Kit and method for detecting cow mastitis pathogenic bacteria

A technology for dairy cow mastitis and pathogenic bacteria, applied in biochemical equipment and methods, microbiological measurement/testing, DNA/RNA fragments, etc., can solve problems such as false positive interference, long time, complicated methods of dairy cow mastitis, etc. Achieve high specificity, low cost, and meet the needs of pathogenic bacteria detection

Active Publication Date: 2022-04-05
SHANGHAI ANIMAL EPIDEMIC PREVENTION & CONTROL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Aiming at the above-mentioned technical problems in the prior art, the present invention provides a kit for detecting pathogenic bacteria of dairy cow mastitis and a method thereof, the described kit and method for detecting pathogenic bacteria of dairy cow mastitis The method should solve technical problems such as complicated method for detecting dairy cow mastitis in the prior art, long time, false positive interference, etc.

Method used

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  • Kit and method for detecting cow mastitis pathogenic bacteria
  • Kit and method for detecting cow mastitis pathogenic bacteria
  • Kit and method for detecting cow mastitis pathogenic bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] The purpose of the present invention is to provide a visual isothermal amplification detection kit for 12 kinds of common pathogenic bacteria of dairy cow mastitis and its application, which shortens the detection time, improves specificity and sensitivity, and improves the convenience of operation , Reduced costs.

[0023] The present invention first designs 4 primers respectively according to the conserved sequences of 12 common cow mastitis pathogenic bacteria, including F3 (forward outer primer), B3 (reverse outer primer), FIP (forward inner primer), BIP ( Reverse inner primer), the sequences of all primers are as follows:

[0024] Primers used to detect Staphylococcus aureus:

[0025] Upstream inner primer FIP for detection of Staphylococcus aureus:

[0026] 5'-accagaaagtcgccttcgcctgtagcggtgaaatgcgc-3' (shown in SEQ ID NO.1)

[0027] The downstream inner primer BIP used to detect Staphylococcus aureus:

[0028] 5'-actgacgctgatgtgcgaaagctagcactcatcgtttacggc-3' (...

Embodiment 2

[0110] Embodiment 2: The establishment of LAMP reaction system

[0111] In the present invention, through continuous experimental optimization, a relatively stable amplification system was obtained as follows: 2×LampPCR Master Mix 12.5 μL, Bst 3.0 DNA polymerase 0.5 μL, internal primers FIP, BIP (10 μM) each 2 μL , external primers F3 and B3 (10 μM) each 0.5 μL, template 2 μL, ddH2O 5 μL, the system can be heated at 64 °C for 1 h in a metal bath or water bath, and MnCl is added to the system at the same time 2 Solution (15mM) 1μL, calcein (500μM) 3μL, so that the reaction results can be judged by the naked eye, positive results are displayed in fluorescent green, and negative results are displayed in brownish yellow.

Embodiment 3

[0112] Example 3 Sensitivity and Stability Verification Test of LAMP Primer Set

[0113] 1. Sensitivity verification test: serially dilute 12 bacterial liquid DNA samples with known concentrations in a 10-fold gradient, starting from 4×10 3 ~4×10 6 In the copies / mL interval, 5 μL of each order of magnitude dilution was taken as the amplification template.

[0114] The test conditions are the same as in Example 1. The results showed: the copy number in the diluent was 4×10 3 copies / mL, the obvious discoloration can still be seen at this time (see figure 1 ), proving that the lowest dilution that can be detected by the LAMP method is 3 copies / mL, with good sensitivity, each reaction can detect a minimum of 20 copies of DNA.

[0115] 2. Stability verification test: on the 0th, 5th, 10th, and 15th day, respectively, four groups of primers provided by the present invention were divided and stored at -20°C. Afterwards, on the 20th day, the LAMP amplification test was carried ...

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Abstract

The invention provides a kit for detecting dairy cow mastitis pathogenic bacteria. Comprising a primer for amplifying staphylococcus aureus, a primer for amplifying staphylococcus epidermidis, a primer for amplifying streptococcus agalactiae, a primer for amplifying streptococcus dysgalactiae, a primer for amplifying streptococcus uberis, a primer for amplifying enterococcus, a primer for amplifying pseudomonas aeruginosa, a primer for amplifying enterobacter and 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 cow mastitis by adopting the kit. The method has the advantages of being convenient to operate, free of special instruments and low in cost, not only can provide powerful help for accurate diagnosis and treatment of a farm, but also has the advantages of being high in detection efficiency and low in cost, is very suitable for basic-level use and has the condition of large-scale application and popularization.

Description

technical field [0001] The invention belongs to the field of biomolecular detection, and relates to a kit, in particular to a kit for detecting pathogenic bacteria of cow mastitis and a method thereof. Background technique [0002] Dairy cow mastitis is the number one disease that has plagued and threatened the dairy industry in recent years. It can cause a decrease in the milk volume and milk quality of sick milk. In severe cases, it can also lead to the loss of dairy cow production performance, and premature elimination of adult dairy cows, causing huge losses. Economic losses. There are many factors that lead to cow mastitis, more than 80% of which are caused by pathogenic microorganism infection. Antibiotics are mainly used in clinical treatment. The use of a large number of antibiotics not only increases the cost of treatment, but also makes bacteria resistant to antibiotics. It also causes a large number of antibiotic residues in dairy products, which seriously threat...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/14C12Q1/10C12Q1/04C12N15/11
CPCY02A50/30
Inventor 王晓旭王建赵洪进徐锋沈莉萍齐新永赵晓明杨德全唐聪圣张玉杰
Owner SHANGHAI ANIMAL EPIDEMIC PREVENTION & CONTROL CENT
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