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LAMP primer of staphylococcus aureus and application method of LAMP primer

A Staphylococcus aureus technology, applied in the detection field of Staphylococcus aureus, to achieve the effect of simple and easy result judgment

Inactive Publication Date: 2015-01-21
河北省食品检验研究院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is still a lack of an effective LAMP detection method for Staphylococcus aureus at home and abroad.

Method used

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  • LAMP primer of staphylococcus aureus and application method of LAMP primer
  • LAMP primer of staphylococcus aureus and application method of LAMP primer
  • LAMP primer of staphylococcus aureus and application method of LAMP primer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Design of LAMP primers

[0029] The primers were synthesized by Bao Biological Engineering (Dalian) Co., Ltd., and the sequences of the four primers are shown in Table 1.

[0030] Table 1 LAMP primer sequence

[0031]

Embodiment 2

[0032] Example 2: Application of primers in the LAMP detection of Staphylococcus aureus

[0033] (1) Preparation of template DNA: Cultivate various bacterial strains overnight, take 1.5 mL of bacterial liquid and extract whole genome DNA with a bacterial genome DNA extraction kit, and store at -20°C.

[0034] (2) Optimizing the LAMP reaction system and reaction conditions

[0035] 25μLAMP reaction system includes: In terms of final concentration, 20mmol / L Tris-HCl (pH8.8), 10mmol / L KCl, 10mmol / L (NH4) 2 SO 4 , 8mmol / L MgSO 4 , 0.1% v / v of Tween20, 1mol / L of betaine, 1.6μmol / L of FIP and BIP, 0.2mol / L of F3 and B3, 1.4mmol / L of dNTP; 8U Bst large fragment DNA polymerase and 1μL DNA template.

[0036] Reaction conditions: the above mixture was placed at 62.5°C for 45 minutes at a constant temperature, and finally kept at 80°C for 10 minutes to terminate the reaction. A negative control was set for each reaction (sterilized double distilled water was used instead of DNA temp...

Embodiment 3

[0043] Embodiment 3: the contrast of LAMP detection method of the present invention and PCR method

[0044] ①PCR primers: According to the published DNA sequence of Staphylococcus aureus 23srRNA (S.aureus, X68425.1) in GenBank, a pair of primers (P1: 5′-TCTTCAGAAGATGCGGAATA-3′, such as Shown in SEQ ID NO: 5; P2: 5'-TAAGTCAAACGTTAACATACG-3', shown in SEQ ID NO: 6), the length of the amplified product is 420bp, synthesized by Treasure Bioengineering (Dalian) Co., Ltd.

[0045] PCR amplification reaction system: 10×PCR buffer 5μL, Mg 2+ (25mmol / L) 3μL, dNTP (25mmol / L) 4μL, Taq enzyme (2.5U) 0.5μL, primers P1 and P2 each 1μL, template 2μL, sterile double distilled water to make up to 50μL. Cycle conditions: pre-denaturation at 94°C for 2 min, denaturation at 94°C for 1 min, annealing at 56°C for 1 min, extension at 72°C for 1 min, amplification for 30 cycles, and the last cycle of extension at 72°C for 5 min. Take 5 μL of the amplification product and analyze it by 1.5% agarose ...

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Abstract

The invention discloses an LAMP primer of staphylococcus aureus. The LAMP primer comprises two inner primers (FIP and BIP) and two outer primers (F3 and B3), namely, F3 as shown in the sequence SEQ ID NO:1, B3 as shown in the sequence SEQ ID NO:2, FIP as shown in the sequence SEQ ID NO:3 and BIP as shown in the sequence SEQ ID NO:4. Aiming at the specific nuc gene of the staphylococcus aureus, a group of primers is designed, and a sensitive and specific staphylococcus aureus LAMP detection method is established by using the primers. The experiment shows that the detection sensitivity and the detection limit of the LAMP detection method are 100 times of those of a PCR method; whether green fluorescence is generated or not can be observed with naked eyes so as to confirm whether the reaction is performed, so that the result judgment can be simple, convenient and feasible; compared with a PCR technique, the method disclosed by the invention has the advantages that the time and the labor can be saved, no expensive instruments or tedious electrophoretic analysis is needed, and the application method disclosed by the invention is particularly applicable to use in base levels.

Description

technical field [0001] The invention relates to a LAMP primer of staphylococcus aureus and an application method thereof, which are used for the detection of staphylococcus aureus. Background technique [0002] The laboratory detection methods of Staphylococcus aureus include: traditional isolation culture and biochemical identification, immunological detection technology, molecular detection technology, etc. The traditional detection method is cumbersome to operate, takes a long time to detect, and has low sensitivity; the specificity and sensitivity of the immunological method are relatively low; the PCR method is sensitive, accurate and fast, but because the detection method requires expensive instruments and , has high technical requirements for testing personnel, making it difficult to apply at the grassroots level and areas with weak testing equipment, which limits the popularization and promotion of this method. [0003] Loop-mediated isothermal amplification (LAMP) ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/14C12N15/11
CPCC12Q1/6844C12Q1/689
Inventor 周巍张岩杨岚李永波马超峰李月华刘涛刘红冉
Owner 河北省食品检验研究院
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