CN114277169B - Kit for detecting cow mastitis pathogenic bacteria and method thereof - Google Patents
Kit for detecting cow mastitis pathogenic bacteria and method thereof Download PDFInfo
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Abstract
The invention provides a kit for detecting cow mastitis pathogenic bacteria, which comprises a primer for amplifying staphylococcus aureus, a primer for amplifying staphylococcus epidermidis, a primer for amplifying streptococcus agalactiae, a primer for amplifying streptococcus uberis, a primer for amplifying enterococcus, a primer for amplifying pseudomonas aeruginosa, a primer for amplifying escherichia coli, a primer for amplifying klebsiella pneumoniae, a primer for amplifying listeria monocytogenes and a primer for amplifying mycoplasma bovis. The invention also provides a method for detecting the dairy cow mastitis by adopting the kit. The method has the characteristics of convenience in operation, no need of special instruments and low cost, can provide powerful help for accurate diagnosis and treatment of farms, has the characteristics of high detection efficiency and low cost, is very suitable for basic level use, and has the condition of large-scale popularization and application.
Description
Technical Field
The invention belongs to the field of biomolecule detection, and relates to a kit, in particular to a kit for detecting cow mastitis pathogenic bacteria and a method thereof.
Background
The dairy cow mastitis is a first disease which always threatens the dairy cow industry in recent years, can cause the reduction of the milk quantity and the milk quality of the produced milk, and can also cause the loss of the production performance of the dairy cow due to serious diseases, so that adult dairy cows are eliminated prematurely, and huge economic losses are caused. There are many factors that lead to cow mastitis, of which more than 80% are caused by pathogenic microorganism infection. Clinically, the antibiotics are mainly applied to treatment, and the use of a large amount of antibiotics not only increases the treatment cost and causes the bacteria to have drug resistance, but also causes a large amount of antibiotics to remain in the dairy product, thereby seriously threatening the health of human beings and environmental safety. Pathogenic bacteria of cow mastitis mainly comprise contagious pathogenic microorganisms, including staphylococcus aureus and streptococcus agalactiae with higher bacteria carrying rate, and the clinical detection rate in certain areas is more than 70%. The other is an environmental pathogenic microorganism mainly comprising Escherichia coli and Streptococcus dysgalactiae. Clinically, the traditional Chinese medicine composition mainly shows acute mastitis and recessive mastitis, wherein the recessive mastitis can cause chronic infection of cows so as to cause long-term decline of milk yield, and huge economic loss is caused for the cow breeding industry. Therefore, how to effectively prevent and treat the disease has important significance.
At present, microbiological test is the most accurate method for diagnosing cow mastitis, but traditional bacterial separation culture, culture characteristics, biochemical experiments and drug sensitivity are still required for diagnosisFeel test, and animal pathogenicity test thereof. The method is time-consuming and labor-consuming, and is unfavorable for field detection. The loop-mediated isothermal amplification method is a new type of nucleic acid amplification method, and is characterized by that 4 specific primers are designed for 6 regions of target gene, under the action of strand displacement DNA polymerase, the isothermal amplification at 60-65 deg.C can be implemented for about 15-60 min, so that the invented method can be used for amplification of nucleic acid 9 ～10 10 The double nucleic acid amplification has the characteristics of simple operation, strong specificity, easy detection of products and the like. During DNA synthesis, a large amount of pyrophosphate ions are generated to combine with divalent metal ions to form insoluble salts, and manganese ions and calcein (a fluorescent metal indicator) are added into the reaction solution, so that the substantial change of fluorescence in the amplification reaction process can be intuitively observed within 30-60 min. Because of the high sensitivity of signal recognition, the system does not require expensive specialized equipment to achieve visual analysis of the results. Therefore, the amplification can be identified by observing the white turbidity precipitate or the green fluorescence generation with naked eyes, without complicated electrophoresis and ultraviolet observation. Because the loop-mediated isothermal amplification reaction does not need a PCR instrument and expensive reagents, the method can be used for on-site rapid detection.
At present, the domestic rapid diagnosis research on dairy cow mastitis is less, even if dairy cow mastitis is related to detection products are developed, the detection level is improved, and the current urgent need is to provide high-quality and safe dairy products for people. Up to now, there is no method for identifying a plurality of common cow mastitis pathogenic bacteria by using a loop-mediated isothermal amplification technology, so it has very important significance to provide a primer and a method for identifying common cow mastitis pathogenic bacteria by using a visual isothermal amplification technology. The whole process of the method only needs 60 minutes, and the problems that the traditional detection means for the pathogenic bacteria of the dairy cows are low in specificity, low in sensitivity, complex and time-consuming in operation, incomplete in pathogenic bacteria detection in the prior art and the like are solved.
