KR20160044232A - Methods for diagnosing bovine mastitis using primer for detecting haptoglobin gene, and real-time PCR kit therefor - Google Patents
Methods for diagnosing bovine mastitis using primer for detecting haptoglobin gene, and real-time PCR kit therefor Download PDFInfo
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- KR20160044232A KR20160044232A KR1020140138977A KR20140138977A KR20160044232A KR 20160044232 A KR20160044232 A KR 20160044232A KR 1020140138977 A KR1020140138977 A KR 1020140138977A KR 20140138977 A KR20140138977 A KR 20140138977A KR 20160044232 A KR20160044232 A KR 20160044232A
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6844—Nucleic acid amplification reactions
Abstract
The present invention relates to a method for diagnosing cow's mastitis using a primer for detecting a heptoglobin gene and a real-time PCR kit for the same, and more particularly, to a method for detecting a cow's mastitis using a primer set for detecting a heptoglobin gene using a milk sample or DNA isolated therefrom as a template And a real-time PCR (real-time PCR) using the same. The present invention relates to a method for diagnosing mastitis of a dairy cow producing milk and a real-time PCR kit for facilitating the diagnosis.
According to the present invention, the concentration of a somatic cell having a heptoglobin gene can be quickly and accurately confirmed from a sample by a simple method. Therefore, the diagnosis of cow mastitis and the quality of milk can be very effectively performed not only by microbial infection but also by other causes . In particular, PCR for microorganisms causing milk cow disease can be more accurately diagnosed or analyzed.
The method of the present invention is advantageous in that it is very simple and fast, and can be used very easily in a general farming farm. Thus, if a cow mastitis occurs, the cow can be quickly isolated, such as isolating the affected cow, so that it is possible to prevent the problem of lowering the overall milk quality due to the additional transmission or mixing of milk. Therefore, it is expected that the present invention will be very helpful for the domestic livestock industry as well as for the individual livestock farming industry.
Description
The present invention relates to a method for diagnosing cow's mastitis using a primer for detecting a heptoglobin gene and a real-time PCR kit for the same, and more particularly, to a method for detecting a cow's mastitis using a primer set for detecting a heptoglobin gene using a milk sample or DNA isolated therefrom as a template And a real-time PCR (real-time PCR) using the same. The present invention relates to a method for diagnosing mastitis of a dairy cow producing milk and a real-time PCR kit for facilitating the diagnosis.
Cow mastitis has the highest distribution among bacterial diseases in dairy cows, and it has been investigated as the biggest economic loss in livestock farms due to decrease of flow rate and oil quality. In 1995, a number of studies have been conducted on the prevention, treatment and diagnosis of cow mastitis due to the well known milk cow disease caused by the pulmonary milk fluctuation in 1995.
The major cause of cow mastitis is known to be caused by microbial infections such as bacteria, mycoplasma, yeast, and algae, and the major causative microorganisms include Staphylococcus ( Staphylococcus aureus) aureus , Streptococcus agalactiae , Streptococcus spp ., Streptococcus spp. dysgalactiae ), Streptococcus uberis uberis ), Mycoplasma bovis genus, and Coagulase negative Staphylococcus genus Koaquagrass. Studies have been conducted to quickly and easily diagnose cow mastitis by confirming whether the causative microorganism is single or multiple infection by using a polymerase chain reaction (PCR) method. 2009/083656 A1 and the like are disclosed. The present applicant also developed a method for diagnosing cow mastitis by detecting the major causative microorganisms easily and quickly and accurately, and applied for a patent. However, there is a problem that such diagnosis methods can not be diagnosed when mastitis occurs due to a cause other than microbial infection.
Accordingly, the present inventors have developed a method for quickly and easily diagnosing mastitis caused by a cause other than microbial infection.
Accordingly, it is a main object of the present invention to provide a method for diagnosing cow mastitis quickly, accurately and easily. Especially to diagnose mastitis caused by other causes than microbial infection.
It is another object of the present invention to provide a kit for diagnosing cow mastitis which enables the cow mastitis diagnostic method to be easily carried out.
According to one aspect of the present invention, the present invention provides a method for producing a cow comprising a milk sample or DNA isolated from the sample as a template, an oligonucleotide having the nucleotide sequence of SEQ ID NO: 1, and a cow including an oligonucleotide having the nucleotide sequence of SEQ ID NO: Wherein the real-time PCR is performed using a primer set for detection of a hepatoglobin gene derived from a cow mastitis.
