Method for detecting characteristic fungus Amorphotheca resinae in jet fuel

A technology of jet fuel and fungi, applied in the field of microbial detection, can solve the problems of expensive, complicated operation, long time-consuming, etc., and achieve the effect of short detection time, high sensitivity, and improved early warning ability

Active Publication Date: 2015-05-13
LOGISTICAL ENGINEERING UNIVERSITY OF PLA +2
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Problems solved by technology

The detection method commonly used in the laboratory is polymerase chain reaction (polymerase chain reaction, PCR) method detection, PCR needs expensive experimental equipment, and this method needs gel electrophoresis device and phase system, needs fluorescent dye when gelling Toxic reagents such as ethidium bromide (EB) also need to configure a large volume of electrophoresis buffer, which is complicated and time-consuming

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  • Method for detecting characteristic fungus Amorphotheca resinae in jet fuel
  • Method for detecting characteristic fungus Amorphotheca resinae in jet fuel
  • Method for detecting characteristic fungus Amorphotheca resinae in jet fuel

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Experimental program
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Effect test

Embodiment 1

[0017] Embodiment 1: primer design and synthesis

[0018] The applicant has found in long-term research that, in addition to A.resinae, fungi in jet fuel also have Trichoderma viride, Penicillium funiculosum, Aspergillus niger, and short-stemmed shoots. Aureobasidium pullulans, therefore, after comparing the A.resinae18S gene sequence with the above four bacteria, the specific sequence of A.resinae was screened out to design a LAMP group, and after sensitivity and specificity screening, the A.resinae including exogenous Primers (F3, B3), internal primers (FIP, BIP), and loop primers (LF, LB) included 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.

[0019] Table 1: A. resinae LAMP primer sequences

[0020]

[0021]

Embodiment 2

[0022] Example 2: Detection of A. resinae

[0023] 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, KCl 10mmol / L, MgSO 4 8mmol / L, (NH 4 ) 2 SO 4 10mmol / L, Tween200.1%, Betaine 0.8mol / L, dNTPs 1.4mmol / 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.

[0024] Table 2: LAMP-LFD reaction system

[0025]

[0026] 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 rea...

Embodiment 3

[0037] Example 3: Detection Application of Jet Fuel Samples

[0038] 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 the chelex-100 method was used to Extract the DNA template. And use the designed LAMP-LFD method to detect whether there is A.resinae; the results are shown in Table 3.

[0039] Table 3: Detection results of jet fuel samples by LAMP-LFD and culture method

[0040]

[0041] Note: +. Positive; —. Negative

[0042] 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 ...

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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 Applications(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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