CN116445634A - Bovine mastitis pathogen multiplex detection kit and application thereof - Google Patents
Bovine mastitis pathogen multiplex detection kit and application thereof Download PDFInfo
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- 238000001514 detection method Methods 0.000 title claims abstract description 53
- 244000052769 pathogen Species 0.000 title claims abstract description 26
- 230000001717 pathogenic effect Effects 0.000 title claims abstract description 18
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- 239000007850 fluorescent dye Substances 0.000 claims abstract description 43
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- 241000191967 Staphylococcus aureus Species 0.000 claims abstract description 21
- 241001138504 Mycoplasma bovis Species 0.000 claims abstract description 20
- 241000193985 Streptococcus agalactiae Species 0.000 claims abstract description 20
- 241000194054 Streptococcus uberis Species 0.000 claims abstract description 20
- 229940115922 streptococcus uberis Drugs 0.000 claims abstract description 20
- 241000194042 Streptococcus dysgalactiae Species 0.000 claims abstract description 18
- 229940115920 streptococcus dysgalactiae Drugs 0.000 claims abstract description 18
- 238000000034 method Methods 0.000 claims abstract description 17
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Abstract
The invention discloses a bovine mastitis pathogen multiplex detection kit and application thereof, wherein the kit comprises a specific primer for amplifying 5 gene loci of mycoplasma bovis, streptococcus agalactiae, streptococcus dysgalactiae, streptococcus uberis and staphylococcus aureus and a fluorescent probe. Compared with the prior art, the invention has the following advantages: (1) The kit disclosed by the invention realizes simultaneous detection of multiple indexes of a single sample based on the qPCR technology of the Taqman method, simplifies experimental operation steps, improves detection flux, and meets urgent requirements of rapid screening and accurate detection of multiple indexes; (2) In addition, the purposes of high sensitivity, high specificity, high adaptability and reliability are realized, bacteria are not required to be cultured, and the cow milk sample can be directly used for identification, so that the time and the cost required by pathogen identification are reduced; (3) The kit has high detection efficiency and short time consumption, and can detect 5 common bovine mastitis pathogens simultaneously in the same device for about 2 hours.
Description
Technical Field
The invention belongs to the technical field of molecular biology, relates to detection of 5 infectious disease pathogens of cattle, and particularly relates to a bovine mastitis pathogen multiplex detection kit and application thereof.
Background
Food-borne pathogenic bacteria pollution is an important hidden danger of cow milk quality safety, and cow mastitis is a main pollution source of pathogenic bacteria in cow milk. Cow mastitis is a common multiple disease which damages the production of cows, and the main reasons for the occurrence of the disease are invasion of pathogenic bacteria, environmental factors, the self status of the cows and the like. About 6000 thousands of cows all over the world have mastitis, the incidence of the mastitis of the cows in China is 41% -60%, wherein the incidence of the recessive mastitis of individual cow farms is as high as 46.4% -85.7% mainly, and the method is one of the important reasons for lower unit production level of the cows in China. Bovine mastitis usually causes the milk yield to be reduced, and the cow milk is abandoned, and when the cow milk is seriously lost in lactation capacity, even human health is endangered, so that the bovine mastitis becomes an important factor for the healthy development of the dairy industry and the animal husbandry. However, since the breast and milk of the dairy cow suffering from mastitis are not obviously changed, the dairy cow is difficult to observe by naked eyes, and is not paid attention to enough in many areas, and a necessary detection means is lacked, great economic losses are caused for the dairy industry and the animal husbandry. The method for detecting the multiple pathogen nucleic acid in the cow milk is an urgent task facing the current dairy industry work, and aims at developing accurate, high-sensitivity, high-efficiency and low-cost full-automatic high-sensitivity cow milk aiming at main pathogenic microorganisms of the cow mastitis.
The detection or diagnosis method in the prior art mainly comprises direct smear microscopic observation of pathogens, cell culture inspection, serum enzyme-linked immunosorbent assay and the like, wherein the specificity of the microscopic examination and culture method is the best, but the sensitivity is poor, the requirements on the collection, preservation, transportation and experimental conditions of samples are high, the detection period is long, part of pathogens are extremely difficult to culture and cannot be controlled in quality, and the detection rate of mixed infection is low; the immunological detection sensitivity is low, and the pathogen infection has a window period, so that the detection value of the pathogen with acute morbidity and high mortality is low.
