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Method for detecting Burkholderia pseudomallei by multi-cross isothermal amplification in combination with gold nano-biosensing

A cross constant temperature amplification and kholderia technology, which is applied in the field of microbial detection, can solve the problems of relying on reagents and complicated operation steps, and achieve the effects of good specificity, excellent specificity and amplification effect, and excellent detection sensitivity

Pending Publication Date: 2020-08-11
三亚市人民医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these constant temperature technologies require multiple enzymes to work simultaneously to achieve nucleic acid amplification, relying on expensive reagents and complicated operating steps
Therefore, the practicability, convenience and operability of these methods need to be improved, especially in the field of rapid diagnosis and underdeveloped areas.

Method used

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  • Method for detecting Burkholderia pseudomallei by multi-cross isothermal amplification in combination with gold nano-biosensing
  • Method for detecting Burkholderia pseudomallei by multi-cross isothermal amplification in combination with gold nano-biosensing
  • Method for detecting Burkholderia pseudomallei by multi-cross isothermal amplification in combination with gold nano-biosensing

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Experimental program
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Effect test

Embodiment 1

[0045] Embodiment 1.MCDA amplification

[0046] For specific methods, see CN201610982015.1

[0047] Standard MCDA reaction system: the concentrations of cross primers CP1 and CP2 were 1.6 μM, the concentrations of displacement primers F1 and F2 were 0.4 μM, and the concentrations of amplification primers C1*, C2, R1, R2, D1 and D2* were 0.8μM, 10mM Betain, 6mM MgSO 4 , 1 mM dNTP, 12.5 μL of 2×DNA polymerase buffer, 8 U of Bst strand-displacing DNA polymerase, 1 μL of template, and add deionized water to 25 μL. The entire reaction was kept at 63°C for 1 hour, and the reaction was terminated at 85°C for 5 minutes.

[0048] Design of Biodetector (LFB):

[0049] The design of the biological detector (LFB) is described in the previous patent (see patent CN201610942289.8 for details). The LFB consists of five parts, the back plate, the sample pad, the gold standard pad, the fiber membrane and the absorbent pad. First, the sample pad, gold standard pad, fiber membrane and absorbe...

Embodiment 2

[0052] Example 2. Verify the feasibility of MCDA primers

[0053] The TTS-1 gene exists in all Burkholderia pseudomallei, and its conservation and specificity are good, which can distinguish Burkholderia pseudomallei from other closely related species. Utilize primer design software PrimerExplorerV4 (Eiken Chemical) (http: / / primerexplorer.jp / e / ) and primer design software Primer Premier5.0 to design MCDA primers, and the specific primers obtained are in the NCBI database (http: / / blast. ncbi.nlm.nih.gov / Blast.cgi) for sequence comparison analysis to exclude possible non-specific matches between primers and sequences of other species, and finally obtain optimized MCDA amplification primers. For the location and direction of primer design, see figure 1 , the sequence and modifications are shown in Table 2.

[0054] Test results:

[0055] After MCDA amplification, two detection methods were used for MCDA amplification discrimination. First, a specific nucleic acid amplification...

Embodiment 3

[0058] Embodiment 3. determine the optimal reaction temperature of MCDA technology

[0059] Under standard reaction system conditions, the DNA template for Burkholderia pseudomallei and the corresponding MCDA primer designed were added, and the template concentration was 10 pg / uL. The reaction was carried out under constant temperature conditions (60-67°C), and the results were detected by a real-time turbidimeter, and different dynamic curves were obtained at different temperatures, see image 3 . 65-67°C (E-G) is recommended as the optimal reaction temperature for MCDA primers. In the subsequent verification of the present invention, 66° C. was selected as the constant temperature condition for MCDA amplification. image 3 It represents the temperature dynamic curve of the MCDA primers designed for the detection of Burkholderia pseudomallei for the TTS-1 gene sequence.

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Abstract

The invention discloses a method for detecting Burkholderia pseudomallei by multi-cross isothermal amplification in combination with gold nano-biosensing. Corresponding specific primers is designed based on Burkholderia pseudomallei TTS1 genes, and meanwhile fluorescein is marked at the 5'end of an amplification primer C1 and biotin is marked at the 5'end of an amplification primer D2 to obtain C1* and D1 * newly labeled amplification primers. The method can realize visual detection of the Burkholderia pseudomallei via the gold nano-biosensor, and the method is convenient, rapid, high in sensitivity and good in specificity.

Description

technical field [0001] The invention relates to the technical field of microbial detection, in particular to a method for detecting Burkholderia pseudomallei with multi-cross constant temperature amplification combined with gold nanobiological sensing. Background technique [0002] Burkholderia pseudomallei (Burkholderia pseudomallei) is a Gram-negative bacterium that is club-shaped, does not form capsules and spores, and has more than three flagella at one end. It is the pathogen of melioidosis. The endemic areas are mainly distributed between south and north latitudes of 20°, and are common in northern Australia, northeastern Thailand, and Hainan, Hong Kong, Taiwan, Guangdong and other regions in China. Hainan Province is the main source of melioidosis in my country, and the isolation rate of B.pseudomallei and The number of clinical cases is the highest in the country. Patients with immunocompromised diseases such as diabetes or people who are frequently exposed to soil a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/6804C12Q1/04C12N15/11C12R1/01
CPCC12Q1/689C12Q1/6844C12Q1/6804C12Q2531/119C12Q2565/607C12Q2563/131
Inventor 朱雄王晓霞
Owner 三亚市人民医院
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