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Primers and detection kit for identifying various staphylococci based on pheS gene and application of primers and detection kit

A detection kit and technology for staphylococcus, which are applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve problems such as multiple primers and methods for staphylococcus that have not been reported, and achieve high detection sensitivity , the effect of simple operation

Active Publication Date: 2021-06-18
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

There are no primers and methods for identifying multiple staphylococci in the environment at the species level based on the pheS signature sequence

Method used

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  • Primers and detection kit for identifying various staphylococci based on pheS gene and application of primers and detection kit
  • Primers and detection kit for identifying various staphylococci based on pheS gene and application of primers and detection kit
  • Primers and detection kit for identifying various staphylococci based on pheS gene and application of primers and detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] The design method of 6 kinds of Staphylococcus specific primers, the specific steps are as follows:

[0069] A. Download the pheS gene sequences of Staphylococcus aureus, Staphylococcus worthii, Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus capitis and Staphylococcus haemolyticus from the gene bank;

[0070] B. Comparing the gene sequences, searching for differential sequences, and designing specific primers for 6 kinds of Staphylococci respectively, the primer sequences are shown in Table 1.

[0071]

Embodiment 2

[0073] Staphylococcus Primer Specificity Detection

[0074] A. Utilize the primers of 6 kinds of Staphylococci designed, use the Staphylococcus species corresponding to the primers as the positive control, and the negative control as the lysate. Staphylococcus spp., Staphylococcus hemolyticus, Bacillus, Pantoea, Micrococcus and Pseudomonas overnight cultured bacteria were lysed with lysate, and the supernatant was taken, and the supernatants of different bacteria were used as templates for PCR amplification.

[0075] Table 2 is a list of sample numbers of bacterial strains used for agarose gel electrophoresis

[0076] Numbering sample 1 Staphylococcus epidermidis 2 lysate 3 Staphylococcus wausii 4 Staphylococcus capitis 5 Staphylococcus aureus 6 Staphylococcus hominis 7 Staphylococcus haemolyticus 8 Bacillus 9 Pantoea 10 Micrococcus 11 Pseudomonas

[0077] B, PCR product is detected by agarose gel...

Embodiment 3

[0089] Staphylococcus sensitivity detection method, the specific steps are as follows:

[0090] A. After overnight shaking culture of Staphylococcus aureus, Staphylococcus worthii, Staphylococcus epidermidis, Staphylococcus hominis and Staphylococcus hemolyticus, the concentrations obtained by plate counting were Staphylococcus aureus (4.95×10 9 CFU / ml), Staphylococcus worbachii (3×10 9 CFU / ml), Staphylococcus epidermidis (4.5×10 9 CFU / ml), Human Staphylococcus (1×10 9 CFU / ml), Staphylococcus hemolyticus (2.7×10 9 CFU / ml). It was serially diluted 10 times, and the dilutions were 10 0 、10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 .

[0091] B, with the bacterial liquid of different concentration as template, carry out corresponding bacterial species respectively with the primer of specific amplification Staphylococcus aureus, Staphylococcus wordleri, Staphylococcus epidermidis, Staphylococcus human and Staphylococcus hemolyticus designed in embodiment 2 For PCR amplific...

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Abstract

The invention provides primers and a detection kit for identifying various staphylococci based on a pheS gene and application of the primers and the detection kit, and belongs to the technical field of bacterial identification. The invention provides a primer group for identifying various staphylococci based on a pheS gene. The primer group comprises the following two or more primer pairs: a primer pair for specifically amplifying staphylococcus aureus, a primer pair for specifically amplifying staphylococcus warneri, a primer pair for specifically amplifying staphylococcus epidermidis, a primer pair for specifically amplifying staphylococcus hominis, a primer pair for specifically amplifying staphylococcus capitis, and a primer pair for specifically amplifying staphylococcus haemolyticus. The primer group has the characteristics of good amplification specificity, high detection sensitivity and simultaneous identification of various staphylococci. A method for detecting various staphylococci in the environment based on the primer group can realize simultaneous identification of various staphylococci, and has the characteristics of short detection time, low detection cost and an accurate and reliable detection result.

Description

technical field [0001] The invention belongs to the technical field of bacterial identification, and in particular relates to a primer for identifying various staphylococci based on the pheS gene, a detection kit and an application thereof. Background technique [0002] Staphylococcus ( Staphylococcus ) is a group of Gram-positive bacteria, 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. . 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 phenylalanyl-tRNA synthetase α subunit gene (pheS) has a high degree of homogeneity among the strains of the same species, but the sequence similarity of this gene is poor in different species, and it is a rapidly evolving gene. Studies...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/14C12N15/11
CPCC12Q1/686C12Q1/689C12Q2565/125
Inventor 梁旭东
Owner 至微生物智能科技(厦门)有限公司
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