Disclosure of Invention
The technical problems to be solved are as follows: in order to overcome the defects of the prior art, the method realizes the simultaneous detection of multiple indexes of a single sample based on the qPCR technology of the Taqman method, simplifies experimental operation steps, improves detection flux, and meets urgent requirements of rapid screening and accurate detection of multiple indexes; in addition, the purposes of high sensitivity, high specificity, high adaptability and reliability are realized, bacteria are not required to be cultured, and the cow milk sample can be directly used for identification, so that the time and the cost required by pathogen identification are reduced; in view of the above, the invention provides a bovine mastitis pathogen multiplex assay kit and application thereof.
The technical scheme is as follows: the kit comprises a specific primer for amplifying 5 gene loci of mycoplasma bovis, streptococcus agalactiae, streptococcus dysgalactiae, streptococcus uberis and staphylococcus aureus and a fluorescent probe; wherein, 5 gene loci are: the kit comprises a specific primer and a fluorescent probe for amplifying 5 gene loci of mycoplasma bovis, streptococcus agalactiae, streptococcus dysgalactiae, streptococcus uberis and staphylococcus aureus; wherein, 5 gene loci are: the DPI gene of mycoplasma bovis, the sip gene of streptococcus agalactiae, the isp gene of streptococcus dysgalactiae, the sodA gene of streptococcus uberis and the nur gene of staphylococcus aureus; specific primers and fluorescent probes of the pig internal reference gene ACTB gene.
Preferably, the specific primer and the fluorescent probe have the following sequences: mbo, SEQ ID No.1-3; sag, SEQ ID NO.4-6; sdy, SEQ ID NO.7-9; sub, SEQ ID No.10-12; SA, SEQ ID NO.13-15; bov, SEQ ID No.16-18.
Preferably, the kit comprises a PCR reaction liquid I, a PCR reaction liquid II and a PCR reaction liquid III; wherein the PCR reaction liquid I comprises specific primers shown in SEQ ID NO.1-2 and SEQ ID NO.4-5, and fluorescent probes shown in SEQ ID NO.3 and SEQ ID NO. 6; the PCR reaction liquid II comprises specific primers shown in SEQ ID NO.7-8 and SEQ ID NO.10-11, and fluorescent probes shown in SEQ ID NO.9 and SEQ ID NO. 12; the PCR reaction solution III comprises specific primers shown as SEQ ID NO.13-14 and SEQ ID NO.16-17, and fluorescent probes shown as SEQ ID NO.15 and SEQ ID NO. 18.
Further, in the detection process, the PCR reaction liquid I, the PCR reaction liquid II and the PCR reaction liquid III are respectively added into different sample holes of the nucleic acid detection shell.
Preferably, the final concentrations of the specific primer and the fluorescent probe in the amplification system are: 0.2. Mu.M.
Preferably, the kit comprises: dNTP 0.2mM, taq enzyme 2.5U, mgCl 2 4mM.
Preferably, the 5 'end of SEQ ID NO.3 is marked by using a fluorescent dye 6-FAM, the 5' end of SEQ ID NO.6 is marked by using a fluorescent dye ROX, the 5 'end of SEQ ID NO.9 is marked by using a fluorescent dye 6-FAM, the 5' end of SEQ ID NO.12 is marked by using a fluorescent dye ROX, the 5 'end of SEQ ID NO.15 is marked by using a fluorescent dye 6-FAM, and the 5' end of SEQ ID NO.18 is marked by using a fluorescent dye ROX.
The use of any of the above kits for simultaneous detection of mycoplasma bovis, streptococcus agalactiae, streptococcus dysgalactiae, streptococcus uberis and staphylococcus aureus in a single sample.
Preferably, the detection sensitivity of the kit for mycoplasma bovis, streptococcus agalactiae, streptococcus dysgalactiae, streptococcus uberis and staphylococcus aureus is up to 500 copies/reaction.
Preferably, the specific method for simultaneously detecting mycoplasma bovis, streptococcus agalactiae, streptococcus dysgalactiae, streptococcus uberis and staphylococcus aureus aiming at a single sample comprises the following steps: adding a sample to be detected into a sample hole of a nucleic acid extraction shell, obtaining purified nucleic acid in the sample after the sample passes through the whole nucleic acid extraction process, transferring the obtained purified nucleic acid into a nucleic acid detection shell filled with a detection freeze-drying reagent, re-dissolving the freeze-drying reagent into a complete nucleic acid amplification system after contacting the liquid purified nucleic acid, finally detecting the sample by adding a sealing reagent, and automatically judging a detection result by a detection instrument after the whole nucleic acid amplification process is finished.
