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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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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 sensitively detect Staphylococcus aureus from food, but because PCR technology has high requirements on the quality of template DNA, the required equipment such as thermal cycling and gel electrophoresis is expensive, and it is difficult to be promoted at the grassroots level and on-site rapid determination

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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Embodiment 1

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

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

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

[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 als...

Embodiment 2

[0055] Embodiment 2: LAMP sensitivity experiment

[0056] Inoculate the standard strain of Staphylococcus aureus in the nutrient broth and culture at 36°C±1°C for 18 hours. Bacterial DNA in cultured products was extracted using Magen Bacterial Gene DNA Extraction Kit. The DNA extracted from the standard strain of Staphylococcus aureus was serially diluted 10 times, and five gradient concentrations of 1ng / μl, 100pg / μl, 10pg / μl, 1pg / μl, and 100fg / μl were used as templates and negative controls (killed Bacterial ultrapure water) The detection method was established according to the reaction system and conditions of Example 1 to determine the sensitivity of the detection method of the kit.

[0057] see figure 1 , the results showed that: after the Staphylococcus aureus genomic DNA was diluted 10 times, the established Staphylococcus aureus LAMP detection kit could detect 1pg / μl Staphylococcus aureus DNA in the sample.

Embodiment 3

[0058] Embodiment 3: LAMP specific experiment

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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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