Primer for detecting sulfate reducing bacteria in jet fuel, application and detection method

A detection method and jet fuel technology, which are applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc. very good effect

Inactive Publication Date: 2020-07-03
LOGISTICAL ENGINEERING UNIVERSITY OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above methods have problems such as complex operation or not suitable for jet fuel sample detection, so it is necessary to establish a method for the detection of sulfate-reducing bacteria in jet fuel that is suitable for rapid on-site inspection and is easy to operate.

Method used

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  • Primer for detecting sulfate reducing bacteria in jet fuel, application and detection method
  • Primer for detecting sulfate reducing bacteria in jet fuel, application and detection method
  • Primer for detecting sulfate reducing bacteria in jet fuel, application and detection method

Examples

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Effect test

example 1

[0042] Example 1: Primer Design and Synthesis

[0043] According to the dsrB gene sequence of sulfate-reducing bacteria, a set of specific primers were designed, including outer primers (F3, B3), inner primers (FIP, BIP), and a loop primer (LB, LF). The 5' end of LF was labeled with fluorescein isothiocyanate (FITC) as a probe. The primer sequences are shown in Table 1,

[0044] Table 1 Primers for sulfate-reducing bacteria

[0045]

[0046] According to the primer sequences in Table 1, LAMP primers were synthesized in Dalian Bao Biology Co., Ltd.

example 2

[0047] Example 2: Optimization of reaction conditions for the amplification of sulfate-reducing bacteria

[0048] The amplification conditions were optimized using the genomic DNA of the sulfate-reducing bacteria Desulfotomaculum ruminis as a template. The concentrations of primers prepared during LAMP reaction were: 0.2 μmol / L for F3 and B3, 1.6 μmol / L for FIP and BIP, and 0.8 μmol / L for LF and LB. LAMP premix is ​​20mmol / L Tris-HCl (pH 8.8), 10mmol / L KCl, 8mmol / L MgSO 4 , 10mmol / L (NH4) 2 SO 4 , 0.1% Tween 20, 0.8mol / L Betaine, 1.4mmol / L dNTPs, 8U / μL Bst DNA polymerase. The LAMP reaction system of sulfate-reducing bacteria is shown in Table 2.

[0049] Table 2 LAMP reaction system of sulfate-reducing bacteria

[0050]

[0051] In order to obtain the best LAMP amplification efficiency and comprehensively consider the applicable conditions of LFD, the primers provided by the present invention and the LAMP reaction system were used to perform LAMP amplification reaction...

example 3

[0053] Example 3 uses the LAMP-LFD technique of the present invention to analyze the detection sensitivity of sulfate-reducing bacteria.

[0054] 1. Using the extracted Desulfotomaculum ruminis DNA as a template, dilute to 10 times according to a 10-fold equal gradient. -8 Gradient (concentrations are 121, 12.1, 1.21ng / μL, 121, 12.1, 1.21pg / μL, 121, 12.1, 1.21fg / μL). Different dilutions of DNA were used as PCR, LAMP and LAMP-LFD reaction templates.

[0055] 2. Use LAMP-LFD, LAMP and PCR methods to detect sulfate-reducing bacteria, and compare the sensitivity of the three methods.

[0056] 2.1 Amplify with the above-mentioned desulfovibrio (Desulfotomaculum ruminis) DNA through gradient dilution as the template of the LAMP reaction, according to the primers in the example 1 and the reaction system and temperature and time in the example 2, analyze the LAMP with the LFD method provided by the invention reaction product, see figure 1 .

[0057] 2.2 Amplify the above-mentioned...

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Abstract

The invention belongs to the technical field of biological detection, and discloses a primer for detecting sulfate reducing bacteria in jet fuel, application and a detection method. The primer comprises outer primers F3 and B3, inner primers FIP and BIP and two loop primers LB and LF; the sequence of the outer primer F3 is shown as SEQ ID NO. 1, the sequence of the outer primer B3 is shown as SEQID NO. 2, the sequence of the inner primer FIP is shown as SEQ ID NO. 3, the sequence of the inner primer BIP is shown as SEQ ID NO. 4, the sequence of the loop primer LB is shown as SEQ ID NO. 5, andthe sequence of the loop primer LF is shown as SEQ ID NO. 6. The method has the advantages of being high in sensitivity and specificity.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a primer for detecting sulfate-reducing bacteria in jet fuel, an application and a detection method. Background technique [0002] The description of the background technology of the present invention belongs to the relevant technology related to the present invention, and is only used to illustrate and facilitate the understanding of the content of the present invention. prior art on the filing date. [0003] Sulfate-Reducing Bacteria (SRB) is a class of facultative anaerobic microorganisms that can reduce sulfate ions and other oxidized sulfur to sulfide and obtain energy from it. Sulfate-reducing bacteria have tenacious vitality and are widely distributed in nature. They are often found in soil, ocean and oil field pipelines. At the same time, sulfate-reducing bacteria are also the most corrosive and widely distributed microorganisms. There are many kinds of sul...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6844C12Q1/04C12N15/11
CPCC12Q1/6895C12Q1/6844C12Q2531/119C12Q2537/1376C12Q2565/625
Inventor 苏鹏熊云李泽振范林君牛明明孙新枫朱鹏
Owner LOGISTICAL ENGINEERING UNIVERSITY OF PLA
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