Specific methods for extracting nucleic acids in the nucleic acid extraction procedure include, but are not limited to, extracting nucleic acids using a magnetic bead method. Pretreatment methods for milk samples include, but are not limited to: 500. Mu.L of physiological saline is added into 1mL of cow milk sample, after uniform mixing, the mixture is centrifuged at 12000rpm/min at 4 ℃ for 10 minutes, the supernatant is removed to leave sediment, 200. Mu.L of physiological saline is added into the sediment, and the mixture is blown and uniformly mixed.
The idea of the primer combination screening in the kit is as follows:
(1) Downloading typical sequences of mycoplasma bovis, streptococcus agalactiae, streptococcus uberis and staphylococcus aureus, comparing the downloaded sequences by using gene comparison software, selecting genes with good conservation and good specificity, and finally screening out candidate genes of the pathogens.
(2) Primer and probe designs are carried out aiming at candidate genes of each pathogen, and 10 sets of primer and probe combinations are designed for each pathogen.
(3) A total of 60 sequences of 20 sets of Primer probe sets of two pathogens (staphylococcus aureus and internal reference genes in reaction solution III) in each reaction solution are subjected to Primer interaction evaluation by Primer Premier 5 software, and finally 6 sets of Primer probe sets with the weakest interaction are preferred.
The beneficial effects are that: (1) The kit disclosed by the invention realizes simultaneous detection of multiple indexes of a single sample based on the qPCR technology of the Taqman method, simplifies experimental operation steps, improves detection flux, and meets urgent requirements of rapid screening and accurate detection of multiple indexes; (2) In addition, the purposes of high sensitivity, high specificity, high adaptability and reliability are realized, bacteria are not required to be cultured, and the cow milk sample can be directly used for identification, so that the time and the cost required by pathogen identification are reduced; (3) The kit has high detection efficiency and short time consumption, and can detect 5 common bovine mastitis pathogens simultaneously in the same device for about 2 hours.
Drawings
FIG. 1 is a schematic diagram of a detection device that may be used in the use of the kit of the present invention.
Detailed Description
The following examples further illustrate the invention but are not to be construed as limiting the invention. Modifications and substitutions to the method, steps or conditions of the invention without departing from the spirit and nature of the invention are intended to be within the scope of the invention. The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.
Example 1
The kit comprises a specific primer for amplifying 5 gene loci of mycoplasma bovis, streptococcus agalactiae, streptococcus dysgalactiae, streptococcus uberis and staphylococcus aureus and a fluorescent probe; wherein, 5 gene loci are: the kit comprises a specific primer and a fluorescent probe for amplifying 5 gene loci of mycoplasma bovis, streptococcus agalactiae, streptococcus dysgalactiae, streptococcus uberis and staphylococcus aureus; wherein, 5 gene loci are: the DPI gene of mycoplasma bovis, the sip gene of streptococcus agalactiae, the isp gene of streptococcus dysgalactiae, the sodA gene of streptococcus uberis and the nur gene of staphylococcus aureus; specific primers and fluorescent probes of the pig internal reference gene ACTB gene.