Disclosure of Invention
Aiming at the technical problems in the prior art, the invention provides a kit for detecting cow mastitis pathogenic bacteria and a method thereof, and the kit for detecting cow mastitis pathogenic bacteria and the method thereof aim to solve the technical problems of complex method, long time, false positive interference and the like in the prior art.
The invention provides a kit for detecting cow mastitis pathogenic bacteria, which comprises a primer for amplifying staphylococcus aureus, a primer for amplifying staphylococcus epidermidis, a primer for amplifying streptococcus agalactiae, a primer for amplifying streptococcus uberis, a primer for amplifying enterococcus, a primer for amplifying pseudomonas aeruginosa, a primer for amplifying escherichia coli, a primer for amplifying klebsiella pneumoniae, a primer for amplifying listeria monocytogenes and a primer for amplifying mycoplasma bovis; the upstream inner primer FIP for detecting staphylococcus aureus is shown as SEQ ID NO. 1; the downstream inner primer BIP for detecting staphylococcus aureus is shown as SEQ ID NO. 2; the upstream outer primer F3 for detecting staphylococcus aureus is shown as SEQ ID NO. 3; the downstream outer primer B3 for detecting staphylococcus aureus is shown as SEQ ID NO. 4; the upstream inner primer FIP for detecting staphylococcus epidermidis is shown as SEQ ID NO. 5; the downstream inner primer BIP for detecting staphylococcus epidermidis is shown as SEQ ID NO. 6; the upstream outer primer F for detecting staphylococcus epidermidis is shown as SEQ ID NO. 7; the downstream outer primer B3 for detecting staphylococcus epidermidis is shown as SEQ ID NO. 8; an upstream inner primer FIP for detecting Streptococcus agalactiae: as shown in SEQ ID NO. 9; the downstream inner primer BIP for detecting streptococcus agalactiae is shown as SEQ ID NO. 10; upstream outer primer F3 for detecting Streptococcus agalactiae: as shown in SEQ ID NO. 11; the downstream outer primer B3 for detecting streptococcus agalactiae is shown as SEQ ID NO. 12; upstream inner primer FIP for detecting streptococcus agalactiae: as shown in SEQ ID NO. 13; the downstream inner primer BIP for detecting streptococcus agalactiae is shown as SEQ ID NO. 14; the upstream outer primer F3 for detecting streptococcus agalactiae is shown as SEQ ID NO. 15; the downstream outer primer B3 for detecting streptococcus agalactiae is shown as SEQ ID NO. 16; the upstream inner primer FIP for detecting the streptococcus uberis is shown as SEQ ID NO. 17; the downstream inner primer BIP for detecting the streptococcus uberis is shown as SEQ ID NO. 18; the upstream outer primer F3 for detecting streptococcus uberis is shown as SEQ ID NO. 19; the downstream outer primer B3 for detecting the streptococcus uberis is shown as SEQ ID NO. 20; the upstream inner primer FIP for detecting enterococcus is shown as SEQ ID NO. 21; the downstream inner primer BIP for detecting enterococcus is shown as SEQ ID NO. 22; the upstream outer primer F3 for detecting enterococcus is shown as SEQ ID NO. 23; the downstream outer primer B3 for detecting enterococcus is shown as SEQ ID NO. 24; the upstream inner primer FIP for detecting the pseudomonas aeruginosa is shown as SEQ ID NO. 25; the downstream inner primer BIP for detecting the pseudomonas aeruginosa is shown as SEQ ID NO. 26; the upstream outer primer F3 for detecting the pseudomonas aeruginosa is shown as SEQ ID NO. 27; the downstream outer primer B3 for detecting the pseudomonas aeruginosa is shown as SEQ ID NO. 28; the upstream inner primer FIP for detecting the enterobacteria is shown as SEQ ID NO. 29; the downstream inner primer BIP for detecting the enterobacteria is shown as SEQ ID NO. 30; the upstream outer primer F3 for detecting the enterobacteria is shown as SEQ ID NO. 31; the downstream outer primer B3 for detecting the enterobacteria is shown as SEQ ID NO. 32; the upstream inner primer FIP for detecting the escherichia coli is shown as SEQ ID NO. 33; the downstream inner primer BIP for detecting the escherichia coli is shown as SEQ ID NO. 34; the upstream outer primer F3 for detecting the escherichia coli is shown as SEQ ID NO. 35; the downstream outer primer B3 for detecting the escherichia coli is shown as SEQ ID NO. 36; the upstream inner primer FIP for detecting klebsiella pneumoniae is shown as SEQ ID No. 37; the downstream inner primer BIP for detecting klebsiella pneumoniae is shown as SEQ ID No. 38; the upstream outer primer F3 for detecting klebsiella pneumoniae is shown as SEQ ID No. 39; the downstream outer primer B3 for detecting klebsiella pneumoniae is shown as SEQ ID No. 40; the upstream inner primer FIP for detecting the listeria monocytogenes is shown as SEQ ID NO. 41; the downstream inner primer BIP for detecting the listeria monocytogenes is shown as SEQ ID NO. 42; the upstream outer primer F3 for detecting the listeria monocytogenes is shown as SEQ ID NO. 43; the downstream outer primer B3 for detecting the listeria monocytogenes is shown as SEQ ID NO. 44; the upstream inner primer FIP for detecting mycoplasma bovis is shown as SEQ ID NO. 45; the downstream inner primer BIP for detecting mycoplasma bovis is shown as SEQ ID NO. 46; the upstream outer primer F3 for detecting mycoplasma bovis is shown as SEQ ID NO. 47; the downstream outer primer B3 for detecting mycoplasma bovis is shown as SEQ ID NO. 48.
