LAMP primer group and detection kit for staphylococcus aureus and use method of detection kit

A technology for detection kits and staphylococci, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, and microorganism measurement/inspection. It can solve the problems of high price, cumbersome operation, and low sensitivity. The effect of convenient operation and simple conditions

Inactive Publication Date: 2017-02-22
中华人民共和国广州机场出入境检验检疫局 +2
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Problems solved by technology

The traditional culture method for detecting Staphylococcus aureus is cumbersome to operate, takes a long time to detect, and has low sensitivity; the immunological method shortens the detection time, although the sensitivity is high, the specificity is poor
PCR technology can be used to quickly and sens

Method used

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  • LAMP primer group and detection kit for staphylococcus aureus and use method of detection kit
  • LAMP primer group and detection kit for staphylococcus aureus and use method of detection kit

Examples

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Example Embodiment

[0039] Example 1: Design and synthesis of a LAMP primer of Staphylococcus aureus and establishment of a detection kit

[0040] (1) Design and synthesis of LAMP primers

[0041] According to literature reports, using the Staphylococcus aureus nuc gene as the target gene, the primer design software PrimerExplorer 4.0 was used to design six LAMP primers including loop primers. The primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. The sequences of the six primers are as follows:

[0042] SEQ NO.1: SAnuc-F3: CGCTACTAGTTGCTTAGTGTTA

[0043] SEQ NO.2: SAnuc-B3: GCTAAGCCACGTCCATAT

[0044] SEQ NO.3: SAnuc-FIP: ACTGTTGGATCTTCAGAACCACCAAGTCTAAGTAGCTCAGCAA

[0045] SEQ NO. 4: SAnuc-BIP:

[0046] AGCGATTGATGGTGATACGGTTCAGGACCATATTTCTCTACACC

[0047] SEQ NO.5: SAnuc-LF: TACGCCGTTATCTGTTTGTGA

[0048] SEQ NO.6: SAnuc-LB: AGGTCAACCAATGACATTCAGA

[0049] (2) In addition to the above-mentioned LAMP primer set, the Staphylococcus aureus LAMP detection kit also includ...

Example Embodiment

[0055] Example 2: LAMP Sensitivity Experiment

[0056] The standard strain of Staphylococcus aureus was inoculated into the nutrient broth and cultured at 36℃±1℃ for 18 hours. The bacterial DNA in the cultured products was extracted using the Magen bacterial gene DNA extraction kit. The DNA extracted from the standard strain of Staphylococcus aureus was diluted 10-fold, and five gradient concentrations of 1ng / μl, 100pg / μl, 10pg / μl, 1pg / μl, and 100fg / μl were used as templates and negative controls (deactivated DNA). Bacterial ultrapure water) According to the reaction system and conditions of Example 1, a detection method was established to determine the sensitivity of the kit detection method.

[0057] see figure 1 , the results show that: after 10-fold gradient dilution of Staphylococcus aureus genomic DNA, the established Staphylococcus aureus LAMP detection kit can detect 1pg / μl of Staphylococcus aureus DNA in the sample.

Example Embodiment

[0058] Example 3: LAMP specificity experiments

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Abstract

The invention discloses an LAMP primer group and an LAMP detection kit for staphylococcus aureus and a use method of the detection kit. The LAMP primer group for staphylococcus aureus comprises a pair of outer primers, a pair of inner primers and a pair of loop primers. According to the LAMP primer group, the LAMP detection kit and the use method, loop-mediated isothermal amplification primers are designed according to the sequences of the conservative regions of the heat-resistant nuclease genes (nuc) of staphylococcus aureus, the specific regions of target genes are amplified by adopting the LAMP technology, thus the rapid detection for staphylococcus aureus is realized from the molecular level, and an effective and rapid nucleic acid screening detection method is provided for detecting staphylococcus aureus.

Description

[0001] Technical field: [0002] The invention belongs to the field of molecular biology detection methods for food safety, and in particular relates to a LAMP primer set and a detection kit for Staphylococcus aureus. [0003] Background technique: [0004] Staphylococcus aureus ( Staphylococcus aureus ) belongs to the genus Staphylococcus of the Micrococcus family and is a Gram-positive coccus widely distributed in nature, such as: air, water, dust, ground, and human and animal excrement. It is a human purulent infection and bacterial poisoning one of the most common pathogens. If the conditions are suitable, Staphylococcus aureus will multiply in large numbers, produce a certain amount of enterotoxin, and cause food poisoning. In recent years, the U.S. Centers for Disease Control and Prevention reported that the infection caused by Staphylococcus aureus ranks second, second only to Escherichia coli, and the annual food poisoning incidents caused by Staphylococcus aureus in ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/14C12N15/11C12R1/445
CPCC12Q1/689C12Q1/6844C12Q2531/119C12Q2563/107
Inventor 柏建山何淑华黄燕琼赵尚志谢会陈洵石磊
Owner 中华人民共和国广州机场出入境检验检疫局
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