According to another aspect of the present invention, there is provided a method for detecting a cow's mastitis (s) comprising a primer set for detecting a cow-derived heptoglobin gene comprising an oligonucleotide having a nucleotide sequence of SEQ ID NO: 1 and an oligonucleotide having a nucleotide sequence of SEQ ID NO: A real-time PCR kit for diagnosis is provided.
According to the cow mastitis diagnostic method of the present invention, the amount of somatic cells having the heptoglobin gene contained in milk squeezed from a cow can be known. Therefore, not only is it possible to diagnose dairy cow mastitis, but it also provides information necessary for milk quality analysis.
Accordingly, the present invention provides a method for detecting a cow-derived hepatoglobin gene comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 1 and an oligonucleotide having the nucleotide sequence of SEQ ID NO: 2, using the milk sample or DNA isolated from the sample as a template, And real-time PCR is performed using a primer set.
The present invention also provides a real-time PCR kit for analyzing milk quality, which comprises an oligonucleotide having the nucleotide sequence of SEQ ID NO: 1 and a primer set for detecting the heptoglobin gene from cow comprising the oligonucleotide having the nucleotide sequence of SEQ ID NO: 2 to provide.
In the present invention, the quantitative detection target is Haptoglobin (Hp), which is classified as Acute-phase proteins (APP).
In the cow mastitis diagnosis method and the milk quality analysis method of the present invention, it is preferable to use a probe having a nucleotide sequence of SEQ ID NO: 3 as a labeling substance for analyzing the concentration of the PCR amplification product in real time, It is preferable to include a probe having the nucleotide sequence of SEQ ID NO: 3.
In the method for diagnosing cow's mastitis and the method for analyzing milk quality of the present invention, it is preferable to further perform PCR for detecting microorganisms causing cow mastitis for more accurate diagnosis.
PCR for bovine mastitis causing microorganisms detection Staphylococcus aureus (Staphylococcus aureus), Streptococcus Agar Rock tiae (Streptococcus agalactiae), Streptococcus display galactose tiae (Streptococcus dysgalactiae), Streptococcus Ube lease (Streptococcus of uberis), mycoplasma Bovis (mycoplasma bovis), and nose Aquila the voice Staphylococcus (it is desirable to target the coagulase negative Staphylococcus) strain, an oligonucleotide having the nucleotide sequence of SEQ ID NO: 4. to this end, nucleotide and SEQ ID NO: 5 A primer set for detecting Staphylococcus aureus comprising an oligonucleotide having a nucleotide sequence; A primer set for detection of Streptococcus agalactiae comprising an oligonucleotide having a nucleotide sequence of SEQ ID NO: 6 and an oligonucleotide having a nucleotide sequence of SEQ ID NO: 7; An oligonucleotide having a nucleotide sequence of SEQ ID NO: 8 and an oligonucleotide having a nucleotide sequence of SEQ ID NO: 9, and a primer set for detecting streptococcus discocclusia; A primer set for detecting Streptococcus uberis comprising an oligonucleotide having a nucleotide sequence of SEQ ID NO: 10 and an oligonucleotide having a nucleotide sequence of SEQ ID NO: 11; A primer set for detecting mycoplasma vivo comprising an oligonucleotide having a nucleotide sequence of SEQ ID NO: 12 and an oligonucleotide having a nucleotide sequence of SEQ ID NO: 13; And an oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 14 and an oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 15; and a primer set for detection of negative staphylococcus produced by coagulase. It is preferable to use a set. It is preferable that the PCR for detecting the causative microorganism is performed in a reaction different from the real-time PCR.
The real-time PCR kit of the present invention may further include a primer set as described above so as to facilitate both real-time PCR on the heptoglobin gene and microbial detection PCR for the cow mastitis. At this time, it is preferable that the primer set for the real-time PCR of the heptoglobin gene and the primer set for the causative microorganism detection PCR are distinguished from each other so that they can be performed in a separate reaction.
According to the present invention, the concentration of a somatic cell having a heptoglobin gene can be quickly and accurately confirmed from a sample by a simple method. Therefore, the diagnosis of cow mastitis and the quality of milk can be very effectively performed not only by microbial infection but also by other causes . In particular, PCR for microorganisms causing milk cow disease can be more accurately diagnosed or analyzed.
The method of the present invention is advantageous in that it is very simple and fast, and can be used very easily in a general farming farm. Thus, if a cow mastitis occurs, the cow can be quickly isolated, such as isolating the affected cow, so that it is possible to prevent the problem of lowering the overall milk quality due to the additional transmission or mixing of milk. Therefore, it is expected that the present invention will be very helpful for the domestic livestock industry as well as for the individual livestock farming industry.