Further, it also includes 2×LampPCR Master Mix, bst 3.0DNA polymerase, calcein-Mn 2+ Mixing the solution and ddH 2 O。
The invention also provides a method for detecting dairy cow mastitis by adopting the kit, which comprises the following steps:
(1) Extracting milk samples of cows, and adding the milk samples into an LAMP reaction system containing the primers for reaction for 1 hour at the constant temperature of 64 ℃;
(2) Judging the result after the reaction: calcein MnCL in post-reaction system 2 The mixed solution changes from brown yellow to green fluorescence to indicate the existence of pathogenic bacteria corresponding to the primer pair; no color change before and after the reaction was brown yellow, indicating that no pathogenic bacteria corresponding to the primer pair were present.
Specifically, the reaction system for detecting each pathogenic bacteria specifically includes: primers FIP and BIP 0.8uM, F3 and B3 0.2uM, milk sample to be tested 2ul, mgCl 2 6mM,10 Xreaction buffer 2.5ul, calcein MnCl 2 2.5ul of solution, dNTP1mM, ddH 2 O2 ul, bst DNA polymerase 320U/ML.
Wherein, calcein MnCl 2 The solution is composed of 1mM calcein solution and 4mM MnCl 2 Mixing the solution in equal volume to obtain calcein with final concentration of 50um and MnCl 2 The final concentration of the solution was 200uM. The fluorescent indicator does not influence the LAMP reaction, is added into a system before the reaction, and directly judges the result according to the color change after one-step reaction, thereby avoiding false positive interference caused by adding the fluorescent indicator after uncovering.
The method for detecting whether the sample to be detected has mastitis or not, wherein the sample can be milk or mammary secretion of a dairy cow or can be a blood sample, and the method comprises the step of detecting by using the visualized isothermal amplification primer set for detecting the mastitis of the dairy cow.
The detection method of the invention is easy to operate, has low cost and can be widely applied. Compared with the prior art, the invention has the following beneficial effects:
(1) The invention provides a method for rapidly and accurately judging specificity of cow mastitis infection with low cost, which has the characteristics of convenient operation, no need of special instruments and low cost, can provide powerful help for accurate diagnosis and treatment of farms, has the characteristics of high detection efficiency and low cost, is very suitable for basic level use, and has the conditions of large-scale popularization and application.
(2) According to the invention, by designing the inner primers FIP and BIP and the outer primers F3 and B3 which are different from the existing primer sequences, the sensitivity of nucleic acid amplification reaction is greatly improved, and experiments show that: the optimal reaction temperature is constant at 64 ℃ and the optimal reaction time is 60 minutes by adopting the specific primer designed by the invention, and compared with the original nucleic acid amplification reaction, the time required by the amplification reaction is shortened by at least more than half. Meanwhile, the primer designed based on the invention has high specificity, and can judge whether the target gene sequence exists or not only according to the existence of the amplification reaction product, so that the on-site nucleic acid detection can be realized rapidly, and the current requirements of cow mastitis pathogenic bacteria detection are completely met.
Drawings
FIG. 1 shows that the number of copies in the dilution is 4X 10 3 At copies/mL, a clear discoloration can still be seen.
FIG. 2 shows that the LAMP reaction products of primers and DNA at various storage times are all green.
Figure 3 shows that both the lactobacillus control group and the bifidobacteria control group are brown yellow, demonstrating no amplification.
Detailed Description
The following examples facilitate a better understanding of the present invention, but are not intended to limit the same. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below are commercially available unless otherwise specified.
Example 1
The invention aims to provide a visual isothermal amplification detection kit for 12 common pathogenic bacteria of dairy cow mastitis and application thereof, which shortens detection time, improves specificity and sensitivity, improves operation convenience and reduces cost.
