Method for quantitative determination of escherichia coli RNA, and specialized standard substance and application thereof

A quantitative detection technology for Escherichia coli, which is applied in the direction of DNA/RNA fragments, recombinant DNA technology, microbial measurement/inspection, etc. It can solve the problems of not considering the reverse transcription efficiency, underestimating the RNA content, etc., and achieve a linear detection range Wide, simple preparation method, good purity effect

Inactive Publication Date: 2013-02-20
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the use of DNA as a standard did not take into account the efficiency of reverse transcription, resulting in 84%-98.6% less detected RNA compared to the actual
Therefore, using DNA as a standard for detecting RNA will greatly underestimate the actual RNA content in the sample

Method used

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  • Method for quantitative determination of escherichia coli RNA, and specialized standard substance and application thereof
  • Method for quantitative determination of escherichia coli RNA, and specialized standard substance and application thereof
  • Method for quantitative determination of escherichia coli RNA, and specialized standard substance and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1, the synthesis of primer and the preparation of standard

[0039] 1. Primer design and synthesis

[0040] The genomic DNA of Escherichia coli was analyzed, the partial sequence of uidA gene was selected, and two pairs of primers (primer pair A and primer pair B) were designed according to the sequence.

[0041] Primer pair A is composed of uidA-T7-F and uidA-T7-R, and the target sequence is the double-stranded DNA molecule shown in sequence 1 of the sequence listing, which is 921bp.

[0042] uidA-T7-F (sequence 2): 5'-taatacgactcactataggggcgttacaagaaagcc-3';

[0043] uidA-T7-R (SEQ ID NO: 3): 5-gcatctcttcagcgtaagggtaatgcga-3'.

[0044] Primer pair B is composed of uidA-F and uidA-R, and the target sequence is the double-stranded DNA molecule shown in the 257th to 443rd nucleotides from the 5' end of sequence 1 in the sequence listing, which is 187bp.

[0045] uidA-F (sequence 4): 5'-cgatgtcacgccgtatgttatt-3';

[0046] uidA-R (SEQ ID NO: 5): 5'-ggtgtagag...

Embodiment 2

[0057] Embodiment 2, gradient dilution of standard substance

[0058] Method for detecting RNA copy number: Use an ultra-micro nucleic acid protein analyzer (NanoDrop ND-2000C, the United States) to measure the RNA concentration, and then calculate the RNA copy number according to the following formula:

[0059]

[0060] 6.02×10 23 is Avogadro's constant; 340 (Da) is the relative molecular mass of one base of RNA.

[0061] The standard substance prepared in Example 1 was sequentially diluted with sterile water to obtain dilutions 1 to 8, in which the concentration of RNA was: 9.6186×10 8 Copy number / μL, 9.6186×10 7 Copy number / μL, 9.6186×10 6 Copy number / μL, 9.6186×10 5 Copy number / μL, 9.6186×10 4 Copy number / μL, 9.6186×10 3 Copy number / μL, 9.6186×10 2 Copy number / μL and 9.6186×10 1 Copy number / μL.

Embodiment 3

[0062] Embodiment 3, the establishment of the method for the quantitative detection Escherichia coli living bacteria RNA

[0063] 1. Optimization of primer annealing temperature

[0064] 1. Using the cDNA after reverse transcription of the standard product obtained in Example 1 as a template, use the primers synthesized in Example 1 to perform PCR amplification on B.

[0065] The PCR reaction system is (20μL): 10×PCR Buffer 2μL, 25mM MgCl 2 1.6 μL, 1.6 μL of 10 mM dNTP, 1 μL of upstream primer (20 μM), 1 μL of downstream primer (20 μM), 2 μL of template (about 100 ng), 0.4 μL of Taq DNA polymerase (5U / μL), 10.4 μL of deionized water.

[0066] PCR reaction program: 95°C for 10min; 95°C for 30s, annealing for 20s, 72°C for 20s, 40 cycles; 72°C for 5min; store at 4°C after the reaction. 51°C, 52°C, 53.7°C, 54.4°C, and 55.5°C were used as annealing temperatures, respectively.

[0067] 2. Perform 2% agarose gel electrophoresis on the PCR amplified product of step 1, the results ...

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PUM

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Abstract

The invention discloses a method for quantitative determination of escherichia coli RNA, and a specialized standard substance and an application thereof. The method comprises the steps of (1) making a standard curve according to the following method, preparing standard substance diluents with different concentrations by using a RNA standard substance; reversely transcribing the standard substance diluents with different concentrations into cDNA; performing real-time fluorescence quantification PCR by using cDNA as templates; making the standard curve equation by using RNA copy number (or data-processed copy number, for example, log-base 10 of the copy number) corresponding to cDNA in the real-time fluorescence quantification PCR system and Ct values; and (2) extracting a total RNA of the escherichia coli, reversely transcribing the extracted total RNA into cDNA; performing the real-time fluorescence quantification PCR by using cDNA as the templates; and substituting the Ct values to the standard curve equation to obtain the RNA content of the escherichia coli. The method can be used for detecting small amount of the escherichia coli in the environment rapidly, and provides technical support for rapid detection of the active escherichia coli.

Description

technical field [0001] The invention relates to a method for quantitatively detecting Escherichia coli RNA, a special standard product and application thereof. Background technique [0002] Escherichia coli (Escherichia coli) is a foodborne pathogen that can cause symptoms such as diarrhea, nausea, and hemorrhagic colitis. In recent years, infectious diseases caused by Escherichia coli have been reported in many countries, and have become a worldwide environmental safety problem. [0003] At present, the traditional detection method of E. coli is the culture method. However, recent studies have found that bacteria can enter a "viable but nonculturable (VBNC)" state under environmental pressure (such as high temperature), which cannot be detected by conventional culture methods, but still maintains metabolic activity and pathogenicity. pathogenicity and are able to restore their culturability under conditions of removal of environmental stress, posing a serious microbiologi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/10C12Q1/06C12N15/11G01N21/64
Inventor 何苗林怡雯李丹吴舒旭
Owner TSINGHUA UNIV
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