virus, bovine polyoma virus and rabies virus negative samples to obtain PCR products, mixing the probe solutions of the first, second, third, fourth, fifth and sixth coupling microspheres to form a probe solution of mixed coupling microspheres, then adding the PCR products into the probe solution of the mixed coupling microspheres, reading signals in a Luminex instrument, and determining a threshold value according to the signals;
s4, obtaining a PCR product to be detected by DNA extraction and PCR amplification of a sample to be detected, adding the PCR product to be detected into a probe solution of the mixed coupling microspheres, reading a signal in a Luminex instrument, and comparing the signal with a threshold value to judge whether the sample to be detected is negative or positive.
7. The method according to claim 6, wherein the Tm value of the reovirus probe, bovine parvovirus probe, bluetongue virus probe, bovine diarrhea virus probe, bovine polyoma virus probe, rabies virus probe is maintained at 56 ℃ to 58 ℃.
8. The method of claim 6, wherein the specific method for obtaining the PCR product to be tested comprises the following steps:
p1, SEQ ID NO: 1-2 for detecting bovine parvovirus SEQ ID NO: 4-5, and is used for detecting the nucleotide sequence shown in SEQ ID NO: 7-8, and is used for detecting the nucleotide sequence shown in SEQ ID NO: 10-11, for detecting the nucleotide sequence shown in SEQ ID NO: 13-14, and the nucleotide sequence shown in SEQ ID NO: 16-17, wherein the nucleotide sequence shown in SEQ ID NO: the 5' ends of 1, 5, 7, 10, 13 and 16 are modified with biotin labels; and
p2, amplifying by using the specific primer pair to respectively obtain a reovirus target gene, a bovine parvovirus target gene, a bluetongue virus target gene, a bovine diarrhea virus target gene, a bovine polyoma virus target gene, a rabies virus target gene and a multiple PCR product containing the reovirus target gene, the bovine parvovirus target gene, the bluetongue virus target gene, the bovine diarrhea virus target gene, the bovine polyoma virus target gene and the rabies virus target gene.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112430686A (en) * | 2020-11-24 | 2021-03-02 | 内蒙古农业大学 | Kit, primer and probe for simultaneously detecting BVDV-1, BVDV-2 and BVDV-3 |
| CN113252892A (en) * | 2021-06-22 | 2021-08-13 | 北京市肝病研究所 | Probe and kit for improving detection sensitivity of pathogenic microorganism antigen |
| CN116144841A (en) * | 2022-12-26 | 2023-05-23 | 苏州药明检测检验有限责任公司 | Primer and probe combination for detecting bovine polyoma virus and application thereof |
| 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 |
| CN117305519A (en) * | 2023-10-19 | 2023-12-29 | 武汉珈创生物技术股份有限公司 | Primer probe combination for Niu Duo tumor virus triple qPCR detection and detection method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103695566A (en) * | 2014-01-14 | 2014-04-02 | 徐超 | Multiplex PCR (polymerase chain reaction) primer, probe and gene chip for detecting bluetongue virus, foot and mouth disease virus and bovine viral diarrhea virus |
| CN105002301A (en) * | 2015-07-31 | 2015-10-28 | 中华人民共和国北京出入境检验检疫局 | Multiple-connection probe amplification detection kit, primer and probe for simultaneously detecting five cow disease viruses |
-
2019
- 2019-11-29 CN CN201911200522.5A patent/CN110904272A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103695566A (en) * | 2014-01-14 | 2014-04-02 | 徐超 | Multiplex PCR (polymerase chain reaction) primer, probe and gene chip for detecting bluetongue virus, foot and mouth disease virus and bovine viral diarrhea virus |
| CN105002301A (en) * | 2015-07-31 | 2015-10-28 | 中华人民共和国北京出入境检验检疫局 | Multiple-connection probe amplification detection kit, primer and probe for simultaneously detecting five cow disease viruses |
Cited By (7)
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|---|---|---|---|---|
| CN112430686A (en) * | 2020-11-24 | 2021-03-02 | 内蒙古农业大学 | Kit, primer and probe for simultaneously detecting BVDV-1, BVDV-2 and BVDV-3 |
| CN113252892A (en) * | 2021-06-22 | 2021-08-13 | 北京市肝病研究所 | Probe and kit for improving detection sensitivity of pathogenic microorganism antigen |
| CN113252892B (en) * | 2021-06-22 | 2021-11-02 | 北京市肝病研究所 | Probe and kit for improving detection sensitivity of pathogenic microorganism antigen |
| CN116144841A (en) * | 2022-12-26 | 2023-05-23 | 苏州药明检测检验有限责任公司 | Primer and probe combination for detecting bovine polyoma virus and application thereof |
| 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 |
| CN117305519A (en) * | 2023-10-19 | 2023-12-29 | 武汉珈创生物技术股份有限公司 | Primer probe combination for Niu Duo tumor virus triple qPCR detection and detection method thereof |
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