According to the invention, 4 primers are designed according to the conserved sequences of 12 common cow mastitis pathogens, wherein the sequences of the primers comprise F3 (forward outer primer), B3 (reverse outer primer), FIP (forward inner primer) and BIP (reverse inner primer) respectively as follows:
primers for detection of staphylococcus aureus:
upstream inner primer FIP for detecting Staphylococcus aureus:
5'-accagaaagtcgccttcgcctgtagcggtgaaatgcgc-3' (SEQ ID NO. 1)
Downstream inner primer BIP for detection of staphylococcus aureus:
5'-actgacgctgatgtgcgaaagctagcactcatcgtttacggc-3' (SEQ ID NO. 2)
Upstream outer primer F3 for detection of Staphylococcus aureus: 5'-gtgcagaagaggaaagtgga-3' (SEQ ID NO. 3)
Downstream outer primer B3 for detection of staphylococcus aureus: 5'-cgttagctgcagcactaagg-3' (SEQ ID NO. 4)
Primers for staphylococcus epidermidis:
upstream inner primer FIP for detecting Staphylococcus epidermidis:
5'-cctgtcactctgtcccccgaacttgacatcctctgacccct-3' (SEQ ID NO. 5)
Downstream inner primer BIP for detection of staphylococcus epidermidis:
5'-ggttaagtcccgcaacgagcgtgtcaccggcagtcaact-3' (SEQ ID NO. 6)
Upstream outer primer F3 for detecting Staphylococcus epidermidis: 5'-aacgcgaagaaccttaccaa-3' (SEQ ID NO. 7)
Downstream outer primer B3 for detection of staphylococcus epidermidis: 5'-tcatccccaccttc-3' (SEQ ID NO. 8)
Primer of streptococcus agalactiae:
an upstream inner primer FIP for detecting Streptococcus agalactiae:
5'-cagcttagttatcccaaatcccatagaagcaatcactttttcaactca-3' (SEQ ID NO. 9)
Downstream inner primer BIP for detection of streptococcus agalactiae:
5'-attcgcattttagatccatttgctt-gcctttacatcgttaacttgag-3' (shown in SEQ ID NO. 10)
Upstream outer primer F3 for detecting Streptococcus agalactiae: 5'-agaagccttaacagatgtga-3' (SEQ ID NO. 11)
Downstream outer primer B3 for detection of streptococcus agalactiae: 5'-caggataagttaaaaccttttgttc-3' (SEQ ID NO. 12)
Primer of streptococcus dysgalactiae:
upstream inner primer FIP for detecting streptococcus agalactiae:
5'-tcgcaactaaatcatttgcaatgtc-agcggtcattgtacctgt-3' (shown in SEQ ID NO. 13)
Downstream inner primer BIP for detecting streptococcus agalactiae:
5'-ctgacaatgctactccggga-cgtatgcatcatttagtaaacgata-3' (SEQ ID NO. 14)
Upstream outer primer F3 for detecting streptococcus agalactiae: 5'-gtttctgcagaaactatacctg-3' (SEQ ID NO. 15)
Downstream outer primer B3 for detecting streptococcus agalactiae: 5'-agtcttttgattccaacacat-3' (SEQ ID NO. 16)
Primer of streptococcus uberis:
upstream inner primer FIP for detecting Streptococcus mammary:
5 '-agtcgcgaattgaactttatactca-aaacgggttgaagaacct-3' (SEQ ID NO. 17)
Downstream inner primer BIP for detecting streptococcus uberis:
5'-attccacccactacctatttttgt-tcgaaactcgtcagaggt-3' (SEQ ID NO. 18)
Upstream outer primer F3 for detecting Streptococcus mammary: 5'-aagggcatgttattctcaga-3' (SEQ ID NO. 19)
Downstream outer primer B3 for detecting streptococcus uberis: 5'-caaaagcttttcttctgcaatt-3' (SEQ ID NO. 20)
Primers for enterococcus:
upstream inner primer FIP for detection of enterococci:
5'-tagctgcagcactgaagggcgtagtccacgccgtaaacg-3' (SEQ ID NO. 21)
Downstream inner primer BIP for detection of enterococci:
5'-ctggggagtacgaccgcaagcatgctccaccgcttgtg-3' (SEQ ID NO. 22)
Upstream outer primer F3 for detection of enterococci: 5'-gtggggagcaaacaggatt-3' (SEQ ID NO. 23)
Downstream outer primer B3 for detection of enterococci: 5'-cctggtaaggttcttcgcg-3' (SEQ ID NO. 24)
Primer of pseudomonas aeruginosa:
upstream inner primer FIP for detecting Pseudomonas aeruginosa: 5'-ttggccaccttgcccttctcgggccaggtgcatgcctt-3' (SEQ ID NO. 25)
Downstream inner primer BIP for detecting Pseudomonas aeruginosa 5 '-tgcccctaccaccagtgga-agtcggcgcccatctc-3' (shown in SEQ ID NO. 26)
Upstream outer primer F3 for detecting Pseudomonas aeruginosa: 5'-cggtcctggtgatccgtg-3' (SEQ ID NO. 27)
Downstream outer primer B3 for detecting pseudomonas aeruginosa: 5'-gccgtactccttcatgtcga-3' (SEQ ID NO. 28)