Figure 1 is an amplification plot of genomic DNA that is serially diluted 10-fold in Haptoglobin ranging from 1,000,000,000 to 0 genome eqivalents using real-time PCR. Delta Rn plots on the Y-axis, and the number of amplification cycles on the X-axis.
A, 1,000,000,000; B, 1,00,000,000; C, 10,000,000; D, 1,000,000; E, 1.00, 000, F, 10,000; G, 1,000; G, 100; H, 10; I, 10; J, 0; DNA genome eqivalents of haptoglobin.
Figure 2 is a standard curve of DNA that is serially diluted 10-fold of the heptoglobin (ATCC CRT-1733) prepared using the average results of three real-time real-time PCR experiments. The theshold cycle (Ct) is plotted on the Y-axis, and the Log genome eqivalents are plotted on the X-axis.
FIG. 3 is a graph showing the results of analysis of microbial infection caused by milk cow mastitis and the quantitative analysis of heptoglobin using real-time PCR.
The primer set of the present invention may comprise an oligonucleotide primer having the nucleotide sequence of SEQ ID NO: 1 and an oligonucleotide primer having the nucleotide sequence of SEQ ID NO: 2.
In the present invention, a "primer" refers to a single-stranded oligonucleotide complementary to a nucleic acid strand to be amplified, and can serve as a starting point for the synthesis of a primer extension product. The length and sequence of the primer should allow the synthesis of the extension product to begin. The specific length and sequence of the primer will depend on the primer usage conditions such as temperature and ionic strength, as well as the complexity of the desired DNA or RNA target.
The real-time PCR kit of the present invention may comprise the primer set and a reagent for performing an amplification reaction. Reagents for carrying out the amplification reaction may include DNA polymerase, dNTPs, dUTP, buffer solution, sterile distilled water. In addition, the kit of the present invention may further include a user's manual describing optimal reaction performing conditions.
The method of the present invention may include separate extraction of genomic DNA from a sample of interest. The target sample may be unique, for example, an electrodeposited milk from a cow. As a method for separating and extracting the genomic DNA from the sample, a method known in the art can be used. For example, a Wizard® Genomic DNA Purification Kit (Promega) can be used. Using the separated genomic DNA as a template, an amplification reaction can be performed using the primer set of the present invention to amplify the target sequence. The target sequence is the TXNL4B gene of heptoglobin (Hp).
Methods for amplifying a target nucleic acid include polymerase chain reaction (PCR), ligase chain reaction, nucleic acid sequence-based amplification, transcription-based amplification system, Strand displacement amplification or amplification with Q [beta] replicase, or any other suitable method for amplifying nucleic acid molecules known in the art. Among them, PCR is a method of amplifying a target nucleic acid from a pair of primers that specifically bind to a target nucleic acid using a polymerase. Such PCR methods are well known in the art, and commercially available kits may be used.
The method of the invention may comprise detecting the amplification product. Detection of the amplification product can be performed quantitatively through real-time PCR. In the method of the present invention, the amplified target sequence may be labeled with a detectable labeling substance. In one embodiment, the labeling material can be, but is not limited to, a fluorescent, phosphorescent or radioactive substance. Preferably, the labeling substance is a fluorescent probe, SYBR Green I, Cy-5 or Cy-3. In the case of a fluorescent probe, the probe is hybridized to the template before the PCR, and as the PCR proceeds, the probe is released from the template and fluorescence is emitted. The fluorescent reporter dye is labeled with 6-FAM (carboxyfluorescein) at the 5 'end and MGB (minor groove binder) at the end of the non-fluorescent quencher 3'. In the case of SYBR Green I, PCR is carried out by mixing the above-mentioned dye in the PCR reaction mixture, and SYBR Green I is inserted into the PCR product and fluoresced. In the case of Cy-5 or Cy-3, when the target sequence is amplified, Cy-5 or Cy-3 is labeled at the 5'-end of the primer and PCR can be carried out so that the target sequence can be labeled with a detectable fluorescent labeling substance.
As used herein, "probe" refers to a single-stranded nucleic acid sequence that hybridizes with a complementary single-stranded target sequence to form a double-stranded molecule (hybrid).
As used herein, an oligonucleotide used as a probe may comprise a nucleotide analogue, such as phosphorothioate, alkylphosphorothioate or peptide nucleic acid, And may include an intercalating agent.
Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.