The specific primer and the fluorescent probe have the following sequences: mbo, SEQ ID No.1-3; sag, SEQ ID No.4-6; sdy, SEQ ID NO.7-9; sub, SEQ ID No.10-12; SA, SEQ ID NO.13-15; bov, SEQ ID No.16-18. The sequences are shown in Table 1:
table 1 specific primers and fluorescent probe sequence listing
| Name of the name | Primer probe sequence (5 '-3') | SEQ ID NO. |
| Mbo-F | CTGGTGATACATCCGATAGACTAA | 1 |
| Mbo-R | GTGAGCTGATTCATAGTCATTTTC | 2 |
| Mbo-P | 6-FAM-GGTATTGGGCCTAAAAAGGCTGTGAG-DBQ1 | 3 |
| Sag-F | CAGCAGTTCCTGTGACTACGAC | 4 |
| Sag-R | CATTTGTTGTTGAAGCTGGTTG | 5 |
| Sag-P | ROX-AGTTACAAGCGACTGAAGTTAAGAGCGTT-BHQ2 | 6 |
| Sdy-F | CTGCCTTGTATTTCCAGTCCC | 7 |
| Sdy-R | ACGATGTTTTGAGTTATCAAGCAT | 8 |
| Sdy-P | 6-FAM-GGATCGAATTGGTAGCCCAGTTCTTTG-DBQ1 | 9 |
| Sub-F | TTCCGAAGTGATTTCTGTTTTC | 10 |
| Sub-R | GTGGCGTTATTATCTGATGTGTC | 11 |
| Sub-P | ROX-CCAAAAAAGTGCATGGTTAAGATGTCCG-BHQ2 | 12 |
| SA-F | ATACACCTGAAACAAAGCATCCT | 13 |
| SA-R | ATATACGCTAAGCCACGTCCA | 14 |
| SA-P | 6-FAM-TATGGTCCTGAAGCAAGTGCATTTACG-DBQ1 | 15 |
| Bov-F | TGTTGGCGTAGAGGTCCTTG | 16 |
| Bov-R | CCTTCCTTCCTGGGTGAGTG | 17 |
| Bov-P | ROX-CATGATGGAATTGAAGGTAGTTTCGTGAA-BHQ2 | 18 |
The kit comprises a PCR reaction liquid I, a PCR reaction liquid II and a PCR reaction liquid III; wherein, the PCR reaction liquid I comprises specific primers shown in SEQ ID NO.1-2 and SEQ ID NO.4-5, and fluorescent probes shown in SEQ ID NO.3 and SEQ ID NO. 6; the PCR reaction liquid II comprises specific primers shown in SEQ ID NO.7-8 and SEQ ID NO.10-11, and fluorescent probes shown in SEQ ID NO.9 and SEQ ID NO. 12; the PCR reaction solution III comprises specific primers shown as SEQ ID NO.13-14 and SEQ ID NO.16-17, and fluorescent probes shown as SEQ ID NO.15 and SEQ ID NO. 18.
In the detection process, the PCR reaction liquid I, the PCR reaction liquid II and the PCR reaction liquid III can be respectively added into different sample holes.
The final concentrations of the specific primer and the fluorescent probe in the amplification system are as follows: 0.2. Mu.M.
The kit comprises: dNTP 0.2mM, taq enzyme 2.5U, mgCl 2 4mM.
The 5 'end of SEQ ID NO.3 is marked by using fluorescent dye 6-FAM, the 5' end of SEQ ID NO.6 is marked by using fluorescent dye ROX, the 5 'end of SEQ ID NO.9 is marked by using fluorescent dye 6-FAM, the 5' end of SEQ ID NO.12 is marked by using fluorescent dye ROX, the 5 'end of SEQ ID NO.15 is marked by using fluorescent dye 6-FAM, and the 5' end of SEQ ID NO.18 is marked by using fluorescent dye ROX.
Example 2 specificity test
The simulated sample prepared by mixing cow milk with mycoplasma bovis, streptococcus agalactiae, streptococcus dysgalactiae, streptococcus uberis, staphylococcus aureus, streptococcus lactiae, lactobacillus acidophilus, escherichia coli, salmonella, corynebacterium bovis, klebsiella, brucella, hemolytic streptococcus and streptococcus pyogenes is pretreated and then added into a sample hole of the detection device, and the detection result is shown in table 2 after being put into an instrument for nucleic acid extraction and detection.
TABLE 2 results of specific assays
In table 2, + represents positive detection result; -negative for the detection result.
The results in Table 2 show that the present invention can specifically detect Mycoplasma bovis, streptococcus agalactiae, streptococcus dysgalactiae, streptococcus uberis and Staphylococcus aureus without cross-reacting with other pathogenic microorganisms such as Streptococcus lactis, lactobacillus acidophilus, escherichia coli, salmonella, corynebacterium bovis, klebsiella, brucella, streptococcus hemolyticus and Streptococcus pyogenes.
Example 3 sensitivity detection assay
The plasmid DNA containing the respective targets of mycoplasma bovis, streptococcus agalactiae, streptococcus dysgalactiae, streptococcus uberis and staphylococcus aureus is diluted by sterilized purified water to a certain copy number multiple ratio dilution, and then is respectively added into detection holes of a detection device for detection.
The detection result shows that the detection sensitivity of the detection method for mycoplasma bovis, streptococcus agalactiae, streptococcus uberis and staphylococcus aureus is 500 copies/reaction.
Claims (9)
1. The bovine mastitis pathogen multiplex detection kit is characterized by comprising a specific primer and a fluorescent probe for amplifying 5 gene loci of mycoplasma bovis, streptococcus agalactiae, streptococcus dysgalactiae, streptococcus uberis and staphylococcus aureus; wherein, 5 gene loci are: the DPI gene of mycoplasma bovis, the sip gene of streptococcus agalactiae, the isp gene of streptococcus dysgalactiae, the sodA gene of streptococcus uberis and the nur gene of staphylococcus aureus; specific primers and fluorescent probes of the pig internal reference gene ACTB gene.