Primers for enterobacteria:
upstream inner primer FIP for detecting Enterobacter:
5'-gtcagtctttgtccagggggc-caggtgtagcggtgaaatgc-3' (SEQ ID NO. 29)
Downstream inner primer BIP for detection of Enterobacter:
5'-gctcaggtgcgaaagcgtgg-acctccaagtcgacatcgtt-3' (shown in SEQ ID NO. 30)
Upstream outer primer F3 for detection of Enterobacter: 5'-gtcttgtagaggggggtaga-3' (SEQ ID NO. 31)
Downstream outer primer B3 for detection of enterobacteria: 5'-cgttagctccggaagcca-3' (SEQ ID NO. 32)
Primers for escherichia coli:
upstream inner primer FIP for detecting Escherichia coli:
5'-ggcacgagcgttaagcagct-taacgcgctagaaaaaggcg-3' (shown in SEQ ID NO. 33)
Downstream inner primer BIP for detecting Escherichia coli:
5'-cggcgcaaaaacctttgctga-acgcagcgtttttccctg-3' (shown in SEQ ID NO. 34)
Upstream outer primer F3 for detecting Escherichia coli: 5'-gcgaccagtgaaaaatgtcc-3' (SEQ ID NO. 35)
Downstream outer primer B3 for detecting Escherichia coli: 5'-cgctcaccaactgataacca-3' (SEQ ID NO. 36)
Primer of klebsiella pneumoniae:
upstream inner primer FIP for detecting klebsiella pneumoniae:
5'-tggtcatcctctcagaccagctgctagtaggtggggtaacgg-3' (SEQ ID NO. 37)
Downstream inner primer BIP for detecting klebsiella pneumoniae:
5'-tggaactgagacacggtccagaatggctgcatcaggcttg-3' (SEQ ID NO. 38)
Upstream outer primer F3 for detecting klebsiella pneumoniae: 5'-gatgtgcccagatgggatt-3' (SEQ ID NO. 39)
Downstream outer primer B3 for detecting klebsiella pneumoniae: 5'-gccttcttcacacacgcg-3' (SEQ ID NO. 40)
Primers for listeria monocytogenes:
upstream inner primer FIP for detection of Listeria monocytogenes:
5’-tgaacaatttcgttaccttcaggat-tcgatcactctggaggatac-3 ， (shown in SEQ ID NO. 41)
Downstream inner primer BIP for detection of listeria monocytogenes:
5'- -ggagcgaaaacaataaaagcaagct-gcgtaaacattaatatttctcgc-3' (shown in SEQ ID NO. 42)
Upstream outer primer F3 for detection of Listeria monocytogenes: 5'-ttcaaaagcttatacagatggaa-3' (SEQ ID NO. 43)
Downstream outer primer B3 for detection of listeria monocytogenes: 5'-aagctaaaccagtgcattc-3' (SEQ ID NO. 44)
Primer of mycoplasma bovis:
upstream inner primer FIP for detecting mycoplasma bovis:
5'-accccgctaaacatcatcgcc-gatcggagtgcgcaacat-3' (shown in SEQ ID NO. 45)
Downstream inner primer BIP for detecting mycoplasma bovis:
5'-agagattgatccgccacactgg-ctactgctgcctcccgta-3' (shown in SEQ ID NO. 46)
Upstream outer primer F3 for detecting mycoplasma bovis: 5'-aggaagcgtttgcttcgc-3' (SEQ ID NO. 47)
Downstream outer primer B3 for detecting mycoplasma bovis: 5'-gctccatcagactttcgtcc-3' (SEQ ID NO. 48)
The visual isothermal amplification detection kit for 12 common pathogenic bacteria of dairy cow mastitis comprises the primer set and a detection reagent.
Preferably, the detection reagent comprises the following components: f3 (forward outer primer), B3 (reverse outer primer), FIP (forward inner primer), BIP (reverse inner primer), 2X Lamp PCR Master Mix, bst 3.0DNA polymerase, calcein-Mn 2+ The solution and ddH2O were mixed.
According to a specific embodiment of the present invention, a method for detecting or aiding in the detection of whether a sample to be tested is infected with mastitis, comprises the steps of:
the reaction steps are as follows: performing loop-mediated isothermal amplification by using the detection reagent or the detection kit according to any one of the above from a milk sample to be detected as a template to obtain an amplification product; preferably, the reaction conditions of the loop-mediated isothermal amplification are: keeping the temperature at 64 ℃ and reacting for 60min; a confirmation step: the amplified product presents green fluorescence, and then the sample to be detected infects corresponding cow mastitis pathogenic bacteria; and if the amplified product is brown yellow, the sample to be detected is not infected with 12 cow mastitis pathogenic bacteria in the detection range of the kit.
