Primer for detecting chaetomium globosum in jet fuel, application and detection method
A technology of Chaetomium globosa and detection method, which is applied in the field of application and detection, detection of Chaetomium globosa primers in jet fuel, can solve the problems that the sensitivity and specificity cannot meet the needs of growing development, etc., to ensure the safety of aircraft flight, High sensitivity and good specific effect
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example 1
[0041] Example 1: Primer Design and Synthesis
[0042] A set of specific primers were designed according to the β protein gene sequence of Chaetomium globosum, including outer primers (F3, B3), inner primers (FIP, BIP), and a loop primer (LB). The 5' end of FIP was biotinylated. The primer sequences are shown in Table 1,
[0043] Table 1 Primers for Chaetomium globosum
[0044]
[0045] According to the primer sequences in Table 1, LAMP primers were synthesized in Dalian Bao Biology Co., Ltd.
example 2
[0046] Example 2: Optimization of reaction conditions for amplification of Chaetomium globosum
[0047] The amplification conditions were optimized using the genomic DNA of Chaetomium globosum as a template. The concentrations of primers prepared during LAMP reaction were: 0.2 μmol / L for each of F3 and B3, 1.6 μmol / L for each of FIP and BIP, and 0.8 μmol / L for each of LB. LAMP premix is 20mmol / LTris-HCl (pH8.8), 10mmol / LKCl, 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 Chaetomium globosum is shown in Table 2.
[0048] Table 2 LAMP reaction system of Chaetomium globosum
[0049]
[0050] 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 reactions at three different temperatures (61°C, 63...
example 3
[0052] Example 3 The LAMP-LFD technique of the present invention is used to determine the sensitivity of Chaetomium globosum.
[0053] 1. Using the extracted Chaetomium globosum DNA as a template, dilute to 10 times according to 10-fold equal gradient -8 Gradient (concentrations are 335, 33.5, 3.35ng / μL, 335, 33.5, 3.35pg / μL, 335, 33.5, 3.35fg / μL). Different dilutions of DNA were used as PCR, LAMP and LAMP-LFD reaction templates.
[0054] 2. Use LAMP, LAMP-LFD and PCR methods to detect Chaetomium globosum, and compare the sensitivity of the three methods.
[0055] 2.1 Amplify with the above-mentioned Chaetomium globosum (Chaetomium globosum) genomic DNA through gradient dilution as the template of the LAMP reaction, according to the primers in Example 1 and the reaction system and temperature and time in Example 2, use the LFD method provided by the invention Analyze the LAMP reaction product, see the results figure 1 .
[0056] 2.2 Amplify the above-mentioned Chaetomium g...
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