A method and its application of one-time accurate quantification of alkane hydroxylase gene alkb

An alkane hydroxylase, sub-accurate technology, applied in the field of molecular ecology and biology, can solve the problems of poor accuracy and comparability of quantitative results, differences in primer amplification efficiency, etc.

Active Publication Date: 2016-09-07
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The purpose of the present invention is to solve the problem of poor accuracy and comparability of quantitative results caused by factors such as primer amplification efficiency difference, non-specific amplification, primer dimer, etc. Method and application of alkane hydroxylase gene alkB

Method used

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  • A method and its application of one-time accurate quantification of alkane hydroxylase gene alkb
  • A method and its application of one-time accurate quantification of alkane hydroxylase gene alkb
  • A method and its application of one-time accurate quantification of alkane hydroxylase gene alkb

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Quantitative method for real-time fluorescent PCR with multiple primers based on universal sequence adapters

[0041] Step 1: Design and specificity verification of universal primers

[0042] Randomly combine stop codons and rare codons of Escherichia coli into a primer sequence of about 20bp, use 6.0, Primer Premier 5.0 and other primer analysis software evaluate the quality of primers, and carry out comparison analysis in Genbank database, therefrom preferred two primer sequences UP-1 (SEQ ID No.1), UP-2 (SEQ ID No. 2) As a general sequence for multiplex real-time fluorescent quantitative PCR. Use this universal sequence to test a microbial genome positive quality control ( figure 2 , 1 lane) and 5 randomly selected environmental gene samples ( figure 2 , lanes 2 to 6) for ordinary PCR amplification, the amplification results showed that the universal primers had no non-specific amplification on environmental genome samples, and basically no primer-di...

Embodiment 2

[0078] Example 2: Comparison of multiple primer PCR amplification effects based on universal sequences and common multiple primer PCR

[0079] When performing multiple PCR amplification, the PCR process is performed with reference to the multiple real-time fluorescent quantitative PCR method based on the universal sequence provided in Example 1 of the present invention, except that the primers used in common multiple PCR are specific primers that remove the universal sequence linker, and No universal sequences were added as primers during PCR. The amplification results of templates containing the same amount of 7 alkB genes showed that the amplification efficiencies of different alkB genes by ordinary multiplex PCR were quite different, and even some alkB genes were not amplified effectively under the mutual inhibition of primers ( Figure 5 , lanes 1 to 7); and the multiplex PCR method based on universal primers provided by the present invention has substantially the same amp...

Embodiment 3

[0080]Example 3: Comparison of the coverage of clone libraries prepared by multiple primers based on universal sequence adapters and degenerate primers

[0081] The designed universal sequence adapter specific primers UJSPs and degenerate primer alkBwf / r were used to amplify the alkB fragment from hydrocarbon oxidizing bacteria and non-hydrocarbon oxidizing bacteria respectively, and the amplification effects of the two methods were compared.

[0082] Formation water samples from oil reservoirs and 9 strains of hydrocarbon oxidizing bacteria (Marineella MCCC 1A00091, Acinetobacter haemolyticus CCTCC NK2.BH-7, Acinetobacter haemolyticus CCTCC NK2. Rhodococcus CGMCC4.1818, Rhodococcus roseus CGMCC 4.1480, Rhodococcus erythropolis CCTCC NK2.53968, Rhodococcus erythropolis CCTCC NK2.53968, Rhodococcus erythropolis NK2.CCTCC T7-2, Rhodococcus erythroflates CCTCC NK2.T1- 3) alkB fragment was amplified in a mixed bacterial sample composed of two strains of non-hydrocarbon oxidizing b...

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Abstract

The invention discloses a method for accurately quantifying an alkane hydroxylase gene alkB in one step and an application of the method for accurately quantifying the alkane hydroxylase gene alkB in one step. In the environment detecting process, the genes for coding the alkane hydroxylase gene alkB are various and difficult to be accurately quantified in one step. The method comprises the following steps: firstly, pre-amplifying a target gene by a special primer of a common sequence joint, so that the pre-amplifying product of the target gene has the common sequence section; and then, realizing quantitative analysis of the pre-amplifying product by taking the common sequence as a primer. The method can be used for successfully solving the problems of poor quantitative result accuracy and poor quantitative result comparability due to primer amplification efficiency difference, nonspecific amplification, primer dimer and the like in the common multiplex real time quantitative PCR (polymerase chain reaction) technology, and further can be used for accurately performing one-step quantifying on the gross of a plurality of target genes in the PCR system through one-step PCR reaction. The method is simple, convenient and quick in operation, high in specificity, accurate and reliable in detected result and high in detection flexibility, and has lowest detection concentration of 1*10<2> copies / ml.

Description

technical field [0001] The invention belongs to the field of molecular ecological technology and biotechnology, in particular, it relates to a multiple real-time fluorescence quantitative PCR method based on a universal sequence, and its application in the quantitative detection of alkane hydroxylase gene alkB. Background technique [0002] There are a large number of hydrocarbon-degrading strains in oil reservoirs and oil-contaminated environments. They can grow and metabolize petroleum hydrocarbons as carbon sources and produce biological surface active substances, thereby achieving enhanced oil recovery or removing petroleum pollutants in the environment. It has been found that more than 100 genera of microorganisms can directly or assist in the degradation of one or more petroleum hydrocarbons, such microorganisms mainly include: Pseudomonas, Acinetobacter, Achromobacter, Nocardia, Bacillus, Micrococcus, Actinomycet, Aureobasidium, Candida, Rhodotorula and Penicillium et...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6851C12Q1/6855C12Q2525/191C12Q2531/113C12Q2545/113C12Q2563/107
Inventor 马挺李国强高梦黎田会梅高配科陈兆会
Owner NANKAI UNIV
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