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Primer group, kit and method for identifying staphylococcus in environment at in-species level

A technology for staphylococcus and staphylococcus wornerii, which is applied in biochemical equipment and methods, microbial determination/inspection, recombinant DNA technology, etc., can solve the problems of small identification range, unfavorable promotion, high price, etc., and achieves short detection time. , high sensitivity, low cost effect

Active Publication Date: 2021-06-15
至微生物智能科技(厦门)有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Chinese invention patent 201610139846.2 discloses a combination of gyrB primers for the detection of Staphylococcus aureus in experimental animals. This primer can be used for the identification of Staphylococcus aureus based on LAMP isothermal amplification. This method has a small identification range and can only be used for one type of Staphylococcus. Identification, and the LAMP isothermal amplification reagent used is expensive, which is not conducive to popularization

Method used

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  • Primer group, kit and method for identifying staphylococcus in environment at in-species level
  • Primer group, kit and method for identifying staphylococcus in environment at in-species level
  • Primer group, kit and method for identifying staphylococcus in environment at in-species level

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Species Identification of Environmental Staphylococci

[0071] Specific steps are as follows:

[0072] A. Use a swab to sample bacteria in different locations in the environment (experimental benches, refrigerators, etc.) (the sampling liquid is lysate), and the sampling area is 25cm 2 .

[0073] B. Gradiently dilute the sample solution to 10 -6 , take 10 respectively -4 , 10 -5 , 10 -6 10 μl of bacterial solutions of three concentrations were spread on LB solid medium, and cultured overnight in a 37°C incubator. After colonies grew, single colonies were picked and streaked three times for isolation and culture to obtain pure bacteria.

[0074] C. Pick a single bacterial colony from the purified streak culture, and perform PCR amplification with 16S rDNA universal primers 27F and 1492R.

[0075] 27F primer sequence: 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID No.13),

[0076] 1492R primer sequence: 5'-CGGYTACCTTGTTACGACTT-3' (SEQ ID No.14);

[0077] D. Sequencing the PCR...

Embodiment 2

[0082] Design of 6 Specific Primers for Staphylococcus

[0083] Specific steps are as follows:

[0084] A. Download the gyrB gene sequences of Staphylococcus aureus, Staphylococcus worthii, Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus capitis and Staphylococcus haemolyticus from the Genebank gene bank.

[0085] B. Compare the gene sequences, search for differential sequences, and design specific primers for 6 kinds of staphylococci respectively. The primer sequences are as follows:

[0086] Staphylococcus aureus

[0087] F1: 5'-ACTTAAAAGAAGTTGGCAC-3' (SEQ ID No. 1)

[0088] R1: 5'-GTTTGACCTTCGAATTGAGGATC-3' (SEQ ID No. 2)

[0089] Staphylococcus wausii

[0090] F2: 5'-AGACGAAGATGATATCAGAC-3' (SEQ ID No. 3)

[0091] R2: 5'-GTTTGACCTTCGAATTGAGGATC-3' (SEQ ID No. 4)

[0092] Staphylococcus epidermidis

[0093] F3: 5'-AAGATGAAAGAGAAGAGGAAG-3' (SEQ ID No. 5)

[0094] R3: 5'-GTTGGTTGCATATCCACTG-3' (SEQ ID No. 6)

[0095] Staphylococcus hominis

[0096...

Embodiment 3

[0105] Staphylococcus Primer Generic Specificity Detection

[0106] A. Taking the primers of Staphylococcus aureus, Staphylococcus haemolyticus, Staphylococcus worthii and Staphylococcus human as examples, verify the inter-genus specificity of Staphylococcus primers. The bacteria of the genus Pantoea, Micrococcus, Pseudomonas, and Bacillus were respectively used as templates for PCR amplification; the positive control was the Staphylococcus strain (P) corresponding to the primers, and the negative control was the lysate ( N), M is Maeker, and the size of Maeker fragments from top to bottom is 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp.

[0107] B. PCR products were detected by agarose gel electrophoresis.

[0108] C. The results of agarose gel electrophoresis are shown in figure 2 , Table 2 is a list of strain numbers used in agarose gel electrophoresis, from figure 2 It can be seen that different Staphylococcus primers can only amplify the corresponding Staphylococcus, ind...

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Abstract

The invention provides a primer group, kit and method for identifying staphylococcus in an environment at an in-species level, and belongs to the technical field of molecular biological detection. The primer group comprises primers F1 and R1 for amplifying staphylococcus aureus, primers F2 and R2 for amplifying staphylococcus warneri, primers F3 and R3 for amplifying staphylococcus epidermidis, primers F4 and R4 for amplifying staphylococcus hominis, primers F5 and R5 for amplifying staphylococcus capitis, and primers F6 and R6 for amplifying staphylococcus haemolyticus; and the nucleotide sequences of the primers F1, R1, F2, R2, F3, R3, F4, R4, F5, R5, F6 and R6 are respectively as shown in SEQ ID No. 1 to SEQ ID No. 12. The primer group, kit and method are easy to operate and high in sensitivity, and can complete the detection of environmental staphylococcus in a short time; and the method is low in cost, can identify various staphylococcus species, and is convenient for popularization and application.

Description

technical field [0001] The invention belongs to the technical field of molecular biological detection, and in particular relates to a primer set, a kit and a method for identifying staphylococci in an environment at the species level. Background technique [0002] Staphylococcus belongs to Gram-positive bacteria and is the most common pyogenic coccus. The elderly, pregnant women, and newborns with low body resistance are most susceptible to Staphylococcus infection, causing pemphigus, conjunctivitis, sepsis, etc. disease. In addition, staphylococcus can also cause infectious diseases in poultry, such as foot pad swelling, omphalitis and staphylococcal sepsis, etc., causing greater losses to the poultry industry. [0003] The 16S rDNA gene is the DNA sequence corresponding to the coding rRNA on bacteria and exists in the genome of all bacteria. 16S rDNA is highly conservative and specific. With the emergence of PCR technology and the continuous improvement of nucleic acid r...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/686C12Q1/14C12N15/11
CPCC12Q1/686C12Q1/689C12Q2600/166C12Q2545/113C12Q2565/125
Inventor 梁旭东
Owner 至微生物智能科技(厦门)有限公司
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