Example 2: establishment of LAMP reaction System
In the invention, a stable amplification system is obtained by continuous experimental optimization, wherein the amplification system comprises 2X Lamp PCR Master Mix 12.5.5 mu L, 0.5 mu L of Bst 3.0DNA polymerase, 2 mu L of inner primers FIP and BIP (10 mu M), 0.5 mu L of outer primers F3 and B3 (10 mu M) and 2 mu L, ddH O5 mu L of template, the system is heated at the constant temperature of 64 ℃ in a metal bath or a water bath kettle for 1 h, and MnCl is added into the system 2 1. Mu.L of the solution (15 mM) and 3. Mu.L of calcein (500. Mu.M) were visually distinguishable, and positive results were shown as fluorescent green and negative results as brown yellow.
Example 3 sensitivity and stability verification test of LAMP primer group
1. Sensitivity verification test: serial dilutions of DNA samples of 12 bacterial solutions of known concentrations were performed at 10-fold gradients from 4X 10 3 ～4×10 6 The copies/mL interval takes 5. Mu.L of diluent of each order of magnitude as the amplification template.
The test conditions were the same as in example 1. The results show that: copy number in dilution of 4X 10 3 The visible discoloration was still visible at the points of the copies/mL (see FIG. 1), demonstrating the lowest dilution detectable by LAMP<4×10 3 The copies/mL has good sensitivity, and 20 copies of DNA can be detected at the minimum in each reaction.
2, stability verification test: four groups of primers provided by the invention are respectively packaged on days 0, 5, 10 and 15 and stored at-20 ℃. Then, the LAMP amplification test was carried out on day 20 (test method and procedure are the same as in example 1), and the results of the LAMP test on the same group of DNA samples and four groups of primers were compared to determine the stability of the primers. The results of four sets of experiments are shown in FIG. 2. The results show that the LAMP reaction products of the primers and the DNA under different storage time are green, and the primer group provided by the invention has good stability.
Example 4 further demonstrates experiments on LAMP primer set specificity.
Clinically common nonpathogenic bacteria are taken as detection objects (including lactobacillus and bifidobacterium). LAMP detection was performed with the primer set provided by the kit to verify the specificity of the reaction, and the results are shown in the Lactobacillus control group and the Bifidobacterium control group of FIG. 3.
The results show that the lactobacillus control group and the bifidobacterium control group are brown yellow, and no amplification is proved; the positive control groups under the same reaction conditions are changed into fluorescent green, and the primer group has good specificity.
The invention can rapidly and effectively realize nucleic acid detection by designing the inner primers (FIP and BIP) and the outer primers (F3 and B3) of 12 common pathogenic bacteria of cow mastitis with different sequences from the existing primers. The invention can provide powerful help for accurate diagnosis and treatment of dairy cow mastitis, and has the characteristics of high detection efficiency and low cost, and is also very suitable for basic use, and is more suitable for large-scale popularization and application.
The above embodiment is only one of the preferred embodiments of the present invention, and should not be used to limit the scope of the present invention, but all the insubstantial modifications or color changes made in the main design concept and spirit of the present invention are still consistent with the present invention, and all the technical problems to be solved are included in the scope of the present invention.