2. The bovine mastitis pathogen multiplex assay kit of claim 1, wherein the specific primer and fluorescent probe sequences are as follows: mbo, SEQ ID No.1-3; sag, SEQ ID No.4-6; sdy, SEQ ID NO.7-9;
Sub、SEQ ID NO.10-12；SA、SEQ ID NO.13-15；Bov、SEQ ID NO.16-18。
3. the bovine mastitis pathogen multiplex assay kit of claim 1, wherein the kit comprises PCR reaction solution i, PCR reaction solution ii, and PCR reaction solution iii; wherein, the PCR reaction liquid I comprises specific primers shown in SEQ ID NO.1-2 and SEQ ID NO.4-5, and fluorescent probes shown in SEQ ID NO.3 and SEQ ID NO. 6; the PCR reaction liquid II comprises specific primers shown in SEQ ID NO.7-8 and SEQ ID NO.10-11, and fluorescent probes shown in SEQ ID NO.9 and SEQ ID NO. 12; the PCR reaction liquid III comprises specific primers shown in SEQ ID NO.13-14 and SEQ ID NO.16-17, and fluorescent probes shown in SEQ ID NO.15 and SEQ ID NO. 18.
4. The bovine mastitis pathogen multiplex assay kit of claim 1, wherein the final concentration of the specific primers and fluorescent probe in the amplification system are: 0.2. Mu.M.
5. The bovine mastitis pathogen multiplex assay kit of claim 1, wherein said kit comprises:
dNTP 0.2mM, taq enzyme 2.5U, mgCl 2 4mM.
6. The multiple detection kit for swine virulent infectious disease pathogens according to claim 1, wherein the 5 'end of SEQ ID No.3 is marked with a fluorescent dye 6-FAM, the 5' end of SEQ ID No.6 is marked with a fluorescent dye ROX, the 5 'end of SEQ ID No.9 is marked with a fluorescent dye 6-FAM, the 5' end of SEQ ID No.12 is marked with a fluorescent dye ROX, the 5 'end of SEQ ID No.15 is marked with a fluorescent dye 6-FAM, and the 5' end of SEQ ID No.18 is marked with a fluorescent dye ROX.
7. Use of the kit according to any one of claims 1-6 for simultaneous detection of mycoplasma bovis, streptococcus agalactiae, streptococcus dysgalactiae, streptococcus uberis and staphylococcus aureus for a single sample.
8. The use according to claim 7, wherein the kit has a detection sensitivity of up to 500 copies/reaction for mycoplasma bovis, streptococcus agalactiae, streptococcus dysgalactiae, streptococcus uberis and staphylococcus aureus.
9. The use according to claim 7, wherein the specific method for simultaneous detection of mycoplasma bovis, streptococcus agalactiae, streptococcus dysgalactiae, streptococcus uberis and staphylococcus aureus for a single sample is: adding a sample to be detected into a sample hole of a nucleic acid extraction shell, obtaining purified nucleic acid in the sample after the sample passes through the whole nucleic acid extraction process, transferring the obtained purified nucleic acid into a nucleic acid detection shell filled with a detection freeze-drying reagent, re-dissolving the freeze-drying reagent into a complete nucleic acid amplification system after contacting the liquid purified nucleic acid, finally detecting the sample by adding a sealing reagent, and automatically judging a detection result by a detection instrument after the whole nucleic acid amplification process is finished.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117248000A (en) * | 2023-11-20 | 2023-12-19 | 深圳市易瑞生物技术股份有限公司 | Lyoprotectant for multiplex fluorescence PCR, lyoprotectant reaction system, kit containing lyoprotectant and application of lyoprotectant |
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2023
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117248000A (en) * | 2023-11-20 | 2023-12-19 | 深圳市易瑞生物技术股份有限公司 | Lyoprotectant for multiplex fluorescence PCR, lyoprotectant reaction system, kit containing lyoprotectant and application of lyoprotectant |
| CN117248000B (en) * | 2023-11-20 | 2024-03-15 | 深圳市易瑞生物技术股份有限公司 | Lyoprotectant for multiplex fluorescence PCR, lyoprotectant reaction system, kit containing lyoprotectant and application of lyoprotectant |
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