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A method for the detection of the characteristic fungus Amorphotheca resinae in jet fuel

A technology of jet fuel and fungus, which is applied in the field of microbial detection, can solve the problems of expensive, time-consuming, and complicated operations, and achieve the effects of short detection time, improved early warning ability, and high sensitivity

Active Publication Date: 2016-08-10
LOGISTICAL ENGINEERING UNIVERSITY OF PLA +2
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  • Abstract
  • Description
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Problems solved by technology

The detection method commonly used in the laboratory is polymerase chain reaction (polymerase chainreaction, PCR) method detection, and PCR needs expensive experimental equipment, and this method needs gel electrophoresis device and phase system, and fluorescent dye bromine is needed when gelling. Ethidium bromide (EB) and other toxic reagents also need to configure a large volume of electrophoresis buffer, which is complex and time-consuming

Method used

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  • A method for the detection of the characteristic fungus Amorphotheca resinae in jet fuel
  • A method for the detection of the characteristic fungus Amorphotheca resinae in jet fuel
  • A method for the detection of the characteristic fungus Amorphotheca resinae in jet fuel

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Embodiment 1

[0019] Embodiment 1: primer design and synthesis

[0020] The applicant has found in long-term research that, in addition to A.resinae, fungi in jet fuel also include Trichoderma viride, Penicillium funiculosum, Aspergillus niger, and Aureobasidium pullulans (Aureobasidium pullulans), therefore, after comparing the A.resinae18S gene sequence with the above four bacteria, the specific sequence of A.resinae was screened to design the LAMP group, and after sensitivity and specificity screening, the outer primers were obtained (F3, B3), internal primers (FIP, BIP), and loop primers (LF, LB), a total of 6 LAMP primers (Table 1). Among them, FIP is labeled with biotin, and FITC is added to LF. Primers were synthesized by Dalian Bao Biological Company.

[0021] Table 1: A. resinae LAMP primer sequences

[0022]

[0023]

Embodiment 2

[0024] Example 2: Detection of A. resinae

[0025] Amplification conditions were optimized using the genomic DNA of A. resinae as a template. The LAMP method DNA reaction kit was purchased from Rongyan Biotechnology Co., Ltd. The concentration of each primer is: FIP-bio and BIP each 1.6 μmol / L, F3 and B3 each 0.2 μmol / L, LF-fitc and LB each 0.8 μmol / L; buffer composition and concentration: Tris-HCl (pH 8.8) 20mmol / L, KCl10mmol / L, MgSO 4 8mmol / L, (NH 4 ) 2 SO 4 10mmol / L, Tween200.1%, Betaine 0.8mol / L, dNTPs1.4mmol / L; 8U Bst DNA polymerase; A. resinae genomic DNA was extracted with a kit. LFD adopts German Milenia GenLine HybriDetect kit. The LAMP-LFD reaction system is shown in Table 2.

[0026] Table 2: LAMP-LFD reaction system

[0027]

[0028] The volume was adjusted to 25 μL with double distilled water, and no DNA template was added in the negative control experiment. The amplification was carried out at the optimized reaction temperature and time, and the react...

Embodiment 3

[0039] Example 3: Detection Application of Jet Fuel Samples

[0040] Take samples from the bottom of 9 storage tanks, filter 100mL of each oil sample through a 0.45μm filter membrane, collect the filter membrane and mark several samples respectively Jet fuel 1, Jet fuel 2, Jet fuel 3, Jet fuel 4, Jet fuel Fuel 5, Jet fuel 6, Jet fuel 7, Jet fuel 8, Jet fuel 9, the collected filter membrane was eluted repeatedly with 500μL 10% chelex-100, the eluate was collected in a 1.5mL EP tube, and extracted by chelex-100 method DNA template. And use the designed LAMP-LFD method to detect whether there is A.resinae; the results are shown in Table 3.

[0041] Table 3: Detection results of jet fuel samples by LAMP-LFD and culture method

[0042]

[0043] Note: +. Positive; —. Negative

[0044] The above results show that the results obtained by the primer set and detection method of the present invention are consistent with the results of bacterial culture, which proves the reliability...

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Abstract

The invention provides a method for detecting characteristic fungus Amorphotheca resinae in a jet fuel. According to the method, a set of LAMP primers is designed, a detection system with a reaction temperature of 63 DEG C and a time of 35min and directly observed by an LFD test strip is established, and then specificity and sensitivity evaluations are carried out. Results show that the lowest detection lines of detecting A.resinae genome DNA and a pure culture by applying an LAMP-LFD method are 26fg / muL and 8chu / mL respectively; in specificity confirmatory experiments, four fungi namely trichoderma viride, penicillium funiculosum, aspergillus niger and aureobasidium pullulans found in the jet fuel, and A.resinae are used as specificity test strains, and the LAMP-LFD method detection only shows a positive reaction for A.resinae. The detection method disclosed by the invention is capable of rapidly detecting A.resinae in the jet fuel, and visualized in result. The method is short in time consumption, simple and convenient to operate, high in specificity, high in sensitivity and independent of special detection equipment, as well as expected to be used for on-site field detection for characteristic fungus A.resinae in the jet fuel as a new detection method.

Description

technical field [0001] The invention belongs to the technical field of microorganism detection, in particular to a method for detecting the characteristic fungus Amorphotheca resinae in jet fuel. Background technique [0002] Amorphotheca resinae, the Chinese name for Cladosporium resinae, is a fungus that can grow in jet fuel. Studies have found that A.resinae is related to suspended matter in reserve jet fuel, and suspended matter is an important indicator affecting the cleanliness of jet fuel. A. resinae is the main contaminating bacteria in jet fuel, 93% of the contamination cases are caused by this fungus. The detection of A. resinae is to improve the early warning capability of jet fuel contamination. At present, the International Air Transport Association (The International Air Transport Association, IATA) recommends 4 detection methods for microorganisms in aircraft fuel tanks, but it is not common to detect A. resinae. MicrobMoiter2 and Easicult Combi are a kind o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/645
CPCC12Q1/6844C12Q2531/119C12Q2565/625
Inventor 熊云和倩倩朱鹏严小军范建忠黄海龙龙泉芝杨浩苏鹏周剑陈然李晓然
Owner LOGISTICAL ENGINEERING UNIVERSITY OF PLA
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