Sequence listing
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 17
agtcgcgaat tgaactttat actcaaaacg ggttgaagaa cct 43
<210> 18
<211> 42
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 18
attccaccca ctacctattt ttgttcgaaa ctcgtcagag gt 42
<210> 19
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 19
aagggcatgt tattctcaga 20
<210> 20
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 20
caaaagcttt tcttctgcaa tt 22
<210> 21
<211> 39
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 21
tagctgcagc actgaagggc gtagtccacg ccgtaaacg 39
<210> 22
<211> 38
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 22
ctggggagta cgaccgcaag catgctccac cgcttgtg 38
<210> 23
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 23
gtggggagca aacaggatt 19
<210> 24
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 24
cctggtaagg ttcttcgcg 19
<210> 25
<211> 38
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 25
ttggccacct tgcccttctc gggccaggtg catgcctt 38
<210> 26
<211> 35
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 26
tgcccctacc accagtggaa gtcggcgccc atctc 35
<210> 27
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 27
cggtcctggt gatccgtg 18
<210> 28
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 28
gccgtactcc ttcatgtcga 20
<210> 29
<211> 41
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 29
gtcagtcttt gtccaggggg ccaggtgtag cggtgaaatg c 41
<210> 30
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 30
gctcaggtgc gaaagcgtgg acctccaagt cgacatcgtt 40
<210> 31
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 31
gtcttgtaga ggggggtaga 20
<210> 32
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 32
cgttagctcc ggaagcca 18
<210> 33
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 33
ggcacgagcg ttaagcagct taacgcgcta gaaaaaggcg 40
<210> 34
<211> 39
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 34
cggcgcaaaa acctttgctg aacgcagcgt ttttccctg 39
<210> 35
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 35
gcgaccagtg aaaaatgtcc 20
<210> 36
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 36
cgctcaccaa ctgataacca 20
<210> 37
<211> 42
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 37
tggtcatcct ctcagaccag ctgctagtag gtggggtaac gg 42
<210> 38
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 38
tggaactgag acacggtcca gaatggctgc atcaggcttg 40
<210> 39
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 39
gatgtgccca gatgggatt 19
<210> 40
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 40
gccttcttca cacacgcg 18
<210> 41
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 41
tgaacaattt cgttaccttc aggattcgat cactctggag gatac 45
<210> 42
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 42
ggagcgaaaa caataaaagc aagctgcgta aacattaata tttctcgc 48
<210> 43
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 43
ttcaaaagct tatacagatg gaa 23
<210> 44
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 44
aagctaaacc agtgcattc 19
<210> 45
<211> 39
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 45
accccgctaa acatcatcgc cgatcggagt gcgcaacat 39
<210> 46
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 46
agagattgat ccgccacact ggctactgct gcctcccgta 40
<210> 47
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 47
aggaagcgtt tgcttcgc 18
<210> 48
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 48
gctccatcag actttcgtcc 20
Claims (2)
1. A kit for detecting a bovine mastitis pathogen, comprising: including a primer for amplifying staphylococcus aureus, a primer for amplifying staphylococcus epidermidis, a primer for amplifying streptococcus agalactiae, a primer for amplifying streptococcus uberis, a primer for amplifying enterococcus, a primer for amplifying pseudomonas aeruginosa, a primer for amplifying enterobacter, a primer for amplifying escherichia coli, a primer for amplifying klebsiella pneumoniae, a primer for amplifying listeria monocytogenes, and a primer for amplifying mycoplasma bovis; the upstream inner primer FIP for detecting staphylococcus aureus is shown as SEQ ID NO. 1; the downstream inner primer BIP for detecting staphylococcus aureus is shown as SEQ ID NO. 2; the upstream outer primer F3 for detecting staphylococcus aureus is shown as SEQ ID NO. 3; the downstream outer primer B3 for detecting staphylococcus aureus is shown as SEQ ID NO. 4; the upstream inner primer FIP for detecting staphylococcus epidermidis is shown as SEQ ID NO. 5; the downstream inner primer BIP for detecting staphylococcus epidermidis is shown as SEQ ID NO. 6; the upstream outer primer F for detecting staphylococcus epidermidis is shown as SEQ ID NO. 7; the downstream outer primer B3 for detecting staphylococcus epidermidis is shown as SEQ ID NO. 8; an upstream inner primer FIP for detecting Streptococcus agalactiae: as shown in SEQ ID NO. 9; the downstream inner primer BIP for detecting streptococcus agalactiae is shown as SEQ ID NO. 10; upstream outer primer F3 for detecting Streptococcus agalactiae: as shown in SEQ ID NO. 11; the downstream outer primer B3 for detecting streptococcus agalactiae is shown as SEQ ID NO. 12; upstream inner primer FIP for detecting streptococcus agalactiae: as shown in SEQ ID NO. 13; the downstream inner primer BIP for detecting streptococcus agalactiae is shown as SEQ ID NO. 14; the upstream outer primer