Example 1. Primer and probe design for detection of heptoglobin gene
Table 1 shows the primers and probes designed for the detection and quantification of the heptoglobin gene.
* a FAM, 6-carboxyfluorescein (report dye).
The primers and probes were designed for amplification of specific DNA fragments of heptoglobin TXNL4B using the OligoAnalyzer 3.1 program (Integrated DNA Technologies, Inc.) considering the reaction temperature, amplification product size, and non-specific binding exclusion . Based on the above design, each primer and probe was custom-made through COSMO Genetech (Seoul, Korea). A fluorescence reporter dye, 6-FAM (carboxyfluorescein) was attached to the 5 'end of the probe, and quencher MGB (minor groove binder) were labeled.
Example 2. Generation of real-time PCR standard curve
Template DNA extraction for PCR from Bos taurus endothelial cell (ATCC CRL-1733) was performed using QIAGEN's Gentra Puregene Cell Kit. 200㎕ the cell lysates (
The extracted DNA was quantified using Qubit ™ Fluoromet and Qubit-it ™ dsDNA HS assay kit (Invitrogen, Carlsbad, CA, USA). The final concentration of the purified DNA is 3㎕ 10 × 10 8, 10 × 10 7, 10 × 10 6, 10 × 10 5 , 10 × 10 4 , 10 × 10 3 , 10 × 10 2 , 100, 10 and 0 genome equivalents. The DNA genome equivalent was calculated by the following equation.
DNA genome equivalent = (A x 6.023 x 10 23 ) (660 x B) -1
Where A is the concentration of DNA and B is the length of the DNA.
Example 3. Establishment of real-time PCR conditions
The amplification reaction was adjusted to final volume by 20 μl and each sample was repeated 3 times. To one real-time PCR reaction tube was added 10.0 [mu] l of Mastermix, 4.0 pM primer, 2.0 pM probe, 3 [mu] l of sample DNA and 5 [mu] l of sterile nuclease free water. Thermal cycling was performed using the two-step PCR protocol as follows.
2 minutes at 50 DEG C, 10 minutes at 94 DEG C After 40 cycles of 94 DEG C for 30 seconds and 63 DEG C for 1 minute and 65 DEG C for 40 seconds, the fluorescence signal was measured in each cycle at the end of the extension step.
Inclusivity and Exclusivity of Primer Pair for Heptoglobin
Real - time PCR was performed on the microbial strains related to cow mastitis and DNA of heptoglobin in order to verify and evaluate the specificity of primers and probes. The results are shown in Table 2. The primers and probes of the present invention were found to specifically amplify only the heptoglobin gene.
Real-time PCR results of quantitative DNA of heptoglobin gene
Amplification curves and real time PCR standard curves were obtained from genomic DNA sequentially diluted 1/10 of the cow endothelial cell (ATCC CRT-1733) and are shown in FIGS. 1, 2 and 3. Rn is related to the concentration of the PCR product, and the larger the Rn, the higher the concentration of the PCR product. Five individual experiments were repeated three times and one amplification curve was presented in FIG. Quantitative genomic DNA of cow endothelial cells was shown from 10 × 10 8 to 0 genome equivalent in one individual experiment that was repeated three times using real - time PCR. As shown in FIG. 1 and Table 3, the DNA of the genome equivalent of the genome DNA of the cow endothelial cell was not amplified, and the amplification curve of the DNA was shown as shown from 10 × 10 8 .
The threshold cycle (Ct) value of each of the quantitative DNAs diluted sequentially by 1/10 was almost the same in three repeated experiments. The Ct value is the number of cycles in which a fluorescent signal detectable in a real-time PCR reaction appears. In other words, the larger the initial DNA concentration, the higher the amount of amplification at low Ct value, the more fluorescence signal can be detected, and the smaller the initial DNA concentration, the larger the Ct value. The mean value and standard deviation of the threshold cycle based on the DNA genome equivalent of the cow endothelial genomic DNA are shown in Table 3.
equivalents *
Deviation
Genomic DNA of cow endothelial cell (ATCC CRT-1733)
** N.D: not detected
The real-time PCR standard curve of the quantified cow endothelial cell DNA was repeated three times and the average result was linearly correlated with the number of log cells in FIG. The Ct value of the standard curve showed a high correlation coefficient of 0.9985 and the calculated slope value was -3.418.
