Primer pair and standard substance for detecting mycobacteria and application thereof

A technology of mycobacteria and primer pairs, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of gas phase mass spectrometry and liquid phase mass spectrometry, which are expensive and not suitable for routine detection, and achieve detection results Accurate, rapid detection, good specificity

Inactive Publication Date: 2013-05-08
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

GC-MS and LC-MS analysis require expensive instrumentation and are not suitable for routine detection

Method used

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  • Primer pair and standard substance for detecting mycobacteria and application thereof
  • Primer pair and standard substance for detecting mycobacteria and application thereof
  • Primer pair and standard substance for detecting mycobacteria and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1, the preparation of specific primer pair and standard plasmid

[0032] 1. Preparation of standard plasmids

[0033] 1. Analyze the hsp65 gene sequences of all mycobacteria in NCBI, and obtain the DNA fragment shown in sequence 1 of the sequence table based on the analysis results. Sequence 1 has more than 98% homology with the hsp65 genes of various mycobacteria in NCBI, and has no homology with other genes. See Table 1 for partial homology comparison results.

[0034] Table 1 Homology comparison results between sequence 1 and some mycobacterial hsp65 genes

[0035]

[0036] 2. Synthesize the double-stranded DNA molecule shown in sequence 1 of the sequence listing.

[0037] 3. Insert the double-stranded DNA molecule synthesized in step 2 18-T Vector (TaKaRa Code: D101A) to obtain the recombinant plasmid pMD-18T-hsp65 (standard plasmid).

[0038] 2. Preparation of specific primer pairs

[0039] A pair of specific primers were designed based on seque...

Embodiment 2

[0043] Embodiment 2, establishment of mycobacterium quantitative PCR detection method

[0044] Mycobacterium fortuitum subsp.fortuitum: CGMCC number 1.513.

[0045] 1. Optimization of annealing temperature

[0046] 1. Extract the genomic DNA of Mycobacterium fortuitum.

[0047] 2. Using the genomic DNA extracted in step 1 as a template, using a primer pair composed of hsp65-F and hsp65-R, using Premix ExTaq TM (Code:DRR041S) and perform quantitative qPCR according to the instructions.

[0048] qPCR reaction system (20 μL): Premix ExTaq TM 10 μL, hsp65-F, hsp65-R, genomic DNA, make up the volume to 20 μL with double distilled water. The initial concentrations of hsp65-F and hsp65-R in the reaction system were both 0.2μmol / L.

[0049] The reaction program of qPCR: (95°C, 5min) × 1 cycle; (95°C, 5s, annealing 30s, 72°C, 30s) × 40 cycles; fluorescence is collected during annealing; the process of melting curve is : 95°C, 1min, 65°C, 1min, starting from 65°C, the temperat...

Embodiment 3

[0057] Embodiment 3, the specificity experiment of specific primer pair

[0058] 1. Experimental samples

[0059] The experimental samples are as follows:

[0060] Escherichia coli (Escherichia coli): CGMCC number is 1.1369.

[0061] Salmonella typhimurium (Salmonella typhimurium): CGMCC number is 1.1194.

[0062] Enterococcus faecalis: CGMCC number is 1.2135.

[0063] Mycobacterium fortuitum subsp.fortuitum: CGMCC number 1.513.

[0064] Mycobacterium smegmatis (Mycobacterium smegmatis): CGMCC number is 1.2621.

[0065] Mycobacterium phlei: CGMCC number is 4.1180.

[0066] Mycobacterium diernhoferi: CGMCC number is 4.1179.

[0067] Mycobacterium vaccae: CGMCC number 4.1181.

[0068] 2. Specificity experiment

[0069] 1. Extract the genomic DNA of each experimental sample.

[0070] 2. Using the genomic DNA extracted in step 1 as a template, using a primer pair composed of hsp65-F and hsp65-R, using Premix ExTaq TM Quantitative qPCR was performed according to the ins...

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Abstract

The invention discloses a primer pair and a standard substance for detecting mycobacteria and applications thereof. The primer pair is formed by DNA (Deoxyribose Nucleic Acid) respectively shown in a sequence 2 and a sequence 3. The standard substance is a double-stranded DNA molecule shown in the sequence 1 or a plasmid containing the double-stranded DNA molecule shown in the sequence 1 in a sequence table. The special primer pair having the good specificity for the mycobacteria and the standard substance having the generality for mycobacterial strains are provided, so that the special primer pair and the standard substance can be used for the auxiliary identification of the mycobacteria and the quantitative detection of the content of the mycobacteria in a liquid sample solution to be detected or a solid sample solution to be detected by means of fluorescence. The primer pair and the standard substance have the advantages of being high in sensitivity, good in specificity, rapid in detection and the like, and can detect bacteria which cannot be cultured, thereby obtaining a more accurate detection result. Therefore, the technical support is provided for the safety of drinking water and recycled water and the like.

Description

technical field [0001] The present invention relates to a pair of primers and a standard for detecting mycobacteria and their applications. Background technique [0002] Most mycobacteria are opportunistic pathogens and often exist in natural water bodies, engineering water supply systems and indoor water supply and drainage pipes. At present, more than 150 kinds of mycobacteria have been isolated from the environment, and the number is still increasing. They can cause a variety of infectious diseases in the human population. [0003] At present, the culture method is the gold standard method for detecting mycobacteria. It is accurate and reliable, but it takes a long time (4-8 weeks), and many mycobacteria cannot be cultured. one. Fluorescent quantitative PCR (qPCR) method refers to the method of adding fluorescent groups to the PCR reaction system, using fluorescence accumulation to monitor the entire PCR process in real time, and finally quantitatively analyzing the unk...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12R1/32
Inventor 李丹林怡雯何苗
Owner TSINGHUA UNIV
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