F3 for detecting streptococcus agalactiae is shown as SEQ ID NO. 15; the downstream outer primer B3 for detecting streptococcus agalactiae is shown as SEQ ID NO. 16; the upstream inner primer FIP for detecting the streptococcus uberis is shown as SEQ ID NO. 17; the downstream inner primer BIP for detecting the streptococcus uberis is shown as SEQ ID NO. 18; the upstream outer primer F3 for detecting streptococcus uberis is shown as SEQ ID NO. 19; the downstream outer primer B3 for detecting the streptococcus uberis is shown as SEQ ID NO. 20; the upstream inner primer FIP for detecting enterococcus is shown as SEQ ID NO. 21; the downstream inner primer BIP for detecting enterococcus is shown as SEQ ID NO. 22; the upstream outer primer F3 for detecting enterococcus is shown as SEQ ID NO. 23; the downstream outer primer B3 for detecting enterococcus is shown as SEQ ID NO. 24; the upstream inner primer FIP for detecting the pseudomonas aeruginosa is shown as SEQ ID NO. 25; the downstream inner primer BIP for detecting the pseudomonas aeruginosa is shown as SEQ ID NO. 26; the upstream outer primer F3 for detecting the pseudomonas aeruginosa is shown as SEQ ID NO. 27; the downstream outer primer B3 for detecting the pseudomonas aeruginosa is shown as SEQ ID NO. 28; the upstream inner primer FIP for detecting the enterobacteria is shown as SEQ ID NO. 29; the downstream inner primer BIP for detecting the enterobacteria is shown as SEQ ID NO. 30; the upstream outer primer F3 for detecting the enterobacteria is shown as SEQ ID NO. 31; the downstream outer primer B3 for detecting the enterobacteria is shown as SEQ ID NO. 32; the upstream inner primer FIP for detecting the escherichia coli is shown as SEQ ID NO. 33; the downstream inner primer BIP for detecting the escherichia coli is shown as SEQ ID NO. 34; the upstream outer primer F3 for detecting the escherichia coli is shown as SEQ ID NO. 35; the downstream outer primer B3 for detecting the escherichia coli is shown as SEQ ID NO. 36; the upstream inner primer FIP for detecting klebsiella pneumoniae is shown as SEQ ID No. 37; the downstream inner primer BIP for detecting klebsiella pneumoniae is shown as SEQ ID No. 38; the upstream outer primer F3 for detecting klebsiella pneumoniae is shown as SEQ ID No. 39; the downstream outer primer B3 for detecting klebsiella pneumoniae is shown as SEQ ID No. 40; the upstream inner primer FIP for detecting the listeria monocytogenes is shown as SEQ ID NO. 41; the downstream inner primer BIP for detecting the listeria monocytogenes is shown as SEQ ID NO. 42; the upstream outer primer F3 for detecting the listeria monocytogenes is shown as SEQ ID NO. 43; the downstream outer primer B3 for detecting the listeria monocytogenes is shown as SEQ ID NO. 44; the upstream inner primer FIP for detecting mycoplasma bovis is shown as SEQ ID NO. 45; the downstream inner primer BIP for detecting mycoplasma bovis is shown as SEQ ID NO. 46; the upstream outer primer F3 for detecting mycoplasma bovis is shown as SEQ ID NO. 47; the downstream outer primer B3 for detecting mycoplasma bovis is shown as SEQ ID NO. 48.
2. A kit for detecting a bovine mastitis pathogen as defined in claim 1, wherein: also comprises 2X Lamp PCR Master Mix, bst 3.0DNA polymerase, calcein-Mn 2+ Mixing the solution and ddH 2 O。
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2015100282A (en) * | 2013-11-21 | 2015-06-04 | 国立研究開発法人農業・食品産業技術総合研究機構 | Method for detecting bovine mastitis causal microorganism, and primer set and assay kit used for the same |
| CN107022629A (en) * | 2017-05-16 | 2017-08-08 | 广东海大畜牧兽医研究院有限公司 | A kind of multiple PCR detection primer group of mastitis for milk cows pathogen and its application |
| CN107541567A (en) * | 2016-06-29 | 2018-01-05 | 博奥生物集团有限公司 | For detecting the LAMP primer composition and its application of 8 kinds of environment pathogens of mastitis for milk cows |
| WO2019143812A1 (en) * | 2018-01-18 | 2019-07-25 | Biomeme, Inc. | Methods of assaying for the presence of microorganisms |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10253376B2 (en) * | 2013-10-16 | 2019-04-09 | Wisconsin Alumni Research Foundation | DNA-based detection and identification of eight mastitis pathogens |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2015100282A (en) * | 2013-11-21 | 2015-06-04 | 国立研究開発法人農業・食品産業技術総合研究機構 | Method for detecting bovine mastitis causal microorganism, and primer set and assay kit used for the same |
| CN107541567A (en) * | 2016-06-29 | 2018-01-05 | 博奥生物集团有限公司 | For detecting the LAMP primer composition and its application of 8 kinds of environment pathogens of mastitis for milk cows |
| CN107022629A (en) * | 2017-05-16 | 2017-08-08 | 广东海大畜牧兽医研究院有限公司 | A kind of multiple PCR detection primer group of mastitis for milk cows pathogen and its application |
| WO2019143812A1 (en) * | 2018-01-18 | 2019-07-25 | Biomeme, Inc. | Methods of assaying for the presence of microorganisms |
Non-Patent Citations (2)
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| Comparative Diagnosis of Infectious Bacteria in Bovine Milk;Doaa Sedky 等;Journal of Animal Health and Production;第8卷(第4期);171-182 * |
| 乳品中食源性致病菌快速检测技术研究进展;王伟;蔡文;李晓芹;胡文涛;方志娟;蒋云枫;叶湖;;食品安全质量检测学报(09);205-213 * |
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