Method for detecting six functional genes of Cr(VI) reducing complex microorganisms through multiple real-time fluorescent PCR

A real-time fluorescent, functional gene technology, applied in the field of microbial detection, can solve the problems of low sensitivity, time-consuming and labor-intensive, and inapplicable expression quantification.

Active Publication Date: 2017-05-24
CENT SOUTH UNIV
View PDF0 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although Northern blotting can detect the size of the target RNA, its operation is cumbersome, time-consuming and labor-intensive; in situ hydridisation is complex; RNAse protection assays are mainly suitable for mapping transcription start and end sites and intron/exon boundaries, And it is used to distinguish related mRNAs of similar size, which is not suitable for quantification of expression; c

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for detecting six functional genes of Cr(VI) reducing complex microorganisms through multiple real-time fluorescent PCR
  • Method for detecting six functional genes of Cr(VI) reducing complex microorganisms through multiple real-time fluorescent PCR
  • Method for detecting six functional genes of Cr(VI) reducing complex microorganisms through multiple real-time fluorescent PCR

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] The preparation of embodiment 1 plasmid standard

[0058] Step 1: Primer Design and Synthesis

[0059] Corresponding primers were designed according to the DNA sequence of the gene. The designed common PCR primers (Table 1) for six genes of HcrR, OmcA, ChrR, 16s rDNA (BB), 16s rDNA (MR-1) and 16s rDNA (F1) were provided by Shanghai Synthesized by Sangon Biotechnology Co., Ltd., the synthetic amount is 2OD.

[0060] Common PCR primers involved in the present invention in table 1

[0061]

[0062] Step 2: Bacterial Total RNA Extraction

[0063] Take 1 mL of the culture fluid of the three strains containing specific genes and place them in a 1.5 mL centrifuge tube, and use the RNAprep PureCell / Bacteria Kit (Tiangen (Beijing)) to extract the bacterial total RNA.

[0064] Step 3: Synthesize cDNA by reverse transcription

[0065]Reverse transcription reaction: Add 1 μL of the total RNA template prepared in the previous step to the RNase-Free 0.2mL PCR tube in turn, per...

Embodiment 2

[0070] Embodiment 2 Multiplex real-time fluorescent PCR detects the specificity experiment of six kinds of specific genes

[0071] Specific detection of a single gene in a multiplex system:

[0072] According to the reaction system of Roche LightCycler Nano 32-well fluorescent detection polymerase chain reaction (PCR) instrument, 4 identical multiplex PCR reaction solutions were prepared for the Cr(VI) reduced gene, and 1 μL of known amounts of HcrR and OmcA were added separately. , ChrR standard plasmids, and set up a negative control; for 16s rDNA, prepare 4 identical multiplex PCR reaction solutions, respectively add 1 μL of known amounts of 16s rDNA (BB), 16s rDNA (MR-1), 16s rDNA Set up a negative control for the standard plasmid of rDNA (F1).

[0073] The Cr(VI) reducing gene reaction system is 2×FastStart Essential DNA Green Master 25 μL, the final concentrations of the upstream and downstream primers of HcrR, OmcA, and ChrR genes are 0.05 μM, 0.2 μM, and 0.2 μM, respe...

Embodiment 3

[0076] Embodiment 3 Multiplex real-time fluorescent PCR detects the stability experiment of six kinds of specific genes

[0077] The six genes were operated as follows:

[0078] Take 3 known plasmid standards and a negative control (water without RNase enzyme), and perform repeated tests within and between batches, respectively. The intra-batch repeat test is to carry out the real-time fluorescence detection PCR experiment on 3 samples at the same time; the inter-batch repeat test is to carry out the real-time fluorescence detection PCR experiment at 3 different times (interval 3 days). The average coefficient of variation (CV) of repeated experiments within and between batches of several genes was less than 1%.

[0079] Experimental results show that the detection method of the present invention has good stability.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention provides a method for detecting six functional genes of Cr(VI) reducing complex microorganisms through multiple real-time fluorescent PCR. The complex microorganisms include a Pannonibacter phragmitetus BB microorganism with the collection number of CGMCC No. 3052, a Shewanella oneidensis MR-1 microorganism with the collection number of ATCC No. 700550 and a Pseudomonas putida F1 microorganism with the collection number of ATCC No. 700007. The six functional genes include a Cr (VI) reducing functional gene HcrR of the BB microorganism, a Cr (VI) reductase gene OmcA of the MR-1 microorganism and a Cr (VI) reductase gene ChrR of the F1 microorganism as well as 16s rDNA genes of the three microorganisms. The invention provides a simple, quick and low-cost method for detecting dynamic changes of expressions of the six functional genes by a multiple fluorescent PCR technology. The detection method is high in sensivity and specificity and the detection result is accurate.

Description

technical field [0001] The invention relates to the technical field of microorganism detection, in particular to a method for detecting six functional genes of Cr(VI)-reduced composite flora by multiplex real-time fluorescent PCR. Background technique [0002] In heavy metal pollution, Cr(VI) poses a great threat to human, animal and plant life safety due to its high toxicity, strong oxidative property and high carcinogenicity, among which Cr(VI) soil pollution is the most prominent problem. According to statistics, the area of ​​soil seriously polluted by Cr(VI) in my country is as high as 5 million square meters, and the amount of polluted earth is about 15 million cubic meters. Therefore, reasonable and effective remediation of Cr(VI)-contaminated soil in my country is imminent. [0003] At present, the reduction of Cr(VI) by microorganisms has become a hotspot in the remediation of Cr(VI)-contaminated soils. The composite Cr(VI)-reducing flora of Pannonibacterphragmite...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/6851C12Q2561/113C12Q2537/143C12Q2563/107C12Q2545/113
Inventor 廖骐丁春连柴立元杨志辉闵小波颜旭唐崇俭王海鹰杨卫春李青竹
Owner CENT SOUTH UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap