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A primer set, kit and method for identifying environmental bacteria at the genus level

An environmental and bacterial technology, applied in biochemical equipment and methods, microbial measurement/inspection, recombinant DNA technology, etc., can solve the problems of inability to detect bacteria extensively and comprehensively, and achieve low cost, short time consumption, and simple operation Effect

Active Publication Date: 2021-08-10
至微生物智能科技(厦门)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the above-mentioned methods have great limitations in the detection range, and cannot detect bacteria in the environment more extensively and comprehensively.

Method used

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  • A primer set, kit and method for identifying environmental bacteria at the genus level
  • A primer set, kit and method for identifying environmental bacteria at the genus level
  • A primer set, kit and method for identifying environmental bacteria at the genus level

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Species Identification and Classification of Environmental Bacteria

[0063] Specific steps are as follows:

[0064] A. Use swabs to sample bacteria from different locations in the environment (experimental benches, instrument surfaces, etc.) (sampling liquid is bacterial lysate), numbered 1~48 in sequence.

[0065] 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.

[0066] C. Pick the single bacterial colony of the purified streak culture, carry out PCR amplification with 16S rDNA universal primers 27F and 1492R, and detect the PCR amplification results by agarose gel electrophoresis (see the electrophoresis results in figure 2 , where 1~48 are 48 collected samples, M is M...

Embodiment 2

[0083] Design and detection of genus-level specific primers

[0084] Specific steps are as follows:

[0085] A, according to the sequencing results of 48 kinds of bacteria in Example 1, and search for differential sequences, design 16S rDNA bacteria-specific primers, wherein R1 is a specific reverse primer for Pantoea, and R2 is Staphylococcus, Bacillus, Micrococcus, Pseudomonas A specific reverse primer common to both Monas and Bacillus.

[0086] Staphylococcus specific forward primer F1: 5'-ATGTGTAAGTAACTATGCACGTCT-3' (SEQ ID No.1)

[0087] Bacillus-specific forward primer F2 5'-CTAGTTGAATAAGCTGGCACCT-3' (SEQ ID No.2)

[0088] Pantoea specific forward primer F3 5'-CAGCGATTCGGTCGGGAACTCA-3' (SEQ ID No.3)

[0089] Micrococcus specific bacteria forward primer F4 5'-AGCGAAAGTGACGGTACCTGCAGAAG-3' (SEQ IDNo.4)

[0090] Pseudomonas specific forward primer F5 5'-GCAGTAAGTTAATACCTTGCTGTTT-3' (SEQ ID No.5)

[0091] Aerobicella specific forward primer F6 5'-ATTGTAGAGTAACTGCTACAGTCT-...

Embodiment 3

[0114] Identifying Environmental Bacteria at the Genus Level

[0115] Specific steps are as follows:

[0116] A. Use a swab to sample bacteria from different locations in the environment (1, table 2, window 3, glass 4, small trash can 5, doorknob 6, floor) (the sampling liquid is lysate).

[0117] B. Using the collected sample as a template, carry out PCR amplification with the forward primer and reverse primer in Example 2, respectively.

[0118] C. Perform agarose gel electrophoresis on the PCR product.

[0119] D. Results of agarose gel electrophoresis ( Figure 5 ) for analysis. From Figure 5 It can be seen that the provided primer pairs are specific and can be used to directly amplify bacterial templates sampled at different locations in the environment, and the bacterial species contained in different locations in the environment are different.

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Abstract

The invention provides a primer set, kit and method for identifying environmental microorganisms at the genus level, belonging to the technical field of environmental bacteria detection and identification. The primer set includes amplified Staphylococcus, Bacillus, Pantoea, Micrococcus, Specific forward primers F1~F6 for Pseudomonas and Aerococci, amplified Pantoea-specific reverse primer R1 and specific reverse primer R2 shared by Staphylococcus, Bacillus, Micrococcus, Pseudomonas and Aerococcus The nucleotide sequence of described forward primer F1~F6 is as shown in SEQ ID No.1~SEQ ID No.6, and the nucleotide sequence of described reverse primer R1 is as shown in SEQ ID No.7, so The nucleotide sequence of the reverse primer R2 is shown in SEQ ID No.8. The primer set, kit and method provided by the invention can quickly, effectively and comprehensively detect environmental bacteria at the genus level.

Description

technical field [0001] The invention belongs to the technical field of environmental bacteria detection and identification, and in particular relates to a primer set, a kit and a method for identifying environmental microorganisms at the genus level. Background technique [0002] Bacteria are one of the major groups of organisms and the most numerous of all organisms. Bacteria in the environment are pathogenic, such as Staphylococcus aureus, Salmonella, Pseudomonas aeruginosa, etc., and there are also beneficial bacteria such as Bifidobacterium, Lactobacillus, etc. Different species of bacteria have different pathogenicity and beneficial effects. There are different distinctions, which are closely related to the survival and development of human beings. Therefore, it is of great significance to provide a method for identifying environmental bacteria at the genus level. [0003] The 16S rRNA gene is the DNA sequence encoding rRNA in bacteria, which exists in the genomes of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/14C12Q1/04C12N15/11
CPCC12Q1/686C12Q1/689C12Q2600/16C12Q2600/166C12Q2537/143C12Q2565/125C12Q2545/113
Inventor 梁旭东
Owner 至微生物智能科技(厦门)有限公司
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