Extraction of template DNA for PCR execution from crude oil
To extract template DNA for PCR, 1,000 ㎕ of crude oil was centrifuged at 13,000 rpm for 5 minutes to remove the milk fat layer. 1,000 μl of PBS buffer was added to the precipitate thus obtained, gently mixed and centrifuged at 7,000 rpm for 2 minutes to remove the supernatant. After repeating the above procedure twice, 400 μl of Lysis buffer was added to the precipitate from which the supernatant was removed, and the reaction was carried out at 60 ° C for 10 minutes to obtain a cell lysate. For template DNA extraction, QIAGEN Gentra Puregene Cell Kit was used. 400 μl of cell lysate was transferred to a spin column, centrifuged at low speed to remove cell suspension, and washed twice with washing buffer. In order to recover the DNA bound to the column, 100 μl of elution buffer was added, followed by reaction at 60 ° C for 5 minutes, followed by centrifugation at 13,000 rpm for 1 minute to recover the DNA.
Identification of microbial PCR and quantitation of heptoglobin gene
The pre-processing crude oil supplied from the dairy cow farm was used as the target sample, and the presence of microbial infection in the major causes of cow mastitis was confirmed by using Multiplex PCR kit (Clean-zoa DP6). This kit contains the primers shown in Table 4 below and includes Staphylococcus aureus , Streptococcus agalactiae , Streptococcus dysgalactiae , Streptococcus aureus, Detection of Streptococcus uberis , Mycoplasma bovis and coagulase negative Staphylococcus (CNS) strains is possible.
size
Of the total 43 crude oil samples, 12 (27.9%) samples showed no causative microorganism infection, and 31 (72.1%) were infected with at least one microorganism causing mastitis. The results are shown in Table 5 Respectively. Real time PCR of the amount of heptoglobin gene was performed from the same 43 samples by the method of Example 3, and the values converted from the standard amplification curve of FIG. 2 into the genome equivalent based on the measured Ct values are shown in Table 6 Respectively.
S. aureus: n = 12, 20.7%
M. bovis: n = 11, 19.0%
CNS: n = 16, 27.6%
E. coli: n = 5, 8.6%
S. uberis + CNS: n = 10, 17.3%
<110> LEE, byoung jig <120> Methods for diagnosing bovine mastitis using primer detecting haptoglobin gene, and real-time PCR kit <130> P141015-01 <160> 15 <170> Kopatentin 2.0 <210> 1 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> forward primer for haptoglobin gene, B-Hp01F <400> 1 atggtctccc agcataacct catctc 26 <210> 2 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for haptoglobin gene, B-HpO2R <400> 2 gagaaccacc ttctccacct ctacaa 26 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> probe for haptoglobin gene, BT-Hp probe <400> 3 gaggttatgc tgggagacc 19 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> forward primer for Staphylococcus aureus, SAp1 <400> 4 ggacgagaag cttgcttctc tgatg 25 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for Staphylococcus aureus, SAp2 <400> 5 gcttaatgcg ttagctgcag cacta 25 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> forward primer for Streptococcus agalactiae, Sagp1 <400> 6 gcgtagaacg ctgatgtttg gtgt 24 <210> 7 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for Streptococcus agalactiae, Sagp2 <400> 7 ccactcctac caacgttctt ctctaacaac 30 <210> 8 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> forward primer for Streptococcus dysgalactiae, SDp1 <400> 8 aatgcaactg catcactatg agatggac 28 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Streptococcus dysgalactiae, SDp2 <400> 9 gtggattttc cactcccacc atca 24 <210> 10 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> forward primer for Streptococcus uberis, SUp1 <400> 10 cgcatgacaa tagggtacac atgtaccta 29 <210> 11 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for Streptococcus uberis, SUp2 <400> 11 gggaaccctg accgagca 18 <210> 12 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> forward primer for Mycoplasma bovis, MBp1 <400> 12 cagctcgtgt tgtcggtgat gttattg 27 <210> 13 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for Mycoplasma bovis, MBp2 <400> 13 gcgggatcat catcaataga gcctaca 27 <210> 14 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> forward primer for coagulase negative Staphylococcus, CNSp1 <400> 14 atattgaacc gcatggttca atagtgaaag ac 32 <210> 15 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for coagulase negative Staphylococcus, CNSp2 <400> 15 atagacgggg acccgcac 18
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106841636A (en) * | 2017-01-24 | 2017-06-13 | 北京美正生物科技有限公司 | A kind of enzyme-linked immunosorbent assay kit for detecting cow subclinical mastitis and its production and use |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106841636A (en) * | 2017-01-24 | 2017-06-13 | 北京美正生物科技有限公司 | A kind of enzyme-linked immunosorbent assay kit for detecting cow subclinical mastitis and its production and use |
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